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PMC:2800179 / 865-17770 JSONTXT

Annnotations TAB JSON ListView MergeView

ICD10

Id Subject Object Predicate Lexical cue
T7121 15830-15838 http://purl.bioontology.org/ontology/ICD10/T14.9 denotes injuries

GO-CC

Id Subject Object Predicate Lexical cue
T601 29-33 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T602 72-77 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T603 159-164 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T604 408-413 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T606 320-328 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T607 320-328 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T1992 1819-1823 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1993 1955-1959 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1994 2359-2363 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1995 2395-2399 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1996 2879-2883 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1997 3235-3239 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T2807 5688-5692 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T3977 7797-7801 http://purl.obolibrary.org/obo/GO_0005623 denotes Cell
T3978 8354-8358 http://purl.obolibrary.org/obo/GO_0005623 denotes Cell
T3979 8512-8520 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T3980 8512-8520 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T4227 8593-8597 http://purl.obolibrary.org/obo/GO_0005623 denotes Cell
T4228 8894-8898 http://purl.obolibrary.org/obo/GO_0005623 denotes Cell
T4229 9040-9045 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4547 9289-9293 http://purl.obolibrary.org/obo/GO_0005623 denotes Cell
T4548 9366-9371 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4549 9503-9508 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4550 9625-9630 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4551 9873-9878 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4552 10090-10095 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4553 9289-9301 http://purl.obolibrary.org/obo/GO_0009986 denotes Cell Surface
T4554 9659-9669 http://purl.obolibrary.org/obo/GO_0019815 denotes antibodies
T4555 9759-9767 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T4556 9659-9669 http://purl.obolibrary.org/obo/GO_0042571 denotes antibodies
T4557 9759-9767 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T4715 10139-10144 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4716 10374-10378 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T5447 11882-11887 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6420 12163-12168 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6421 13180-13185 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6422 12720-12728 http://purl.obolibrary.org/obo/GO_0016020 denotes membrane
T6423 13545-13553 http://purl.obolibrary.org/obo/GO_0016020 denotes membrane
T6424 13593-13602 http://purl.obolibrary.org/obo/GO_0016020 denotes Membranes
T6425 13689-13698 http://purl.obolibrary.org/obo/GO_0016020 denotes Membranes
T6426 13926-13935 http://purl.obolibrary.org/obo/GO_0016020 denotes Membranes
T6427 14459-14468 http://purl.obolibrary.org/obo/GO_0016020 denotes membranes
T6428 12783-12791 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T6429 12884-12892 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T6430 13747-13755 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T6431 13805-13813 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T6432 14228-14236 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T6433 12783-12791 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T6434 12884-12892 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T6435 13747-13755 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T6436 13805-13813 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T6437 14228-14236 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody

GO-MF

Id Subject Object Predicate Lexical cue
T599 320-328 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T1991 1904-1908 http://purl.obolibrary.org/obo/GO_0005153 denotes IL-8
T3641 7526-7529 http://purl.obolibrary.org/obo/GO_0004601 denotes MPO
T3642 7585-7588 http://purl.obolibrary.org/obo/GO_0004601 denotes MPO
T3643 7710-7713 http://purl.obolibrary.org/obo/GO_0004601 denotes MPO
T3976 8512-8520 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T4226 9200-9202 http://purl.obolibrary.org/obo/GO_0033968 denotes CA
T4544 9588-9595 http://purl.obolibrary.org/obo/GO_0005488 denotes binding
T4545 9659-9669 http://purl.obolibrary.org/obo/GO_0003823 denotes antibodies
T4546 9759-9767 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T5200 10443-10445 http://purl.obolibrary.org/obo/GO_0003964 denotes RT
T5201 10624-10637 http://purl.obolibrary.org/obo/GO_0003899 denotes transcriptase
T5202 10624-10637 http://purl.obolibrary.org/obo/GO_0003968 denotes transcriptase
T5203 10624-10637 http://purl.obolibrary.org/obo/GO_0034062 denotes transcriptase
T5442 12034-12053 http://purl.obolibrary.org/obo/GO_0045289 denotes Luciferase activity
T5443 12034-12053 http://purl.obolibrary.org/obo/GO_0047077 denotes Luciferase activity
T5444 12034-12053 http://purl.obolibrary.org/obo/GO_0047712 denotes Luciferase activity
T5445 12034-12053 http://purl.obolibrary.org/obo/GO_0050248 denotes Luciferase activity
T5446 12034-12053 http://purl.obolibrary.org/obo/GO_0050397 denotes Luciferase activity
T6413 12783-12791 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T6414 12884-12892 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T6415 13747-13755 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T6416 13805-13813 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T6417 14228-14236 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T6418 12819-12821 http://purl.obolibrary.org/obo/GO_0033968 denotes CA
T6419 14499-14502 http://purl.obolibrary.org/obo/GO_0004707 denotes ERK
T7120 15365-15389 http://purl.obolibrary.org/obo/GO_0004601 denotes myeloperoxidase activity
T15792 16566-16569 http://purl.obolibrary.org/obo/GO_0004601 denotes MPO

GO-BP

Id Subject Object Predicate Lexical cue
T585 0-8 http://purl.obolibrary.org/obo/GO_0009056 denotes Degraded
T586 503-511 http://purl.obolibrary.org/obo/GO_0009056 denotes degraded
T589 376-385 http://purl.obolibrary.org/obo/GO_0046903 denotes secretion
T595 29-47 http://purl.obolibrary.org/obo/GO_0008283 denotes cell proliferation
T596 81-89 http://purl.obolibrary.org/obo/GO_0051318 denotes G1 phase
T597 240-260 http://purl.obolibrary.org/obo/GO_0070487 denotes monocyte aggregation
T598 448-464 http://purl.obolibrary.org/obo/GO_0051092 denotes NF-κB activation
T1955 668-677 http://purl.obolibrary.org/obo/GO_0051923 denotes sulphated
T1956 2501-2510 http://purl.obolibrary.org/obo/GO_0051923 denotes sulphated
T1957 2736-2745 http://purl.obolibrary.org/obo/GO_0051923 denotes sulphated
T1958 1177-1185 http://purl.obolibrary.org/obo/GO_0009056 denotes degraded
T1959 3656-3664 http://purl.obolibrary.org/obo/GO_0009056 denotes degraded
T1960 1217-1229 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T1961 1708-1720 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T1962 2114-2126 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T1963 2557-2569 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T1964 2636-2648 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T1965 2673-2685 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T1966 2840-2852 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T1967 2400-2427 http://purl.obolibrary.org/obo/GO_0006954 denotes response to an inflammatory
T1968 3585-3606 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammatory response
T1969 1583-1592 http://purl.obolibrary.org/obo/GO_0007586 denotes digestion
T1970 1904-1919 http://purl.obolibrary.org/obo/GO_0032677 denotes IL-8 production
T1971 1904-1919 http://purl.obolibrary.org/obo/GO_0032637 denotes IL-8 production
T1972 1933-1959 http://purl.obolibrary.org/obo/GO_0061582 denotes intestinal epithelial cell
T1973 1933-1959 http://purl.obolibrary.org/obo/GO_0060574 denotes intestinal epithelial cell
T1974 1933-1959 http://purl.obolibrary.org/obo/GO_0060575 denotes intestinal epithelial cell
T1975 1933-1959 http://purl.obolibrary.org/obo/GO_0060576 denotes intestinal epithelial cell
T1976 1993-2014 http://purl.obolibrary.org/obo/GO_0051092 denotes κB (NF-κB) activation
T1977 3473-3489 http://purl.obolibrary.org/obo/GO_0051092 denotes NF-κB activation
T1978 2038-2051 http://purl.obolibrary.org/obo/GO_0006351 denotes transcription
T1979 2078-2097 http://purl.obolibrary.org/obo/GO_0010467 denotes expression of genes
T1980 2359-2372 http://purl.obolibrary.org/obo/GO_0007155 denotes cell-adhesion
T1981 2359-2382 http://purl.obolibrary.org/obo/GO_0060352 denotes cell-adhesion molecules
T1982 2388-2408 http://purl.obolibrary.org/obo/GO_0002418 denotes immune cell response
T1983 2388-2408 http://purl.obolibrary.org/obo/GO_0002443 denotes immune cell response
T1984 2400-2436 http://purl.obolibrary.org/obo/GO_0002437 denotes response to an inflammatory stimulus
T1985 2859-2878 http://purl.obolibrary.org/obo/GO_0001865 denotes T-lymphocyte and NK
T1986 2859-2878 http://purl.obolibrary.org/obo/GO_0001866 denotes T-lymphocyte and NK
T1987 2859-2878 http://purl.obolibrary.org/obo/GO_0051132 denotes T-lymphocyte and NK
T1988 3235-3253 http://purl.obolibrary.org/obo/GO_0008283 denotes cell proliferation
T1989 3321-3341 http://purl.obolibrary.org/obo/GO_0070487 denotes monocyte aggregation
T1990 3379-3388 http://purl.obolibrary.org/obo/GO_0046903 denotes secretion
T2804 3795-3803 http://purl.obolibrary.org/obo/GO_0009056 denotes Degraded
T2805 3836-3844 http://purl.obolibrary.org/obo/GO_0009056 denotes degraded
T2806 5305-5313 http://purl.obolibrary.org/obo/GO_0051923 denotes sulphate
T3085 5941-5949 http://purl.obolibrary.org/obo/GO_0009056 denotes Degraded
T3086 5993-6001 http://purl.obolibrary.org/obo/GO_0007631 denotes drinking
T3087 6359-6367 http://purl.obolibrary.org/obo/GO_0007631 denotes drinking
T3634 7150-7162 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T3635 7255-7267 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T3636 7377-7386 http://purl.obolibrary.org/obo/GO_0051923 denotes sulphated
T3637 7526-7529 http://purl.obolibrary.org/obo/GO_0004601 denotes MPO
T3638 7585-7588 http://purl.obolibrary.org/obo/GO_0004601 denotes MPO
T3639 7710-7713 http://purl.obolibrary.org/obo/GO_0004601 denotes MPO
T3640 7742-7753 http://purl.obolibrary.org/obo/GO_0009056 denotes degradation
T3974 8261-8277 http://purl.obolibrary.org/obo/GO_0098743 denotes cell aggregation
T3975 8266-8288 http://purl.obolibrary.org/obo/GO_0070487 denotes aggregation, monocytes
T4223 8593-8603 http://purl.obolibrary.org/obo/GO_0007049 denotes Cell Cycle
T4224 8640-8646 http://purl.obolibrary.org/obo/GO_0040007 denotes growth
T4225 9200-9202 http://purl.obolibrary.org/obo/GO_0033968 denotes CA
T4714 10374-10386 http://purl.obolibrary.org/obo/GO_0001906 denotes cell killing
T5196 10443-10445 http://purl.obolibrary.org/obo/GO_0003964 denotes RT
T5197 10624-10637 http://purl.obolibrary.org/obo/GO_0003899 denotes transcriptase
T5198 10624-10637 http://purl.obolibrary.org/obo/GO_0003968 denotes transcriptase
T5199 10624-10637 http://purl.obolibrary.org/obo/GO_0034062 denotes transcriptase
T5436 11586-11599 http://purl.obolibrary.org/obo/GO_0006351 denotes Transcription
T5437 12034-12053 http://purl.obolibrary.org/obo/GO_0045289 denotes Luciferase activity
T5438 12034-12053 http://purl.obolibrary.org/obo/GO_0047077 denotes Luciferase activity
T5439 12034-12053 http://purl.obolibrary.org/obo/GO_0047712 denotes Luciferase activity
T5440 12034-12053 http://purl.obolibrary.org/obo/GO_0050248 denotes Luciferase activity
T5441 12034-12053 http://purl.obolibrary.org/obo/GO_0050397 denotes Luciferase activity
T6409 12309-12314 http://purl.obolibrary.org/obo/GO_0019835 denotes lysis
T6410 12469-12477 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T6411 12819-12821 http://purl.obolibrary.org/obo/GO_0033968 denotes CA
T6412 14499-14502 http://purl.obolibrary.org/obo/GO_0004707 denotes ERK
T7112 14901-14909 http://purl.obolibrary.org/obo/GO_0009056 denotes Degraded
T7113 14977-14985 http://purl.obolibrary.org/obo/GO_0009056 denotes degraded
T7114 14929-14941 http://purl.obolibrary.org/obo/GO_0006954 denotes Inflammation
T7115 15328-15340 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T7116 15516-15528 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T7117 15605-15617 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T7118 15365-15389 http://purl.obolibrary.org/obo/GO_0004601 denotes myeloperoxidase activity
T7119 16001-16010 http://purl.obolibrary.org/obo/GO_0051923 denotes sulphated
T8025 0-8 http://purl.obolibrary.org/obo/GO_0009056 denotes Degraded
T8026 16867-16883 http://purl.obolibrary.org/obo/GO_0032680 denotes TNF-α Production
T8035 16867-16883 http://purl.obolibrary.org/obo/GO_0032640 denotes TNF-α Production
T15785 16366-16374 http://purl.obolibrary.org/obo/GO_0009056 denotes Degraded
T15786 16448-16456 http://purl.obolibrary.org/obo/GO_0009056 denotes degraded
T15787 16619-16627 http://purl.obolibrary.org/obo/GO_0009056 denotes degraded
T15788 16665-16673 http://purl.obolibrary.org/obo/GO_0009056 denotes degraded
T15789 16393-16405 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T15790 16520-16532 http://purl.obolibrary.org/obo/GO_0006954 denotes inflammation
T15791 16566-16569 http://purl.obolibrary.org/obo/GO_0004601 denotes MPO

2_test

Id Subject Object Predicate Lexical cue
20072622-3943102-93233346 1326-1327 3943102 denotes 4
20072622-2570843-93233347 1346-1347 2570843 denotes 5
20072622-3943102-93233348 1418-1419 3943102 denotes 4
20072622-11675262-93233349 1423-1424 11675262 denotes 6
20072622-17034924-93233350 1427-1428 17034924 denotes 8
20072622-8849638-93233351 1644-1646 8849638 denotes 11
20072622-9178362-93233352 1650-1652 9178362 denotes 12
20072622-17095757-93233353 2016-2018 17095757 denotes 13
20072622-3140380-93233354 2128-2130 3140380 denotes 15
20072622-8011280-93233355 2134-2136 8011280 denotes 16
20072622-9136827-93233356 2231-2233 9136827 denotes 17
20072622-1316857-93233357 2656-2658 1316857 denotes 18
20072622-7557106-93233358 2662-2664 7557106 denotes 19
20072622-8741008-93233359 2810-2812 8741008 denotes 20
20072622-8741008-93233360 2895-2897 8741008 denotes 20
20072622-4212170-93233361 6412-6414 4212170 denotes 23
20072622-1688816-93233362 6769-6771 1688816 denotes 24
20072622-9824605-93233363 7307-7309 9824605 denotes 25
20072622-10940278-93233364 7313-7315 10940278 denotes 26
20072622-6092199-93233365 7567-7569 6092199 denotes 27
20072622-8482919-93233366 8252-8254 8482919 denotes 28
20072622-3782828-93233367 10394-10396 3782828 denotes 29
20072622-8625304-93233368 11942-11944 8625304 denotes 30
20072622-19342628-93233369 14517-14519 19342628 denotes 32

pmc-enju-pas

Id Subject Object Predicate Lexical cue
T220 0-8 VB denotes Degraded
T221 9-12 NN denotes CGN
T222 13-22 VB denotes inhibited
T223 23-28 NN denotes THP-1
T224 29-33 NN denotes cell
T225 34-47 NN denotes proliferation
T226 48-50 FW denotes in
T227 51-56 FW denotes vitro
T228 56-57 -COMMA- denotes ,
T229 58-67 VB denotes arresting
T230 68-71 DT denotes the
T231 72-77 NN denotes cells
T232 78-80 IN denotes in
T233 81-83 NN denotes G1
T234 84-89 NN denotes phase
T235 91-93 IN denotes In
T236 94-102 NN denotes addition
T237 102-103 -COMMA- denotes ,
T238 104-108 NN denotes dCGN
T239 109-118 VB denotes increased
T240 119-125 NN denotes ICAM-1
T241 126-136 NN denotes expression
T242 137-139 IN denotes in
T243 140-144 CC denotes both
T244 145-148 NN denotes PBM
T245 149-152 CC denotes and
T246 153-158 NN denotes THP-1
T247 159-164 NN denotes cells
T248 165-169 IN denotes with
T249 170-171 DT denotes a
T250 172-177 JJ denotes major
T251 178-184 NN denotes effect
T252 185-189 VB denotes seen
T253 190-195 IN denotes after
T254 196-198 CD denotes 40
T255 199-202 NN denotes kDa
T256 203-207 NN denotes dCGN
T257 208-216 NN denotes exposure
T258 218-222 RB denotes Also
T259 222-223 -COMMA- denotes ,
T260 224-228 NN denotes dCGN
T261 229-239 VB denotes stimulated
T262 240-248 NN denotes monocyte
T263 249-260 NN denotes aggregation
T264 261-263 FW denotes in
T265 264-269 FW denotes vitro
T266 270-274 WDT denotes that
T267 275-278 VB denotes was
T268 279-288 VB denotes prevented
T269 289-291 IN denotes by
T270 292-302 NN denotes incubation
T271 303-307 IN denotes with
T272 308-319 JJ denotes anti-ICAM-1
T273 320-328 NN denotes antibody
T274 330-337 RB denotes Finally
T275 337-338 -COMMA- denotes ,
T276 339-343 NN denotes dCGN
T277 344-354 VB denotes stimulated
T278 355-360 NN denotes TNF-α
T279 361-371 NN denotes expression
T280 372-375 CC denotes and
T281 376-385 NN denotes secretion
T282 386-388 IN denotes by
T283 389-393 CC denotes both
T284 394-397 NN denotes PBM
T285 398-401 CC denotes and
T286 402-407 NN denotes THP-1
T287 408-413 NN denotes cells
T288 415-418 PDT denotes All
T289 419-424 DT denotes these
T290 425-432 NN denotes effects
T291 433-437 VB denotes were
T292 438-444 VB denotes linked
T293 445-447 TO denotes to
T294 448-453 NN denotes NF-κB
T295 454-464 NN denotes activation
T296 466-471 DT denotes These
T297 472-476 NN denotes data
T298 477-485 RB denotes strongly
T299 486-493 VB denotes suggest
T300 494-498 IN denotes that
T301 499-502 DT denotes the
T302 503-511 JJ denotes degraded
T303 512-517 NN denotes forms
T304 518-520 IN denotes of
T305 521-524 NN denotes CGN
T306 525-529 VB denotes have
T307 530-531 DT denotes a
T308 532-542 JJ denotes pronounced
T309 543-549 NN denotes effect
T310 550-552 IN denotes on
T311 553-562 NN denotes monocytes
T312 562-563 -COMMA- denotes ,
T313 564-578 JJ denotes characteristic
T314 579-581 IN denotes of
T315 582-584 DT denotes an
T316 585-597 JJ denotes inflammatory
T317 598-607 NN denotes phenotype
T776 623-634 NN denotes Carrageenan
T777 635-636 -LRB- denotes (
T778 636-639 NN denotes CGN
T779 639-640 -RRB- denotes )
T780 641-643 VB denotes is
T781 644-645 DT denotes a
T782 646-650 JJ denotes high
T783 651-660 JJ denotes molecular
T784 661-667 NN denotes weight
T785 668-677 VB denotes sulphated
T786 678-692 NN denotes polysaccharide
T787 693-694 -LRB- denotes (
T788 694-695 SYM denotes >
T789 695-698 CD denotes 200
T790 699-702 NN denotes kDa
T791 702-703 -RRB- denotes )
T792 704-711 VB denotes derived
T793 712-716 IN denotes from
T794 717-720 JJ denotes red
T795 721-726 NN denotes algae
T796 727-728 -LRB- denotes (
T797 728-740 NNP denotes Rhodophyceae
T798 740-741 -RRB- denotes )
T799 743-748 CD denotes Three
T800 749-753 JJ denotes main
T801 754-759 NN denotes forms
T802 760-762 IN denotes of
T803 763-766 NN denotes CGN
T804 767-771 VB denotes have
T805 772-776 VB denotes been
T806 777-787 VB denotes identified
T807 787-788 -COLON- denotes :
T808 789-794 NN denotes kappa
T809 794-795 -COMMA- denotes ,
T810 796-800 NN denotes iota
T811 800-801 -COMMA- denotes ,
T812 802-805 CC denotes and
T813 806-812 NN denotes lambda
T814 814-818 PRP denotes They
T815 819-825 VB denotes differ
T816 826-830 IN denotes from
T817 831-835 DT denotes each
T818 836-841 JJ denotes other
T819 842-844 IN denotes in
T820 845-855 NN denotes sulphation
T821 856-862 NN denotes degree
T822 863-866 CC denotes and
T823 867-877 NN denotes solubility
T824 878-879 -LRB- denotes [
T825 879-880 CD denotes 1
T826 880-881 -RRB- denotes ]
T827 881-882 -COMMA- denotes ,
T828 883-884 -LRB- denotes [
T829 884-885 CD denotes 2
T830 885-886 -RRB- denotes ]
T831 888-894 JJ denotes Native
T832 895-898 NN denotes CGN
T833 899-901 VB denotes is
T834 902-909 VB denotes thought
T835 910-912 TO denotes to
T836 913-915 VB denotes be
T837 916-924 JJ denotes harmless
T838 925-928 CC denotes and
T839 929-931 VB denotes is
T840 932-938 RB denotes widely
T841 939-943 VB denotes used
T842 944-946 IN denotes as
T843 947-948 DT denotes a
T844 949-953 NN denotes food
T845 954-962 JJ denotes additive
T846 963-965 TO denotes to
T847 966-973 VB denotes improve
T848 974-981 NN denotes texture
T849 983-985 PRP denotes It
T850 986-988 VB denotes is
T851 989-993 RB denotes also
T852 994-998 VB denotes used
T853 999-1001 IN denotes in
T854 1002-1011 NN denotes cosmetics
T855 1012-1015 CC denotes and
T856 1016-1031 NN denotes pharmaceuticals
T857 1033-1040 RB denotes However
T858 1040-1041 -COMMA- denotes ,
T859 1042-1046 NN denotes acid
T860 1047-1056 NN denotes treatment
T861 1057-1059 IN denotes at
T862 1060-1064 JJ denotes high
T863 1065-1076 NN denotes temperature
T864 1077-1078 -LRB- denotes (
T865 1078-1082 NN denotes 80°C
T866 1082-1083 -RRB- denotes )
T867 1084-1092 VB denotes triggers
T868 1093-1096 NN denotes CGN
T869 1097-1107 NN denotes hydrolysis
T870 1108-1110 TO denotes to
T871 1111-1116 VB denotes lower
T872 1117-1126 JJ denotes molecular
T873 1127-1133 NN denotes weight
T874 1134-1135 -LRB- denotes (
T875 1135-1136 SYM denotes <
T876 1136-1138 CD denotes 50
T877 1139-1142 NN denotes kDa
T878 1142-1143 -RRB- denotes )
T879 1144-1153 NN denotes compounds
T880 1154-1159 VB denotes known
T881 1160-1162 IN denotes as
T882 1163-1173 NN denotes poligeenan
T883 1174-1176 CC denotes or
T884 1177-1185 VB denotes degraded
T885 1186-1189 NN denotes CGN
T886 1190-1191 -LRB- denotes (
T887 1191-1195 NN denotes dCGN
T888 1195-1196 -RRB- denotes )
T889 1198-1203 DT denotes These
T890 1204-1209 NN denotes dCGNs
T891 1210-1216 VB denotes induce
T892 1217-1229 NN denotes inflammation
T893 1230-1233 CC denotes and
T894 1234-1238 VB denotes have
T895 1239-1243 VB denotes been
T896 1244-1250 RB denotes widely
T897 1251-1255 VB denotes used
T898 1256-1258 IN denotes as
T899 1259-1265 NN denotes models
T900 1266-1268 IN denotes of
T901 1269-1276 NN denotes colitis
T902 1277-1279 IN denotes in
T903 1280-1287 JJ denotes several
T904 1288-1295 NN denotes species
T905 1295-1296 -COMMA- denotes ,
T906 1297-1306 VB denotes including
T907 1307-1311 NN denotes rats
T908 1312-1313 -LRB- denotes [
T909 1313-1314 CD denotes 3
T910 1314-1315 -RRB- denotes ]
T911 1315-1316 -COMMA- denotes ,
T912 1317-1324 NN denotes rabbits
T913 1325-1326 -LRB- denotes [
T914 1326-1327 CD denotes 4
T915 1327-1328 -RRB- denotes ]
T916 1329-1332 CC denotes and
T917 1333-1339 NN denotes guinea
T918 1340-1344 NN denotes pigs
T919 1345-1346 -LRB- denotes [
T920 1346-1347 CD denotes 5
T921 1347-1348 -RRB- denotes ]
T922 1350-1353 DT denotes The
T923 1354-1358 NN denotes role
T924 1359-1361 IN denotes of
T925 1362-1366 NN denotes dCGN
T926 1367-1369 IN denotes as
T927 1370-1371 DT denotes a
T928 1372-1387 JJ denotes tumor-promoting
T929 1388-1394 NN denotes factor
T930 1395-1402 VB denotes remains
T931 1403-1416 JJ denotes controversial
T932 1417-1418 -LRB- denotes [
T933 1418-1419 CD denotes 4
T934 1419-1420 -RRB- denotes ]
T935 1420-1421 -COMMA- denotes ,
T936 1422-1423 -LRB- denotes [
T937 1423-1424 CD denotes 6
T938 1424-1425 -RRB- denotes ]
T939 1425-1426 -LRB- denotes –
T940 1426-1427 CD denotes [
T941 1427-1428 -RRB- denotes 8
T942 1431-1439 IN denotes Although
T943 1440-1443 DT denotes the
T944 1444-1450 JJ denotes native
T945 1451-1455 NN denotes form
T946 1456-1458 VB denotes is
T947 1459-1466 VB denotes thought
T948 1467-1469 TO denotes to
T949 1470-1472 VB denotes be
T950 1473-1481 JJ denotes harmless
T951 1482-1485 IN denotes for
T952 1486-1491 JJ denotes human
T953 1492-1503 NN denotes consumption
T954 1503-1504 -COMMA- denotes ,
T955 1505-1510 JJ denotes small
T956 1511-1518 NN denotes amounts
T957 1519-1521 IN denotes of
T958 1522-1526 NN denotes dCGN
T959 1527-1530 VB denotes are
T960 1531-1539 RB denotes probably
T961 1540-1548 VB denotes produced
T962 1549-1551 IN denotes by
T963 1552-1556 NN denotes acid
T964 1557-1567 NN denotes hydrolysis
T965 1568-1574 IN denotes during
T966 1575-1582 JJ denotes gastric
T967 1583-1592 NN denotes digestion
T968 1593-1594 -LRB- denotes [
T969 1594-1595 CD denotes 9
T970 1595-1596 -RRB- denotes ]
T971 1596-1597 -COMMA- denotes ,
T972 1598-1599 -LRB- denotes [
T973 1599-1601 CD denotes 10
T974 1601-1602 -RRB- denotes ]
T975 1603-1605 CC denotes or
T976 1606-1617 NN denotes interaction
T977 1618-1622 IN denotes with
T978 1623-1633 JJ denotes intestinal
T979 1634-1642 NN denotes bacteria
T980 1643-1644 -LRB- denotes [
T981 1644-1646 CD denotes 11
T982 1646-1647 -RRB- denotes ]
T983 1647-1648 -COMMA- denotes ,
T984 1649-1650 -LRB- denotes [
T985 1650-1652 CD denotes 12
T986 1652-1653 -RRB- denotes ]
T987 1655-1662 IN denotes Whereas
T988 1663-1666 DT denotes the
T989 1667-1674 NN denotes effects
T990 1675-1677 IN denotes of
T991 1678-1684 JJ denotes native
T992 1685-1688 CC denotes and
T993 1689-1693 NN denotes dCGN
T994 1694-1696 IN denotes on
T995 1697-1707 JJ denotes intestinal
T996 1708-1720 NN denotes inflammation
T997 1721-1725 VB denotes have
T998 1726-1730 VB denotes been
T999 1731-1742 RB denotes extensively
T1000 1743-1751 VB denotes analyzed
T1001 1752-1754 IN denotes in
T1002 1755-1761 NN denotes animal
T1003 1762-1768 NN denotes models
T1004 1768-1769 -COMMA- denotes ,
T1005 1770-1774 RB denotes only
T1006 1775-1778 JJ denotes few
T1007 1779-1786 NN denotes studies
T1008 1787-1791 VB denotes have
T1009 1792-1796 VB denotes been
T1010 1797-1806 VB denotes conducted
T1011 1807-1812 VB denotes using
T1012 1813-1818 JJ denotes human
T1013 1819-1823 NN denotes cell
T1014 1824-1829 NN denotes lines
T1015 1831-1837 JJ denotes Recent
T1016 1838-1845 NN denotes studies
T1017 1846-1850 VB denotes have
T1018 1851-1856 VB denotes shown
T1019 1857-1858 DT denotes a
T1020 1859-1863 NN denotes link
T1021 1864-1871 IN denotes between
T1022 1872-1880 NN denotes exposure
T1023 1881-1883 TO denotes to
T1024 1884-1890 JJ denotes native
T1025 1891-1895 NN denotes form
T1026 1896-1899 NN denotes CGN
T1027 1900-1903 CC denotes and
T1028 1904-1908 NN denotes IL-8
T1029 1909-1919 NN denotes production
T1030 1920-1922 IN denotes by
T1031 1923-1926 DT denotes the
T1032 1927-1932 JJ denotes human
T1033 1933-1943 JJ denotes intestinal
T1034 1944-1954 JJ denotes epithelial
T1035 1955-1959 NN denotes cell
T1036 1960-1964 NN denotes line
T1037 1964-1965 -COMMA- denotes ,
T1038 1966-1972 NN denotes NCM460
T1039 1972-1973 -COMMA- denotes ,
T1040 1974-1977 IN denotes via
T1041 1978-1985 JJ denotes Nuclear
T1042 1986-1995 NN denotes Factor-κB
T1043 1996-1997 -LRB- denotes (
T1044 1997-2002 NN denotes NF-κB
T1045 2002-2003 -RRB- denotes )
T1046 2004-2014 NN denotes activation
T1047 2015-2016 -LRB- denotes [
T1048 2016-2018 CD denotes 13
T1049 2018-2019 -RRB- denotes ]
T1050 2019-2020 -COMMA- denotes ,
T1051 2021-2022 -LRB- denotes [
T1052 2022-2024 CD denotes 14
T1053 2024-2025 -RRB- denotes ]
T1054 2027-2032 NN denotes NF-κB
T1055 2033-2035 VB denotes is
T1056 2036-2037 DT denotes a
T1057 2038-2051 NN denotes transcription
T1058 2052-2058 NN denotes factor
T1059 2059-2063 WDT denotes that
T1060 2064-2073 VB denotes regulates
T1061 2074-2077 DT denotes the
T1062 2078-2088 NN denotes expression
T1063 2089-2091 IN denotes of
T1064 2092-2097 NN denotes genes
T1065 2098-2108 VB denotes associated
T1066 2109-2113 IN denotes with
T1067 2114-2126 NN denotes inflammation
T1068 2127-2128 -LRB- denotes [
T1069 2128-2130 CD denotes 15
T1070 2130-2131 -RRB- denotes ]
T1071 2131-2132 -COMMA- denotes ,
T1072 2133-2134 -LRB- denotes [
T1073 2134-2136 CD denotes 16
T1074 2136-2137 -RRB- denotes ]
T1075 2139-2149 NN denotes Macrophage
T1076 2150-2162 NN denotes infiltration
T1077 2163-2166 CC denotes and
T1078 2167-2179 NN denotes accumulation
T1079 2180-2182 VB denotes is
T1080 2183-2184 DT denotes a
T1081 2185-2191 JJ denotes common
T1082 2192-2206 NN denotes characteristic
T1083 2207-2209 IN denotes of
T1084 2210-2220 JJ denotes intestinal
T1085 2221-2229 NN denotes diseases
T1086 2230-2231 -LRB- denotes [
T1087 2231-2233 CD denotes 17
T1088 2233-2234 -RRB- denotes ]
T1089 2236-2247 NN denotes Macrophages
T1090 2248-2257 VB denotes represent
T1091 2258-2260 CD denotes 10
T1092 2260-2261 NN denotes %
T1093 2262-2264 IN denotes of
T1094 2265-2270 JJ denotes total
T1095 2271-2277 NN denotes lamina
T1096 2278-2285 NN denotes propria
T1097 2286-2291 NN denotes cells
T1098 2291-2292 -COMMA- denotes ,
T1099 2293-2300 VB denotes secrete
T1100 2301-2302 DT denotes a
T1101 2303-2307 JJ denotes wide
T1102 2308-2313 NN denotes range
T1103 2314-2316 IN denotes of
T1104 2317-2329 RB denotes biologically
T1105 2330-2336 JJ denotes active
T1106 2337-2346 NN denotes compounds
T1107 2347-2350 CC denotes and
T1108 2351-2358 VB denotes express
T1109 2359-2372 NN denotes cell-adhesion
T1110 2373-2382 NN denotes molecules
T1111 2384-2387 DT denotes The
T1112 2388-2394 JJ denotes immune
T1113 2395-2399 NN denotes cell
T1114 2400-2408 NN denotes response
T1115 2409-2411 TO denotes to
T1116 2412-2414 DT denotes an
T1117 2415-2427 JJ denotes inflammatory
T1118 2428-2436 NN denotes stimulus
T1119 2437-2442 VB denotes seems
T1120 2443-2445 TO denotes to
T1121 2446-2448 VB denotes be
T1122 2449-2458 VB denotes amplified
T1123 2459-2461 CC denotes or
T1124 2462-2470 RB denotes directly
T1125 2471-2480 VB denotes generated
T1126 2481-2483 IN denotes by
T1127 2484-2489 NN denotes cells
T1128 2490-2497 VB denotes exposed
T1129 2498-2500 TO denotes to
T1130 2501-2510 JJ denotes sulphated
T1131 2511-2526 NN denotes polysaccharides
T1132 2527-2531 JJ denotes such
T1133 2532-2534 IN denotes as
T1134 2535-2547 NN denotes carrageenans
T1135 2549-2555 RB denotes Indeed
T1136 2555-2556 -COMMA- denotes ,
T1137 2557-2569 NN denotes inflammation
T1138 2570-2577 VB denotes induced
T1139 2578-2580 IN denotes by
T1140 2581-2585 NN denotes dCGN
T1141 2586-2589 VB denotes was
T1142 2590-2600 VB denotes associated
T1143 2601-2605 IN denotes with
T1144 2606-2617 NN denotes recruitment
T1145 2618-2620 IN denotes of
T1146 2621-2632 NN denotes macrophages
T1147 2633-2635 TO denotes to
T1148 2636-2648 NN denotes inflammation
T1149 2649-2654 NN denotes sites
T1150 2655-2656 -LRB- denotes [
T1151 2656-2658 CD denotes 18
T1152 2658-2659 -RRB- denotes ]
T1153 2659-2660 -COMMA- denotes ,
T1154 2661-2662 -LRB- denotes [
T1155 2662-2664 CD denotes 19
T1156 2664-2665 -RRB- denotes ]
T1157 2667-2671 RB denotes Also
T1158 2671-2672 -COMMA- denotes ,
T1159 2673-2685 NN denotes inflammation
T1160 2686-2693 VB denotes induced
T1161 2694-2696 IN denotes by
T1162 2697-2704 NNP denotes Dextran
T1163 2705-2713 NNP denotes Sulphate
T1164 2714-2720 NNP denotes Sodium
T1165 2721-2722 -LRB- denotes (
T1166 2722-2725 NN denotes DSS
T1167 2725-2726 -RRB- denotes )
T1168 2726-2727 -COMMA- denotes ,
T1169 2728-2735 DT denotes another
T1170 2736-2745 VB denotes sulphated
T1171 2746-2754 NN denotes compound
T1172 2754-2755 -COMMA- denotes ,
T1173 2756-2759 VB denotes was
T1174 2760-2768 RB denotes directly
T1175 2769-2779 VB denotes associated
T1176 2780-2784 IN denotes with
T1177 2785-2796 NN denotes macrophages
T1178 2797-2808 NN denotes recruitment
T1179 2809-2810 -LRB- denotes [
T1180 2810-2812 CD denotes 20
T1181 2812-2813 -RRB- denotes ]
T1182 2813-2814 -COMMA- denotes ,
T1183 2815-2820 IN denotes since
T1184 2821-2824 NN denotes DSS
T1185 2825-2830 RB denotes still
T1186 2831-2839 VB denotes provoked
T1187 2840-2852 NN denotes inflammation
T1188 2853-2858 IN denotes after
T1189 2859-2871 NN denotes T-lymphocyte
T1190 2872-2875 CC denotes and
T1191 2876-2878 NN denotes NK
T1192 2879-2883 NN denotes cell
T1193 2884-2893 NN denotes depletion
T1194 2894-2895 -LRB- denotes [
T1195 2895-2897 CD denotes 20
T1196 2897-2898 -RRB- denotes ]
T1197 2900-2908 IN denotes Although
T1198 2909-2921 NN denotes inflammation
T1199 2922-2925 MD denotes can
T1200 2926-2928 VB denotes be
T1201 2929-2936 VB denotes induced
T1202 2937-2939 IN denotes by
T1203 2940-2944 NN denotes dCGN
T1204 2944-2945 -COMMA- denotes ,
T1205 2946-2951 EX denotes there
T1206 2952-2955 VB denotes are
T1207 2956-2958 DT denotes no
T1208 2959-2963 NN denotes data
T1209 2964-2966 IN denotes on
T1210 2967-2972 JJ denotes human
T1211 2973-2981 NN denotes monocyte
T1212 2982-2991 NN denotes responses
T1213 2992-2994 TO denotes to
T1214 2995-2999 NN denotes dCGN
T1215 3000-3008 NN denotes exposure
T1216 3010-3019 RB denotes Therefore
T1217 3019-3020 -COMMA- denotes ,
T1218 3021-3023 TO denotes to
T1219 3024-3035 VB denotes investigate
T1220 3036-3039 DT denotes the
T1221 3040-3047 NN denotes effects
T1222 3048-3050 IN denotes of
T1223 3051-3055 NN denotes dCGN
T1224 3056-3058 IN denotes on
T1225 3059-3064 JJ denotes human
T1226 3065-3074 NN denotes monocytes
T1227 3074-3075 -COMMA- denotes ,
T1228 3076-3082 JJ denotes normal
T1229 3083-3093 JJ denotes Peripheral
T1230 3094-3099 NN denotes Blood
T1231 3100-3109 NN denotes Monocytes
T1232 3110-3111 -LRB- denotes (
T1233 3111-3114 NN denotes PBM
T1234 3114-3115 -RRB- denotes )
T1235 3116-3119 CC denotes and
T1236 3120-3127 JJ denotes tumoral
T1237 3128-3147 NN denotes monocyte/macrophage
T1238 3148-3153 NN denotes THP-1
T1239 3154-3159 NN denotes cells
T1240 3160-3164 VB denotes were
T1241 3165-3172 VB denotes exposed
T1242 3173-3175 TO denotes to
T1243 3176-3178 CD denotes 10
T1244 3179-3182 NN denotes kDa
T1245 3183-3186 CC denotes and
T1246 3187-3189 CD denotes 40
T1247 3190-3193 NN denotes kDa
T1248 3194-3198 NN denotes dCGN
T1249 3200-3202 PRP denotes We
T1250 3203-3208 VB denotes found
T1251 3209-3213 IN denotes that
T1252 3214-3218 NN denotes dCGN
T1253 3219-3228 VB denotes inhibited
T1254 3229-3234 NN denotes THP-1
T1255 3235-3239 NN denotes cell
T1256 3240-3253 NN denotes proliferation
T1257 3254-3256 FW denotes in
T1258 3257-3262 FW denotes vitro
T1259 3262-3263 -COMMA- denotes ,
T1260 3264-3273 VB denotes increased
T1261 3274-3280 NN denotes ICAM-1
T1262 3281-3291 NN denotes expression
T1263 3291-3292 -COMMA- denotes ,
T1264 3293-3303 VB denotes stimulated
T1265 3304-3320 JJ denotes ICAM-1-dependent
T1266 3321-3329 NN denotes monocyte
T1267 3330-3341 NN denotes aggregation
T1268 3341-3342 -COMMA- denotes ,
T1269 3343-3346 CC denotes and
T1270 3347-3357 VB denotes stimulated
T1271 3358-3363 NN denotes TNF-α
T1272 3364-3374 NN denotes expression
T1273 3375-3378 CC denotes and
T1274 3379-3388 NN denotes secretion
T1275 3390-3395 DT denotes These
T1276 3396-3405 NN denotes responses
T1277 3406-3410 VB denotes were
T1278 3411-3415 RB denotes more
T1279 3416-3426 JJ denotes pronounced
T1280 3427-3432 IN denotes after
T1281 3433-3435 CD denotes 40
T1282 3436-3439 NN denotes kDa
T1283 3440-3444 NN denotes dCGN
T1284 3445-3453 NN denotes exposure
T1285 3454-3457 CC denotes and
T1286 3458-3462 VB denotes were
T1287 3463-3469 VB denotes linked
T1288 3470-3472 TO denotes to
T1289 3473-3478 NN denotes NF-κB
T1290 3479-3489 NN denotes activation
T1291 3491-3493 IN denotes In
T1292 3494-3502 NN denotes addition
T1293 3502-3503 -COMMA- denotes ,
T1294 3504-3507 DT denotes the
T1295 3508-3510 CD denotes 40
T1296 3511-3514 NN denotes kDa
T1297 3515-3519 NN denotes dCGN
T1298 3519-3520 -COMMA- denotes ,
T1299 3521-3524 CC denotes but
T1300 3525-3528 RB denotes not
T1301 3529-3532 DT denotes the
T1302 3533-3535 CD denotes 10
T1303 3536-3539 NN denotes kDa
T1304 3540-3544 NN denotes dCGN
T1305 3545-3552 VB denotes induced
T1306 3553-3555 FW denotes in
T1307 3556-3560 FW denotes vivo
T1308 3561-3568 NN denotes colitis
T1309 3569-3571 IN denotes as
T1310 3572-3577 VB denotes shown
T1311 3578-3580 IN denotes by
T1312 3581-3584 DT denotes the
T1313 3585-3597 JJ denotes inflammatory
T1314 3598-3606 NN denotes response
T1315 3607-3609 IN denotes in
T1316 3610-3613 DT denotes the
T1317 3614-3617 NN denotes rat
T1318 3618-3623 NN denotes colon
T1319 3625-3630 DT denotes These
T1320 3631-3638 NN denotes results
T1321 3639-3646 VB denotes suggest
T1322 3647-3651 IN denotes that
T1323 3652-3655 DT denotes the
T1324 3656-3664 JJ denotes degraded
T1325 3665-3670 NN denotes forms
T1326 3671-3673 IN denotes of
T1327 3674-3677 NN denotes CGN
T1328 3678-3682 VB denotes have
T1329 3683-3685 DT denotes an
T1330 3686-3695 JJ denotes important
T1331 3696-3702 NN denotes effect
T1332 3703-3705 IN denotes on
T1333 3706-3715 NN denotes monocytes
T1334 3716-3725 VB denotes resulting
T1335 3726-3728 IN denotes in
T1336 3729-3731 DT denotes an
T1337 3732-3744 JJ denotes inflammatory
T1338 3745-3754 NN denotes phenotype
T2063 3780-3791 NN denotes Preparation
T2064 3792-3794 IN denotes of
T2065 3795-3803 NNP denotes Degraded
T2066 3804-3815 NNP denotes Carrageenan
T2067 3816-3819 CD denotes Two
T2068 3820-3832 NN denotes preparations
T2069 3833-3835 IN denotes of
T2070 3836-3844 VB denotes degraded
T2071 3845-3856 NN denotes carrageenan
T2072 3857-3861 IN denotes with
T2073 3862-3865 JJ denotes low
T2074 3865-3866 -COMMA- denotes ,
T2075 3867-3868 -LRB- denotes (
T2076 3868-3871 CD denotes ∼10
T2077 3872-3875 NN denotes kDa
T2078 3875-3876 -COLON- denotes ;
T2079 3877-3880 NN denotes C10
T2080 3880-3881 -RRB- denotes )
T2081 3881-3882 -COMMA- denotes ,
T2082 3883-3886 CC denotes and
T2083 3887-3893 NN denotes medium
T2084 3893-3894 -COMMA- denotes ,
T2085 3895-3896 -LRB- denotes (
T2086 3896-3899 CD denotes ∼40
T2087 3900-3903 NN denotes kDa
T2088 3903-3904 -COLON- denotes ;
T2089 3905-3908 NN denotes C40
T2090 3908-3909 -RRB- denotes )
T2091 3910-3919 JJ denotes molecular
T2092 3920-3926 NN denotes weight
T2093 3927-3931 VB denotes were
T2094 3932-3940 VB denotes prepared
T2095 3941-3945 IN denotes from
T2096 3946-3952 JJ denotes native
T2097 3953-3969 NN denotes iota-carrageenan
T2098 3970-3979 VB denotes extracted
T2099 3980-3984 IN denotes from
T2100 3985-3992 NNP denotes Euchema
T2101 3993-4001 NN denotes spinosum
T2102 4002-4003 -LRB- denotes (
T2103 4003-4013 RB denotes generously
T2104 4014-4022 VB denotes provided
T2105 4023-4025 IN denotes by
T2106 4026-4032 NNP denotes Sanofi
T2107 4033-4043 NNP denotes Biosystems
T2108 4044-4052 NNP denotes Industry
T2109 4052-4053 -COMMA- denotes ,
T2110 4054-4074 NNP denotes Boulogne-Billancourt
T2111 4074-4075 -COMMA- denotes ,
T2112 4076-4082 NNP denotes France
T2113 4082-4083 -RRB- denotes )
T2114 4085-4091 JJ denotes Native
T2115 4092-4103 NN denotes carrageenan
T2116 4104-4107 VB denotes was
T2117 4108-4117 VB denotes dissolved
T2118 4118-4120 IN denotes in
T2119 4121-4130 JJ denotes distilled
T2120 4131-4136 NN denotes water
T2121 4137-4138 -LRB- denotes (
T2122 4138-4139 CD denotes 5
T2123 4139-4140 NN denotes %
T2124 4141-4144 NN denotes w/v
T2125 4144-4145 -RRB- denotes )
T2126 4146-4151 IN denotes under
T2127 4152-4160 JJ denotes vigorous
T2128 4161-4169 VB denotes stirring
T2129 4170-4173 CC denotes and
T2130 4174-4180 VB denotes heated
T2131 4181-4183 TO denotes to
T2132 4184-4189 NNP denotes 60°C.
T2133 4190-4194 NNP denotes Then
T2134 4194-4195 -COMMA- denotes ,
T2135 4196-4199 DT denotes the
T2136 4200-4211 NN denotes carrageenan
T2137 4212-4220 NN denotes solution
T2138 4221-4224 VB denotes was
T2139 4225-4234 VB denotes submitted
T2140 4235-4237 TO denotes to
T2141 4238-4241 CD denotes two
T2142 4242-4251 JJ denotes different
T2143 4252-4262 NN denotes treatments
T2144 4263-4265 TO denotes to
T2145 4266-4272 VB denotes obtain
T2146 4273-4277 CC denotes both
T2147 4278-4281 JJ denotes low
T2148 4282-4285 CC denotes and
T2149 4286-4292 JJ denotes medium
T2150 4293-4302 JJ denotes molecular
T2151 4303-4309 NN denotes weight
T2152 4310-4319 NN denotes fractions
T2153 4321-4328 RB denotes Briefly
T2154 4328-4329 -COMMA- denotes ,
T2155 4330-4333 IN denotes for
T2156 4334-4337 DT denotes the
T2157 4338-4341 JJ denotes low
T2158 4342-4351 JJ denotes molecular
T2159 4352-4358 NN denotes weight
T2160 4359-4367 NN denotes fraction
T2161 4367-4368 -COMMA- denotes ,
T2162 4369-4380 NN denotes carrageenan
T2163 4381-4389 NN denotes solution
T2164 4390-4393 VB denotes was
T2165 4394-4404 VB denotes hydrolyzed
T2166 4405-4409 IN denotes with
T2167 4410-4413 CD denotes 0.3
T2168 4413-4414 NN denotes %
T2169 4415-4416 -LRB- denotes (
T2170 4416-4419 NN denotes v/v
T2171 4419-4420 -RRB- denotes )
T2172 4421-4433 VB denotes concentrated
T2173 4434-4443 JJ denotes sulphuric
T2174 4444-4448 NN denotes acid
T2175 4449-4452 IN denotes for
T2176 4453-4455 CD denotes 15
T2177 4456-4459 NN denotes min
T2178 4460-4462 IN denotes at
T2179 4463-4468 NNP denotes 80°C.
T2180 4469-4474 NNP denotes After
T2181 4475-4489 NN denotes neutralization
T2182 4490-4494 IN denotes with
T2183 4495-4499 NN denotes NaOH
T2184 4500-4502 NN denotes 4N
T2185 4502-4503 -COMMA- denotes ,
T2186 4504-4507 DT denotes the
T2187 4508-4516 NN denotes solution
T2188 4517-4520 VB denotes was
T2189 4521-4526 NN denotes ultra
T2190 4527-4535 VB denotes filtered
T2191 4536-4543 IN denotes through
T2192 4544-4545 DT denotes a
T2193 4546-4552 JJ denotes hollow
T2194 4553-4558 NN denotes fibre
T2195 4559-4568 NN denotes cartridge
T2196 4569-4573 IN denotes with
T2197 4574-4576 NN denotes MW
T2198 4577-4584 JJ denotes cut-off
T2199 4585-4586 CD denotes 5
T2200 4587-4590 NN denotes kDa
T2201 4590-4591 -COMMA- denotes ,
T2202 4592-4593 -LRB- denotes (
T2203 4593-4599 NNP denotes Amicon
T2204 4600-4603 NNP denotes Inc
T2205 4603-4604 -COMMA- denotes ,
T2206 4605-4612 NNP denotes Beverly
T2207 4612-4613 -COMMA- denotes ,
T2208 4614-4617 NNP denotes USA
T2209 4617-4618 -RRB- denotes )
T2210 4620-4623 IN denotes For
T2211 4624-4627 DT denotes the
T2212 4628-4634 NN denotes medium
T2213 4635-4644 JJ denotes molecular
T2214 4645-4651 NN denotes weight
T2215 4652-4660 NN denotes fraction
T2216 4660-4661 -COMMA- denotes ,
T2217 4662-4665 DT denotes the
T2218 4666-4677 NN denotes carrageenan
T2219 4678-4686 NN denotes solution
T2220 4687-4690 VB denotes was
T2221 4691-4701 VB denotes hydrolyzed
T2222 4702-4706 IN denotes with
T2223 4707-4710 CD denotes 0.3
T2224 4710-4711 NN denotes %
T2225 4712-4713 -LRB- denotes (
T2226 4713-4716 NN denotes v/v
T2227 4716-4717 -RRB- denotes )
T2228 4718-4730 VB denotes concentrated
T2229 4731-4740 JJ denotes sulphuric
T2230 4741-4745 NN denotes acid
T2231 4746-4749 IN denotes for
T2232 4750-4752 CD denotes 30
T2233 4753-4756 NN denotes min
T2234 4757-4759 IN denotes at
T2235 4760-4765 NNP denotes 60°C.
T2236 4766-4771 IN denotes After
T2237 4772-4786 NN denotes neutralization
T2238 4786-4787 -COMMA- denotes ,
T2239 4788-4791 DT denotes the
T2240 4792-4803 NN denotes supernatant
T2241 4804-4807 VB denotes was
T2242 4808-4813 AFX denotes ultra
T2243 4814-4822 VB denotes filtered
T2244 4823-4824 -LRB- denotes (
T2245 4824-4826 NN denotes MW
T2246 4827-4834 JJ denotes cut-off
T2247 4835-4838 CD denotes 100
T2248 4839-4842 NN denotes kDa
T2249 4842-4843 -RRB- denotes )
T2250 4845-4848 DT denotes The
T2251 4849-4857 NN denotes filtrate
T2252 4858-4861 VB denotes was
T2253 4862-4871 VB denotes submitted
T2254 4872-4874 TO denotes to
T2255 4875-4876 DT denotes a
T2256 4877-4883 JJ denotes second
T2257 4884-4889 NN denotes ultra
T2258 4890-4900 NN denotes filtration
T2259 4901-4902 -LRB- denotes (
T2260 4902-4904 NN denotes MW
T2261 4905-4912 NN denotes cut-off
T2262 4913-4914 CD denotes 5
T2263 4915-4918 NN denotes kDa
T2264 4918-4919 -RRB- denotes )
T2265 4921-4925 DT denotes Both
T2266 4926-4938 NN denotes preparations
T2267 4939-4941 IN denotes of
T2268 4942-4946 NN denotes dCGN
T2269 4947-4951 VB denotes were
T2270 4952-4964 VB denotes precipitated
T2271 4965-4969 IN denotes with
T2272 4970-4971 CD denotes 4
T2273 4972-4979 NN denotes volumes
T2274 4980-4982 IN denotes of
T2275 4983-4985 CD denotes 95
T2276 4985-4986 NN denotes %
T2277 4987-4994 NN denotes ethanol
T2278 4994-4995 -COMMA- denotes ,
T2279 4996-5001 VB denotes dried
T2280 5002-5004 IN denotes at
T2281 5005-5009 NN denotes room
T2282 5010-5021 NN denotes temperature
T2283 5022-5025 CC denotes and
T2284 5026-5032 NN denotes ground
T2285 5033-5035 TO denotes to
T2286 5036-5041 JJ denotes small
T2287 5042-5051 NN denotes particles
T2288 5052-5053 -LRB- denotes (
T2289 5053-5054 CD denotes 1
T2290 5055-5057 NN denotes mm
T2291 5058-5060 IN denotes in
T2292 5061-5069 NN denotes diameter
T2293 5069-5070 -RRB- denotes )
T2294 5072-5077 VB denotes Using
T2295 5078-5092 NN denotes gel-permeation
T2296 5093-5107 NN denotes chromatography
T2297 5108-5110 IN denotes in
T2298 5111-5122 NN denotes combination
T2299 5123-5127 IN denotes with
T2300 5128-5133 JJ denotes light
T2301 5134-5144 NN denotes scattering
T2302 5145-5157 NN denotes measurements
T2303 5158-5159 -LRB- denotes (
T2304 5159-5162 VB denotes see
T2305 5163-5169 NNP denotes Viebke
T2306 5170-5172 FW denotes et
T2307 5173-5176 FW denotes al.
T2308 5177-5178 -LRB- denotes [
T2309 5178-5180 CD denotes 21
T2310 5180-5181 -RRB- denotes ]
T2311 5181-5182 -RRB- denotes )
T2312 5182-5183 -COMMA- denotes ,
T2313 5184-5186 PRP denotes it
T2314 5187-5190 VB denotes was
T2315 5191-5200 VB denotes confirmed
T2316 5201-5205 IN denotes that
T2317 5206-5209 DT denotes the
T2318 5210-5213 JJ denotes low
T2319 5214-5222 NN denotes fraction
T2320 5223-5226 VB denotes had
T2321 5227-5229 DT denotes an
T2322 5230-5237 JJ denotes average
T2323 5238-5247 JJ denotes molecular
T2324 5248-5254 NN denotes weight
T2325 5255-5257 IN denotes of
T2326 5258-5260 CD denotes 10
T2327 5261-5264 NN denotes kDa
T2328 5264-5265 -COMMA- denotes ,
T2329 5266-5269 CC denotes and
T2330 5270-5273 DT denotes the
T2331 5274-5280 NN denotes medium
T2332 5281-5289 NN denotes fraction
T2333 5290-5292 IN denotes of
T2334 5293-5295 CD denotes 40
T2335 5296-5299 NN denotes kDa
T2336 5301-5304 DT denotes The
T2337 5305-5313 NN denotes sulphate
T2338 5314-5321 NN denotes content
T2339 5322-5324 IN denotes of
T2340 5325-5340 NN denotes polysaccharides
T2341 5341-5343 IN denotes in
T2342 5344-5348 DT denotes both
T2343 5349-5358 NN denotes fractions
T2344 5359-5362 VB denotes was
T2345 5363-5371 VB denotes measured
T2346 5372-5381 VB denotes following
T2347 5382-5385 DT denotes the
T2348 5386-5392 NN denotes method
T2349 5393-5395 IN denotes of
T2350 5396-5404 NNP denotes Quemener
T2351 5405-5407 FW denotes et
T2352 5408-5411 FW denotes al.
T2353 5412-5413 -LRB- denotes [
T2354 5413-5415 CD denotes 22
T2355 5415-5416 -RRB- denotes ]
T2356 5418-5425 RB denotes Finally
T2357 5425-5426 -COMMA- denotes ,
T2358 5427-5430 DT denotes the
T2359 5431-5438 NN denotes absence
T2360 5439-5441 IN denotes of
T2361 5442-5456 NN denotes polysaccharide
T2362 5457-5466 NN denotes structure
T2363 5467-5480 NN denotes modifications
T2364 5481-5483 IN denotes in
T2365 5484-5487 DT denotes the
T2366 5488-5491 CD denotes two
T2367 5492-5501 NN denotes fractions
T2368 5502-5505 VB denotes was
T2369 5506-5515 VB denotes confirmed
T2370 5516-5521 VB denotes using
T2371 5522-5528 NN denotes 2H-NMR
T2372 5529-5541 NN denotes spectroscopy
T2373 5543-5546 DT denotes The
T2374 5547-5554 NN denotes absence
T2375 5555-5557 IN denotes of
T2376 5558-5561 NN denotes LPS
T2377 5562-5575 NN denotes contamination
T2378 5576-5578 IN denotes in
T2379 5579-5582 DT denotes the
T2380 5583-5586 CD denotes two
T2381 5587-5596 NN denotes fractions
T2382 5597-5600 VB denotes was
T2383 5601-5610 VB denotes confirmed
T2384 5611-5616 VB denotes using
T2385 5617-5620 DT denotes the
T2386 5621-5630 NN denotes e-Toxate®
T2387 5631-5634 NN denotes kit
T2388 5635-5636 -LRB- denotes (
T2389 5636-5641 NNP denotes Sigma
T2390 5641-5642 -COMMA- denotes ,
T2391 5643-5645 NNP denotes St
T2392 5646-5653 NNP denotes Quentin
T2393 5654-5663 NNP denotes Fallavier
T2394 5663-5664 -COMMA- denotes ,
T2395 5665-5671 NNP denotes France
T2396 5671-5672 -RRB- denotes )
T2397 5674-5680 IN denotes Before
T2398 5681-5684 NN denotes use
T2399 5685-5687 IN denotes in
T2400 5688-5692 NN denotes cell
T2401 5693-5700 NN denotes culture
T2402 5700-5701 -COMMA- denotes ,
T2403 5702-5705 DT denotes the
T2404 5706-5709 CD denotes two
T2405 5710-5719 NN denotes fractions
T2406 5720-5724 VB denotes were
T2407 5725-5734 VB denotes dissolved
T2408 5735-5737 IN denotes in
T2409 5738-5746 JJ denotes complete
T2410 5747-5753 NN denotes medium
T2411 5754-5760 IN denotes during
T2412 5761-5763 CD denotes 30
T2413 5764-5767 NN denotes min
T2414 5768-5770 IN denotes at
T2415 5771-5775 NN denotes 56°C
T2818 5785-5786 -COMMA- denotes ,
T2819 5787-5796 NNP denotes Chemicals
T2820 5797-5800 CC denotes and
T2821 5801-5805 NNP denotes Diet
T2822 5806-5810 JJ denotes Male
T2823 5811-5817 NNP denotes Wistar
T2824 5818-5822 NN denotes rats
T2825 5823-5824 -LRB- denotes (
T2826 5824-5827 CD denotes 150
T2827 5828-5829 NN denotes g
T2828 5830-5837 JJ denotes average
T2829 5838-5844 NN denotes weight
T2830 5844-5845 -RRB- denotes )
T2831 5846-5850 VB denotes were
T2832 5851-5857 VB denotes housed
T2833 5858-5863 IN denotes under
T2834 5864-5872 JJ denotes standard
T2835 5873-5883 NN denotes conditions
T2836 5884-5887 CC denotes and
T2837 5888-5891 VB denotes fed
T2838 5892-5894 NN denotes ad
T2839 5895-5902 NN denotes libitum
T2840 5903-5907 IN denotes with
T2841 5908-5916 JJ denotes standard
T2842 5917-5923 NN denotes rodent
T2843 5924-5934 NN denotes laboratory
T2844 5935-5939 NN denotes chow
T2845 5941-5949 VB denotes Degraded
T2846 5950-5967 NN denotes iota-carrageenans
T2847 5968-5972 VB denotes were
T2848 5973-5985 VB denotes administered
T2849 5986-5988 IN denotes in
T2850 5989-5992 DT denotes the
T2851 5993-6001 NN denotes drinking
T2852 6002-6007 NN denotes water
T2853 6008-6009 -LRB- denotes (
T2854 6009-6010 CD denotes 5
T2855 6010-6011 NN denotes %
T2856 6012-6015 NN denotes w/v
T2857 6015-6016 -RRB- denotes )
T2858 6017-6020 IN denotes for
T2859 6021-6023 CD denotes 55
T2860 6024-6028 NN denotes days
T2861 6029-6031 TO denotes to
T2862 6032-6033 CD denotes 2
T2863 6034-6040 NN denotes groups
T2864 6041-6043 IN denotes of
T2865 6044-6047 CD denotes six
T2866 6048-6055 NN denotes animals
T2867 6056-6060 DT denotes each
T2868 6062-6065 DT denotes The
T2869 6066-6071 JJ denotes first
T2870 6072-6077 NN denotes group
T2871 6078-6086 VB denotes received
T2872 6087-6090 DT denotes the
T2873 6091-6094 JJ denotes low
T2874 6095-6104 JJ denotes molecular
T2875 6105-6111 NN denotes weight
T2876 6112-6123 NN denotes carrageenan
T2877 6124-6125 -LRB- denotes (
T2878 6125-6127 CD denotes 10
T2879 6128-6131 NN denotes kDa
T2880 6132-6136 NN denotes dCGN
T2881 6136-6137 -RRB- denotes )
T2882 6138-6141 CC denotes and
T2883 6142-6145 DT denotes the
T2884 6146-6152 NN denotes second
T2885 6153-6161 VB denotes received
T2886 6162-6165 DT denotes the
T2887 6166-6172 NN denotes medium
T2888 6173-6182 JJ denotes molecular
T2889 6183-6189 NN denotes weight
T2890 6190-6201 NN denotes carrageenan
T2891 6202-6203 -LRB- denotes (
T2892 6203-6205 CD denotes 40
T2893 6206-6209 NN denotes kDa
T2894 6210-6214 NN denotes dCGN
T2895 6214-6215 -RRB- denotes )
T2896 6217-6219 DT denotes An
T2897 6220-6230 JJ denotes additional
T2898 6231-6236 NN denotes group
T2899 6237-6239 IN denotes of
T2900 6240-6244 CD denotes four
T2901 6245-6249 NN denotes rats
T2902 6250-6254 VB denotes were
T2903 6255-6265 VB denotes maintained
T2904 6266-6268 IN denotes on
T2905 6269-6276 JJ denotes regular
T2906 6277-6280 NN denotes tap
T2907 6281-6286 NN denotes water
T2908 6287-6288 -LRB- denotes (
T2909 6288-6295 NN denotes control
T2910 6296-6301 NN denotes group
T2911 6301-6302 -RRB- denotes )
T2912 6304-6306 TO denotes To
T2913 6307-6315 VB denotes increase
T2914 6316-6328 NN denotes palatability
T2915 6329-6332 CD denotes 0.2
T2916 6332-6333 NN denotes %
T2917 6334-6341 NN denotes sucrose
T2918 6342-6345 VB denotes was
T2919 6346-6351 VB denotes added
T2920 6352-6354 TO denotes to
T2921 6355-6358 DT denotes the
T2922 6359-6367 NN denotes drinking
T2923 6368-6373 NN denotes water
T2924 6374-6376 IN denotes of
T2925 6377-6380 DT denotes all
T2926 6381-6387 NN denotes groups
T2927 6388-6389 -LRB- denotes (
T2928 6389-6392 NNP denotes Van
T2929 6393-6396 NNP denotes der
T2930 6397-6402 NNP denotes Waaji
T2931 6403-6405 FW denotes et
T2932 6406-6409 FW denotes al.
T2933 6409-6410 -COMMA- denotes ,
T2934 6411-6412 -LRB- denotes [
T2935 6412-6414 CD denotes 23
T2936 6414-6415 -RRB- denotes ]
T2937 6415-6416 -RRB- denotes )
T2938 6418-6423 JJ denotes Fresh
T2939 6424-6435 NN denotes carrageenan
T2940 6436-6445 NN denotes solutions
T2941 6446-6450 VB denotes were
T2942 6451-6459 VB denotes prepared
T2943 6460-6465 RB denotes daily
T3130 6468-6478 NN denotes Evaluation
T3131 6479-6481 IN denotes of
T3132 6482-6489 NN denotes Colitis
T3133 6490-6494 NNP denotes Body
T3134 6495-6501 NN denotes weight
T3135 6501-6502 -COMMA- denotes ,
T3136 6503-6509 NN denotes liquid
T3137 6510-6513 CC denotes and
T3138 6514-6518 NN denotes food
T3139 6519-6530 NN denotes consumption
T3140 6530-6531 -COMMA- denotes ,
T3141 6532-6540 NN denotes diarrhea
T3142 6541-6544 CC denotes and
T3143 6545-6551 JJ denotes rectal
T3144 6552-6560 NN denotes bleeding
T3145 6561-6562 -LRB- denotes (
T3146 6562-6570 VB denotes detected
T3147 6571-6573 IN denotes by
T3148 6574-6577 NN denotes eye
T3149 6578-6588 NN denotes inspection
T3150 6588-6589 -RRB- denotes )
T3151 6590-6594 VB denotes were
T3152 6595-6603 VB denotes recorded
T3153 6604-6614 IN denotes throughout
T3154 6615-6618 DT denotes the
T3155 6619-6626 NN denotes feeding
T3156 6627-6633 NN denotes period
T3157 6635-6640 IN denotes After
T3158 6641-6643 CD denotes 55
T3159 6644-6648 NN denotes days
T3160 6648-6649 -COMMA- denotes ,
T3161 6650-6657 NN denotes animals
T3162 6658-6662 VB denotes were
T3163 6663-6673 VB denotes sacrificed
T3164 6674-6676 IN denotes by
T3165 6677-6685 JJ denotes cervical
T3166 6686-6697 NN denotes dislocation
T3167 6699-6702 DT denotes The
T3168 6703-6709 NN denotes length
T3169 6710-6712 IN denotes of
T3170 6713-6716 DT denotes the
T3171 6717-6722 NN denotes colon
T3172 6723-6726 VB denotes was
T3173 6727-6735 VB denotes measured
T3174 6736-6738 IN denotes as
T3175 6739-6748 VB denotes described
T3176 6749-6751 IN denotes by
T3177 6752-6760 NNP denotes Okayashu
T3178 6761-6763 FW denotes et
T3179 6764-6767 FW denotes al.
T3180 6768-6769 -LRB- denotes [
T3181 6769-6771 CD denotes 24
T3182 6771-6772 -RRB- denotes ]
T3183 6774-6778 RB denotes Then
T3184 6778-6779 -COMMA- denotes ,
T3185 6780-6784 DT denotes each
T3186 6785-6790 NN denotes colon
T3187 6791-6794 VB denotes was
T3188 6795-6802 VB denotes ligated
T3189 6803-6805 IN denotes in
T3190 6806-6814 NN denotes sections
T3191 6815-6817 IN denotes of
T3192 6818-6819 CD denotes 2
T3193 6820-6822 NN denotes cm
T3194 6823-6826 CC denotes and
T3195 6827-6828 CD denotes 1
T3196 6829-6831 TO denotes to
T3197 6832-6833 CD denotes 2
T3198 6834-6836 NN denotes ml
T3199 6837-6839 IN denotes of
T3200 6840-6842 CD denotes 10
T3201 6842-6843 NN denotes %
T3202 6844-6852 NN denotes formalin
T3203 6853-6856 VB denotes was
T3204 6857-6864 VB denotes infused
T3205 6865-6869 IN denotes into
T3206 6870-6873 DT denotes the
T3207 6874-6884 JJ denotes intestinal
T3208 6885-6890 NN denotes lumen
T3209 6892-6895 DT denotes The
T3210 6896-6906 RB denotes moderately
T3211 6907-6916 VB denotes distended
T3212 6917-6924 NN denotes segment
T3213 6925-6928 VB denotes was
T3214 6929-6938 VB denotes sectioned
T3215 6939-6942 CC denotes and
T3216 6943-6948 VB denotes fixed
T3217 6949-6951 IN denotes in
T3218 6952-6954 CD denotes 10
T3219 6954-6955 NN denotes %
T3220 6956-6964 NN denotes formalin
T3221 6966-6969 DT denotes The
T3222 6970-6979 VB denotes following
T3223 6980-6983 NN denotes day
T3224 6983-6984 -COMMA- denotes ,
T3225 6985-6988 DT denotes the
T3226 6989-6999 JJ denotes intestinal
T3227 7000-7007 NN denotes content
T3228 7008-7011 VB denotes was
T3229 7012-7019 VB denotes removed
T3230 7020-7022 IN denotes by
T3231 7023-7032 NN denotes vortexing
T3232 7034-7037 DT denotes The
T3233 7038-7043 VB denotes fixed
T3234 7044-7051 NN denotes segment
T3235 7052-7055 VB denotes was
T3236 7056-7060 VB denotes kept
T3237 7061-7063 IN denotes in
T3238 7064-7066 CD denotes 10
T3239 7066-7067 NN denotes %
T3240 7068-7076 NN denotes formalin
T3241 7077-7079 IN denotes at
T3242 7080-7083 NN denotes 4°C
T3243 7084-7089 IN denotes until
T3244 7090-7093 DT denotes the
T3245 7094-7102 NN denotes paraffin
T3246 7103-7112 NN denotes embedding
T3247 7113-7122 NN denotes procedure
T3248 7124-7126 TO denotes To
T3249 7127-7135 VB denotes evaluate
T3250 7136-7139 DT denotes the
T3251 7140-7146 NN denotes degree
T3252 7147-7149 IN denotes of
T3253 7150-7162 NN denotes inflammation
T3254 7162-7163 -COMMA- denotes ,
T3255 7164-7168 DT denotes this
T3256 7169-7176 NN denotes segment
T3257 7177-7179 IN denotes of
T3258 7180-7185 NN denotes colon
T3259 7186-7189 VB denotes was
T3260 7190-7196 VB denotes opened
T3261 7197-7211 RB denotes longitudinally
T3262 7212-7215 CC denotes and
T3263 7216-7227 JJ denotes macroscopic
T3264 7228-7231 CC denotes and
T3265 7232-7244 JJ denotes histological
T3266 7245-7251 NN denotes scores
T3267 7252-7254 IN denotes of
T3268 7255-7267 NN denotes inflammation
T3269 7268-7272 VB denotes were
T3270 7273-7281 VB denotes recorded
T3271 7282-7284 IN denotes as
T3272 7285-7295 RB denotes previously
T3273 7296-7305 VB denotes described
T3274 7306-7307 -LRB- denotes [
T3275 7307-7309 CD denotes 25
T3276 7309-7310 -RRB- denotes ]
T3277 7310-7311 -COMMA- denotes ,
T3278 7312-7313 -LRB- denotes [
T3279 7313-7315 CD denotes 26
T3280 7315-7316 -RRB- denotes ]
T3281 7318-7321 DT denotes The
T3282 7322-7331 NN denotes toluidine
T3283 7332-7336 JJ denotes blue
T3284 7337-7345 NN denotes staining
T3285 7346-7349 VB denotes was
T3286 7350-7354 VB denotes used
T3287 7355-7358 IN denotes for
T3288 7359-7373 NN denotes identification
T3289 7374-7376 IN denotes of
T3290 7377-7386 VB denotes sulphated
T3291 7387-7402 NN denotes polysaccharides
T3292 7403-7405 IN denotes in
T3293 7406-7409 DT denotes the
T3294 7410-7420 JJ denotes intestinal
T3295 7421-7427 NN denotes mucosa
T3296 7429-7431 IN denotes On
T3297 7432-7435 DT denotes the
T3298 7436-7439 NN denotes day
T3299 7440-7442 IN denotes of
T3300 7443-7452 NN denotes sacrifice
T3301 7452-7453 -COMMA- denotes ,
T3302 7454-7455 DT denotes a
T3303 7456-7461 JJ denotes fresh
T3304 7462-7468 NN denotes sample
T3305 7469-7471 IN denotes of
T3306 7472-7476 DT denotes each
T3307 7477-7482 NN denotes colon
T3308 7483-7484 -LRB- denotes (
T3309 7484-7486 CD denotes 50
T3310 7487-7489 NN denotes mg
T3311 7489-7490 -RRB- denotes )
T3312 7491-7494 VB denotes was
T3313 7495-7504 VB denotes collected
T3314 7505-7508 IN denotes for
T3315 7509-7524 NN denotes myeloperoxidase
T3316 7525-7526 -LRB- denotes (
T3317 7526-7529 NN denotes MPO
T3318 7529-7530 -RRB- denotes )
T3319 7531-7536 NN denotes assay
T3320 7537-7546 VB denotes according
T3321 7547-7549 TO denotes to
T3322 7550-7557 NNP denotes Krawisz
T3323 7558-7560 FW denotes et
T3324 7561-7564 FW denotes al.
T3325 7564-7565 -COMMA- denotes ,
T3326 7566-7567 -LRB- denotes [
T3327 7567-7569 CD denotes 27
T3328 7569-7570 -RRB- denotes ]
T3329 7572-7575 DT denotes The
T3330 7576-7581 NN denotes level
T3331 7582-7584 IN denotes of
T3332 7585-7588 NN denotes MPO
T3333 7588-7589 -COMMA- denotes ,
T3334 7590-7596 RB denotes mainly
T3335 7597-7606 VB denotes expressed
T3336 7607-7609 IN denotes by
T3337 7610-7621 NN denotes neutrophils
T3338 7621-7622 -COMMA- denotes ,
T3339 7623-7632 VB denotes indicates
T3340 7633-7636 DT denotes the
T3341 7637-7641 NN denotes rate
T3342 7642-7644 IN denotes of
T3343 7645-7656 NN denotes recruitment
T3344 7657-7659 IN denotes of
T3345 7660-7671 NN denotes neutrophils
T3346 7672-7674 TO denotes to
T3347 7675-7678 DT denotes the
T3348 7679-7689 JJ denotes intestinal
T3349 7690-7696 NN denotes mucosa
T3350 7698-7701 CD denotes One
T3351 7702-7706 NN denotes unit
T3352 7707-7709 IN denotes of
T3353 7710-7713 NN denotes MPO
T3354 7714-7722 NN denotes activity
T3355 7723-7734 VB denotes corresponds
T3356 7735-7737 TO denotes to
T3357 7738-7741 DT denotes the
T3358 7742-7753 NN denotes degradation
T3359 7754-7756 IN denotes of
T3360 7757-7758 CD denotes 1
T3361 7759-7763 NN denotes µmol
T3362 7764-7766 IN denotes of
T3363 7767-7775 NN denotes peroxide
T3364 7776-7779 IN denotes per
T3365 7780-7786 NN denotes minute
T3366 7787-7789 IN denotes at
T3367 7790-7794 NN denotes 25°C
T3676 7797-7801 NNP denotes Cell
T3677 7802-7809 NNP denotes Culture
T3678 7810-7813 DT denotes All
T3679 7814-7820 NN denotes tissue
T3680 7821-7828 NN denotes culture
T3681 7829-7837 NN denotes reagents
T3682 7838-7842 VB denotes were
T3683 7843-7847 IN denotes from
T3684 7848-7858 NN denotes Invitrogen
T3685 7859-7860 -LRB- denotes (
T3686 7860-7865 NNP denotes Cergy
T3687 7866-7874 NNP denotes Pontoise
T3688 7874-7875 -COMMA- denotes ,
T3689 7876-7882 NNP denotes France
T3690 7882-7883 -RRB- denotes )
T3691 7885-7890 NN denotes THP-1
T3692 7891-7896 JJ denotes human
T3693 7897-7906 JJ denotes monocytic
T3694 7907-7912 NN denotes cells
T3695 7913-7917 VB denotes were
T3696 7918-7928 VB denotes maintained
T3697 7929-7931 IN denotes in
T3698 7932-7941 NN denotes RPMI-1640
T3699 7942-7954 VB denotes supplemented
T3700 7955-7959 IN denotes with
T3701 7960-7962 CD denotes 10
T3702 7962-7963 NN denotes %
T3703 7964-7967 NN denotes FCS
T3704 7967-7968 -COMMA- denotes ,
T3705 7969-7970 CD denotes 2
T3706 7971-7973 NN denotes mM
T3707 7974-7975 NN denotes L
T3708 7976-7986 NN denotes -glutamine
T3709 7986-7987 -COMMA- denotes ,
T3710 7988-7990 CD denotes 50
T3711 7991-7995 NN denotes U/ml
T3712 7996-8006 NN denotes penicillin
T3713 8007-8010 CC denotes and
T3714 8011-8013 CD denotes 50
T3715 8014-8019 NN denotes mg/ml
T3716 8020-8032 NN denotes streptomycin
T3717 8033-8035 IN denotes at
T3718 8036-8040 NN denotes 37°C
T3719 8041-8043 IN denotes in
T3720 8044-8045 DT denotes a
T3721 8046-8047 CD denotes 5
T3722 8047-8048 NN denotes %
T3723 8049-8052 NN denotes CO2
T3724 8053-8062 NN denotes incubator
T3725 8064-8069 JJ denotes Human
T3726 8070-8080 JJ denotes peripheral
T3727 8081-8086 NN denotes blood
T3728 8087-8098 JJ denotes mononuclear
T3729 8099-8104 NN denotes cells
T3730 8105-8109 VB denotes were
T3731 8110-8118 VB denotes obtained
T3732 8119-8123 IN denotes from
T3733 8124-8135 VB denotes heparinized
T3734 8136-8141 NN denotes blood
T3735 8142-8144 IN denotes by
T3736 8145-8159 JJ denotes Ficoll-Hypaque
T3737 8160-8167 NN denotes density
T3738 8168-8176 NN denotes gradient
T3739 8178-8187 NN denotes Monocytes
T3740 8188-8192 VB denotes were
T3741 8193-8197 RB denotes then
T3742 8198-8206 VB denotes isolated
T3743 8207-8209 IN denotes by
T3744 8210-8219 NN denotes adherence
T3745 8220-8222 TO denotes to
T3746 8223-8230 NN denotes culture
T3747 8231-8237 NN denotes flasks
T3748 8238-8240 IN denotes as
T3749 8241-8250 VB denotes described
T3750 8251-8252 -LRB- denotes [
T3751 8252-8254 CD denotes 28
T3752 8254-8255 -RRB- denotes ]
T3753 8257-8260 IN denotes For
T3754 8261-8265 NN denotes cell
T3755 8266-8277 NN denotes aggregation
T3756 8277-8278 -COMMA- denotes ,
T3757 8279-8288 NN denotes monocytes
T3758 8289-8293 VB denotes were
T3759 8294-8302 VB denotes cultured
T3760 8303-8305 IN denotes in
T3761 8306-8309 DT denotes the
T3762 8310-8318 NN denotes presence
T3763 8319-8321 CC denotes or
T3764 8322-8329 NN denotes absence
T3765 8330-8332 IN denotes of
T3766 8333-8336 NN denotes C10
T3767 8337-8339 CC denotes or
T3768 8340-8343 NN denotes C40
T3769 8344-8347 IN denotes for
T3770 8348-8350 CD denotes 72
T3771 8351-8352 NN denotes h
T3772 8354-8358 NN denotes Cell
T3773 8359-8367 NN denotes colonies
T3774 8368-8372 VB denotes were
T3775 8373-8382 VB denotes monitored
T3776 8383-8388 IN denotes under
T3777 8389-8391 DT denotes an
T3778 8392-8400 VB denotes inverted
T3779 8401-8406 NN denotes phase
T3780 8407-8415 NN denotes contrast
T3781 8416-8426 NN denotes microscope
T3782 8427-8434 VB denotes coupled
T3783 8435-8442 IN denotes through
T3784 8443-8444 DT denotes a
T3785 8445-8450 NN denotes video
T3786 8451-8457 NN denotes camera
T3787 8458-8460 TO denotes to
T3788 8461-8462 DT denotes a
T3789 8463-8471 NN denotes computer
T3790 8473-8475 IN denotes In
T3791 8476-8480 DT denotes some
T3792 8481-8486 NN denotes wells
T3793 8486-8487 -COMMA- denotes ,
T3794 8488-8500 VB denotes neutralizing
T3795 8501-8511 JJ denotes monoclonal
T3796 8512-8520 NN denotes antibody
T3797 8521-8523 TO denotes to
T3798 8524-8530 NN denotes ICAM-1
T3799 8531-8532 -LRB- denotes (
T3800 8532-8535 CD denotes 2.5
T3801 8536-8541 NN denotes µg/ml
T3802 8541-8542 -RRB- denotes )
T3803 8543-8544 -LRB- denotes (
T3804 8544-8548 NNP denotes Tebu
T3805 8548-8549 -COMMA- denotes ,
T3806 8550-8552 NNP denotes Le
T3807 8553-8559 NNP denotes Perray
T3808 8560-8562 NNP denotes en
T3809 8563-8571 NNP denotes Yvelines
T3810 8571-8572 -COMMA- denotes ,
T3811 8573-8579 NNP denotes France
T3812 8579-8580 -RRB- denotes )
T3813 8581-8584 VB denotes was
T3814 8585-8590 VB denotes added
T3986 8593-8597 NN denotes Cell
T3987 8598-8603 NN denotes Cycle
T3988 8604-8612 NN denotes Analysis
T3989 8613-8618 NN denotes THP-1
T3990 8619-8624 NN denotes cells
T3991 8625-8627 IN denotes in
T3992 8628-8639 JJ denotes exponential
T3993 8640-8646 NN denotes growth
T3994 8647-8652 NN denotes phase
T3995 8653-8657 VB denotes were
T3996 8658-8665 VB denotes exposed
T3997 8666-8668 TO denotes to
T3998 8669-8677 JJ denotes complete
T3999 8678-8684 NN denotes medium
T4000 8685-8687 IN denotes in
T4001 8688-8691 DT denotes the
T4002 8692-8700 NN denotes presence
T4003 8701-8703 CC denotes or
T4004 8704-8711 NN denotes absence
T4005 8712-8714 IN denotes of
T4006 8715-8727 NN denotes carrageenans
T4007 8728-8731 IN denotes for
T4008 8732-8734 CD denotes 24
T4009 8735-8736 NN denotes h
T4010 8737-8743 IN denotes before
T4011 8744-8749 VB denotes being
T4012 8750-8757 VB denotes stained
T4013 8758-8762 IN denotes with
T4014 8763-8772 NN denotes propidium
T4015 8773-8779 NN denotes iodide
T4016 8780-8785 VB denotes using
T4017 8786-8789 DT denotes the
T4018 8790-8798 JJ denotes DNA-Prep
T4019 8799-8806 NNP denotes Coulter
T4020 8807-8810 NN denotes kit
T4021 8811-8820 VB denotes according
T4022 8821-8823 TO denotes to
T4023 8824-8827 DT denotes the
T4024 8828-8840 NN denotes manufacturer
T4025 8840-8842 POS denotes 's
T4026 8843-8854 NN denotes instruction
T4027 8855-8856 -LRB- denotes (
T4028 8856-8871 NNP denotes Beckman-Coulter
T4029 8871-8872 -COMMA- denotes ,
T4030 8873-8883 NNP denotes Villepinte
T4031 8883-8884 -COMMA- denotes ,
T4032 8885-8891 NNP denotes France
T4033 8891-8892 -RRB- denotes )
T4034 8894-8898 NN denotes Cell
T4035 8899-8902 NN denotes DNA
T4036 8903-8910 NN denotes content
T4037 8911-8914 VB denotes was
T4038 8915-8919 RB denotes then
T4039 8920-8928 VB denotes analyzed
T4040 8929-8931 IN denotes by
T4041 8932-8936 NN denotes flow
T4042 8937-8946 NN denotes cytometry
T4043 8947-8952 VB denotes using
T4044 8953-8955 DT denotes an
T4045 8956-8961 NN denotes EPICS
T4046 8962-8965 NN denotes XL2
T4047 8966-8967 -LRB- denotes (
T4048 8967-8982 NN denotes Beckman-Coulter
T4049 8982-8983 -RRB- denotes )
T4050 8985-8988 NN denotes Raw
T4051 8989-8993 NN denotes data
T4052 8994-8997 IN denotes for
T4053 8998-9001 DT denotes the
T4054 9002-9014 NN denotes distribution
T4055 9015-9017 IN denotes of
T4056 9018-9021 NN denotes DNA
T4057 9022-9029 NN denotes content
T4058 9030-9032 IN denotes of
T4059 9033-9039 CD denotes 30,000
T4060 9040-9045 NN denotes cells
T4061 9046-9055 VB denotes retrieved
T4062 9056-9060 IN denotes from
T4063 9061-9064 DT denotes the
T4064 9065-9074 NN denotes cytometer
T4065 9075-9079 VB denotes were
T4066 9080-9089 VB denotes expressed
T4067 9090-9092 IN denotes as
T4068 9093-9096 DT denotes the
T4069 9097-9107 NN denotes percentage
T4070 9108-9110 IN denotes of
T4071 9111-9116 NN denotes G0/G1
T4072 9117-9124 IN denotes through
T4073 9125-9129 NN denotes G2/M
T4074 9130-9141 NN denotes populations
T4075 9143-9153 NNP denotes Multicycle
T4076 9154-9156 NN denotes AV
T4077 9157-9165 NN denotes software
T4078 9166-9167 -LRB- denotes (
T4079 9167-9174 NNP denotes Phoenix
T4080 9175-9179 NNP denotes Flow
T4081 9180-9187 NNP denotes Systems
T4082 9187-9188 -COMMA- denotes ,
T4083 9189-9192 NNP denotes San
T4084 9193-9198 NNP denotes Diego
T4085 9198-9199 -COMMA- denotes ,
T4086 9200-9202 NN denotes CA
T4087 9202-9203 -RRB- denotes )
T4088 9204-9207 VB denotes was
T4089 9208-9212 VB denotes used
T4090 9213-9215 TO denotes to
T4091 9216-9224 VB denotes generate
T4092 9225-9228 NN denotes DNA
T4093 9229-9236 NN denotes content
T4094 9237-9246 NN denotes frequency
T4095 9247-9257 NN denotes histograms
T4096 9258-9261 CC denotes and
T4097 9262-9272 VB denotes facilitate
T4098 9273-9277 NN denotes data
T4099 9278-9286 NN denotes analysis
T4242 9289-9293 NNP denotes Cell
T4243 9294-9301 NNP denotes Surface
T4244 9302-9309 NNP denotes Antigen
T4245 9310-9320 NN denotes Expression
T4246 9321-9329 NN denotes Analysis
T4247 9330-9340 JJ denotes Peripheral
T4248 9341-9346 NN denotes Blood
T4249 9347-9356 NN denotes Monocytes
T4250 9357-9359 CC denotes or
T4251 9360-9365 NN denotes THP-1
T4252 9366-9371 NN denotes cells
T4253 9372-9376 VB denotes were
T4254 9377-9384 VB denotes exposed
T4255 9385-9387 TO denotes to
T4256 9388-9396 JJ denotes complete
T4257 9397-9403 NN denotes medium
T4258 9404-9406 IN denotes in
T4259 9407-9410 DT denotes the
T4260 9411-9419 NN denotes presence
T4261 9420-9422 CC denotes or
T4262 9423-9430 NN denotes absence
T4263 9431-9433 IN denotes of
T4264 9434-9445 NN denotes carrageenan
T4265 9446-9449 IN denotes for
T4266 9450-9452 CD denotes 36
T4267 9453-9454 NN denotes h
T4268 9456-9461 IN denotes After
T4269 9462-9465 CD denotes two
T4270 9466-9472 NN denotes washes
T4271 9473-9475 IN denotes in
T4272 9476-9479 NNP denotes PBS
T4273 9480-9487 IN denotes without
T4274 9488-9492 NN denotes Ca2+
T4275 9493-9496 CC denotes and
T4276 9497-9501 NN denotes Mg2+
T4277 9501-9502 -COMMA- denotes ,
T4278 9503-9508 NN denotes cells
T4279 9509-9513 VB denotes were
T4280 9514-9523 VB denotes incubated
T4281 9524-9526 IN denotes in
T4282 9527-9530 NN denotes PBS
T4283 9531-9541 VB denotes containing
T4284 9542-9545 CD denotes 0.1
T4285 9545-9546 NN denotes %
T4286 9547-9554 NN denotes gelatin
T4287 9555-9558 CC denotes and
T4288 9559-9560 CD denotes 8
T4289 9560-9561 NN denotes %
T4290 9562-9564 NN denotes AB
T4291 9565-9570 JJ denotes human
T4292 9571-9576 NN denotes serum
T4293 9577-9579 TO denotes to
T4294 9580-9587 VB denotes prevent
T4295 9588-9595 NN denotes binding
T4296 9596-9598 TO denotes to
T4297 9599-9601 NN denotes Fc
T4298 9602-9611 NN denotes receptors
T4299 9613-9617 RB denotes Then
T4300 9617-9618 -COMMA- denotes ,
T4301 9619-9624 NN denotes 5×105
T4302 9625-9630 NN denotes cells
T4303 9631-9635 VB denotes were
T4304 9636-9645 VB denotes incubated
T4305 9646-9650 IN denotes with
T4306 9651-9658 JJ denotes primary
T4307 9659-9669 NN denotes antibodies
T4308 9670-9672 IN denotes at
T4309 9673-9676 NN denotes 4°C
T4310 9677-9680 IN denotes for
T4311 9681-9683 CD denotes 30
T4312 9684-9687 NN denotes min
T4313 9689-9692 CD denotes Two
T4314 9693-9698 JJ denotes other
T4315 9699-9705 NN denotes washes
T4316 9706-9708 IN denotes in
T4317 9709-9712 NNP denotes PBS
T4318 9713-9721 VB denotes preceded
T4319 9722-9732 NN denotes incubation
T4320 9733-9737 IN denotes with
T4321 9738-9753 JJ denotes FITC-conjugated
T4322 9754-9758 NN denotes goat
T4323 9759-9767 NN denotes antibody
T4324 9768-9778 JJ denotes anti-mouse
T4325 9779-9782 NN denotes IgG
T4326 9783-9790 VB denotes diluted
T4327 9791-9797 CD denotes 1/1000
T4328 9798-9800 IN denotes at
T4329 9801-9804 NN denotes 4°C
T4330 9805-9808 IN denotes for
T4331 9809-9811 CD denotes 30
T4332 9812-9815 NN denotes min
T4333 9816-9817 -LRB- denotes (
T4334 9817-9821 NN denotes Tebu
T4335 9821-9822 -RRB- denotes )
T4336 9824-9829 IN denotes After
T4337 9830-9833 CD denotes two
T4338 9834-9844 JJ denotes additional
T4339 9845-9851 NN denotes washes
T4340 9851-9852 -COMMA- denotes ,
T4341 9853-9861 NN denotes analysis
T4342 9862-9864 IN denotes of
T4343 9865-9872 VB denotes stained
T4344 9873-9878 NN denotes cells
T4345 9879-9882 VB denotes was
T4346 9883-9892 VB denotes performed
T4347 9893-9895 IN denotes on
T4348 9896-9898 DT denotes an
T4349 9899-9904 NN denotes EPICS
T4350 9905-9908 NN denotes XL2
T4351 9909-9910 -LRB- denotes (
T4352 9910-9925 NN denotes Beckman-Coulter
T4353 9925-9926 -RRB- denotes )
T4354 9928-9931 DT denotes The
T4355 9932-9936 NN denotes cell
T4356 9937-9947 NN denotes population
T4357 9948-9951 VB denotes was
T4358 9952-9957 VB denotes gated
T4359 9958-9967 VB denotes according
T4360 9968-9970 TO denotes to
T4361 9971-9974 PRP-DOLLAR- denotes its
T4362 9975-9982 JJ denotes forward
T4363 9983-9986 CC denotes and
T4364 9987-9997 JJ denotes wide-angle
T4365 9998-10003 NN denotes light
T4366 10004-10014 NN denotes scattering
T4367 10016-10020 NN denotes Data
T4368 10021-10025 VB denotes were
T4369 10026-10035 VB denotes expressed
T4370 10036-10038 IN denotes as
T4371 10039-10043 JJ denotes mean
T4372 10044-10052 JJ denotes relative
T4373 10053-10065 NN denotes fluorescence
T4374 10066-10075 NN denotes intensity
T4375 10076-10077 -LRB- denotes (
T4376 10077-10080 NN denotes MFI
T4377 10080-10081 -RRB- denotes )
T4378 10082-10084 IN denotes of
T4379 10085-10089 CD denotes 3000
T4380 10090-10095 NN denotes cells
T4587 10098-10101 NN denotes TNF
T4588 10102-10110 NNP denotes Activity
T4589 10111-10119 NNP denotes Bioassay
T4590 10120-10129 NN denotes Monocytes
T4591 10130-10132 CC denotes or
T4592 10133-10138 NN denotes THP-1
T4593 10139-10144 NN denotes cells
T4594 10145-10149 VB denotes were
T4595 10150-10158 VB denotes cultured
T4596 10159-10163 IN denotes with
T4597 10164-10166 CC denotes or
T4598 10167-10174 IN denotes without
T4599 10175-10184 JJ denotes different
T4600 10185-10199 NN denotes concentrations
T4601 10200-10202 IN denotes of
T4602 10203-10207 NN denotes CGNs
T4603 10208-10210 CC denotes or
T4604 10211-10214 NN denotes LPS
T4605 10215-10216 -LRB- denotes (
T4606 10216-10226 NN denotes Salmonella
T4607 10227-10234 NN denotes typhosa
T4608 10234-10235 -COMMA- denotes ,
T4609 10236-10241 NNP denotes Sigma
T4610 10241-10242 -RRB- denotes )
T4611 10243-10246 IN denotes for
T4612 10247-10249 CD denotes 24
T4613 10250-10251 NN denotes h
T4614 10252-10254 CC denotes or
T4615 10255-10258 DT denotes the
T4616 10259-10268 VB denotes indicated
T4617 10269-10273 NN denotes time
T4618 10275-10287 RB denotes Biologically
T4619 10288-10294 JJ denotes active
T4620 10295-10302 NN denotes TNF-α/β
T4621 10303-10305 IN denotes in
T4622 10306-10312 NN denotes tissue
T4623 10313-10320 NN denotes culture
T4624 10321-10332 NN denotes supernatant
T4625 10333-10336 VB denotes was
T4626 10337-10345 VB denotes measured
T4627 10346-10351 VB denotes using
T4628 10352-10355 DT denotes the
T4629 10356-10360 NN denotes WEHI
T4630 10361-10364 CD denotes 164
T4631 10365-10370 NN denotes clone
T4632 10371-10378 NN denotes 13-cell
T4633 10379-10386 NN denotes killing
T4634 10387-10392 NN denotes assay
T4635 10393-10394 -LRB- denotes [
T4636 10394-10396 CD denotes 29
T4637 10396-10397 -RRB- denotes ]
T4638 10399-10402 NN denotes TNF
T4639 10403-10417 NN denotes concentrations
T4640 10418-10421 VB denotes are
T4641 10422-10431 VB denotes expressed
T4642 10432-10434 IN denotes as
T4643 10435-10440 NN denotes pg/ml
T4750 10443-10449 NN denotes RT-PCR
T4751 10450-10458 NN denotes Analysis
T4752 10459-10464 JJ denotes Total
T4753 10465-10468 NN denotes RNA
T4754 10469-10473 IN denotes from
T4755 10474-10483 NN denotes monocytes
T4756 10484-10487 VB denotes was
T4757 10488-10496 VB denotes isolated
T4758 10497-10502 VB denotes using
T4759 10503-10509 NN denotes TRIzol
T4760 10510-10518 NN denotes Reagent™
T4761 10519-10520 -LRB- denotes (
T4762 10520-10530 NN denotes Invitrogen
T4763 10530-10531 -RRB- denotes )
T4764 10531-10535 NN denotes . cD
T4765 10535-10538 VB denotes NA
T4766 10538-10547 VB denotes was gener
T4767 10547-10549 IN denotes at
T4768 10549-10550 CD denotes e
T4769 10550-10552 NN denotes d
T4770 10552-10554 IN denotes on
T4771 10555-10560 JJ denotes 1 µg
T4772 10560-10563 NN denotes of
T4773 10563-10565 IN denotes to
T4774 10565-10566 DT denotes t
T4775 10566-10574 NN denotes al RNA i
T4776 10574-10580 NN denotes n a re
T4777 10580-10582 IN denotes ac
T4778 10582-10584 CD denotes ti
T4779 10584-10586 NN denotes on
T4780 10587-10588 -COMMA- denotes v
T4781 10588-10593 VB denotes olume
T4782 10594-10599 NN denotes of 20
T4783 10600-10607 JJ denotes µl, usi
T4784 10607-10620 NN denotes ng M-MLV reve
T4785 10620-10621 -LRB- denotes r
T4786 10621-10631 NN denotes se transcr
T4787 10631-10632 -RRB- denotes i
T4788 10652-10655 NN denotes PCR
T4789 10656-10659 VB denotes was
T4790 10660-10664 VB denotes done
T4791 10665-10667 IN denotes in
T4792 10668-10671 DT denotes the
T4793 10672-10678 JJ denotes linear
T4794 10679-10684 NN denotes range
T4795 10685-10687 IN denotes of
T4796 10688-10701 NN denotes amplification
T4797 10702-10703 -LRB- denotes (
T4798 10703-10713 VB denotes determined
T4799 10714-10717 IN denotes for
T4800 10718-10722 DT denotes each
T4801 10723-10729 NN denotes primer
T4802 10730-10739 NN denotes pair-cDNA
T4803 10740-10751 NN denotes combination
T4804 10751-10752 -RRB- denotes )
T4805 10754-10762 JJ denotes Standard
T4806 10763-10766 NN denotes PCR
T4807 10767-10776 NN denotes reactions
T4808 10777-10781 VB denotes were
T4809 10782-10791 VB denotes performed
T4810 10792-10796 IN denotes with
T4811 10797-10798 CD denotes 1
T4812 10799-10801 NN denotes µl
T4813 10802-10804 IN denotes of
T4814 10805-10808 DT denotes the
T4815 10809-10813 NN denotes cDNA
T4816 10814-10822 NN denotes solution
T4817 10822-10823 -COMMA- denotes ,
T4818 10824-10826 CD denotes 50
T4819 10827-10829 NN denotes µM
T4820 10830-10832 IN denotes of
T4821 10833-10837 DT denotes each
T4822 10838-10844 NN denotes primer
T4823 10845-10853 NN denotes solution
T4824 10853-10854 -COMMA- denotes ,
T4825 10855-10857 CD denotes 10
T4826 10858-10860 NN denotes mM
T4827 10861-10863 IN denotes of
T4828 10864-10868 DT denotes each
T4829 10869-10873 NN denotes dNTP
T4830 10873-10874 -COMMA- denotes ,
T4831 10875-10877 CD denotes 25
T4832 10878-10880 NN denotes mM
T4833 10881-10886 NN denotes MgCl2
T4834 10886-10887 -COMMA- denotes ,
T4835 10888-10891 NNP denotes 10X
T4836 10892-10900 NNP denotes Goldstar
T4837 10901-10904 NN denotes DNA
T4838 10905-10915 NN denotes polymerase
T4839 10916-10924 NN denotes reaction
T4840 10925-10931 NN denotes buffer
T4841 10931-10932 -COMMA- denotes ,
T4842 10933-10936 CC denotes and
T4843 10937-10940 CD denotes 0.5
T4844 10941-10946 NN denotes units
T4845 10947-10949 IN denotes of
T4846 10950-10958 NNP denotes Goldstar
T4847 10959-10962 NN denotes DNA
T4848 10963-10973 NN denotes polymerase
T4849 10974-10975 -LRB- denotes (
T4850 10975-10985 NNP denotes Eurogentec
T4851 10985-10986 -COMMA- denotes ,
T4852 10987-10994 NNP denotes Seraing
T4853 10994-10995 -COMMA- denotes ,
T4854 10996-11003 NNP denotes Belgium
T4855 11003-11004 -RRB- denotes )
T4856 11006-11011 JJ denotes First
T4857 11012-11015 NN denotes PCR
T4858 11016-11021 NN denotes cycle
T4859 11022-11031 VB denotes consisted
T4860 11032-11034 IN denotes of
T4861 11035-11036 CD denotes 1
T4862 11037-11040 NN denotes min
T4863 11041-11043 IN denotes at
T4864 11044-11048 NN denotes 92°C
T4865 11048-11049 -COMMA- denotes ,
T4866 11050-11051 CD denotes 1
T4867 11052-11055 NN denotes min
T4868 11056-11058 IN denotes at
T4869 11059-11063 NN denotes 58°C
T4870 11064-11067 CC denotes and
T4871 11068-11069 CD denotes 1
T4872 11070-11073 NN denotes min
T4873 11074-11076 IN denotes at
T4874 11077-11081 NN denotes 72°C
T4875 11081-11082 -COLON- denotes ;
T4876 11083-11087 RB denotes then
T4877 11088-11092 DT denotes each
T4878 11093-11096 NN denotes PCR
T4879 11097-11102 NN denotes cycle
T4880 11103-11112 VB denotes consisted
T4881 11113-11115 IN denotes of
T4882 11116-11118 CD denotes 40
T4883 11119-11122 NN denotes sec
T4884 11123-11125 IN denotes at
T4885 11126-11130 NN denotes 92°C
T4886 11130-11131 -COMMA- denotes ,
T4887 11132-11134 CD denotes 40
T4888 11135-11138 NN denotes sec
T4889 11139-11141 IN denotes at
T4890 11142-11146 NN denotes 58°C
T4891 11147-11150 CC denotes and
T4892 11151-11153 CD denotes 50
T4893 11154-11157 NN denotes sec
T4894 11158-11160 IN denotes at
T4895 11161-11166 NNP denotes 72°C.
T4896 11167-11171 NN denotes cDNA
T4897 11172-11175 IN denotes for
T4898 11176-11183 NN denotes β-actin
T4899 11184-11187 VB denotes was
T4900 11188-11197 VB denotes amplified
T4901 11198-11201 IN denotes for
T4902 11202-11204 CD denotes 28
T4903 11205-11211 NN denotes cycles
T4904 11212-11217 VB denotes using
T4905 11218-11221 DT denotes the
T4906 11222-11228 NN denotes oligos
T4907 11228-11229 -COLON- denotes :
T4908 11230-11235 NN denotes sense
T4909 11236-11261 NN denotes 5′-GGCATCGTGATGGACTCCG-3′
T4910 11262-11265 CC denotes and
T4911 11266-11275 JJ denotes antisense
T4912 11276-11301 JJ denotes 5′GCTGGAAGGTGGACAGCGA-3′.
T4913 11302-11306 NN denotes cDNA
T4914 11307-11310 IN denotes for
T4915 11311-11316 NN denotes TNF-α
T4916 11317-11320 VB denotes was
T4917 11321-11330 VB denotes amplified
T4918 11331-11334 IN denotes for
T4919 11335-11337 CD denotes 35
T4920 11338-11344 NN denotes cycles
T4921 11345-11350 VB denotes using
T4922 11351-11354 DT denotes the
T4923 11355-11361 NN denotes oligos
T4924 11361-11362 -COLON- denotes :
T4925 11363-11368 NN denotes sense
T4926 11369-11395 NN denotes 5′-AAGCCTGTAGCCCATGTTGT-3′
T4927 11396-11399 CC denotes and
T4928 11400-11409 JJ denotes antisense
T4929 11410-11437 JJ denotes 5′-CAGATAGATGGGCTCATACC-3′.
T4930 11438-11442 NN denotes cDNA
T4931 11443-11446 IN denotes for
T4932 11447-11453 NN denotes ICAM-1
T4933 11454-11457 VB denotes was
T4934 11458-11467 VB denotes amplified
T4935 11468-11471 IN denotes for
T4936 11472-11474 CD denotes 35
T4937 11475-11481 NN denotes cycles
T4938 11482-11487 VB denotes using
T4939 11488-11491 DT denotes the
T4940 11492-11498 NN denotes oligos
T4941 11499-11504 VB denotes sense
T4942 11505-11533 NN denotes 5′-GTAGCAGCCGCAGTCATAATGG-3′
T4943 11534-11537 CC denotes and
T4944 11538-11547 JJ denotes antisense
T4945 11548-11552 NN denotes 5′-A
T4946 11553-11577 NN denotes TGCTGTTGTATCTGACTGAGG-3′
T5239 11580-11585 NN denotes NF-kB
T5240 11586-11599 NN denotes Transcription
T5241 11600-11608 NNP denotes Reporter
T5242 11609-11613 NNP denotes Gene
T5243 11614-11619 NNP denotes Assay
T5244 11620-11623 DT denotes The
T5245 11624-11631 NN denotes plasmid
T5246 11632-11641 NN denotes 3XMHC-luc
T5247 11642-11643 -LRB- denotes (
T5248 11643-11644 DT denotes a
T5249 11645-11653 JJ denotes generous
T5250 11654-11658 NN denotes gift
T5251 11659-11663 IN denotes from
T5252 11664-11668 NNP denotes Drs.
T5253 11669-11671 NNP denotes J.
T5254 11672-11680 NNP denotes Westwick
T5255 11681-11684 CC denotes and
T5256 11685-11689 NNP denotes D.A.
T5257 11690-11697 NNP denotes Brenner
T5258 11697-11698 -COMMA- denotes ,
T5259 11699-11709 NNP denotes University
T5260 11710-11712 IN denotes of
T5261 11713-11718 NNP denotes North
T5262 11719-11727 NNP denotes Carolina
T5263 11727-11728 -COMMA- denotes ,
T5264 11729-11735 NNP denotes Chapel
T5265 11736-11740 NNP denotes Hill
T5266 11740-11741 -RRB- denotes )
T5267 11742-11750 VB denotes contains
T5268 11751-11756 CD denotes three
T5269 11757-11763 NN denotes copies
T5270 11764-11766 IN denotes of
T5271 11767-11783 JJ denotes NF-κB-responsive
T5272 11784-11791 NN denotes element
T5273 11792-11796 IN denotes from
T5274 11797-11800 DT denotes the
T5275 11801-11804 NN denotes MHC
T5276 11805-11810 NN denotes class
T5277 11811-11812 CD denotes I
T5278 11813-11818 NN denotes locus
T5279 11818-11819 -COMMA- denotes ,
T5280 11820-11826 VB denotes placed
T5281 11827-11835 RB denotes upstream
T5282 11836-11838 IN denotes of
T5283 11839-11842 DT denotes the
T5284 11843-11853 NN denotes luciferase
T5285 11854-11858 NN denotes gene
T5286 11860-11865 JJ denotes Human
T5287 11866-11875 JJ denotes monocytic
T5288 11876-11881 NN denotes THP-1
T5289 11882-11887 NN denotes cells
T5290 11888-11892 VB denotes were
T5291 11893-11904 RB denotes transiently
T5292 11905-11916 VB denotes transfected
T5293 11917-11919 IN denotes as
T5294 11920-11930 RB denotes previously
T5295 11931-11940 VB denotes described
T5296 11941-11942 -LRB- denotes [
T5297 11942-11944 CD denotes 30
T5298 11944-11945 -RRB- denotes ]
T5299 11945-11946 -COMMA- denotes ,
T5300 11947-11950 CC denotes and
T5301 11951-11955 RB denotes then
T5302 11956-11964 VB denotes cultured
T5303 11965-11968 IN denotes for
T5304 11969-11970 CD denotes 4
T5305 11971-11972 NN denotes h
T5306 11973-11978 RB denotes alone
T5307 11979-11981 CC denotes or
T5308 11982-11986 IN denotes with
T5309 11987-11997 VB denotes increasing
T5310 11998-12011 NN denotes concentration
T5311 12012-12014 IN denotes of
T5312 12015-12021 CC denotes either
T5313 12022-12025 NN denotes C10
T5314 12026-12028 CC denotes or
T5315 12029-12032 NN denotes C40
T5316 12034-12044 NN denotes Luciferase
T5317 12045-12053 NN denotes activity
T5318 12054-12057 VB denotes was
T5319 12058-12068 VB denotes determined
T5320 12069-12074 VB denotes using
T5321 12075-12076 DT denotes a
T5322 12077-12088 NN denotes luminometer
T5323 12089-12090 -LRB- denotes (
T5324 12090-12099 NN denotes Monolight
T5325 12100-12104 CD denotes 2010
T5326 12105-12116 NNP denotes Luminometer
T5327 12116-12117 -COMMA- denotes ,
T5328 12118-12121 NNP denotes Ann
T5329 12122-12127 NNP denotes Arbor
T5330 12127-12128 -COMMA- denotes ,
T5331 12129-12131 NNP denotes MI
T5332 12131-12132 -RRB- denotes )
T5492 12135-12142 NNP denotes Western
T5493 12143-12147 NNP denotes Blot
T5494 12148-12156 NN denotes Analysis
T5495 12157-12162 NN denotes THP-1
T5496 12163-12168 NN denotes cells
T5497 12169-12173 VB denotes were
T5498 12174-12184 VB denotes stimulated
T5499 12185-12188 IN denotes for
T5500 12189-12196 JJ denotes various
T5501 12197-12204 NN denotes lengths
T5502 12205-12207 IN denotes of
T5503 12208-12212 NN denotes time
T5504 12213-12217 IN denotes with
T5505 12218-12221 CD denotes 0.1
T5506 12222-12227 NN denotes mg/ml
T5507 12228-12231 NN denotes C10
T5508 12232-12234 CC denotes or
T5509 12235-12238 NN denotes C40
T5510 12238-12239 -COMMA- denotes ,
T5511 12240-12242 CC denotes or
T5512 12243-12245 CD denotes 10
T5513 12246-12251 NN denotes µg/ml
T5514 12252-12255 NN denotes LPS
T5515 12257-12262 NN denotes Cells
T5516 12263-12267 VB denotes were
T5517 12268-12272 RB denotes then
T5518 12273-12281 VB denotes pelleted
T5519 12281-12282 -COMMA- denotes ,
T5520 12283-12289 VB denotes washed
T5521 12290-12293 CC denotes and
T5522 12294-12305 VB denotes homogenised
T5523 12306-12308 IN denotes in
T5524 12309-12314 NN denotes lysis
T5525 12315-12321 NN denotes buffer
T5526 12322-12323 -LRB- denotes (
T5527 12323-12325 CD denotes 10
T5528 12326-12328 NN denotes mM
T5529 12329-12334 NNP denotes Hepes
T5530 12334-12335 -COMMA- denotes ,
T5531 12336-12338 NN denotes pH
T5532 12339-12342 CD denotes 7.9
T5533 12342-12343 -COMMA- denotes ,
T5534 12344-12347 CD denotes 150
T5535 12348-12350 NN denotes mM
T5536 12351-12355 NN denotes NaCl
T5537 12355-12356 -COMMA- denotes ,
T5538 12357-12358 CD denotes 1
T5539 12359-12361 NN denotes mM
T5540 12362-12366 NN denotes EDTA
T5541 12366-12367 -COMMA- denotes ,
T5542 12368-12371 CD denotes 0.6
T5543 12371-12372 NN denotes %
T5544 12373-12378 NN denotes NP-40
T5545 12378-12379 -COMMA- denotes ,
T5546 12380-12383 CC denotes and
T5547 12384-12387 CD denotes 0.5
T5548 12388-12390 NN denotes mM
T5549 12391-12395 NN denotes PMSF
T5550 12395-12396 -RRB- denotes )
T5551 12397-12399 IN denotes on
T5552 12400-12403 NN denotes ice
T5553 12405-12416 NN denotes Homogenates
T5554 12417-12421 VB denotes were
T5555 12422-12431 VB denotes sonicated
T5556 12431-12432 -COMMA- denotes ,
T5557 12433-12444 VB denotes centrifuged
T5558 12445-12447 IN denotes at
T5559 12448-12454 CD denotes 10,000
T5560 12455-12458 NN denotes rpm
T5561 12459-12461 TO denotes to
T5562 12462-12468 VB denotes remove
T5563 12469-12477 JJ denotes cellular
T5564 12478-12484 NN denotes debris
T5565 12484-12485 -COMMA- denotes ,
T5566 12486-12489 CC denotes and
T5567 12490-12501 NN denotes supernatant
T5568 12502-12511 VB denotes collected
T5569 12513-12520 NN denotes Protein
T5570 12521-12534 NN denotes concentration
T5571 12535-12538 VB denotes was
T5572 12539-12549 VB denotes determined
T5573 12550-12555 VB denotes using
T5574 12556-12559 DT denotes the
T5575 12560-12562 NN denotes DC
T5576 12563-12570 NN denotes Protein
T5577 12571-12576 NN denotes Assay
T5578 12577-12578 -LRB- denotes (
T5579 12578-12585 NN denotes Bio-Rad
T5580 12585-12586 -RRB- denotes )
T5581 12588-12596 NN denotes Proteins
T5582 12597-12599 IN denotes in
T5583 12600-12607 NN denotes samples
T5584 12608-12609 -LRB- denotes (
T5585 12609-12611 CD denotes 15
T5586 12612-12614 -COLON- denotes µg
T5587 12615-12620 JJ denotes total
T5588 12621-12629 NN denotes proteins
T5589 12629-12630 -RRB- denotes )
T5590 12631-12635 VB denotes were
T5591 12636-12644 VB denotes resolved
T5592 12645-12647 IN denotes in
T5593 12648-12649 DT denotes a
T5594 12650-12660 VB denotes denaturing
T5595 12661-12663 CD denotes 12
T5596 12663-12664 NN denotes %
T5597 12665-12679 NN denotes polyacrylamide
T5598 12680-12683 NN denotes gel
T5599 12684-12687 CC denotes and
T5600 12688-12699 VB denotes transferred
T5601 12700-12702 TO denotes to
T5602 12703-12704 DT denotes a
T5603 12705-12719 NN denotes nitrocellulose
T5604 12720-12728 NN denotes membrane
T5605 12730-12735 JJ denotes I-κBα
T5606 12736-12743 NN denotes protein
T5607 12744-12747 VB denotes was
T5608 12748-12756 VB denotes detected
T5609 12757-12762 VB denotes using
T5610 12763-12764 DT denotes a
T5611 12765-12771 NN denotes rabbit
T5612 12772-12782 JJ denotes polyclonal
T5613 12783-12791 NN denotes antibody
T5614 12792-12793 -LRB- denotes (
T5615 12793-12798 NNP denotes Santa
T5616 12799-12803 NNP denotes Cruz
T5617 12804-12817 NNP denotes Biotechnology
T5618 12817-12818 -COMMA- denotes ,
T5619 12819-12821 NN denotes CA
T5620 12821-12822 -RRB- denotes )
T5621 12823-12831 VB denotes followed
T5622 12832-12834 IN denotes by
T5623 12835-12836 DT denotes a
T5624 12837-12848 JJ denotes horseradish
T5625 12849-12867 JJ denotes peroxidase-coupled
T5626 12868-12872 NN denotes goat
T5627 12873-12883 JJ denotes polyclonal
T5628 12884-12892 NN denotes antibody
T5629 12893-12900 IN denotes against
T5630 12901-12907 NN denotes rabbit
T5631 12908-12910 NN denotes Ig
T5632 12911-12912 -LRB- denotes (
T5633 12912-12918 NNP denotes Caltag
T5634 12919-12931 NNP denotes Laboratories
T5635 12931-12932 -RRB- denotes )
T5636 12934-12941 RB denotes Finally
T5637 12941-12942 -COMMA- denotes ,
T5638 12943-12946 NN denotes IκB
T5639 12947-12952 NN denotes bands
T5640 12953-12957 VB denotes were
T5641 12958-12966 VB denotes revealed
T5642 12967-12972 VB denotes using
T5643 12973-12976 DT denotes the
T5644 12977-12981 NN denotes ECL™
T5645 12982-12991 NN denotes detection
T5646 12992-12998 NN denotes system
T5647 12999-13000 -LRB- denotes (
T5648 13000-13008 NNP denotes Amersham
T5649 13009-13018 NNP denotes Pharmacia
T5650 13019-13026 NNP denotes Biotech
T5651 13026-13027 -COMMA- denotes ,
T5652 13028-13031 NNP denotes Les
T5653 13032-13037 NNP denotes Ullis
T5654 13037-13038 -COMMA- denotes ,
T5655 13039-13045 NNP denotes France
T5656 13045-13046 -RRB- denotes )
T5657 13047-13056 VB denotes according
T5658 13057-13059 TO denotes to
T5659 13060-13063 DT denotes the
T5660 13064-13077 NN denotes manufacturers
T5661 13077-13078 POS denotes '
T5662 13079-13090 NN denotes instruction
T5663 13092-13100 NN denotes Antibody
T5664 13101-13103 TO denotes to
T5665 13104-13113 NN denotes α-Tubulin
T5666 13114-13115 -LRB- denotes (
T5667 13115-13120 NNP denotes Santa
T5668 13121-13125 NNP denotes Cruz
T5669 13125-13126 -RRB- denotes )
T5670 13127-13130 VB denotes was
T5671 13131-13134 NN denotes use
T5672 13135-13137 IN denotes as
T5673 13138-13145 VB denotes loading
T5674 13146-13153 NN denotes control
T5675 13155-13158 IN denotes For
T5676 13159-13166 JJ denotes nuclear
T5677 13167-13172 NN denotes NF-κB
T5678 13172-13173 -COMMA- denotes ,
T5679 13174-13179 NN denotes THP-1
T5680 13180-13185 NN denotes cells
T5681 13186-13190 VB denotes were
T5682 13191-13201 VB denotes stimulated
T5683 13202-13206 IN denotes with
T5684 13207-13208 CD denotes 1
T5685 13209-13214 NN denotes mg/ml
T5686 13215-13218 NN denotes C10
T5687 13219-13221 CC denotes or
T5688 13222-13225 NN denotes C40
T5689 13226-13229 IN denotes for
T5690 13230-13232 CD denotes 30
T5691 13233-13240 NN denotes minutes
T5692 13241-13243 IN denotes at
T5693 13244-13249 NNP denotes 37°C.
T5694 13250-13255 NNP denotes Cells
T5695 13256-13260 VB denotes were
T5696 13261-13265 RB denotes then
T5697 13266-13274 VB denotes pelleted
T5698 13275-13278 CC denotes and
T5699 13279-13285 NN denotes nuclei
T5700 13286-13295 VB denotes separated
T5701 13296-13298 IN denotes as
T5702 13299-13308 VB denotes described
T5703 13309-13310 -LRB- denotes [
T5704 13310-13312 CD denotes 31
T5705 13312-13313 -RRB- denotes ]
T5706 13315-13321 NN denotes Nuclei
T5707 13322-13326 VB denotes were
T5708 13327-13333 VB denotes washed
T5709 13334-13337 CC denotes and
T5710 13338-13349 VB denotes homogenized
T5711 13350-13358 RB denotes directly
T5712 13359-13361 IN denotes in
T5713 13362-13369 VB denotes loading
T5714 13370-13371 -LRB- denotes (
T5715 13371-13377 NNP denotes Laemli
T5716 13377-13378 -RRB- denotes )
T5717 13379-13385 NN denotes buffer
T5718 13386-13389 CC denotes and
T5719 13390-13396 VB denotes heated
T5720 13397-13400 IN denotes for
T5721 13401-13402 CD denotes 5
T5722 13403-13410 NN denotes minutes
T5723 13411-13413 IN denotes at
T5724 13414-13420 NNP denotes 100°C.
T5725 13421-13429 NNP denotes Proteins
T5726 13430-13432 IN denotes in
T5727 13433-13440 NN denotes samples
T5728 13441-13445 VB denotes were
T5729 13446-13454 VB denotes resolved
T5730 13455-13457 IN denotes in
T5731 13458-13459 DT denotes a
T5732 13460-13470 VB denotes denaturing
T5733 13471-13472 CD denotes 8
T5734 13472-13473 NN denotes %
T5735 13474-13488 NN denotes polyacrylamide
T5736 13489-13492 NN denotes gel
T5737 13493-13496 CC denotes and
T5738 13497-13508 VB denotes transferred
T5739 13509-13511 TO denotes to
T5740 13512-13513 DT denotes a
T5741 13514-13528 NN denotes polyvinylidine
T5742 13529-13537 NN denotes fluoride
T5743 13538-13539 -LRB- denotes (
T5744 13539-13543 NN denotes PVDF
T5745 13543-13544 -RRB- denotes )
T5746 13545-13553 NN denotes membrane
T5747 13554-13555 -LRB- denotes (
T5748 13555-13566 NN denotes Immobilon-P
T5749 13566-13567 -COLON- denotes ;
T5750 13568-13577 NNP denotes Millipore
T5751 13577-13578 -COMMA- denotes ,
T5752 13579-13586 NNP denotes Bedford
T5753 13586-13587 -COMMA- denotes ,
T5754 13588-13590 JJ denotes MA
T5755 13590-13591 -RRB- denotes )
T5756 13593-13602 NN denotes Membranes
T5757 13603-13607 VB denotes were
T5758 13608-13617 VB denotes incubated
T5759 13618-13620 IN denotes in
T5760 13621-13629 VB denotes blocking
T5761 13630-13636 NN denotes buffer
T5762 13637-13638 -LRB- denotes (
T5763 13638-13639 CD denotes 1
T5764 13639-13640 NN denotes %
T5765 13641-13644 NN denotes BSA
T5766 13644-13645 -COMMA- denotes ,
T5767 13646-13648 IN denotes in
T5768 13649-13652 NNP denotes PBS
T5769 13652-13653 -RRB- denotes )
T5770 13654-13657 IN denotes for
T5771 13658-13661 CD denotes two
T5772 13662-13667 NN denotes hours
T5773 13668-13670 IN denotes at
T5774 13671-13675 NN denotes room
T5775 13676-13687 NN denotes temperature
T5776 13689-13698 NN denotes Membranes
T5777 13699-13703 VB denotes were
T5778 13704-13716 RB denotes subsequently
T5779 13717-13723 VB denotes probed
T5780 13724-13728 IN denotes with
T5781 13729-13732 DT denotes the
T5782 13733-13746 JJ denotes corresponding
T5783 13747-13755 NN denotes antibody
T5784 13756-13758 IN denotes in
T5785 13759-13767 VB denotes blocking
T5786 13768-13774 NN denotes buffer
T5787 13774-13775 -COMMA- denotes ,
T5788 13776-13785 RB denotes overnight
T5789 13787-13793 NN denotes Rabbit
T5790 13794-13804 JJ denotes polyclonal
T5791 13805-13813 NN denotes antibody
T5792 13814-13824 JJ denotes anti-NF-κB
T5793 13825-13828 NN denotes p50
T5794 13829-13836 NN denotes subunit
T5795 13837-13838 -LRB- denotes (
T5796 13838-13839 -SHARP- denotes #
T5797 13840-13846 NN denotes sc-114
T5798 13846-13847 -RRB- denotes )
T5799 13848-13850 CC denotes or
T5800 13851-13861 JJ denotes anti-NF-κB
T5801 13862-13865 NN denotes p65
T5802 13866-13873 NN denotes subunit
T5803 13874-13875 -LRB- denotes (
T5804 13875-13876 -SHARP- denotes #
T5805 13877-13883 NN denotes sc-109
T5806 13883-13884 -RRB- denotes )
T5807 13885-13889 IN denotes from
T5808 13890-13895 NNP denotes Santa
T5809 13896-13900 NNP denotes Cruz
T5810 13901-13914 NNP denotes Biotechnology
T5811 13915-13919 VB denotes were
T5812 13920-13924 VB denotes used
T5813 13926-13935 NN denotes Membranes
T5814 13936-13940 VB denotes were
T5815 13941-13947 VB denotes washed
T5816 13948-13951 CD denotes six
T5817 13952-13957 NN denotes times
T5818 13958-13960 IN denotes in
T5819 13961-13964 NNP denotes PBS
T5820 13965-13969 IN denotes with
T5821 13970-13974 CD denotes 0.05
T5822 13974-13975 NN denotes %
T5823 13976-13981 CD denotes Tween
T5824 13982-13984 CD denotes 20
T5825 13984-13985 -COMMA- denotes ,
T5826 13986-13987 CD denotes 5
T5827 13988-13995 NN denotes minutes
T5828 13996-14000 DT denotes each
T5829 14001-14005 NN denotes time
T5830 14005-14006 -COMMA- denotes ,
T5831 14007-14010 CC denotes and
T5832 14011-14020 VB denotes incubated
T5833 14021-14025 IN denotes with
T5834 14026-14027 DT denotes a
T5835 14028-14034 CD denotes 1/3000
T5836 14035-14043 NN denotes dilution
T5837 14044-14046 IN denotes of
T5838 14047-14061 JJ denotes HRP-conjugated
T5839 14062-14063 NN denotes F
T5840 14063-14064 -LRB- denotes (
T5841 14064-14066 NN denotes ab
T5842 14066-14067 -DQE- denotes '
T5843 14067-14068 -RRB- denotes )
T5844 14068-14069 CD denotes 2
T5845 14070-14074 NN denotes goat
T5846 14075-14086 JJ denotes anti-rabbit
T5847 14087-14090 NN denotes IgG
T5848 14091-14093 IN denotes in
T5849 14094-14095 CD denotes 5
T5850 14095-14096 NN denotes %
T5851 14097-14103 JJ denotes nonfat
T5852 14104-14107 JJ denotes dry
T5853 14108-14112 NN denotes milk
T5854 14113-14116 CC denotes and
T5855 14117-14121 CD denotes 0.05
T5856 14121-14122 NN denotes %
T5857 14123-14128 CD denotes Tween
T5858 14129-14131 CD denotes 20
T5859 14132-14134 IN denotes in
T5860 14135-14138 NNP denotes PBS
T5861 14139-14142 IN denotes for
T5862 14143-14144 CD denotes 1
T5863 14145-14149 NN denotes hour
T5864 14150-14152 IN denotes at
T5865 14153-14157 NN denotes room
T5866 14158-14169 NN denotes temperature
T5867 14171-14176 IN denotes After
T5868 14177-14184 VB denotes washing
T5869 14185-14188 CD denotes six
T5870 14189-14193 JJ denotes more
T5871 14194-14199 NN denotes times
T5872 14200-14202 IN denotes in
T5873 14203-14206 NNP denotes PBS
T5874 14207-14211 IN denotes with
T5875 14212-14216 CD denotes 0.05
T5876 14216-14217 NN denotes %
T5877 14218-14223 CD denotes Tween
T5878 14224-14226 CD denotes 20
T5879 14226-14227 -COMMA- denotes ,
T5880 14228-14245 JJ denotes antibody-reactive
T5881 14246-14254 NN denotes proteins
T5882 14255-14259 VB denotes were
T5883 14260-14268 VB denotes detected
T5884 14269-14274 VB denotes using
T5885 14275-14276 DT denotes a
T5886 14277-14294 NN denotes chemiluminescence
T5887 14295-14304 NN denotes substrate
T5888 14305-14306 -LRB- denotes (
T5889 14306-14317 NNP denotes SuperSignal
T5890 14317-14318 -COLON- denotes ;
T5891 14319-14325 NNP denotes Pierce
T5892 14325-14326 -COMMA- denotes ,
T5893 14327-14335 NNP denotes Rockford
T5894 14335-14336 -COMMA- denotes ,
T5895 14337-14339 NN denotes IL
T5896 14339-14340 -RRB- denotes )
T5897 14341-14350 VB denotes according
T5898 14351-14353 TO denotes to
T5899 14354-14357 DT denotes the
T5900 14358-14370 NN denotes manufacturer
T5901 14370-14372 POS denotes 's
T5902 14373-14385 NN denotes instructions
T5903 14387-14389 TO denotes To
T5904 14390-14397 VB denotes confirm
T5905 14398-14402 IN denotes that
T5906 14403-14413 JJ denotes equivalent
T5907 14414-14421 NN denotes amounts
T5908 14422-14424 IN denotes of
T5909 14425-14432 NN denotes protein
T5910 14433-14437 VB denotes were
T5911 14438-14444 VB denotes loaded
T5912 14445-14447 IN denotes in
T5913 14448-14452 DT denotes each
T5914 14453-14457 NN denotes line
T5915 14457-14458 -COMMA- denotes ,
T5916 14459-14468 NN denotes membranes
T5917 14469-14473 VB denotes were
T5918 14474-14478 RB denotes also
T5919 14479-14486 JJ denotes Western
T5920 14487-14494 VB denotes blotted
T5921 14495-14498 IN denotes for
T5922 14499-14502 NN denotes ERK
T5923 14503-14505 IN denotes as
T5924 14506-14515 VB denotes described
T5925 14516-14517 -LRB- denotes [
T5926 14517-14519 CD denotes 32
T5927 14519-14520 -RRB- denotes ]
T6465 14523-14531 NN denotes Analysis
T6466 14532-14534 IN denotes of
T6467 14535-14540 NN denotes NF-κB
T6468 14541-14551 NN denotes Activation
T6469 14552-14554 IN denotes by
T6470 14555-14559 NN denotes Flow
T6471 14560-14569 NN denotes Cytometry
T6472 14570-14577 JJ denotes Nuclear
T6473 14578-14588 NN denotes activation
T6474 14589-14591 IN denotes of
T6475 14592-14597 NN denotes NF−κΒ
T6476 14598-14600 IN denotes by
T6477 14601-14605 NN denotes flow
T6478 14606-14615 NN denotes cytometry
T6479 14616-14619 VB denotes was
T6480 14620-14629 VB denotes performed
T6481 14630-14632 IN denotes as
T6482 14633-14642 VB denotes described
T6483 14643-14644 -LRB- denotes [
T6484 14644-14646 CD denotes 31
T6485 14646-14647 -RRB- denotes ]
T6520 14650-14661 JJ denotes Statistical
T6521 14662-14670 NN denotes Analysis
T6522 14671-14674 DT denotes The
T6523 14675-14682 NN denotes results
T6524 14683-14687 VB denotes were
T6525 14688-14697 VB denotes expressed
T6526 14698-14700 IN denotes as
T6527 14701-14704 DT denotes the
T6528 14705-14709 JJ denotes mean
T6529 14710-14715 NN denotes value
T6530 14716-14717 VB denotes ±
T6531 14718-14724 NNP denotes S.E.M.
T6532 14725-14727 IN denotes of
T6533 14728-14738 JJ denotes individual
T6534 14739-14750 NN denotes experiments
T6535 14752-14755 DT denotes The
T6536 14756-14767 JJ denotes statistical
T6537 14768-14780 NN denotes significance
T6538 14781-14783 IN denotes of
T6539 14784-14787 DT denotes the
T6540 14788-14799 NN denotes differences
T6541 14800-14807 IN denotes between
T6542 14808-14812 JJ denotes mean
T6543 14813-14819 NN denotes values
T6544 14820-14823 VB denotes was
T6545 14824-14832 VB denotes assessed
T6546 14833-14835 IN denotes by
T6547 14836-14839 DT denotes the
T6548 14840-14847 NN denotes Student
T6549 14847-14849 POS denotes 's
T6550 14850-14856 NN denotes t-test
T6551 14857-14860 CC denotes and
T6552 14861-14869 NN denotes analysis
T6553 14870-14872 IN denotes of
T6554 14873-14881 NN denotes variance
T6555 14882-14883 -LRB- denotes (
T6556 14883-14888 NN denotes ANOVA
T6557 14888-14889 -RRB- denotes )
T6623 14901-14909 VB denotes Degraded
T6624 14910-14913 NNP denotes CGN
T6625 14914-14920 NNP denotes Induce
T6626 14921-14928 NNP denotes Colonic
T6627 14929-14941 NNP denotes Inflammation
T6628 14942-14945 DT denotes All
T6629 14946-14950 NN denotes rats
T6630 14951-14960 VB denotes developed
T6631 14961-14969 NN denotes diarrhea
T6632 14970-14976 IN denotes during
T6633 14977-14985 VB denotes degraded
T6634 14986-14997 NN denotes carrageenan
T6635 14998-15012 NN denotes administration
T6636 15013-15016 CC denotes and
T6637 15017-15022 JJ denotes gross
T6638 15023-15031 NN denotes evidence
T6639 15032-15034 IN denotes of
T6640 15035-15040 NN denotes blood
T6641 15041-15044 VB denotes was
T6642 15045-15055 RB denotes frequently
T6643 15056-15064 VB denotes detected
T6644 15065-15067 IN denotes in
T6645 15068-15071 DT denotes the
T6646 15072-15078 NN denotes stools
T6647 15080-15085 NN denotes Colon
T6648 15086-15092 NN denotes length
T6649 15093-15105 RB denotes dramatically
T6650 15106-15115 VB denotes decreased
T6651 15116-15118 IN denotes in
T6652 15119-15122 DT denotes all
T6653 15123-15130 VB denotes treated
T6654 15131-15135 NN denotes rats
T6655 15136-15140 IN denotes with
T6656 15141-15142 DT denotes a
T6657 15143-15147 RB denotes more
T6658 15148-15158 JJ denotes pronounced
T6659 15159-15165 NN denotes effect
T6660 15166-15171 VB denotes being
T6661 15172-15180 VB denotes observed
T6662 15181-15183 IN denotes in
T6663 15184-15187 DT denotes the
T6664 15188-15190 CD denotes 40
T6665 15191-15194 NN denotes kDa
T6666 15195-15199 NN denotes dCGN
T6667 15200-15207 VB denotes treated
T6668 15208-15213 NN denotes group
T6669 15214-15215 -LRB- denotes (
T6670 15215-15219 NN denotes Fig.
T6671 15220-15222 NN denotes 1A
T6672 15222-15223 -RRB- denotes )
T6673 15225-15236 RB denotes Furthermore
T6674 15236-15237 -COMMA- denotes ,
T6675 15238-15247 VB denotes prolonged
T6676 15248-15256 NN denotes exposure
T6677 15257-15259 TO denotes to
T6678 15260-15262 CD denotes 40
T6679 15263-15266 NN denotes kDa
T6680 15267-15271 NN denotes dCGN
T6681 15272-15280 VB denotes resulted
T6682 15281-15283 IN denotes in
T6683 15284-15288 JJ denotes high
T6684 15289-15300 JJ denotes macroscopic
T6685 15301-15304 CC denotes and
T6686 15305-15317 JJ denotes histological
T6687 15318-15324 NN denotes scores
T6688 15325-15327 IN denotes of
T6689 15328-15340 NN denotes inflammation
T6690 15341-15342 -LRB- denotes (
T6691 15342-15346 NNP denotes Fig.
T6692 15347-15349 NN denotes 1B
T6693 15349-15350 -COMMA- denotes ,
T6694 15351-15352 NN denotes C
T6695 15352-15353 -RRB- denotes )
T6696 15355-15359 RB denotes Only
T6697 15360-15364 JJ denotes weak
T6698 15365-15380 NN denotes myeloperoxidase
T6699 15381-15389 NN denotes activity
T6700 15390-15393 VB denotes was
T6701 15394-15402 VB denotes detected
T6702 15403-15405 IN denotes in
T6703 15406-15410 CC denotes both
T6704 15411-15418 NN denotes control
T6705 15419-15422 CC denotes and
T6706 15423-15435 JJ denotes dCGN-treated
T6707 15436-15442 NN denotes groups
T6708 15443-15444 -LRB- denotes (
T6709 15444-15448 NNP denotes Fig.
T6710 15449-15451 NN denotes 1D
T6711 15451-15452 -RRB- denotes )
T6712 15452-15453 -COMMA- denotes ,
T6713 15454-15464 VB denotes indicating
T6714 15465-15469 IN denotes that
T6715 15470-15482 NN denotes granulocytes
T6716 15483-15486 VB denotes did
T6717 15487-15490 RB denotes not
T6718 15491-15495 VB denotes play
T6719 15496-15497 DT denotes a
T6720 15498-15503 JJ denotes major
T6721 15504-15508 NN denotes role
T6722 15509-15511 IN denotes in
T6723 15512-15515 DT denotes the
T6724 15516-15528 NN denotes inflammation
T6725 15529-15531 IN denotes at
T6726 15532-15536 DT denotes that
T6727 15537-15542 NN denotes stage
T6728 15544-15556 JJ denotes Histological
T6729 15557-15568 NN denotes examination
T6730 15569-15577 VB denotes revealed
T6731 15578-15585 JJ denotes various
T6732 15586-15593 NN denotes degrees
T6733 15594-15596 IN denotes of
T6734 15597-15604 JJ denotes mucosal
T6735 15605-15617 NN denotes inflammation
T6736 15619-15623 NN denotes Rats
T6737 15624-15631 VB denotes treated
T6738 15632-15636 IN denotes with
T6739 15637-15639 CD denotes 10
T6740 15640-15643 NN denotes kDa
T6741 15644-15648 NN denotes dCGN
T6742 15649-15655 VB denotes showed
T6743 15656-15661 NN denotes edema
T6744 15661-15662 -COMMA- denotes ,
T6745 15663-15673 NN denotes epithelium
T6746 15674-15681 NN denotes atrophy
T6747 15682-15685 CC denotes and
T6748 15686-15692 JJ denotes slight
T6749 15693-15703 NN denotes lymphocyte
T6750 15704-15716 NN denotes infiltration
T6751 15717-15718 -LRB- denotes (
T6752 15718-15722 NN denotes data
T6753 15723-15726 RB denotes not
T6754 15727-15732 VB denotes shown
T6755 15732-15733 -RRB- denotes )
T6756 15735-15740 DT denotes These
T6757 15741-15749 NN denotes symptoms
T6758 15750-15754 VB denotes were
T6759 15755-15762 RB denotes totally
T6760 15763-15769 JJ denotes absent
T6761 15770-15772 IN denotes in
T6762 15773-15776 DT denotes the
T6763 15777-15782 NN denotes colon
T6764 15783-15785 IN denotes of
T6765 15786-15793 NN denotes control
T6766 15794-15798 NN denotes rats
T6767 15799-15800 -LRB- denotes (
T6768 15800-15804 NNP denotes Fig.
T6769 15805-15807 NN denotes 1E
T6770 15807-15808 -RRB- denotes )
T6771 15810-15814 RB denotes More
T6772 15815-15821 JJ denotes severe
T6773 15822-15829 JJ denotes mucosal
T6774 15830-15838 NN denotes injuries
T6775 15839-15848 VB denotes including
T6776 15849-15859 NN denotes ulceration
T6777 15859-15860 -COMMA- denotes ,
T6778 15861-15873 JJ denotes hyperplastic
T6779 15874-15884 NN denotes epithelium
T6780 15884-15885 -COMMA- denotes ,
T6781 15886-15891 NN denotes crypt
T6782 15892-15902 NN denotes distortion
T6783 15903-15906 CC denotes and
T6784 15907-15908 DT denotes a
T6785 15909-15915 JJ denotes strong
T6786 15916-15926 NN denotes macrophage
T6787 15927-15939 NN denotes infiltration
T6788 15939-15940 -COMMA- denotes ,
T6789 15941-15945 VB denotes were
T6790 15946-15954 VB denotes observed
T6791 15955-15957 IN denotes in
T6792 15958-15961 DT denotes the
T6793 15962-15964 CD denotes 40
T6794 15965-15968 NN denotes kDa
T6795 15969-15981 JJ denotes dCGN-treated
T6796 15982-15986 NN denotes rats
T6797 15987-15988 -LRB- denotes (
T6798 15988-15992 NNP denotes Fig.
T6799 15993-15995 NN denotes 1F
T6800 15995-15996 -RRB- denotes )
T6801 15998-16000 DT denotes No
T6802 16001-16010 VB denotes sulphated
T6803 16011-16026 NN denotes polysaccharides
T6804 16027-16031 VB denotes were
T6805 16032-16040 VB denotes detected
T6806 16041-16043 IN denotes by
T6807 16044-16053 NN denotes toluidine
T6808 16054-16058 JJ denotes blue
T6809 16059-16067 NN denotes staining
T6810 16068-16070 IN denotes of
T6811 16071-16076 NN denotes colon
T6812 16077-16083 NN denotes mucosa
T6813 16084-16088 IN denotes from
T6814 16089-16093 NN denotes rats
T6815 16094-16101 VB denotes treated
T6816 16102-16106 IN denotes with
T6817 16107-16113 CC denotes either
T6818 16114-16117 DT denotes the
T6819 16118-16120 CD denotes 10
T6820 16121-16123 CC denotes or
T6821 16124-16126 CD denotes 40
T6822 16127-16130 NN denotes kDa
T6823 16131-16135 NN denotes dCGN
T6824 16136-16137 -LRB- denotes (
T6825 16137-16140 RB denotes not
T6826 16141-16146 VB denotes shown
T6827 16146-16147 -RRB- denotes )
T6828 16149-16157 IN denotes Although
T6829 16158-16160 PRP denotes we
T6830 16161-16164 MD denotes can
T6831 16164-16167 RB denotes not
T6832 16168-16175 VB denotes exclude
T6833 16176-16180 IN denotes that
T6834 16181-16185 NN denotes dCGN
T6835 16186-16189 NN denotes mat
T6836 16190-16193 RB denotes not
T6837 16194-16198 VB denotes have
T6838 16199-16207 VB denotes retained
T6839 16208-16210 IN denotes in
T6840 16211-16214 DT denotes the
T6841 16215-16222 NN denotes section
T6842 16223-16229 IN denotes during
T6843 16230-16233 DT denotes the
T6844 16234-16243 NN denotes histology
T6845 16244-16253 NN denotes procedure
T6846 16253-16254 -COMMA- denotes ,
T6847 16255-16259 DT denotes this
T6848 16260-16269 VB denotes indicates
T6849 16270-16274 IN denotes that
T6850 16275-16280 DT denotes these
T6851 16281-16289 NN denotes polymers
T6852 16290-16293 MD denotes may
T6853 16294-16297 RB denotes not
T6854 16298-16302 VB denotes have
T6855 16303-16307 VB denotes been
T6856 16308-16320 VB denotes phagocytosed
T7286 0-8 VB denotes Degraded
T7287 9-12 NN denotes CGN
T7288 13-16872 NNP denotes inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced-TNF-α
T7289 16873-16883 NN denotes Production
T7290 16884-16886 IN denotes by
T7291 16887-16896 NN denotes Monocytes
T7292 16897-16899 IN denotes In
T7293 16900-16905 NNP denotes Vitro
T15571 16366-16374 VB denotes Degraded
T15572 16375-16378 NN denotes CGN
T15573 16379-16386 VB denotes induced
T15574 16387-16392 NN denotes colon
T15575 16393-16405 NN denotes inflammation
T15576 16406-16408 IN denotes in
T15577 16409-16413 NN denotes rats
T15578 16415-16425 NN denotes Histograms
T15579 16426-16433 VB denotes showing
T15580 16434-16437 DT denotes the
T15581 16438-16444 NN denotes effect
T15582 16445-16447 IN denotes of
T15583 16448-16456 VB denotes degraded
T15584 16457-16460 NN denotes CGN
T15585 16461-16463 IN denotes on
T15586 16463-16464 -COLON- denotes :
T15587 16465-16470 NN denotes colon
T15588 16471-16477 NN denotes length
T15589 16478-16479 -LRB- denotes (
T15590 16479-16480 NN denotes A
T15591 16480-16481 -RRB- denotes )
T15592 16481-16482 -COLON- denotes ;
T15593 16483-16494 JJ denotes macroscopic
T15594 16495-16496 -LRB- denotes (
T15595 16496-16497 NN denotes B
T15596 16497-16498 -RRB- denotes )
T15597 16499-16502 CC denotes and
T15598 16503-16515 JJ denotes histological
T15599 16516-16517 -LRB- denotes (
T15600 16517-16518 NN denotes C
T15601 16518-16519 -RRB- denotes )
T15602 16520-16532 NN denotes inflammation
T15603 16533-16538 NN denotes score
T15604 16539-16541 IN denotes of
T15605 16542-16547 NN denotes colon
T15606 16547-16548 -COLON- denotes ;
T15607 16549-16564 NN denotes Myeloperoxidase
T15608 16565-16566 -LRB- denotes (
T15609 16566-16569 NN denotes MPO
T15610 16569-16570 -RRB- denotes )
T15611 16571-16579 NN denotes activity
T15612 16580-16581 -LRB- denotes (
T15613 16581-16582 NN denotes D
T15614 16582-16583 -RRB- denotes )
T15615 16585-16592 NN denotes Control
T15616 16593-16597 NN denotes rats
T15617 16598-16599 -LRB- denotes (
T15618 16599-16604 JJ denotes white
T15619 16605-16609 NN denotes bars
T15620 16609-16610 -RRB- denotes )
T15621 16610-16611 -COLON- denotes ;
T15622 16612-16614 CD denotes 10
T15623 16615-16618 NN denotes kDa
T15624 16619-16627 VB denotes degraded
T15625 16628-16639 JJ denotes CGN-treated
T15626 16640-16644 NN denotes rats
T15627 16645-16646 -LRB- denotes (
T15628 16646-16650 JJ denotes grey
T15629 16651-16655 NN denotes bars
T15630 16655-16656 -RRB- denotes )
T15631 16656-16657 -COLON- denotes ;
T15632 16658-16660 CD denotes 40
T15633 16661-16664 NN denotes kDa
T15634 16665-16673 VB denotes degraded
T15635 16674-16685 JJ denotes CGN-treated
T15636 16686-16690 NN denotes rats
T15637 16691-16692 -LRB- denotes (
T15638 16692-16697 JJ denotes black
T15639 16698-16702 NN denotes bars
T15640 16702-16703 -RRB- denotes )
T15641 16703-16704 SYM denotes .
T15642 16705-16706 NN denotes *
T15643 16707-16708 SYM denotes p
T15644 16708-16712 CD denotes <0.0
T15645 16712-16716 IN denotes 5 fr
T15646 16716-16724 NN denotes om contr
T15647 16724-16726 SYM denotes ol
T15648 16726-16727 NN denotes .
T15649 16728-16729 SYM denotes *
T15650 16729-16733 CD denotes * p<
T15651 16733-16737 IN denotes 0.01
T15652 16738-16745 NN denotes from co
T15653 16752-16764 JJ denotes Histological
T15654 16765-16773 NN denotes analysis
T15655 16774-16776 IN denotes of
T15656 16777-16782 NN denotes colon
T15657 16783-16787 IN denotes from
T15658 16788-16795 NN denotes control
T15659 16796-16800 NN denotes rats
T15660 16801-16802 -LRB- denotes (
T15661 16802-16803 NN denotes E
T15662 16803-16804 -RRB- denotes )
T15663 16804-16805 -COMMA- denotes ,
T15664 16806-16809 CC denotes and
T15665 16810-16814 IN denotes from
T15666 16815-16817 CD denotes 40
T15667 16818-16821 NN denotes kDa
T15668 16822-16834 JJ denotes dCGN-treated
T15669 16835-16839 NN denotes rats
T15670 16840-16841 -LRB- denotes (
T15671 16841-16842 NN denotes F
T15672 16842-16843 -RRB- denotes )
T2817 5778-5785 NN denotes Animals
R160 T221 T220 arg2Of CGN,Degraded
R161 T221 T222 arg1Of CGN,inhibited
R162 T221 T229 arg1Of CGN,arresting
R163 T222 T227 arg1Of inhibited,vitro
R164 T222 T228 arg1Of inhibited,","
R165 T222 T229 modOf inhibited,arresting
R166 T225 T222 arg2Of proliferation,inhibited
R167 T225 T223 arg1Of proliferation,THP-1
R168 T225 T224 arg1Of proliferation,cell
R169 T227 T226 arg1Of vitro,in
R170 T231 T229 arg2Of cells,arresting
R171 T231 T230 arg1Of cells,the
R172 T231 T232 arg1Of cells,in
R173 T234 T232 arg2Of phase,in
R174 T234 T233 arg1Of phase,G1
R175 T236 T235 arg2Of addition,In
R176 T238 T239 arg1Of dCGN,increased
R177 T239 T235 arg1Of increased,In
R178 T239 T237 arg1Of increased,","
R179 T239 T242 arg1Of increased,in
R180 T239 T248 arg1Of increased,with
R181 T241 T239 arg2Of expression,increased
R182 T241 T240 arg1Of expression,ICAM-1
R183 T244 T245 arg1Of PBM,and
R184 T245 T242 arg2Of and,in
R185 T245 T243 arg1Of and,both
R186 T247 T245 arg2Of cells,and
R187 T247 T246 arg1Of cells,THP-1
R188 T251 T248 arg2Of effect,with
R189 T251 T249 arg1Of effect,a
R190 T251 T250 arg1Of effect,major
R191 T251 T252 arg2Of effect,seen
R192 T252 T253 arg1Of seen,after
R193 T257 T253 arg2Of exposure,after
R194 T257 T254 arg1Of exposure,40
R195 T257 T255 arg1Of exposure,kDa
R196 T257 T256 arg1Of exposure,dCGN
R197 T260 T261 arg1Of dCGN,stimulated
R198 T261 T258 arg1Of stimulated,Also
R199 T261 T259 arg1Of stimulated,","
R200 T263 T261 arg2Of aggregation,stimulated
R201 T263 T262 arg1Of aggregation,monocyte
R202 T263 T265 arg1Of aggregation,vitro
R203 T263 T266 arg1Of aggregation,that
R204 T263 T267 arg1Of aggregation,was
R205 T263 T268 arg2Of aggregation,prevented
R206 T265 T264 arg1Of vitro,in
R207 T268 T267 arg2Of prevented,was
R208 T270 T268 arg1Of incubation,prevented
R209 T270 T269 arg2Of incubation,by
R210 T270 T271 arg1Of incubation,with
R211 T273 T271 arg2Of antibody,with
R212 T273 T272 arg1Of antibody,anti-ICAM-1
R213 T276 T277 arg1Of dCGN,stimulated
R214 T277 T274 arg1Of stimulated,Finally
R215 T277 T275 arg1Of stimulated,","
R216 T277 T282 arg1Of stimulated,by
R217 T279 T280 arg1Of expression,and
R218 T280 T277 arg2Of and,stimulated
R219 T280 T278 arg1Of and,TNF-α
R220 T281 T280 arg2Of secretion,and
R221 T284 T285 arg1Of PBM,and
R222 T285 T282 arg2Of and,by
R223 T285 T283 arg1Of and,both
R224 T287 T285 arg2Of cells,and
R225 T287 T286 arg1Of cells,THP-1
R226 T290 T288 arg1Of effects,All
R227 T290 T289 arg1Of effects,these
R228 T290 T291 arg1Of effects,were
R229 T290 T292 arg2Of effects,linked
R230 T292 T291 arg2Of linked,were
R231 T292 T293 arg1Of linked,to
R232 T295 T293 arg2Of activation,to
R233 T295 T294 arg1Of activation,NF-κB
R234 T297 T296 arg1Of data,These
R235 T297 T299 arg1Of data,suggest
R236 T299 T298 arg1Of suggest,strongly
R237 T303 T301 arg1Of forms,the
R238 T303 T302 arg1Of forms,degraded
R239 T303 T304 arg1Of forms,of
R240 T303 T306 arg1Of forms,have
R241 T305 T304 arg2Of CGN,of
R242 T306 T299 arg2Of have,suggest
R243 T306 T300 arg1Of have,that
R244 T309 T306 arg2Of effect,have
R245 T309 T307 arg1Of effect,a
R246 T309 T308 arg1Of effect,pronounced
R247 T309 T310 arg1Of effect,on
R248 T309 T312 arg1Of effect,","
R249 T309 T313 arg1Of effect,characteristic
R250 T311 T310 arg2Of monocytes,on
R251 T313 T314 arg1Of characteristic,of
R252 T317 T314 arg2Of phenotype,of
R253 T317 T315 arg1Of phenotype,an
R254 T317 T316 arg1Of phenotype,inflammatory
R579 T776 T777 arg1Of Carrageenan,(
R580 T776 T780 arg1Of Carrageenan,is
R581 T778 T777 arg2Of CGN,(
R582 T779 T777 arg3Of ),(
R583 T784 T780 arg2Of weight,is
R584 T784 T781 arg1Of weight,a
R585 T784 T782 arg1Of weight,high
R586 T784 T783 arg1Of weight,molecular
R587 T784 T785 arg2Of weight,sulphated
R588 T786 T785 arg3Of polysaccharide,sulphated
R589 T786 T787 arg1Of polysaccharide,(
R590 T786 T792 arg2Of polysaccharide,derived
R591 T789 T788 arg1Of 200,>
R592 T790 T787 arg2Of kDa,(
R593 T790 T789 arg1Of kDa,200
R594 T791 T787 arg3Of ),(
R595 T792 T793 arg1Of derived,from
R596 T795 T793 arg2Of algae,from
R597 T795 T794 arg1Of algae,red
R598 T795 T796 arg1Of algae,(
R599 T797 T796 arg2Of Rhodophyceae,(
R600 T798 T796 arg3Of ),(
R601 T801 T799 arg1Of forms,Three
R602 T801 T800 arg1Of forms,main
R603 T801 T802 arg1Of forms,of
R604 T801 T804 arg1Of forms,have
R605 T801 T805 arg1Of forms,been
R606 T801 T806 arg2Of forms,identified
R607 T803 T802 arg2Of CGN,of
R608 T806 T804 arg2Of identified,have
R609 T806 T805 arg2Of identified,been
R610 T806 T807 arg1Of identified,:
R611 T808 T809 arg1Of kappa,","
R612 T809 T812 arg1Of ",",and
R613 T810 T809 arg2Of iota,","
R614 T812 T806 arg3Of and,identified
R615 T812 T811 arg1Of and,","
R616 T813 T812 arg2Of lambda,and
R617 T814 T815 arg1Of They,differ
R618 T815 T816 arg1Of differ,from
R619 T815 T819 arg1Of differ,in
R620 T815 T827 arg1Of differ,","
R621 T815 T828 arg1Of differ,[
R622 T818 T816 arg2Of other,from
R623 T818 T817 arg1Of other,each
R624 T821 T820 arg1Of degree,sulphation
R625 T821 T822 arg1Of degree,and
R626 T822 T819 arg2Of and,in
R627 T823 T822 arg2Of solubility,and
R628 T823 T824 arg1Of solubility,[
R629 T825 T824 arg2Of 1,[
R630 T826 T824 arg3Of ],[
R631 T829 T828 arg2Of 2,[
R632 T830 T828 arg3Of ],[
R633 T832 T831 arg1Of CGN,Native
R634 T832 T833 arg1Of CGN,is
R635 T832 T834 arg2Of CGN,thought
R636 T832 T836 arg1Of CGN,be
R637 T832 T837 arg1Of CGN,harmless
R638 T832 T839 arg1Of CGN,is
R639 T832 T841 arg2Of CGN,used
R640 T834 T833 arg2Of thought,is
R641 T834 T838 arg1Of thought,and
R642 T836 T834 arg3Of be,thought
R643 T836 T835 arg1Of be,to
R644 T837 T836 arg2Of harmless,be
R645 T841 T838 arg2Of used,and
R646 T841 T839 arg2Of used,is
R647 T841 T840 arg1Of used,widely
R648 T841 T842 arg1Of used,as
R649 T844 T842 arg2Of food,as
R650 T844 T843 arg1Of food,a
R651 T844 T845 arg1Of food,additive
R652 T847 T845 arg2Of improve,additive
R653 T847 T846 arg1Of improve,to
R654 T848 T847 arg2Of texture,improve
R655 T849 T850 arg1Of It,is
R656 T849 T852 arg2Of It,used
R657 T852 T850 arg2Of used,is
R658 T852 T851 arg1Of used,also
R659 T852 T853 arg1Of used,in
R660 T854 T855 arg1Of cosmetics,and
R661 T855 T853 arg2Of and,in
R662 T856 T855 arg2Of pharmaceuticals,and
R663 T860 T859 arg1Of treatment,acid
R664 T860 T861 arg1Of treatment,at
R665 T860 T867 arg1Of treatment,triggers
R666 T863 T861 arg2Of temperature,at
R667 T863 T862 arg1Of temperature,high
R668 T863 T864 arg1Of temperature,(
R669 T865 T864 arg2Of 80°C,(
R670 T866 T864 arg3Of ),(
R671 T867 T857 arg1Of triggers,However
R672 T867 T858 arg1Of triggers,","
R673 T867 T870 modOf triggers,to
R674 T869 T867 arg2Of hydrolysis,triggers
R675 T869 T868 arg1Of hydrolysis,CGN
R676 T871 T870 arg1Of lower,to
R677 T876 T875 arg1Of 50,<
R678 T877 T874 arg2Of kDa,(
R679 T877 T876 arg1Of kDa,50
R680 T878 T874 arg3Of ),(
R681 T879 T871 arg2Of compounds,lower
R682 T879 T872 arg1Of compounds,molecular
R683 T879 T873 arg1Of compounds,weight
R684 T879 T874 arg1Of compounds,(
R685 T879 T880 arg2Of compounds,known
R686 T880 T881 arg1Of known,as
R687 T882 T883 arg1Of poligeenan,or
R688 T883 T881 arg2Of or,as
R689 T885 T883 arg2Of CGN,or
R690 T885 T884 arg2Of CGN,degraded
R691 T885 T886 arg1Of CGN,(
R692 T887 T886 arg2Of dCGN,(
R693 T888 T886 arg3Of ),(
R694 T890 T889 arg1Of dCGNs,These
R695 T890 T891 arg1Of dCGNs,induce
R696 T890 T894 arg1Of dCGNs,have
R697 T890 T895 arg1Of dCGNs,been
R698 T890 T897 arg2Of dCGNs,used
R699 T891 T893 arg1Of induce,and
R700 T892 T891 arg2Of inflammation,induce
R701 T897 T893 arg2Of used,and
R702 T897 T894 arg2Of used,have
R703 T897 T895 arg2Of used,been
R704 T897 T896 arg1Of used,widely
R705 T897 T898 arg1Of used,as
R706 T899 T898 arg2Of models,as
R707 T899 T900 arg1Of models,of
R708 T901 T900 arg2Of colitis,of
R709 T901 T902 arg1Of colitis,in
R710 T904 T902 arg2Of species,in
R711 T904 T903 arg1Of species,several
R712 T904 T905 arg1Of species,","
R713 T904 T906 arg1Of species,including
R714 T907 T906 arg2Of rats,including
R715 T907 T908 arg1Of rats,[
R716 T907 T911 arg1Of rats,","
R717 T909 T908 arg2Of 3,[
R718 T910 T908 arg3Of ],[
R719 T912 T913 arg1Of rabbits,[
R720 T912 T916 arg1Of rabbits,and
R721 T914 T913 arg2Of 4,[
R722 T915 T913 arg3Of ],[
R723 T916 T911 arg2Of and,","
R724 T918 T916 arg2Of pigs,and
R725 T918 T917 arg1Of pigs,guinea
R726 T918 T919 arg1Of pigs,[
R727 T920 T919 arg2Of 5,[
R728 T921 T919 arg3Of ],[
R729 T923 T922 arg1Of role,The
R730 T923 T924 arg1Of role,of
R731 T923 T926 arg1Of role,as
R732 T923 T930 arg1Of role,remains
R733 T923 T931 arg1Of role,controversial
R734 T925 T924 arg2Of dCGN,of
R735 T929 T926 arg2Of factor,as
R736 T929 T927 arg1Of factor,a
R737 T929 T928 arg1Of factor,tumor-promoting
R738 T930 T932 arg1Of remains,[
R739 T930 T935 arg1Of remains,","
R740 T930 T936 arg1Of remains,[
R741 T930 T939 arg1Of remains,–
R742 T931 T930 arg2Of controversial,remains
R743 T933 T932 arg2Of 4,[
R744 T934 T932 arg3Of ],[
R745 T937 T936 arg2Of 6,[
R746 T938 T936 arg3Of ],[
R747 T940 T939 arg2Of [,–
R748 T941 T939 arg3Of 8,–
R749 T945 T943 arg1Of form,the
R750 T945 T944 arg1Of form,native
R751 T945 T946 arg1Of form,is
R752 T945 T947 arg2Of form,thought
R753 T945 T949 arg1Of form,be
R754 T945 T950 arg1Of form,harmless
R755 T947 T942 arg2Of thought,Although
R756 T947 T946 arg2Of thought,is
R757 T949 T947 arg3Of be,thought
R758 T949 T948 arg1Of be,to
R759 T950 T949 arg2Of harmless,be
R760 T950 T951 arg1Of harmless,for
R761 T953 T951 arg2Of consumption,for
R762 T953 T952 arg1Of consumption,human
R763 T956 T955 arg1Of amounts,small
R764 T956 T957 arg1Of amounts,of
R765 T956 T959 arg1Of amounts,are
R766 T956 T961 arg2Of amounts,produced
R767 T958 T957 arg2Of dCGN,of
R768 T961 T942 arg1Of produced,Although
R769 T961 T954 arg1Of produced,","
R770 T961 T959 arg2Of produced,are
R771 T961 T960 arg1Of produced,probably
R772 T961 T983 arg1Of produced,","
R773 T961 T984 arg1Of produced,[
R774 T964 T963 arg1Of hydrolysis,acid
R775 T964 T965 arg1Of hydrolysis,during
R776 T964 T972 arg1Of hydrolysis,[
R777 T964 T975 arg1Of hydrolysis,or
R778 T967 T965 arg2Of digestion,during
R779 T967 T966 arg1Of digestion,gastric
R780 T967 T968 arg1Of digestion,[
R781 T967 T971 arg1Of digestion,","
R782 T969 T968 arg2Of 9,[
R783 T970 T968 arg3Of ],[
R784 T973 T972 arg2Of 10,[
R785 T974 T972 arg3Of ],[
R786 T975 T961 arg1Of or,produced
R787 T975 T962 arg2Of or,by
R788 T976 T975 arg2Of interaction,or
R789 T976 T977 arg1Of interaction,with
R790 T979 T977 arg2Of bacteria,with
R791 T979 T978 arg1Of bacteria,intestinal
R792 T979 T980 arg1Of bacteria,[
R793 T981 T980 arg2Of 11,[
R794 T982 T980 arg3Of ],[
R795 T985 T984 arg2Of 12,[
R796 T986 T984 arg3Of ],[
R797 T989 T988 arg1Of effects,the
R798 T989 T990 arg1Of effects,of
R799 T989 T994 arg1Of effects,on
R800 T989 T997 arg1Of effects,have
R801 T989 T998 arg1Of effects,been
R802 T989 T1000 arg2Of effects,analyzed
R803 T991 T992 arg1Of native,and
R804 T993 T990 arg2Of dCGN,of
R805 T993 T991 arg1Of dCGN,native
R806 T996 T994 arg2Of inflammation,on
R807 T996 T995 arg1Of inflammation,intestinal
R808 T1000 T987 arg2Of analyzed,Whereas
R809 T1000 T997 arg2Of analyzed,have
R810 T1000 T998 arg2Of analyzed,been
R811 T1000 T999 arg1Of analyzed,extensively
R812 T1000 T1001 arg1Of analyzed,in
R813 T1003 T1001 arg2Of models,in
R814 T1003 T1002 arg1Of models,animal
R815 T1007 T1005 arg1Of studies,only
R816 T1007 T1006 arg1Of studies,few
R817 T1007 T1008 arg1Of studies,have
R818 T1007 T1009 arg1Of studies,been
R819 T1007 T1010 arg2Of studies,conducted
R820 T1010 T987 arg1Of conducted,Whereas
R821 T1010 T1004 arg1Of conducted,","
R822 T1010 T1008 arg2Of conducted,have
R823 T1010 T1009 arg2Of conducted,been
R824 T1010 T1011 modOf conducted,using
R825 T1014 T1011 arg2Of lines,using
R826 T1014 T1012 arg1Of lines,human
R827 T1014 T1013 arg1Of lines,cell
R828 T1016 T1015 arg1Of studies,Recent
R829 T1016 T1017 arg1Of studies,have
R830 T1016 T1018 arg1Of studies,shown
R831 T1018 T1017 arg2Of shown,have
R832 T1018 T1047 arg1Of shown,[
R833 T1018 T1050 arg1Of shown,","
R834 T1018 T1051 arg1Of shown,[
R835 T1020 T1018 arg2Of link,shown
R836 T1020 T1019 arg1Of link,a
R837 T1020 T1021 arg1Of link,between
R838 T1020 T1030 arg1Of link,by
R839 T1020 T1039 arg1Of link,","
R840 T1020 T1040 arg1Of link,via
R841 T1022 T1023 arg1Of exposure,to
R842 T1022 T1027 arg1Of exposure,and
R843 T1026 T1023 arg2Of CGN,to
R844 T1026 T1024 arg1Of CGN,native
R845 T1026 T1025 arg1Of CGN,form
R846 T1027 T1021 arg2Of and,between
R847 T1029 T1027 arg2Of production,and
R848 T1029 T1028 arg1Of production,IL-8
R849 T1036 T1030 arg2Of line,by
R850 T1036 T1031 arg1Of line,the
R851 T1036 T1032 arg1Of line,human
R852 T1036 T1033 arg1Of line,intestinal
R853 T1036 T1034 arg1Of line,epithelial
R854 T1036 T1035 arg1Of line,cell
R855 T1036 T1037 arg1Of line,","
R856 T1038 T1037 arg2Of NCM460,","
R857 T1044 T1043 arg2Of NF-κB,(
R858 T1045 T1043 arg3Of ),(
R859 T1046 T1040 arg2Of activation,via
R860 T1046 T1041 arg1Of activation,Nuclear
R861 T1046 T1042 arg1Of activation,Factor-κB
R862 T1046 T1043 arg1Of activation,(
R863 T1048 T1047 arg2Of 13,[
R864 T1049 T1047 arg3Of ],[
R865 T1052 T1051 arg2Of 14,[
R866 T1053 T1051 arg3Of ],[
R867 T1054 T1055 arg1Of NF-κB,is
R868 T1058 T1055 arg2Of factor,is
R869 T1058 T1056 arg1Of factor,a
R870 T1058 T1057 arg1Of factor,transcription
R871 T1058 T1059 arg1Of factor,that
R872 T1058 T1060 arg1Of factor,regulates
R873 T1060 T1071 arg1Of regulates,","
R874 T1060 T1072 arg1Of regulates,[
R875 T1062 T1060 arg2Of expression,regulates
R876 T1062 T1061 arg1Of expression,the
R877 T1062 T1063 arg1Of expression,of
R878 T1064 T1063 arg2Of genes,of
R879 T1064 T1065 arg2Of genes,associated
R880 T1065 T1066 arg1Of associated,with
R881 T1067 T1066 arg2Of inflammation,with
R882 T1067 T1068 arg1Of inflammation,[
R883 T1069 T1068 arg2Of 15,[
R884 T1070 T1068 arg3Of ],[
R885 T1073 T1072 arg2Of 16,[
R886 T1074 T1072 arg3Of ],[
R887 T1076 T1077 arg1Of infiltration,and
R888 T1077 T1075 arg1Of and,Macrophage
R889 T1077 T1079 arg1Of and,is
R890 T1078 T1077 arg2Of accumulation,and
R891 T1079 T1086 arg1Of is,[
R892 T1082 T1079 arg2Of characteristic,is
R893 T1082 T1080 arg1Of characteristic,a
R894 T1082 T1081 arg1Of characteristic,common
R895 T1082 T1083 arg1Of characteristic,of
R896 T1085 T1083 arg2Of diseases,of
R897 T1085 T1084 arg1Of diseases,intestinal
R898 T1087 T1086 arg2Of 17,[
R899 T1088 T1086 arg3Of ],[
R900 T1089 T1090 arg1Of Macrophages,represent
R901 T1089 T1099 arg1Of Macrophages,secrete
R902 T1089 T1108 arg1Of Macrophages,express
R903 T1090 T1098 arg1Of represent,","
R904 T1092 T1090 arg2Of %,represent
R905 T1092 T1091 arg1Of %,10
R906 T1092 T1093 arg1Of %,of
R907 T1097 T1093 arg2Of cells,of
R908 T1097 T1094 arg1Of cells,total
R909 T1097 T1095 arg1Of cells,lamina
R910 T1097 T1096 arg1Of cells,propria
R911 T1098 T1107 arg1Of ",",and
R912 T1099 T1098 arg2Of secrete,","
R913 T1102 T1099 arg2Of range,secrete
R914 T1102 T1100 arg1Of range,a
R915 T1102 T1101 arg1Of range,wide
R916 T1102 T1103 arg1Of range,of
R917 T1106 T1103 arg2Of compounds,of
R918 T1106 T1104 arg1Of compounds,biologically
R919 T1106 T1105 arg1Of compounds,active
R920 T1108 T1107 arg2Of express,and
R921 T1110 T1108 arg2Of molecules,express
R922 T1110 T1109 arg1Of molecules,cell-adhesion
R923 T1114 T1111 arg1Of response,The
R924 T1114 T1112 arg1Of response,immune
R925 T1114 T1113 arg1Of response,cell
R926 T1114 T1115 arg1Of response,to
R927 T1114 T1119 arg1Of response,seems
R928 T1114 T1121 arg1Of response,be
R929 T1114 T1122 arg2Of response,amplified
R930 T1114 T1125 arg2Of response,generated
R931 T1118 T1115 arg2Of stimulus,to
R932 T1118 T1116 arg1Of stimulus,an
R933 T1118 T1117 arg1Of stimulus,inflammatory
R934 T1122 T1123 arg1Of amplified,or
R935 T1123 T1119 arg2Of or,seems
R936 T1123 T1120 arg1Of or,to
R937 T1123 T1121 arg2Of or,be
R938 T1125 T1123 arg2Of generated,or
R939 T1125 T1124 arg1Of generated,directly
R940 T1125 T1126 arg1Of generated,by
R941 T1127 T1126 arg2Of cells,by
R942 T1127 T1128 arg2Of cells,exposed
R943 T1128 T1129 arg1Of exposed,to
R944 T1131 T1129 arg2Of polysaccharides,to
R945 T1131 T1130 arg1Of polysaccharides,sulphated
R946 T1131 T1133 arg1Of polysaccharides,as
R947 T1133 T1132 arg1Of as,such
R948 T1134 T1133 arg2Of carrageenans,as
R949 T1137 T1138 arg2Of inflammation,induced
R950 T1137 T1141 arg1Of inflammation,was
R951 T1137 T1142 arg2Of inflammation,associated
R952 T1140 T1138 arg1Of dCGN,induced
R953 T1140 T1139 arg2Of dCGN,by
R954 T1142 T1135 arg1Of associated,Indeed
R955 T1142 T1136 arg1Of associated,","
R956 T1142 T1141 arg2Of associated,was
R957 T1142 T1143 arg1Of associated,with
R958 T1142 T1153 arg1Of associated,","
R959 T1142 T1154 arg1Of associated,[
R960 T1144 T1143 arg2Of recruitment,with
R961 T1144 T1145 arg1Of recruitment,of
R962 T1144 T1147 arg1Of recruitment,to
R963 T1146 T1145 arg2Of macrophages,of
R964 T1149 T1147 arg2Of sites,to
R965 T1149 T1148 arg1Of sites,inflammation
R966 T1149 T1150 arg1Of sites,[
R967 T1151 T1150 arg2Of 18,[
R968 T1152 T1150 arg3Of ],[
R969 T1155 T1154 arg2Of 19,[
R970 T1156 T1154 arg3Of ],[
R971 T1159 T1160 arg2Of inflammation,induced
R972 T1159 T1173 arg1Of inflammation,was
R973 T1159 T1175 arg2Of inflammation,associated
R974 T1164 T1160 arg1Of Sodium,induced
R975 T1164 T1161 arg2Of Sodium,by
R976 T1164 T1162 arg1Of Sodium,Dextran
R977 T1164 T1163 arg1Of Sodium,Sulphate
R978 T1164 T1165 arg1Of Sodium,(
R979 T1164 T1168 arg1Of Sodium,","
R980 T1166 T1165 arg2Of DSS,(
R981 T1167 T1165 arg3Of ),(
R982 T1171 T1168 arg2Of compound,","
R983 T1171 T1169 arg1Of compound,another
R984 T1171 T1170 arg2Of compound,sulphated
R985 T1175 T1157 arg1Of associated,Also
R986 T1175 T1158 arg1Of associated,","
R987 T1175 T1172 arg1Of associated,","
R988 T1175 T1173 arg2Of associated,was
R989 T1175 T1174 arg1Of associated,directly
R990 T1175 T1176 arg1Of associated,with
R991 T1175 T1182 arg1Of associated,","
R992 T1175 T1183 arg1Of associated,since
R993 T1178 T1176 arg2Of recruitment,with
R994 T1178 T1177 arg1Of recruitment,macrophages
R995 T1178 T1179 arg1Of recruitment,[
R996 T1180 T1179 arg2Of 20,[
R997 T1181 T1179 arg3Of ],[
R998 T1184 T1186 arg1Of DSS,provoked
R999 T1186 T1183 arg2Of provoked,since
R1000 T1186 T1185 arg1Of provoked,still
R1001 T1186 T1188 arg1Of provoked,after
R1002 T1186 T1194 arg1Of provoked,[
R1003 T1187 T1186 arg2Of inflammation,provoked
R1004 T1189 T1190 arg1Of T-lymphocyte,and
R1005 T1191 T1190 arg2Of NK,and
R1006 T1193 T1188 arg2Of depletion,after
R1007 T1193 T1189 arg1Of depletion,T-lymphocyte
R1008 T1193 T1191 arg1Of depletion,NK
R1009 T1193 T1192 arg1Of depletion,cell
R1010 T1195 T1194 arg2Of 20,[
R1011 T1196 T1194 arg3Of ],[
R1012 T1198 T1199 arg1Of inflammation,can
R1013 T1198 T1200 arg1Of inflammation,be
R1014 T1198 T1201 arg2Of inflammation,induced
R1015 T1201 T1197 arg2Of induced,Although
R1016 T1201 T1199 arg2Of induced,can
R1017 T1201 T1200 arg2Of induced,be
R1018 T1203 T1201 arg1Of dCGN,induced
R1019 T1203 T1202 arg2Of dCGN,by
R1020 T1205 T1206 arg1Of there,are
R1021 T1206 T1197 arg1Of are,Although
R1022 T1206 T1204 arg1Of are,","
R1023 T1208 T1206 arg2Of data,are
R1024 T1208 T1207 arg1Of data,no
R1025 T1208 T1209 arg1Of data,on
R1026 T1212 T1209 arg2Of responses,on
R1027 T1212 T1210 arg1Of responses,human
R1028 T1212 T1211 arg1Of responses,monocyte
R1029 T1212 T1213 arg1Of responses,to
R1030 T1215 T1213 arg2Of exposure,to
R1031 T1215 T1214 arg1Of exposure,dCGN
R1032 T1219 T1218 arg1Of investigate,to
R1033 T1219 T1240 arg1Of investigate,were
R1034 T1219 T1241 arg2Of investigate,exposed
R1035 T1221 T1219 arg2Of effects,investigate
R1036 T1221 T1220 arg1Of effects,the
R1037 T1221 T1222 arg1Of effects,of
R1038 T1221 T1224 arg1Of effects,on
R1039 T1223 T1222 arg2Of dCGN,of
R1040 T1226 T1225 arg1Of monocytes,human
R1041 T1226 T1227 arg1Of monocytes,","
R1042 T1227 T1235 arg1Of ",",and
R1043 T1231 T1227 arg2Of Monocytes,","
R1044 T1231 T1228 arg1Of Monocytes,normal
R1045 T1231 T1229 arg1Of Monocytes,Peripheral
R1046 T1231 T1230 arg1Of Monocytes,Blood
R1047 T1231 T1232 arg1Of Monocytes,(
R1048 T1233 T1232 arg2Of PBM,(
R1049 T1234 T1232 arg3Of ),(
R1050 T1235 T1224 arg2Of and,on
R1051 T1239 T1235 arg2Of cells,and
R1052 T1239 T1236 arg1Of cells,tumoral
R1053 T1239 T1237 arg1Of cells,monocyte/macrophage
R1054 T1239 T1238 arg1Of cells,THP-1
R1055 T1241 T1216 arg1Of exposed,Therefore
R1056 T1241 T1217 arg1Of exposed,","
R1057 T1241 T1240 arg2Of exposed,were
R1058 T1241 T1242 arg1Of exposed,to
R1059 T1244 T1243 arg1Of kDa,10
R1060 T1244 T1245 arg1Of kDa,and
R1061 T1245 T1242 arg2Of and,to
R1062 T1248 T1245 arg2Of dCGN,and
R1063 T1248 T1246 arg1Of dCGN,40
R1064 T1248 T1247 arg1Of dCGN,kDa
R1065 T1249 T1250 arg1Of We,found
R1066 T1252 T1253 arg1Of dCGN,inhibited
R1067 T1253 T1258 arg1Of inhibited,vitro
R1068 T1256 T1253 arg2Of proliferation,inhibited
R1069 T1256 T1254 arg1Of proliferation,THP-1
R1070 T1256 T1255 arg1Of proliferation,cell
R1071 T1258 T1257 arg1Of vitro,in
R1072 T1262 T1260 arg2Of expression,increased
R1073 T1262 T1261 arg1Of expression,ICAM-1
R1074 T1262 T1264 arg1Of expression,stimulated
R1075 T1262 T1270 arg1Of expression,stimulated
R1076 T1264 T1269 arg1Of stimulated,and
R1077 T1267 T1264 arg2Of aggregation,stimulated
R1078 T1267 T1265 arg1Of aggregation,ICAM-1-dependent
R1079 T1267 T1266 arg1Of aggregation,monocyte
R1080 T1269 T1250 arg2Of and,found
R1081 T1269 T1251 arg1Of and,that
R1082 T1269 T1253 arg3Of and,inhibited
R1083 T1269 T1259 arg1Of and,","
R1084 T1269 T1263 arg1Of and,","
R1085 T1269 T1268 arg1Of and,","
R1086 T1270 T1269 arg2Of stimulated,and
R1087 T1272 T1273 arg1Of expression,and
R1088 T1273 T1270 arg2Of and,stimulated
R1089 T1273 T1271 arg1Of and,TNF-α
R1090 T1274 T1273 arg2Of secretion,and
R1091 T1276 T1275 arg1Of responses,These
R1092 T1276 T1277 arg1Of responses,were
R1093 T1276 T1279 arg1Of responses,pronounced
R1094 T1276 T1286 arg1Of responses,were
R1095 T1276 T1287 arg2Of responses,linked
R1096 T1277 T1280 arg1Of were,after
R1097 T1277 T1285 arg1Of were,and
R1098 T1279 T1277 arg2Of pronounced,were
R1099 T1279 T1278 arg1Of pronounced,more
R1100 T1284 T1280 arg2Of exposure,after
R1101 T1284 T1281 arg1Of exposure,40
R1102 T1284 T1282 arg1Of exposure,kDa
R1103 T1284 T1283 arg1Of exposure,dCGN
R1104 T1287 T1285 arg2Of linked,and
R1105 T1287 T1286 arg2Of linked,were
R1106 T1287 T1288 arg1Of linked,to
R1107 T1290 T1288 arg2Of activation,to
R1108 T1290 T1289 arg1Of activation,NF-κB
R1109 T1292 T1291 arg2Of addition,In
R1110 T1297 T1294 arg1Of dCGN,the
R1111 T1297 T1295 arg1Of dCGN,40
R1112 T1297 T1296 arg1Of dCGN,kDa
R1113 T1297 T1299 arg1Of dCGN,but
R1114 T1299 T1298 arg1Of but,","
R1115 T1299 T1300 arg1Of but,not
R1116 T1299 T1305 arg1Of but,induced
R1117 T1304 T1299 arg2Of dCGN,but
R1118 T1304 T1301 arg1Of dCGN,the
R1119 T1304 T1302 arg1Of dCGN,10
R1120 T1304 T1303 arg1Of dCGN,kDa
R1121 T1305 T1291 arg1Of induced,In
R1122 T1305 T1293 arg1Of induced,","
R1123 T1305 T1309 arg1Of induced,as
R1124 T1307 T1306 arg1Of vivo,in
R1125 T1308 T1305 arg2Of colitis,induced
R1126 T1308 T1307 arg1Of colitis,vivo
R1127 T1310 T1309 arg2Of shown,as
R1128 T1314 T1310 arg1Of response,shown
R1129 T1314 T1311 arg2Of response,by
R1130 T1314 T1312 arg1Of response,the
R1131 T1314 T1313 arg1Of response,inflammatory
R1132 T1314 T1315 arg1Of response,in
R1133 T1318 T1315 arg2Of colon,in
R1134 T1318 T1316 arg1Of colon,the
R1135 T1318 T1317 arg1Of colon,rat
R1136 T1320 T1319 arg1Of results,These
R1137 T1320 T1321 arg1Of results,suggest
R1138 T1325 T1323 arg1Of forms,the
R1139 T1325 T1324 arg1Of forms,degraded
R1140 T1325 T1326 arg1Of forms,of
R1141 T1325 T1328 arg1Of forms,have
R1142 T1327 T1326 arg2Of CGN,of
R1143 T1328 T1321 arg2Of have,suggest
R1144 T1328 T1322 arg1Of have,that
R1145 T1328 T1332 arg1Of have,on
R1146 T1331 T1328 arg2Of effect,have
R1147 T1331 T1329 arg1Of effect,an
R1148 T1331 T1330 arg1Of effect,important
R1149 T1333 T1332 arg2Of monocytes,on
R1150 T1333 T1334 arg1Of monocytes,resulting
R1151 T1334 T1335 arg1Of resulting,in
R1152 T1338 T1335 arg2Of phenotype,in
R1153 T1338 T1336 arg1Of phenotype,an
R1154 T1338 T1337 arg1Of phenotype,inflammatory
R1776 T2063 T2064 arg1Of Preparation,of
R1777 T2066 T2064 arg2Of Carrageenan,of
R1778 T2066 T2065 arg1Of Carrageenan,Degraded
R1779 T2068 T2067 arg1Of preparations,Two
R1780 T2068 T2069 arg1Of preparations,of
R1781 T2068 T2093 modOf preparations,were
R1784 T2071 T2069 arg2Of carrageenan,of
R1786 T2073 T2075 arg1Of low,(
R1787 T2073 T2082 arg1Of low,and
R1788 T2077 T2075 arg2Of kDa,(
R1789 T2077 T2076 arg1Of kDa,∼10
R1790 T2077 T2078 arg1Of kDa,;
R1791 T2079 T2078 arg2Of C10,;
R1792 T2080 T2075 arg3Of ),(
R1794 T2082 T2081 arg1Of and,","
R1795 T2083 T2082 arg2Of medium,and
R1796 T2087 T2085 arg2Of kDa,(
R1797 T2087 T2086 arg1Of kDa,∼40
R1798 T2087 T2088 arg1Of kDa,;
R1799 T2089 T2088 arg2Of C40,;
R1800 T2090 T2085 arg3Of ),(
R1801 T2092 T2085 arg1Of weight,(
R1802 T2092 T2091 arg1Of weight,molecular
R1803 T2092 T2093 arg1Of weight,were
R1804 T2092 T2094 arg2Of weight,prepared
R1805 T2094 T2084 arg1Of prepared,","
R1806 T2094 T2093 arg2Of prepared,were
R1807 T2094 T2095 arg1Of prepared,from
R1808 T2097 T2095 arg2Of iota-carrageenan,from
R1809 T2097 T2096 arg1Of iota-carrageenan,native
R1810 T2097 T2098 arg2Of iota-carrageenan,extracted
R1811 T2098 T2099 arg1Of extracted,from
R1812 T2101 T2099 arg2Of spinosum,from
R1813 T2101 T2100 arg1Of spinosum,Euchema
R1814 T2101 T2102 arg1Of spinosum,(
R1815 T2104 T2102 arg2Of provided,(
R1816 T2104 T2103 arg1Of provided,generously
R1817 T2108 T2104 arg1Of Industry,provided
R1818 T2108 T2105 arg2Of Industry,by
R1819 T2108 T2106 arg1Of Industry,Sanofi
R1820 T2108 T2107 arg1Of Industry,Biosystems
R1821 T2108 T2109 arg1Of Industry,","
R1822 T2110 T2109 arg2Of Boulogne-Billancourt,","
R1823 T2110 T2111 arg1Of Boulogne-Billancourt,","
R1824 T2112 T2111 arg2Of France,","
R1825 T2113 T2102 arg3Of ),(
R1826 T2115 T2114 arg1Of carrageenan,Native
R1827 T2115 T2116 arg1Of carrageenan,was
R1828 T2115 T2117 arg2Of carrageenan,dissolved
R1829 T2115 T2130 arg2Of carrageenan,heated
R1830 T2117 T2118 arg1Of dissolved,in
R1831 T2117 T2129 arg1Of dissolved,and
R1832 T2120 T2118 arg2Of water,in
R1833 T2120 T2119 arg1Of water,distilled
R1834 T2120 T2121 arg1Of water,(
R1835 T2120 T2126 arg1Of water,under
R1836 T2122 T2123 arg1Of 5,%
R1837 T2124 T2121 arg2Of w/v,(
R1838 T2124 T2122 arg1Of w/v,5
R1839 T2125 T2121 arg3Of ),(
R1840 T2128 T2126 arg2Of stirring,under
R1841 T2128 T2127 arg1Of stirring,vigorous
R1842 T2129 T2116 arg2Of and,was
R1843 T2130 T2129 arg2Of heated,and
R1844 T2130 T2131 arg1Of heated,to
R1845 T2133 T2131 arg2Of Then,to
R1846 T2133 T2132 arg1Of Then,60°C.
R1847 T2137 T2135 arg1Of solution,the
R1848 T2137 T2136 arg1Of solution,carrageenan
R1849 T2137 T2138 arg1Of solution,was
R1850 T2137 T2139 arg2Of solution,submitted
R1851 T2139 T2116 modOf submitted,was
R1852 T2139 T2134 arg1Of submitted,","
R1853 T2139 T2138 arg2Of submitted,was
R1854 T2139 T2140 arg1Of submitted,to
R1855 T2143 T2140 arg2Of treatments,to
R1856 T2143 T2141 arg1Of treatments,two
R1857 T2143 T2142 arg1Of treatments,different
R1858 T2145 T2139 arg3Of obtain,submitted
R1859 T2145 T2144 arg1Of obtain,to
R1860 T2147 T2148 arg1Of low,and
R1861 T2148 T2146 arg1Of and,both
R1862 T2149 T2148 arg2Of medium,and
R1863 T2152 T2145 arg2Of fractions,obtain
R1864 T2152 T2147 arg1Of fractions,low
R1865 T2152 T2149 arg1Of fractions,medium
R1866 T2152 T2150 arg1Of fractions,molecular
R1867 T2152 T2151 arg1Of fractions,weight
R1868 T2160 T2155 arg2Of fraction,for
R1869 T2160 T2156 arg1Of fraction,the
R1870 T2160 T2157 arg1Of fraction,low
R1871 T2160 T2158 arg1Of fraction,molecular
R1872 T2160 T2159 arg1Of fraction,weight
R1873 T2163 T2162 arg1Of solution,carrageenan
R1874 T2163 T2164 arg1Of solution,was
R1875 T2163 T2165 arg2Of solution,hydrolyzed
R1876 T2165 T2164 arg2Of hydrolyzed,was
R1877 T2165 T2166 arg1Of hydrolyzed,with
R1878 T2168 T2166 arg2Of %,with
R1879 T2168 T2167 arg1Of %,0.3
R1880 T2168 T2169 arg1Of %,(
R1881 T2168 T2172 arg2Of %,concentrated
R1882 T2170 T2169 arg2Of v/v,(
R1883 T2171 T2169 arg3Of ),(
R1884 T2174 T2172 arg1Of acid,concentrated
R1885 T2174 T2173 arg1Of acid,sulphuric
R1886 T2174 T2175 arg1Of acid,for
R1887 T2174 T2178 arg1Of acid,at
R1888 T2177 T2175 arg2Of min,for
R1889 T2177 T2176 arg1Of min,15
R1890 T2180 T2179 arg1Of After,80°C.
R1891 T2181 T2178 arg2Of neutralization,at
R1892 T2181 T2180 arg1Of neutralization,After
R1893 T2181 T2182 arg1Of neutralization,with
R1894 T2184 T2182 arg2Of 4N,with
R1895 T2184 T2183 arg1Of 4N,NaOH
R1896 T2187 T2186 arg1Of solution,the
R1897 T2187 T2188 arg1Of solution,was
R1898 T2188 T2153 arg1Of was,Briefly
R1899 T2188 T2154 arg1Of was,","
R1900 T2188 T2155 arg1Of was,for
R1901 T2188 T2161 arg1Of was,","
R1902 T2188 T2164 modOf was,was
R1903 T2188 T2185 arg1Of was,","
R1904 T2189 T2188 arg2Of ultra,was
R1905 T2189 T2190 arg2Of ultra,filtered
R1906 T2189 T2201 arg1Of ultra,","
R1907 T2189 T2202 arg1Of ultra,(
R1908 T2190 T2191 arg1Of filtered,through
R1909 T2195 T2191 arg2Of cartridge,through
R1910 T2195 T2192 arg1Of cartridge,a
R1911 T2195 T2193 arg1Of cartridge,hollow
R1912 T2195 T2194 arg1Of cartridge,fibre
R1913 T2195 T2196 arg1Of cartridge,with
R1914 T2200 T2196 arg2Of kDa,with
R1915 T2200 T2197 arg1Of kDa,MW
R1916 T2200 T2198 arg1Of kDa,cut-off
R1917 T2200 T2199 arg1Of kDa,5
R1918 T2204 T2202 arg2Of Inc,(
R1919 T2204 T2203 arg1Of Inc,Amicon
R1920 T2204 T2205 arg1Of Inc,","
R1921 T2206 T2205 arg2Of Beverly,","
R1922 T2206 T2207 arg1Of Beverly,","
R1923 T2208 T2207 arg2Of USA,","
R1924 T2209 T2202 arg3Of ),(
R1925 T2215 T2211 arg1Of fraction,the
R1926 T2215 T2212 arg1Of fraction,medium
R1927 T2215 T2213 arg1Of fraction,molecular
R1928 T2215 T2214 arg1Of fraction,weight
R1929 T2215 T2228 arg1Of fraction,concentrated
R1930 T2219 T2217 arg1Of solution,the
R1931 T2219 T2218 arg1Of solution,carrageenan
R1932 T2219 T2220 arg1Of solution,was
R1933 T2219 T2221 arg2Of solution,hydrolyzed
R1934 T2221 T2220 arg2Of hydrolyzed,was
R1935 T2221 T2222 arg1Of hydrolyzed,with
R1936 T2224 T2222 arg2Of %,with
R1937 T2224 T2223 arg1Of %,0.3
R1938 T2224 T2225 arg1Of %,(
R1939 T2226 T2225 arg2Of v/v,(
R1940 T2227 T2225 arg3Of ),(
R1941 T2228 T2210 arg1Of concentrated,For
R1942 T2228 T2216 arg1Of concentrated,","
R1943 T2228 T2220 modOf concentrated,was
R1944 T2228 T2244 arg1Of concentrated,(
R1945 T2230 T2228 arg2Of acid,concentrated
R1946 T2230 T2229 arg1Of acid,sulphuric
R1947 T2230 T2231 arg1Of acid,for
R1948 T2230 T2234 arg1Of acid,at
R1949 T2230 T2243 arg2Of acid,filtered
R1950 T2233 T2231 arg2Of min,for
R1951 T2233 T2232 arg1Of min,30
R1952 T2235 T2234 arg2Of 60°C.,at
R1953 T2235 T2236 arg1Of 60°C.,After
R1954 T2237 T2236 arg2Of neutralization,After
R1955 T2240 T2239 arg1Of supernatant,the
R1956 T2240 T2241 arg1Of supernatant,was
R1957 T2240 T2242 arg1Of supernatant,ultra
R1958 T2241 T2238 arg1Of was,","
R1959 T2242 T2241 arg2Of ultra,was
R1960 T2243 T2241 arg3Of filtered,was
R1961 T2245 T2244 arg2Of MW,(
R1962 T2245 T2246 arg1Of MW,cut-off
R1963 T2246 T2248 arg1Of cut-off,kDa
R1964 T2248 T2247 arg1Of kDa,100
R1965 T2249 T2244 arg3Of ),(
R1966 T2251 T2250 arg1Of filtrate,The
R1967 T2251 T2252 arg1Of filtrate,was
R1968 T2251 T2253 arg2Of filtrate,submitted
R1969 T2253 T2252 arg2Of submitted,was
R1970 T2253 T2254 arg1Of submitted,to
R1971 T2258 T2254 arg2Of filtration,to
R1972 T2258 T2255 arg1Of filtration,a
R1973 T2258 T2256 arg1Of filtration,second
R1974 T2258 T2257 arg1Of filtration,ultra
R1975 T2258 T2259 arg1Of filtration,(
R1976 T2263 T2259 arg2Of kDa,(
R1977 T2263 T2260 arg1Of kDa,MW
R1978 T2263 T2261 arg1Of kDa,cut-off
R1979 T2263 T2262 arg1Of kDa,5
R1980 T2264 T2259 arg3Of ),(
R1981 T2266 T2265 arg1Of preparations,Both
R1982 T2266 T2267 arg1Of preparations,of
R1983 T2266 T2269 arg1Of preparations,were
R1984 T2266 T2270 arg2Of preparations,precipitated
R1985 T2268 T2267 arg2Of dCGN,of
R1986 T2270 T2269 arg2Of precipitated,were
R1987 T2270 T2271 arg1Of precipitated,with
R1988 T2273 T2271 arg2Of volumes,with
R1989 T2273 T2272 arg1Of volumes,4
R1990 T2273 T2274 arg1Of volumes,of
R1991 T2273 T2279 arg2Of volumes,dried
R1992 T2275 T2276 arg1Of 95,%
R1993 T2277 T2274 arg2Of ethanol,of
R1994 T2277 T2275 arg1Of ethanol,95
R1995 T2279 T2278 arg1Of dried,","
R1996 T2279 T2280 arg1Of dried,at
R1997 T2282 T2281 arg1Of temperature,room
R1998 T2282 T2283 arg1Of temperature,and
R1999 T2283 T2280 arg2Of and,at
R2000 T2284 T2283 arg2Of ground,and
R2001 T2284 T2285 arg1Of ground,to
R2002 T2287 T2285 arg2Of particles,to
R2003 T2287 T2286 arg1Of particles,small
R2004 T2287 T2288 arg1Of particles,(
R2005 T2290 T2288 arg2Of mm,(
R2006 T2290 T2289 arg1Of mm,1
R2007 T2290 T2291 arg1Of mm,in
R2008 T2292 T2291 arg2Of diameter,in
R2009 T2293 T2288 arg3Of ),(
R2010 T2296 T2294 arg1Of chromatography,Using
R2011 T2296 T2295 arg1Of chromatography,gel-permeation
R2012 T2296 T2297 arg1Of chromatography,in
R2013 T2296 T2299 arg1Of chromatography,with
R2014 T2296 T2304 arg1Of chromatography,see
R2015 T2298 T2297 arg2Of combination,in
R2016 T2302 T2299 arg2Of measurements,with
R2017 T2302 T2300 arg1Of measurements,light
R2018 T2302 T2301 arg1Of measurements,scattering
R2019 T2304 T2303 arg1Of see,(
R2020 T2307 T2305 arg1Of al.,Viebke
R2021 T2307 T2306 arg1Of al.,et
R2022 T2307 T2308 arg1Of al.,[
R2023 T2307 T2311 arg1Of al.,)
R2024 T2307 T2312 arg1Of al.,","
R2025 T2307 T2314 arg1Of al.,was
R2026 T2307 T2315 arg2Of al.,confirmed
R2027 T2309 T2308 arg2Of 21,[
R2028 T2310 T2308 arg3Of ],[
R2029 T2313 T2312 arg2Of it,","
R2030 T2315 T2314 arg2Of confirmed,was
R2031 T2316 T2315 arg3Of that,confirmed
R2032 T2319 T2317 arg1Of fraction,the
R2033 T2319 T2318 arg1Of fraction,low
R2034 T2319 T2320 arg1Of fraction,had
R2035 T2320 T2304 arg2Of had,see
R2036 T2320 T2314 modOf had,was
R2037 T2324 T2321 arg1Of weight,an
R2038 T2324 T2322 arg1Of weight,average
R2039 T2324 T2323 arg1Of weight,molecular
R2040 T2324 T2325 arg1Of weight,of
R2041 T2324 T2329 arg1Of weight,and
R2042 T2327 T2325 arg2Of kDa,of
R2043 T2327 T2326 arg1Of kDa,10
R2044 T2329 T2320 arg2Of and,had
R2045 T2329 T2328 arg1Of and,","
R2046 T2332 T2329 arg2Of fraction,and
R2047 T2332 T2330 arg1Of fraction,the
R2048 T2332 T2331 arg1Of fraction,medium
R2049 T2332 T2333 arg1Of fraction,of
R2050 T2335 T2333 arg2Of kDa,of
R2051 T2335 T2334 arg1Of kDa,40
R2052 T2338 T2336 arg1Of content,The
R2053 T2338 T2337 arg1Of content,sulphate
R2054 T2338 T2339 arg1Of content,of
R2055 T2338 T2341 arg1Of content,in
R2056 T2338 T2344 arg1Of content,was
R2057 T2338 T2345 arg2Of content,measured
R2058 T2340 T2339 arg2Of polysaccharides,of
R2059 T2343 T2341 arg2Of fractions,in
R2060 T2343 T2342 arg1Of fractions,both
R2061 T2345 T2344 arg2Of measured,was
R2062 T2345 T2346 arg1Of measured,following
R2063 T2348 T2346 arg2Of method,following
R2064 T2348 T2347 arg1Of method,the
R2065 T2348 T2349 arg1Of method,of
R2066 T2352 T2349 arg2Of al.,of
R2067 T2352 T2350 arg1Of al.,Quemener
R2068 T2352 T2351 arg1Of al.,et
R2069 T2352 T2353 arg1Of al.,[
R2070 T2354 T2353 arg2Of 22,[
R2071 T2355 T2353 arg3Of ],[
R2072 T2359 T2358 arg1Of absence,the
R2073 T2359 T2360 arg1Of absence,of
R2074 T2359 T2368 arg1Of absence,was
R2075 T2359 T2369 arg2Of absence,confirmed
R2076 T2363 T2360 arg2Of modifications,of
R2077 T2363 T2361 arg1Of modifications,polysaccharide
R2078 T2363 T2362 arg1Of modifications,structure
R2079 T2363 T2364 arg1Of modifications,in
R2080 T2367 T2364 arg2Of fractions,in
R2081 T2367 T2365 arg1Of fractions,the
R2082 T2367 T2366 arg1Of fractions,two
R2083 T2369 T2356 arg1Of confirmed,Finally
R2084 T2369 T2357 arg1Of confirmed,","
R2085 T2369 T2368 arg2Of confirmed,was
R2086 T2370 T2369 arg3Of using,confirmed
R2087 T2372 T2370 arg2Of spectroscopy,using
R2088 T2372 T2371 arg1Of spectroscopy,2H-NMR
R2089 T2374 T2373 arg1Of absence,The
R2090 T2374 T2375 arg1Of absence,of
R2091 T2374 T2378 arg1Of absence,in
R2092 T2374 T2382 arg1Of absence,was
R2093 T2374 T2383 arg2Of absence,confirmed
R2094 T2377 T2375 arg2Of contamination,of
R2095 T2377 T2376 arg1Of contamination,LPS
R2096 T2381 T2378 arg2Of fractions,in
R2097 T2381 T2379 arg1Of fractions,the
R2098 T2381 T2380 arg1Of fractions,two
R2099 T2383 T2382 arg2Of confirmed,was
R2100 T2384 T2383 arg3Of using,confirmed
R2101 T2384 T2388 arg1Of using,(
R2102 T2387 T2384 arg2Of kit,using
R2103 T2387 T2385 arg1Of kit,the
R2104 T2387 T2386 arg1Of kit,e-Toxate®
R2105 T2389 T2388 arg2Of Sigma,(
R2106 T2389 T2390 arg1Of Sigma,","
R2107 T2393 T2390 arg2Of Fallavier,","
R2108 T2393 T2391 arg1Of Fallavier,St
R2109 T2393 T2392 arg1Of Fallavier,Quentin
R2110 T2393 T2394 arg1Of Fallavier,","
R2111 T2395 T2394 arg2Of France,","
R2112 T2396 T2388 arg3Of ),(
R2113 T2398 T2397 arg2Of use,Before
R2114 T2398 T2399 arg1Of use,in
R2115 T2401 T2399 arg2Of culture,in
R2116 T2401 T2400 arg1Of culture,cell
R2117 T2405 T2403 arg1Of fractions,the
R2118 T2405 T2404 arg1Of fractions,two
R2119 T2405 T2406 arg1Of fractions,were
R2120 T2405 T2407 arg2Of fractions,dissolved
R2121 T2407 T2397 arg1Of dissolved,Before
R2122 T2407 T2402 arg1Of dissolved,","
R2123 T2407 T2406 arg2Of dissolved,were
R2124 T2407 T2408 arg1Of dissolved,in
R2125 T2407 T2411 arg1Of dissolved,during
R2126 T2410 T2408 arg2Of medium,in
R2127 T2410 T2409 arg1Of medium,complete
R2128 T2413 T2411 arg2Of min,during
R2129 T2413 T2412 arg1Of min,30
R2130 T2413 T2414 arg1Of min,at
R2131 T2415 T2414 arg2Of 56°C,at
R2508 T2817 T2818 arg1Of Animals,","
R2509 T2818 T2820 arg1Of ",",and
R2510 T2819 T2818 arg2Of Chemicals,","
R2511 T2821 T2820 arg2Of Diet,and
R2512 T2824 T2822 arg1Of rats,Male
R2513 T2824 T2823 arg1Of rats,Wistar
R2514 T2824 T2825 arg1Of rats,(
R2515 T2824 T2831 arg1Of rats,were
R2516 T2824 T2832 arg2Of rats,housed
R2517 T2829 T2825 arg2Of weight,(
R2518 T2829 T2826 arg1Of weight,150
R2519 T2829 T2827 arg1Of weight,g
R2520 T2829 T2828 arg1Of weight,average
R2521 T2830 T2825 arg3Of ),(
R2522 T2832 T2831 arg2Of housed,were
R2523 T2832 T2833 arg1Of housed,under
R2524 T2835 T2834 arg1Of conditions,standard
R2525 T2835 T2836 arg1Of conditions,and
R2526 T2836 T2833 arg2Of and,under
R2527 T2836 T2840 arg1Of and,with
R2528 T2839 T2836 arg2Of libitum,and
R2529 T2839 T2837 arg2Of libitum,fed
R2530 T2839 T2838 arg1Of libitum,ad
R2531 T2844 T2840 arg2Of chow,with
R2532 T2844 T2841 arg1Of chow,standard
R2533 T2844 T2842 arg1Of chow,rodent
R2534 T2844 T2843 arg1Of chow,laboratory
R2535 T2846 T2845 arg2Of iota-carrageenans,Degraded
R2536 T2846 T2847 arg1Of iota-carrageenans,were
R2537 T2846 T2848 arg2Of iota-carrageenans,administered
R2538 T2848 T2847 arg2Of administered,were
R2539 T2848 T2849 arg1Of administered,in
R2540 T2848 T2858 arg1Of administered,for
R2541 T2852 T2849 arg2Of water,in
R2542 T2852 T2850 arg1Of water,the
R2543 T2852 T2851 arg1Of water,drinking
R2544 T2852 T2853 arg1Of water,(
R2545 T2854 T2855 arg1Of 5,%
R2546 T2856 T2853 arg2Of w/v,(
R2547 T2856 T2854 arg1Of w/v,5
R2548 T2857 T2853 arg3Of ),(
R2549 T2860 T2858 arg2Of days,for
R2550 T2860 T2859 arg1Of days,55
R2551 T2860 T2861 arg1Of days,to
R2552 T2863 T2861 arg2Of groups,to
R2553 T2863 T2862 arg1Of groups,2
R2554 T2863 T2864 arg1Of groups,of
R2555 T2866 T2864 arg2Of animals,of
R2556 T2866 T2865 arg1Of animals,six
R2557 T2866 T2867 arg1Of animals,each
R2558 T2870 T2868 arg1Of group,The
R2559 T2870 T2869 arg1Of group,first
R2560 T2870 T2871 arg1Of group,received
R2561 T2871 T2882 arg1Of received,and
R2562 T2876 T2871 arg2Of carrageenan,received
R2563 T2876 T2872 arg1Of carrageenan,the
R2564 T2876 T2873 arg1Of carrageenan,low
R2565 T2876 T2874 arg1Of carrageenan,molecular
R2566 T2876 T2875 arg1Of carrageenan,weight
R2567 T2876 T2877 arg1Of carrageenan,(
R2568 T2880 T2877 arg2Of dCGN,(
R2569 T2880 T2878 arg1Of dCGN,10
R2570 T2880 T2879 arg1Of dCGN,kDa
R2571 T2881 T2877 arg3Of ),(
R2572 T2884 T2883 arg1Of second,the
R2573 T2884 T2885 arg1Of second,received
R2574 T2885 T2882 arg2Of received,and
R2575 T2890 T2885 arg2Of carrageenan,received
R2576 T2890 T2886 arg1Of carrageenan,the
R2577 T2890 T2887 arg1Of carrageenan,medium
R2578 T2890 T2888 arg1Of carrageenan,molecular
R2579 T2890 T2889 arg1Of carrageenan,weight
R2580 T2890 T2891 arg1Of carrageenan,(
R2581 T2894 T2891 arg2Of dCGN,(
R2582 T2894 T2892 arg1Of dCGN,40
R2583 T2894 T2893 arg1Of dCGN,kDa
R2584 T2895 T2891 arg3Of ),(
R2585 T2898 T2896 arg1Of group,An
R2586 T2898 T2897 arg1Of group,additional
R2587 T2898 T2899 arg1Of group,of
R2588 T2898 T2902 arg1Of group,were
R2589 T2898 T2903 arg2Of group,maintained
R2590 T2901 T2899 arg2Of rats,of
R2591 T2901 T2900 arg1Of rats,four
R2592 T2903 T2902 arg2Of maintained,were
R2593 T2903 T2904 arg1Of maintained,on
R2594 T2907 T2904 arg2Of water,on
R2595 T2907 T2905 arg1Of water,regular
R2596 T2907 T2906 arg1Of water,tap
R2597 T2907 T2908 arg1Of water,(
R2598 T2910 T2908 arg2Of group,(
R2599 T2910 T2909 arg1Of group,control
R2600 T2911 T2908 arg3Of ),(
R2601 T2913 T2912 arg1Of increase,To
R2602 T2913 T2918 arg1Of increase,was
R2603 T2913 T2919 arg2Of increase,added
R2604 T2915 T2916 arg1Of 0.2,%
R2605 T2917 T2913 arg2Of sucrose,increase
R2606 T2917 T2914 arg1Of sucrose,palatability
R2607 T2917 T2915 arg1Of sucrose,0.2
R2608 T2919 T2918 arg2Of added,was
R2609 T2919 T2920 arg1Of added,to
R2610 T2919 T2927 arg1Of added,(
R2611 T2923 T2920 arg2Of water,to
R2612 T2923 T2921 arg1Of water,the
R2613 T2923 T2922 arg1Of water,drinking
R2614 T2923 T2924 arg1Of water,of
R2615 T2926 T2924 arg2Of groups,of
R2616 T2926 T2925 arg1Of groups,all
R2617 T2930 T2927 arg2Of Waaji,(
R2618 T2930 T2928 arg1Of Waaji,Van
R2619 T2930 T2929 arg1Of Waaji,der
R2620 T2930 T2931 arg1Of Waaji,et
R2621 T2931 T2932 arg1Of et,al.
R2622 T2931 T2933 arg1Of et,","
R2623 T2931 T2934 arg1Of et,[
R2624 T2935 T2934 arg2Of 23,[
R2625 T2936 T2934 arg3Of ],[
R2626 T2937 T2927 arg3Of ),(
R2627 T2940 T2938 arg1Of solutions,Fresh
R2628 T2940 T2939 arg1Of solutions,carrageenan
R2629 T2940 T2941 arg1Of solutions,were
R2630 T2940 T2942 arg2Of solutions,prepared
R2631 T2942 T2941 arg2Of prepared,were
R2632 T2942 T2943 arg1Of prepared,daily
R2772 T3130 T3131 arg1Of Evaluation,of
R2773 T3132 T3131 arg2Of Colitis,of
R2774 T3134 T3133 arg1Of weight,Body
R2775 T3134 T3135 arg1Of weight,","
R2776 T3135 T3140 arg1Of ",",","
R2777 T3136 T3137 arg1Of liquid,and
R2778 T3138 T3137 arg2Of food,and
R2779 T3139 T3135 arg2Of consumption,","
R2780 T3139 T3136 arg1Of consumption,liquid
R2781 T3139 T3138 arg1Of consumption,food
R2782 T3140 T3142 arg1Of ",",and
R2783 T3141 T3140 arg2Of diarrhea,","
R2784 T3142 T3151 arg1Of and,were
R2785 T3142 T3152 arg2Of and,recorded
R2786 T3144 T3142 arg2Of bleeding,and
R2787 T3144 T3143 arg1Of bleeding,rectal
R2788 T3144 T3145 arg1Of bleeding,(
R2789 T3146 T3145 arg2Of detected,(
R2790 T3149 T3146 arg1Of inspection,detected
R2791 T3149 T3147 arg2Of inspection,by
R2792 T3149 T3148 arg1Of inspection,eye
R2793 T3150 T3145 arg3Of ),(
R2794 T3152 T3151 arg2Of recorded,were
R2795 T3152 T3153 arg1Of recorded,throughout
R2796 T3156 T3153 arg2Of period,throughout
R2797 T3156 T3154 arg1Of period,the
R2798 T3156 T3155 arg1Of period,feeding
R2799 T3159 T3157 arg2Of days,After
R2800 T3159 T3158 arg1Of days,55
R2801 T3161 T3162 arg1Of animals,were
R2802 T3161 T3163 arg2Of animals,sacrificed
R2803 T3163 T3157 arg1Of sacrificed,After
R2804 T3163 T3160 arg1Of sacrificed,","
R2805 T3163 T3162 arg2Of sacrificed,were
R2806 T3166 T3163 arg1Of dislocation,sacrificed
R2807 T3166 T3164 arg2Of dislocation,by
R2808 T3166 T3165 arg1Of dislocation,cervical
R2809 T3168 T3167 arg1Of length,The
R2810 T3168 T3169 arg1Of length,of
R2811 T3168 T3172 arg1Of length,was
R2812 T3168 T3173 arg2Of length,measured
R2813 T3171 T3169 arg2Of colon,of
R2814 T3171 T3170 arg1Of colon,the
R2815 T3173 T3172 arg2Of measured,was
R2816 T3173 T3174 arg1Of measured,as
R2817 T3173 T3180 arg1Of measured,[
R2818 T3175 T3174 arg2Of described,as
R2819 T3177 T3175 arg1Of Okayashu,described
R2820 T3177 T3176 arg2Of Okayashu,by
R2821 T3177 T3179 arg1Of Okayashu,al.
R2822 T3179 T3178 arg1Of al.,et
R2823 T3181 T3180 arg2Of 24,[
R2824 T3182 T3180 arg3Of ],[
R2825 T3186 T3185 arg1Of colon,each
R2826 T3186 T3187 arg1Of colon,was
R2827 T3186 T3188 arg2Of colon,ligated
R2828 T3188 T3187 arg2Of ligated,was
R2829 T3188 T3189 arg1Of ligated,in
R2830 T3188 T3194 arg1Of ligated,and
R2831 T3190 T3189 arg2Of sections,in
R2832 T3190 T3191 arg1Of sections,of
R2833 T3193 T3191 arg2Of cm,of
R2834 T3193 T3192 arg1Of cm,2
R2835 T3194 T3183 arg1Of and,Then
R2836 T3194 T3184 arg1Of and,","
R2837 T3195 T3196 arg1Of 1,to
R2838 T3198 T3196 arg2Of ml,to
R2839 T3198 T3197 arg1Of ml,2
R2840 T3200 T3201 arg1Of 10,%
R2841 T3202 T3195 arg1Of formalin,1
R2842 T3202 T3199 arg1Of formalin,of
R2843 T3202 T3200 arg1Of formalin,10
R2844 T3202 T3203 arg1Of formalin,was
R2845 T3202 T3204 arg2Of formalin,infused
R2846 T3204 T3194 arg2Of infused,and
R2847 T3204 T3203 arg2Of infused,was
R2848 T3204 T3205 arg1Of infused,into
R2849 T3208 T3205 arg2Of lumen,into
R2850 T3208 T3206 arg1Of lumen,the
R2851 T3208 T3207 arg1Of lumen,intestinal
R2852 T3211 T3210 arg1Of distended,moderately
R2853 T3212 T3209 arg1Of segment,The
R2854 T3212 T3211 arg1Of segment,distended
R2855 T3212 T3213 arg1Of segment,was
R2856 T3212 T3214 arg2Of segment,sectioned
R2857 T3212 T3216 arg2Of segment,fixed
R2858 T3214 T3215 arg1Of sectioned,and
R2859 T3215 T3213 arg2Of and,was
R2860 T3215 T3217 arg1Of and,in
R2861 T3216 T3215 arg2Of fixed,and
R2862 T3218 T3219 arg1Of 10,%
R2863 T3220 T3217 arg2Of formalin,in
R2864 T3220 T3218 arg1Of formalin,10
R2865 T3223 T3221 arg1Of day,The
R2866 T3223 T3222 arg1Of day,following
R2867 T3227 T3225 arg1Of content,the
R2868 T3227 T3226 arg1Of content,intestinal
R2869 T3227 T3228 arg1Of content,was
R2870 T3227 T3229 arg2Of content,removed
R2871 T3229 T3223 arg1Of removed,day
R2872 T3229 T3224 arg1Of removed,","
R2873 T3229 T3228 arg2Of removed,was
R2874 T3231 T3229 arg1Of vortexing,removed
R2875 T3231 T3230 arg2Of vortexing,by
R2876 T3234 T3232 arg1Of segment,The
R2877 T3234 T3233 arg2Of segment,fixed
R2878 T3234 T3235 arg1Of segment,was
R2879 T3234 T3236 arg2Of segment,kept
R2880 T3236 T3235 arg2Of kept,was
R2881 T3236 T3237 arg1Of kept,in
R2882 T3236 T3241 arg1Of kept,at
R2883 T3236 T3243 arg1Of kept,until
R2884 T3238 T3239 arg1Of 10,%
R2885 T3240 T3237 arg2Of formalin,in
R2886 T3240 T3238 arg1Of formalin,10
R2887 T3242 T3241 arg2Of 4°C,at
R2888 T3247 T3243 arg2Of procedure,until
R2889 T3247 T3244 arg1Of procedure,the
R2890 T3247 T3245 arg1Of procedure,paraffin
R2891 T3247 T3246 arg1Of procedure,embedding
R2892 T3249 T3248 arg1Of evaluate,To
R2893 T3251 T3249 arg2Of degree,evaluate
R2894 T3251 T3250 arg1Of degree,the
R2895 T3251 T3252 arg1Of degree,of
R2896 T3253 T3252 arg2Of inflammation,of
R2897 T3256 T3255 arg1Of segment,this
R2898 T3256 T3257 arg1Of segment,of
R2899 T3256 T3259 arg1Of segment,was
R2900 T3256 T3260 arg2Of segment,opened
R2901 T3258 T3257 arg2Of colon,of
R2902 T3260 T3259 arg2Of opened,was
R2903 T3260 T3261 arg1Of opened,longitudinally
R2904 T3263 T3264 arg1Of macroscopic,and
R2905 T3265 T3264 arg2Of histological,and
R2906 T3266 T3262 arg1Of scores,and
R2907 T3266 T3263 arg1Of scores,macroscopic
R2908 T3266 T3265 arg1Of scores,histological
R2909 T3266 T3267 arg1Of scores,of
R2910 T3266 T3269 arg1Of scores,were
R2911 T3266 T3270 arg2Of scores,recorded
R2912 T3268 T3267 arg2Of inflammation,of
R2913 T3270 T3248 modOf recorded,To
R2914 T3270 T3254 arg1Of recorded,","
R2915 T3270 T3259 modOf recorded,was
R2916 T3270 T3269 arg2Of recorded,were
R2917 T3270 T3271 arg1Of recorded,as
R2918 T3270 T3277 arg1Of recorded,","
R2919 T3270 T3278 arg1Of recorded,[
R2920 T3273 T3272 arg1Of described,previously
R2921 T3275 T3271 arg2Of 25,as
R2922 T3275 T3273 arg1Of 25,described
R2923 T3275 T3274 arg2Of 25,[
R2924 T3276 T3274 arg3Of ],[
R2925 T3279 T3278 arg2Of 26,[
R2926 T3280 T3278 arg3Of ],[
R2927 T3284 T3281 arg1Of staining,The
R2928 T3284 T3282 arg1Of staining,toluidine
R2929 T3284 T3283 arg1Of staining,blue
R2930 T3284 T3285 arg1Of staining,was
R2931 T3284 T3286 arg2Of staining,used
R2932 T3286 T3285 arg2Of used,was
R2933 T3286 T3287 arg1Of used,for
R2934 T3288 T3287 arg2Of identification,for
R2935 T3288 T3289 arg1Of identification,of
R2936 T3288 T3292 arg1Of identification,in
R2937 T3291 T3289 arg2Of polysaccharides,of
R2938 T3291 T3290 arg2Of polysaccharides,sulphated
R2939 T3295 T3292 arg2Of mucosa,in
R2940 T3295 T3293 arg1Of mucosa,the
R2941 T3295 T3294 arg1Of mucosa,intestinal
R2942 T3298 T3296 arg2Of day,On
R2943 T3298 T3297 arg1Of day,the
R2944 T3298 T3299 arg1Of day,of
R2945 T3300 T3299 arg2Of sacrifice,of
R2946 T3304 T3302 arg1Of sample,a
R2947 T3304 T3303 arg1Of sample,fresh
R2948 T3304 T3305 arg1Of sample,of
R2949 T3304 T3312 arg1Of sample,was
R2950 T3304 T3313 arg2Of sample,collected
R2951 T3307 T3305 arg2Of colon,of
R2952 T3307 T3306 arg1Of colon,each
R2953 T3307 T3308 arg1Of colon,(
R2954 T3310 T3308 arg2Of mg,(
R2955 T3310 T3309 arg1Of mg,50
R2956 T3311 T3308 arg3Of ),(
R2957 T3313 T3296 arg1Of collected,On
R2958 T3313 T3301 arg1Of collected,","
R2959 T3313 T3312 arg2Of collected,was
R2960 T3313 T3314 arg1Of collected,for
R2961 T3313 T3320 arg1Of collected,according
R2962 T3313 T3325 arg1Of collected,","
R2963 T3313 T3326 arg1Of collected,[
R2964 T3317 T3316 arg2Of MPO,(
R2965 T3318 T3316 arg3Of ),(
R2966 T3319 T3314 arg2Of assay,for
R2967 T3319 T3315 arg1Of assay,myeloperoxidase
R2968 T3319 T3316 arg1Of assay,(
R2969 T3321 T3320 arg2Of to,according
R2970 T3322 T3321 arg2Of Krawisz,to
R2971 T3322 T3323 arg1Of Krawisz,et
R2972 T3322 T3324 arg1Of Krawisz,al.
R2973 T3327 T3326 arg2Of 27,[
R2974 T3328 T3326 arg3Of ],[
R2975 T3330 T3329 arg1Of level,The
R2976 T3330 T3331 arg1Of level,of
R2977 T3330 T3333 arg1Of level,","
R2978 T3330 T3335 arg2Of level,expressed
R2979 T3330 T3339 arg1Of level,indicates
R2980 T3332 T3331 arg2Of MPO,of
R2981 T3335 T3334 arg1Of expressed,mainly
R2982 T3337 T3335 arg1Of neutrophils,expressed
R2983 T3337 T3336 arg2Of neutrophils,by
R2984 T3339 T3338 arg1Of indicates,","
R2985 T3341 T3339 arg2Of rate,indicates
R2986 T3341 T3340 arg1Of rate,the
R2987 T3341 T3342 arg1Of rate,of
R2988 T3343 T3342 arg2Of recruitment,of
R2989 T3343 T3344 arg1Of recruitment,of
R2990 T3343 T3346 arg1Of recruitment,to
R2991 T3345 T3344 arg2Of neutrophils,of
R2992 T3349 T3346 arg2Of mucosa,to
R2993 T3349 T3347 arg1Of mucosa,the
R2994 T3349 T3348 arg1Of mucosa,intestinal
R2995 T3351 T3350 arg1Of unit,One
R2996 T3351 T3352 arg1Of unit,of
R2997 T3351 T3355 arg1Of unit,corresponds
R2998 T3354 T3352 arg2Of activity,of
R2999 T3354 T3353 arg1Of activity,MPO
R3000 T3355 T3356 arg1Of corresponds,to
R3001 T3358 T3356 arg2Of degradation,to
R3002 T3358 T3357 arg1Of degradation,the
R3003 T3358 T3359 arg1Of degradation,of
R3004 T3361 T3359 arg2Of µmol,of
R3005 T3361 T3360 arg1Of µmol,1
R3006 T3361 T3362 arg1Of µmol,of
R3007 T3361 T3364 arg1Of µmol,per
R3008 T3361 T3366 arg1Of µmol,at
R3009 T3363 T3362 arg2Of peroxide,of
R3010 T3365 T3364 arg2Of minute,per
R3011 T3367 T3366 arg2Of 25°C,at
R3275 T3677 T3676 arg1Of Culture,Cell
R3276 T3681 T3678 arg1Of reagents,All
R3277 T3681 T3679 arg1Of reagents,tissue
R3278 T3681 T3680 arg1Of reagents,culture
R3279 T3681 T3682 arg1Of reagents,were
R3280 T3681 T3683 arg1Of reagents,from
R3281 T3683 T3682 arg2Of from,were
R3282 T3684 T3683 arg2Of Invitrogen,from
R3283 T3684 T3685 arg1Of Invitrogen,(
R3284 T3687 T3685 arg2Of Pontoise,(
R3285 T3687 T3686 arg1Of Pontoise,Cergy
R3286 T3687 T3688 arg1Of Pontoise,","
R3287 T3689 T3688 arg2Of France,","
R3288 T3690 T3685 arg3Of ),(
R3289 T3694 T3691 arg1Of cells,THP-1
R3290 T3694 T3692 arg1Of cells,human
R3291 T3694 T3693 arg1Of cells,monocytic
R3292 T3694 T3695 arg1Of cells,were
R3293 T3694 T3696 arg2Of cells,maintained
R3294 T3696 T3695 arg2Of maintained,were
R3295 T3696 T3697 arg1Of maintained,in
R3296 T3698 T3697 arg2Of RPMI-1640,in
R3297 T3698 T3699 arg2Of RPMI-1640,supplemented
R3298 T3699 T3700 arg1Of supplemented,with
R3299 T3701 T3702 arg1Of 10,%
R3300 T3703 T3701 arg1Of FCS,10
R3301 T3703 T3704 arg1Of FCS,","
R3302 T3704 T3709 arg1Of ",",","
R3303 T3705 T3706 arg1Of 2,mM
R3304 T3708 T3704 arg2Of -glutamine,","
R3305 T3708 T3705 arg1Of -glutamine,2
R3306 T3708 T3707 arg1Of -glutamine,L
R3307 T3709 T3713 arg1Of ",",and
R3308 T3710 T3711 arg1Of 50,U/ml
R3309 T3712 T3709 arg2Of penicillin,","
R3310 T3712 T3710 arg1Of penicillin,50
R3311 T3713 T3700 arg2Of and,with
R3312 T3714 T3715 arg1Of 50,mg/ml
R3313 T3716 T3713 arg2Of streptomycin,and
R3314 T3716 T3714 arg1Of streptomycin,50
R3315 T3716 T3717 arg1Of streptomycin,at
R3316 T3716 T3719 arg1Of streptomycin,in
R3317 T3718 T3717 arg2Of 37°C,at
R3318 T3721 T3722 arg1Of 5,%
R3319 T3724 T3719 arg2Of incubator,in
R3320 T3724 T3720 arg1Of incubator,a
R3321 T3724 T3721 arg1Of incubator,5
R3322 T3724 T3723 arg1Of incubator,CO2
R3323 T3729 T3725 arg1Of cells,Human
R3324 T3729 T3726 arg1Of cells,peripheral
R3325 T3729 T3727 arg1Of cells,blood
R3326 T3729 T3728 arg1Of cells,mononuclear
R3327 T3729 T3730 arg1Of cells,were
R3328 T3729 T3731 arg2Of cells,obtained
R3329 T3731 T3730 arg2Of obtained,were
R3330 T3731 T3732 arg1Of obtained,from
R3331 T3734 T3732 arg2Of blood,from
R3332 T3734 T3733 arg2Of blood,heparinized
R3333 T3738 T3731 arg1Of gradient,obtained
R3334 T3738 T3735 arg2Of gradient,by
R3335 T3738 T3736 arg1Of gradient,Ficoll-Hypaque
R3336 T3738 T3737 arg1Of gradient,density
R3337 T3739 T3740 arg1Of Monocytes,were
R3338 T3739 T3742 arg2Of Monocytes,isolated
R3339 T3742 T3740 arg2Of isolated,were
R3340 T3742 T3741 arg1Of isolated,then
R3341 T3742 T3743 arg1Of isolated,by
R3342 T3742 T3745 arg1Of isolated,to
R3343 T3742 T3748 arg1Of isolated,as
R3344 T3744 T3743 arg2Of adherence,by
R3345 T3747 T3745 arg2Of flasks,to
R3346 T3747 T3746 arg1Of flasks,culture
R3347 T3749 T3748 arg2Of described,as
R3348 T3749 T3750 arg1Of described,[
R3349 T3751 T3750 arg2Of 28,[
R3350 T3752 T3750 arg3Of ],[
R3351 T3755 T3753 arg2Of aggregation,For
R3352 T3755 T3754 arg1Of aggregation,cell
R3353 T3757 T3758 arg1Of monocytes,were
R3354 T3757 T3759 arg2Of monocytes,cultured
R3355 T3759 T3753 arg1Of cultured,For
R3356 T3759 T3756 arg1Of cultured,","
R3357 T3759 T3758 arg2Of cultured,were
R3358 T3759 T3760 arg1Of cultured,in
R3359 T3762 T3763 arg1Of presence,or
R3360 T3763 T3760 arg2Of or,in
R3361 T3763 T3761 arg1Of or,the
R3362 T3763 T3765 arg1Of or,of
R3363 T3764 T3763 arg2Of absence,or
R3364 T3766 T3767 arg1Of C10,or
R3365 T3767 T3765 arg2Of or,of
R3366 T3767 T3769 arg1Of or,for
R3367 T3768 T3767 arg2Of C40,or
R3368 T3771 T3769 arg2Of h,for
R3369 T3771 T3770 arg1Of h,72
R3370 T3773 T3772 arg1Of colonies,Cell
R3371 T3773 T3774 arg1Of colonies,were
R3372 T3773 T3775 arg2Of colonies,monitored
R3373 T3775 T3774 arg2Of monitored,were
R3374 T3775 T3776 arg1Of monitored,under
R3375 T3781 T3776 arg2Of microscope,under
R3376 T3781 T3777 arg1Of microscope,an
R3377 T3781 T3778 arg2Of microscope,inverted
R3378 T3781 T3779 arg1Of microscope,phase
R3379 T3781 T3780 arg1Of microscope,contrast
R3380 T3781 T3782 arg2Of microscope,coupled
R3381 T3782 T3783 arg1Of coupled,through
R3382 T3786 T3783 arg2Of camera,through
R3383 T3786 T3784 arg1Of camera,a
R3384 T3786 T3785 arg1Of camera,video
R3385 T3786 T3787 arg1Of camera,to
R3386 T3789 T3787 arg2Of computer,to
R3387 T3789 T3788 arg1Of computer,a
R3388 T3792 T3790 arg2Of wells,In
R3389 T3792 T3791 arg1Of wells,some
R3390 T3794 T3813 arg1Of neutralizing,was
R3391 T3794 T3814 arg2Of neutralizing,added
R3392 T3796 T3794 arg2Of antibody,neutralizing
R3393 T3796 T3795 arg1Of antibody,monoclonal
R3394 T3796 T3797 arg1Of antibody,to
R3395 T3798 T3797 arg2Of ICAM-1,to
R3396 T3798 T3799 arg1Of ICAM-1,(
R3397 T3798 T3803 arg1Of ICAM-1,(
R3398 T3801 T3799 arg2Of µg/ml,(
R3399 T3801 T3800 arg1Of µg/ml,2.5
R3400 T3802 T3799 arg3Of ),(
R3401 T3804 T3803 arg2Of Tebu,(
R3402 T3804 T3805 arg1Of Tebu,","
R3403 T3804 T3810 arg1Of Tebu,","
R3404 T3809 T3805 arg2Of Yvelines,","
R3405 T3809 T3806 arg1Of Yvelines,Le
R3406 T3809 T3807 arg1Of Yvelines,Perray
R3407 T3809 T3808 arg1Of Yvelines,en
R3408 T3811 T3810 arg2Of France,","
R3409 T3812 T3803 arg3Of ),(
R3410 T3814 T3790 arg1Of added,In
R3411 T3814 T3793 arg1Of added,","
R3412 T3814 T3813 arg2Of added,was
R3563 T3988 T3986 arg1Of Analysis,Cell
R3564 T3988 T3987 arg1Of Analysis,Cycle
R3565 T3990 T3989 arg1Of cells,THP-1
R3566 T3990 T3991 arg1Of cells,in
R3567 T3990 T3995 arg1Of cells,were
R3568 T3990 T3996 arg2Of cells,exposed
R3569 T3990 T4011 arg1Of cells,being
R3570 T3990 T4012 arg2Of cells,stained
R3571 T3990 T4016 arg1Of cells,using
R3572 T3994 T3991 arg2Of phase,in
R3573 T3994 T3992 arg1Of phase,exponential
R3574 T3994 T3993 arg1Of phase,growth
R3575 T3996 T3995 arg2Of exposed,were
R3576 T3996 T3997 arg1Of exposed,to
R3577 T3996 T4000 arg1Of exposed,in
R3578 T3996 T4010 arg1Of exposed,before
R3579 T3999 T3997 arg2Of medium,to
R3580 T3999 T3998 arg1Of medium,complete
R3581 T4002 T4003 arg1Of presence,or
R3582 T4003 T4000 arg2Of or,in
R3583 T4003 T4001 arg1Of or,the
R3584 T4003 T4005 arg1Of or,of
R3585 T4003 T4007 arg1Of or,for
R3586 T4004 T4003 arg2Of absence,or
R3587 T4006 T4005 arg2Of carrageenans,of
R3588 T4009 T4007 arg2Of h,for
R3589 T4009 T4008 arg1Of h,24
R3590 T4012 T4010 arg2Of stained,before
R3591 T4012 T4011 arg2Of stained,being
R3592 T4012 T4013 arg1Of stained,with
R3593 T4012 T4016 modOf stained,using
R3594 T4015 T4013 arg2Of iodide,with
R3595 T4015 T4014 arg1Of iodide,propidium
R3596 T4016 T4021 arg1Of using,according
R3597 T4016 T4027 arg1Of using,(
R3598 T4020 T4016 arg2Of kit,using
R3599 T4020 T4017 arg1Of kit,the
R3600 T4020 T4018 arg1Of kit,DNA-Prep
R3601 T4020 T4019 arg1Of kit,Coulter
R3602 T4022 T4021 arg2Of to,according
R3603 T4024 T4023 arg1Of manufacturer,the
R3604 T4024 T4025 arg2Of manufacturer,'s
R3605 T4026 T4022 arg2Of instruction,to
R3606 T4026 T4025 arg1Of instruction,'s
R3607 T4028 T4029 arg1Of Beckman-Coulter,","
R3608 T4029 T4031 arg1Of ",",","
R3609 T4030 T4029 arg2Of Villepinte,","
R3610 T4031 T4027 arg2Of ",",(
R3611 T4032 T4031 arg2Of France,","
R3612 T4033 T4027 arg3Of ),(
R3613 T4036 T4034 arg1Of content,Cell
R3614 T4036 T4035 arg1Of content,DNA
R3615 T4036 T4037 arg1Of content,was
R3616 T4036 T4039 arg2Of content,analyzed
R3617 T4039 T4037 arg2Of analyzed,was
R3618 T4039 T4038 arg1Of analyzed,then
R3619 T4042 T4039 arg1Of cytometry,analyzed
R3620 T4042 T4040 arg2Of cytometry,by
R3621 T4042 T4041 arg1Of cytometry,flow
R3622 T4042 T4043 arg1Of cytometry,using
R3623 T4046 T4043 arg2Of XL2,using
R3624 T4046 T4044 arg1Of XL2,an
R3625 T4046 T4045 arg1Of XL2,EPICS
R3626 T4046 T4047 arg1Of XL2,(
R3627 T4048 T4047 arg2Of Beckman-Coulter,(
R3628 T4049 T4047 arg3Of ),(
R3629 T4051 T4050 arg1Of data,Raw
R3630 T4051 T4052 arg1Of data,for
R3631 T4051 T4065 arg1Of data,were
R3632 T4051 T4066 arg2Of data,expressed
R3633 T4054 T4052 arg2Of distribution,for
R3634 T4054 T4053 arg1Of distribution,the
R3635 T4054 T4055 arg1Of distribution,of
R3636 T4057 T4055 arg2Of content,of
R3637 T4057 T4056 arg1Of content,DNA
R3638 T4057 T4058 arg1Of content,of
R3639 T4060 T4058 arg2Of cells,of
R3640 T4060 T4059 arg1Of cells,"30,000"
R3641 T4060 T4061 arg2Of cells,retrieved
R3642 T4061 T4062 arg1Of retrieved,from
R3643 T4064 T4062 arg2Of cytometer,from
R3644 T4064 T4063 arg1Of cytometer,the
R3645 T4066 T4065 arg2Of expressed,were
R3646 T4066 T4067 arg1Of expressed,as
R3647 T4069 T4067 arg2Of percentage,as
R3648 T4069 T4068 arg1Of percentage,the
R3649 T4069 T4070 arg1Of percentage,of
R3650 T4069 T4072 arg1Of percentage,through
R3651 T4071 T4070 arg2Of G0/G1,of
R3652 T4074 T4072 arg2Of populations,through
R3653 T4074 T4073 arg1Of populations,G2/M
R3654 T4077 T4075 arg1Of software,Multicycle
R3655 T4077 T4076 arg1Of software,AV
R3656 T4077 T4078 arg1Of software,(
R3657 T4077 T4088 arg1Of software,was
R3658 T4077 T4089 arg2Of software,used
R3659 T4077 T4097 arg1Of software,facilitate
R3660 T4081 T4078 arg2Of Systems,(
R3661 T4081 T4079 arg1Of Systems,Phoenix
R3662 T4081 T4080 arg1Of Systems,Flow
R3663 T4081 T4082 arg1Of Systems,","
R3664 T4084 T4082 arg2Of Diego,","
R3665 T4084 T4083 arg1Of Diego,San
R3666 T4084 T4085 arg1Of Diego,","
R3667 T4086 T4085 arg2Of CA,","
R3668 T4087 T4078 arg3Of ),(
R3669 T4089 T4088 arg2Of used,was
R3670 T4089 T4090 modOf used,to
R3671 T4089 T4096 arg1Of used,and
R3672 T4091 T4090 arg1Of generate,to
R3673 T4095 T4091 arg2Of histograms,generate
R3674 T4095 T4092 arg1Of histograms,DNA
R3675 T4095 T4093 arg1Of histograms,content
R3676 T4095 T4094 arg1Of histograms,frequency
R3677 T4097 T4096 arg2Of facilitate,and
R3678 T4099 T4097 arg2Of analysis,facilitate
R3679 T4099 T4098 arg1Of analysis,data
R3798 T4244 T4242 arg1Of Antigen,Cell
R3799 T4244 T4243 arg1Of Antigen,Surface
R3800 T4246 T4244 arg1Of Analysis,Antigen
R3801 T4246 T4245 arg1Of Analysis,Expression
R3802 T4249 T4247 arg1Of Monocytes,Peripheral
R3803 T4249 T4248 arg1Of Monocytes,Blood
R3804 T4249 T4250 arg1Of Monocytes,or
R3805 T4250 T4253 arg1Of or,were
R3806 T4250 T4254 arg2Of or,exposed
R3807 T4252 T4250 arg2Of cells,or
R3808 T4252 T4251 arg1Of cells,THP-1
R3809 T4254 T4253 arg2Of exposed,were
R3810 T4254 T4255 arg1Of exposed,to
R3811 T4254 T4258 arg1Of exposed,in
R3812 T4257 T4255 arg2Of medium,to
R3813 T4257 T4256 arg1Of medium,complete
R3814 T4260 T4261 arg1Of presence,or
R3815 T4261 T4258 arg2Of or,in
R3816 T4261 T4259 arg1Of or,the
R3817 T4261 T4263 arg1Of or,of
R3818 T4261 T4265 arg1Of or,for
R3819 T4262 T4261 arg2Of absence,or
R3820 T4264 T4263 arg2Of carrageenan,of
R3821 T4267 T4265 arg2Of h,for
R3822 T4267 T4266 arg1Of h,36
R3823 T4270 T4268 arg2Of washes,After
R3824 T4270 T4269 arg1Of washes,two
R3825 T4270 T4271 arg1Of washes,in
R3826 T4270 T4273 arg1Of washes,without
R3827 T4272 T4271 arg2Of PBS,in
R3828 T4274 T4275 arg1Of Ca2+,and
R3829 T4275 T4273 arg2Of and,without
R3830 T4276 T4275 arg2Of Mg2+,and
R3831 T4278 T4279 arg1Of cells,were
R3832 T4278 T4280 arg2Of cells,incubated
R3833 T4280 T4268 arg1Of incubated,After
R3834 T4280 T4277 arg1Of incubated,","
R3835 T4280 T4279 arg2Of incubated,were
R3836 T4280 T4281 arg1Of incubated,in
R3837 T4280 T4293 modOf incubated,to
R3838 T4282 T4281 arg2Of PBS,in
R3839 T4282 T4283 arg1Of PBS,containing
R3840 T4284 T4285 arg1Of 0.1,%
R3841 T4286 T4284 arg1Of gelatin,0.1
R3842 T4286 T4287 arg1Of gelatin,and
R3843 T4287 T4283 arg2Of and,containing
R3844 T4288 T4289 arg1Of 8,%
R3845 T4292 T4287 arg2Of serum,and
R3846 T4292 T4288 arg1Of serum,8
R3847 T4292 T4290 arg1Of serum,AB
R3848 T4292 T4291 arg1Of serum,human
R3849 T4294 T4293 arg1Of prevent,to
R3850 T4295 T4294 arg2Of binding,prevent
R3851 T4295 T4296 arg1Of binding,to
R3852 T4298 T4296 arg2Of receptors,to
R3853 T4298 T4297 arg1Of receptors,Fc
R3854 T4302 T4301 arg1Of cells,5×105
R3855 T4302 T4303 arg1Of cells,were
R3856 T4302 T4304 arg2Of cells,incubated
R3857 T4304 T4299 arg1Of incubated,Then
R3858 T4304 T4300 arg1Of incubated,","
R3859 T4304 T4303 arg2Of incubated,were
R3860 T4304 T4305 arg1Of incubated,with
R3861 T4304 T4308 arg1Of incubated,at
R3862 T4304 T4310 arg1Of incubated,for
R3863 T4307 T4305 arg2Of antibodies,with
R3864 T4307 T4306 arg1Of antibodies,primary
R3865 T4309 T4308 arg2Of 4°C,at
R3866 T4312 T4310 arg2Of min,for
R3867 T4312 T4311 arg1Of min,30
R3868 T4315 T4313 arg1Of washes,Two
R3869 T4315 T4314 arg1Of washes,other
R3870 T4315 T4316 arg1Of washes,in
R3871 T4315 T4318 arg1Of washes,preceded
R3872 T4317 T4316 arg2Of PBS,in
R3873 T4319 T4318 arg2Of incubation,preceded
R3874 T4319 T4320 arg1Of incubation,with
R3875 T4325 T4320 arg2Of IgG,with
R3876 T4325 T4321 arg1Of IgG,FITC-conjugated
R3877 T4325 T4322 arg1Of IgG,goat
R3878 T4325 T4323 arg1Of IgG,antibody
R3879 T4325 T4324 arg1Of IgG,anti-mouse
R3880 T4325 T4326 arg2Of IgG,diluted
R3881 T4326 T4328 arg1Of diluted,at
R3882 T4326 T4330 arg1Of diluted,for
R3883 T4327 T4326 arg3Of 1/1000,diluted
R3884 T4329 T4328 arg2Of 4°C,at
R3885 T4332 T4330 arg2Of min,for
R3886 T4332 T4331 arg1Of min,30
R3887 T4332 T4333 arg1Of min,(
R3888 T4334 T4333 arg2Of Tebu,(
R3889 T4335 T4333 arg3Of ),(
R3890 T4339 T4336 arg2Of washes,After
R3891 T4339 T4337 arg1Of washes,two
R3892 T4339 T4338 arg1Of washes,additional
R3893 T4341 T4342 arg1Of analysis,of
R3894 T4341 T4345 arg1Of analysis,was
R3895 T4341 T4346 arg2Of analysis,performed
R3896 T4344 T4342 arg2Of cells,of
R3897 T4344 T4343 arg2Of cells,stained
R3898 T4346 T4336 arg1Of performed,After
R3899 T4346 T4340 arg1Of performed,","
R3900 T4346 T4345 arg2Of performed,was
R3901 T4346 T4347 arg1Of performed,on
R3902 T4350 T4347 arg2Of XL2,on
R3903 T4350 T4348 arg1Of XL2,an
R3904 T4350 T4349 arg1Of XL2,EPICS
R3905 T4350 T4351 arg1Of XL2,(
R3906 T4352 T4351 arg2Of Beckman-Coulter,(
R3907 T4353 T4351 arg3Of ),(
R3908 T4356 T4354 arg1Of population,The
R3909 T4356 T4355 arg1Of population,cell
R3910 T4356 T4357 arg1Of population,was
R3911 T4356 T4358 arg2Of population,gated
R3912 T4358 T4357 arg2Of gated,was
R3913 T4358 T4359 arg1Of gated,according
R3914 T4360 T4359 arg2Of to,according
R3915 T4362 T4363 arg1Of forward,and
R3916 T4364 T4363 arg2Of wide-angle,and
R3917 T4366 T4360 arg2Of scattering,to
R3918 T4366 T4361 arg1Of scattering,its
R3919 T4366 T4362 arg1Of scattering,forward
R3920 T4366 T4364 arg1Of scattering,wide-angle
R3921 T4366 T4365 arg1Of scattering,light
R3922 T4367 T4368 arg1Of Data,were
R3923 T4367 T4369 arg2Of Data,expressed
R3924 T4369 T4368 arg2Of expressed,were
R3925 T4369 T4370 arg1Of expressed,as
R3926 T4374 T4370 arg2Of intensity,as
R3927 T4374 T4371 arg1Of intensity,mean
R3928 T4374 T4372 arg1Of intensity,relative
R3929 T4374 T4373 arg1Of intensity,fluorescence
R3930 T4374 T4375 arg1Of intensity,(
R3931 T4374 T4378 arg1Of intensity,of
R3932 T4376 T4375 arg2Of MFI,(
R3933 T4377 T4375 arg3Of ),(
R3934 T4380 T4378 arg2Of cells,of
R3935 T4380 T4379 arg1Of cells,3000
R4092 T4589 T4587 arg1Of Bioassay,TNF
R4093 T4589 T4588 arg1Of Bioassay,Activity
R4094 T4590 T4591 arg1Of Monocytes,or
R4095 T4591 T4594 arg1Of or,were
R4096 T4591 T4595 arg2Of or,cultured
R4097 T4593 T4591 arg2Of cells,or
R4098 T4593 T4592 arg1Of cells,THP-1
R4099 T4595 T4594 arg2Of cultured,were
R4100 T4595 T4596 arg1Of cultured,with
R4101 T4595 T4598 arg1Of cultured,without
R4102 T4596 T4597 arg1Of with,or
R4103 T4598 T4597 arg2Of without,or
R4104 T4600 T4596 arg2Of concentrations,with
R4105 T4600 T4598 arg2Of concentrations,without
R4106 T4600 T4599 arg1Of concentrations,different
R4107 T4600 T4601 arg1Of concentrations,of
R4108 T4600 T4611 arg1Of concentrations,for
R4109 T4602 T4603 arg1Of CGNs,or
R4110 T4603 T4601 arg2Of or,of
R4111 T4603 T4605 arg1Of or,(
R4112 T4604 T4603 arg2Of LPS,or
R4113 T4607 T4605 arg2Of typhosa,(
R4114 T4607 T4606 arg1Of typhosa,Salmonella
R4115 T4607 T4608 arg1Of typhosa,","
R4116 T4609 T4608 arg2Of Sigma,","
R4117 T4610 T4605 arg3Of ),(
R4118 T4613 T4612 arg1Of h,24
R4119 T4613 T4614 arg1Of h,or
R4120 T4614 T4611 arg2Of or,for
R4121 T4617 T4614 arg2Of time,or
R4122 T4617 T4615 arg1Of time,the
R4123 T4617 T4616 arg2Of time,indicated
R4124 T4619 T4618 arg1Of active,Biologically
R4125 T4620 T4619 arg1Of TNF-α/β,active
R4126 T4620 T4621 arg1Of TNF-α/β,in
R4127 T4620 T4625 arg1Of TNF-α/β,was
R4128 T4620 T4626 arg2Of TNF-α/β,measured
R4129 T4624 T4621 arg2Of supernatant,in
R4130 T4624 T4622 arg1Of supernatant,tissue
R4131 T4624 T4623 arg1Of supernatant,culture
R4132 T4626 T4625 arg2Of measured,was
R4133 T4627 T4626 arg3Of using,measured
R4134 T4627 T4635 arg1Of using,[
R4135 T4634 T4627 arg2Of assay,using
R4136 T4634 T4628 arg1Of assay,the
R4137 T4634 T4629 arg1Of assay,WEHI
R4138 T4634 T4630 arg1Of assay,164
R4139 T4634 T4631 arg1Of assay,clone
R4140 T4634 T4632 arg1Of assay,13-cell
R4141 T4634 T4633 arg1Of assay,killing
R4142 T4636 T4635 arg2Of 29,[
R4143 T4637 T4635 arg3Of ],[
R4144 T4639 T4638 arg1Of concentrations,TNF
R4145 T4639 T4640 arg1Of concentrations,are
R4146 T4639 T4641 arg2Of concentrations,expressed
R4147 T4641 T4640 arg2Of expressed,are
R4148 T4641 T4642 arg1Of expressed,as
R4149 T4643 T4642 arg2Of pg/ml,as
R4213 T4751 T4750 arg1Of Analysis,RT-PCR
R4214 T4753 T4752 arg1Of RNA,Total
R4215 T4753 T4754 arg1Of RNA,from
R4216 T4753 T4756 arg1Of RNA,was
R4217 T4753 T4757 arg2Of RNA,isolated
R4218 T4755 T4754 arg2Of monocytes,from
R4219 T4757 T4756 arg2Of isolated,was
R4220 T4758 T4757 arg3Of using,isolated
R4221 T4760 T4758 arg2Of Reagent™,using
R4222 T4760 T4759 arg1Of Reagent™,TRIzol
R4223 T4760 T4761 arg1Of Reagent™,(
R4224 T4762 T4761 arg2Of Invitrogen,(
R4225 T4763 T4761 arg3Of ),(
R4226 T4764 T4765 arg1Of . cD,NA
R4227 T4764 T4766 arg2Of . cD,was gener
R4228 T4766 T4756 modOf was gener,was
R4229 T4766 T4765 arg2Of was gener,NA
R4230 T4766 T4767 arg1Of was gener,at
R4231 T4766 T4780 arg1Of was gener,v
R4232 T4769 T4767 arg2Of d ,at
R4233 T4769 T4768 arg1Of d ,e
R4234 T4769 T4770 arg1Of d ,on
R4235 T4769 T4773 arg1Of d ,to
R4236 T4772 T4770 arg2Of of ,on
R4237 T4772 T4771 arg1Of of ,1 µg
R4238 T4776 T4773 arg2Of n a re,to
R4239 T4776 T4774 arg1Of n a re,t
R4240 T4776 T4775 arg1Of n a re,al RNA i
R4241 T4776 T4777 arg1Of n a re,ac
R4242 T4779 T4777 arg2Of on,ac
R4243 T4779 T4778 arg1Of on,ti
R4244 T4781 T4766 arg3Of olume,was gener
R4245 T4784 T4781 arg2Of ng M-MLV reve,olume
R4246 T4784 T4782 arg1Of ng M-MLV reve,of 20
R4247 T4784 T4783 arg1Of ng M-MLV reve,"µl, usi"
R4248 T4784 T4785 arg1Of ng M-MLV reve,r
R4249 T4786 T4785 arg2Of se transcr,r
R4250 T4787 T4785 arg3Of i,r
R4251 T4788 T4789 arg1Of PCR,was
R4252 T4788 T4790 arg2Of PCR,done
R4253 T4790 T4789 arg2Of done,was
R4254 T4790 T4791 arg1Of done,in
R4255 T4794 T4791 arg2Of range,in
R4256 T4794 T4792 arg1Of range,the
R4257 T4794 T4793 arg1Of range,linear
R4258 T4794 T4795 arg1Of range,of
R4259 T4796 T4795 arg2Of amplification,of
R4260 T4796 T4797 arg1Of amplification,(
R4261 T4798 T4797 arg2Of determined,(
R4262 T4798 T4799 arg1Of determined,for
R4263 T4803 T4799 arg2Of combination,for
R4264 T4803 T4800 arg1Of combination,each
R4265 T4803 T4801 arg1Of combination,primer
R4266 T4803 T4802 arg1Of combination,pair-cDNA
R4267 T4804 T4797 arg3Of ),(
R4268 T4807 T4805 arg1Of reactions,Standard
R4269 T4807 T4806 arg1Of reactions,PCR
R4270 T4807 T4808 arg1Of reactions,were
R4271 T4807 T4809 arg2Of reactions,performed
R4272 T4809 T4808 arg2Of performed,were
R4273 T4809 T4810 arg1Of performed,with
R4274 T4809 T4849 arg1Of performed,(
R4275 T4812 T4811 arg1Of µl,1
R4276 T4812 T4813 arg1Of µl,of
R4277 T4812 T4817 arg1Of µl,","
R4278 T4816 T4813 arg2Of solution,of
R4279 T4816 T4814 arg1Of solution,the
R4280 T4816 T4815 arg1Of solution,cDNA
R4281 T4817 T4824 arg1Of ",",","
R4282 T4819 T4817 arg2Of µM,","
R4283 T4819 T4818 arg1Of µM,50
R4284 T4819 T4820 arg1Of µM,of
R4285 T4823 T4820 arg2Of solution,of
R4286 T4823 T4821 arg1Of solution,each
R4287 T4823 T4822 arg1Of solution,primer
R4288 T4824 T4830 arg1Of ",",","
R4289 T4826 T4824 arg2Of mM,","
R4290 T4826 T4825 arg1Of mM,10
R4291 T4826 T4827 arg1Of mM,of
R4292 T4829 T4827 arg2Of dNTP,of
R4293 T4829 T4828 arg1Of dNTP,each
R4294 T4830 T4834 arg1Of ",",","
R4295 T4830 T4842 arg1Of ",",and
R4296 T4831 T4832 arg1Of 25,mM
R4297 T4833 T4830 arg2Of MgCl2,","
R4298 T4833 T4831 arg1Of MgCl2,25
R4299 T4836 T4835 arg1Of Goldstar,10X
R4300 T4840 T4834 arg2Of buffer,","
R4301 T4840 T4836 arg1Of buffer,Goldstar
R4302 T4840 T4837 arg1Of buffer,DNA
R4303 T4840 T4838 arg1Of buffer,polymerase
R4304 T4840 T4839 arg1Of buffer,reaction
R4305 T4842 T4810 arg2Of and,with
R4306 T4842 T4841 arg1Of and,","
R4307 T4844 T4842 arg2Of units,and
R4308 T4844 T4843 arg1Of units,0.5
R4309 T4844 T4845 arg1Of units,of
R4310 T4848 T4845 arg2Of polymerase,of
R4311 T4848 T4846 arg1Of polymerase,Goldstar
R4312 T4848 T4847 arg1Of polymerase,DNA
R4313 T4850 T4851 arg1Of Eurogentec,","
R4314 T4851 T4849 arg2Of ",",(
R4315 T4851 T4853 arg1Of ",",","
R4316 T4852 T4851 arg2Of Seraing,","
R4317 T4854 T4853 arg2Of Belgium,","
R4318 T4855 T4849 arg3Of ),(
R4319 T4858 T4856 arg1Of cycle,First
R4320 T4858 T4857 arg1Of cycle,PCR
R4321 T4858 T4859 arg1Of cycle,consisted
R4322 T4859 T4860 arg1Of consisted,of
R4323 T4859 T4863 arg1Of consisted,at
R4324 T4859 T4875 arg1Of consisted,;
R4325 T4862 T4860 arg2Of min,of
R4326 T4862 T4861 arg1Of min,1
R4327 T4864 T4865 arg1Of 92°C,","
R4328 T4865 T4870 arg1Of ",",and
R4329 T4867 T4865 arg2Of min,","
R4330 T4867 T4866 arg1Of min,1
R4331 T4867 T4868 arg1Of min,at
R4332 T4869 T4868 arg2Of 58°C,at
R4333 T4870 T4863 arg2Of and,at
R4334 T4870 T4873 arg1Of and,at
R4335 T4872 T4870 arg2Of min,and
R4336 T4872 T4871 arg1Of min,1
R4337 T4874 T4873 arg2Of 72°C,at
R4338 T4879 T4877 arg1Of cycle,each
R4339 T4879 T4878 arg1Of cycle,PCR
R4340 T4879 T4880 arg1Of cycle,consisted
R4341 T4880 T4875 arg2Of consisted,;
R4342 T4880 T4876 arg1Of consisted,then
R4343 T4880 T4881 arg1Of consisted,of
R4344 T4880 T4884 arg1Of consisted,at
R4345 T4883 T4881 arg2Of sec,of
R4346 T4883 T4882 arg1Of sec,40
R4347 T4885 T4884 arg2Of 92°C,at
R4348 T4888 T4887 arg1Of sec,40
R4349 T4888 T4889 arg1Of sec,at
R4350 T4888 T4894 arg1Of sec,at
R4351 T4888 T4899 arg1Of sec,was
R4352 T4888 T4900 arg2Of sec,amplified
R4353 T4888 T4941 arg1Of sec,sense
R4354 T4890 T4891 arg1Of 58°C,and
R4355 T4891 T4889 arg2Of and,at
R4356 T4893 T4891 arg2Of sec,and
R4357 T4893 T4892 arg1Of sec,50
R4358 T4896 T4894 arg2Of cDNA,at
R4359 T4896 T4895 arg1Of cDNA,72°C.
R4360 T4896 T4897 arg1Of cDNA,for
R4361 T4898 T4897 arg2Of β-actin,for
R4362 T4900 T4899 arg2Of amplified,was
R4363 T4900 T4901 arg1Of amplified,for
R4364 T4900 T4907 arg1Of amplified,:
R4365 T4903 T4901 arg2Of cycles,for
R4366 T4903 T4902 arg1Of cycles,28
R4367 T4903 T4904 arg1Of cycles,using
R4368 T4906 T4904 arg2Of oligos,using
R4369 T4906 T4905 arg1Of oligos,the
R4370 T4907 T4875 arg3Of :,;
R4371 T4907 T4886 arg1Of :,","
R4372 T4909 T4908 arg1Of 5′-GGCATCGTGATGGACTCCG-3′,sense
R4373 T4909 T4910 arg1Of 5′-GGCATCGTGATGGACTCCG-3′,and
R4374 T4910 T4914 arg1Of and,for
R4375 T4910 T4916 arg1Of and,was
R4376 T4910 T4917 arg2Of and,amplified
R4377 T4913 T4910 arg2Of cDNA,and
R4378 T4913 T4911 arg1Of cDNA,antisense
R4379 T4913 T4912 arg1Of cDNA,5′GCTGGAAGGTGGACAGCGA-3′.
R4380 T4915 T4914 arg2Of TNF-α,for
R4381 T4917 T4916 arg2Of amplified,was
R4382 T4917 T4918 arg1Of amplified,for
R4383 T4917 T4924 arg1Of amplified,:
R4384 T4920 T4918 arg2Of cycles,for
R4385 T4920 T4919 arg1Of cycles,35
R4386 T4920 T4921 arg1Of cycles,using
R4387 T4923 T4921 arg2Of oligos,using
R4388 T4923 T4922 arg1Of oligos,the
R4389 T4926 T4925 arg1Of 5′-AAGCCTGTAGCCCATGTTGT-3′,sense
R4390 T4926 T4927 arg1Of 5′-AAGCCTGTAGCCCATGTTGT-3′,and
R4391 T4927 T4931 arg1Of and,for
R4392 T4927 T4933 arg1Of and,was
R4393 T4927 T4934 arg2Of and,amplified
R4394 T4930 T4927 arg2Of cDNA,and
R4395 T4930 T4928 arg1Of cDNA,antisense
R4396 T4930 T4929 arg1Of cDNA,5′-CAGATAGATGGGCTCATACC-3′.
R4397 T4932 T4931 arg2Of ICAM-1,for
R4398 T4934 T4924 arg2Of amplified,:
R4399 T4934 T4933 arg2Of amplified,was
R4400 T4934 T4935 arg1Of amplified,for
R4401 T4937 T4935 arg2Of cycles,for
R4402 T4937 T4936 arg1Of cycles,35
R4403 T4937 T4938 arg1Of cycles,using
R4404 T4940 T4938 arg2Of oligos,using
R4405 T4940 T4939 arg1Of oligos,the
R4406 T4941 T4907 arg2Of sense,:
R4407 T4941 T4916 modOf sense,was
R4408 T4942 T4943 arg1Of 5′-GTAGCAGCCGCAGTCATAATGG-3′,and
R4409 T4944 T4943 arg2Of antisense,and
R4410 T4946 T4941 arg2Of TGCTGTTGTATCTGACTGAGG-3′,sense
R4411 T4946 T4942 arg1Of TGCTGTTGTATCTGACTGAGG-3′,5′-GTAGCAGCCGCAGTCATAATGG-3′
R4412 T4946 T4944 arg1Of TGCTGTTGTATCTGACTGAGG-3′,antisense
R4413 T4946 T4945 arg1Of TGCTGTTGTATCTGACTGAGG-3′,5′-A
R4654 T5243 T5239 arg1Of Assay,NF-kB
R4655 T5243 T5240 arg1Of Assay,Transcription
R4656 T5243 T5241 arg1Of Assay,Reporter
R4657 T5243 T5242 arg1Of Assay,Gene
R4658 T5246 T5244 arg1Of 3XMHC-luc,The
R4659 T5246 T5245 arg1Of 3XMHC-luc,plasmid
R4660 T5246 T5247 arg1Of 3XMHC-luc,(
R4661 T5246 T5267 arg1Of 3XMHC-luc,contains
R4662 T5250 T5248 arg1Of gift,a
R4663 T5250 T5249 arg1Of gift,generous
R4664 T5250 T5251 arg1Of gift,from
R4665 T5250 T5255 arg1Of gift,and
R4666 T5254 T5251 arg2Of Westwick,from
R4667 T5254 T5252 arg1Of Westwick,Drs.
R4668 T5254 T5253 arg1Of Westwick,J.
R4669 T5255 T5247 arg2Of and,(
R4670 T5257 T5255 arg2Of Brenner,and
R4671 T5257 T5256 arg1Of Brenner,D.A.
R4672 T5257 T5258 arg1Of Brenner,","
R4673 T5257 T5263 arg1Of Brenner,","
R4674 T5259 T5258 arg2Of University,","
R4675 T5259 T5260 arg1Of University,of
R4676 T5262 T5260 arg2Of Carolina,of
R4677 T5262 T5261 arg1Of Carolina,North
R4678 T5265 T5263 arg2Of Hill,","
R4679 T5265 T5264 arg1Of Hill,Chapel
R4680 T5266 T5247 arg3Of ),(
R4681 T5269 T5267 arg2Of copies,contains
R4682 T5269 T5268 arg1Of copies,three
R4683 T5269 T5270 arg1Of copies,of
R4684 T5269 T5273 arg1Of copies,from
R4685 T5269 T5279 arg1Of copies,","
R4686 T5269 T5280 arg2Of copies,placed
R4687 T5272 T5270 arg2Of element,of
R4688 T5272 T5271 arg1Of element,NF-κB-responsive
R4689 T5278 T5273 arg2Of locus,from
R4690 T5278 T5274 arg1Of locus,the
R4691 T5278 T5275 arg1Of locus,MHC
R4692 T5278 T5276 arg1Of locus,class
R4693 T5278 T5277 arg1Of locus,I
R4694 T5280 T5281 arg1Of placed,upstream
R4695 T5281 T5282 arg1Of upstream,of
R4696 T5285 T5282 arg2Of gene,of
R4697 T5285 T5283 arg1Of gene,the
R4698 T5285 T5284 arg1Of gene,luciferase
R4699 T5289 T5286 arg1Of cells,Human
R4700 T5289 T5287 arg1Of cells,monocytic
R4701 T5289 T5288 arg1Of cells,THP-1
R4702 T5289 T5290 arg1Of cells,were
R4703 T5289 T5292 arg2Of cells,transfected
R4704 T5289 T5302 arg2Of cells,cultured
R4705 T5292 T5293 arg1Of transfected,as
R4706 T5292 T5300 arg1Of transfected,and
R4707 T5295 T5294 arg1Of described,previously
R4708 T5297 T5293 arg2Of 30,as
R4709 T5297 T5295 arg1Of 30,described
R4710 T5297 T5296 arg2Of 30,[
R4711 T5298 T5296 arg3Of ],[
R4712 T5300 T5290 arg2Of and,were
R4713 T5300 T5291 arg1Of and,transiently
R4714 T5300 T5299 arg1Of and,","
R4715 T5302 T5300 arg2Of cultured,and
R4716 T5302 T5301 arg1Of cultured,then
R4717 T5302 T5303 arg1Of cultured,for
R4718 T5305 T5303 arg2Of h,for
R4719 T5305 T5304 arg1Of h,4
R4720 T5305 T5306 arg1Of h,alone
R4721 T5305 T5308 arg1Of h,with
R4722 T5306 T5307 arg1Of alone,or
R4723 T5308 T5307 arg2Of with,or
R4724 T5310 T5308 arg2Of concentration,with
R4725 T5310 T5309 arg1Of concentration,increasing
R4726 T5310 T5311 arg1Of concentration,of
R4727 T5313 T5314 arg1Of C10,or
R4728 T5314 T5311 arg2Of or,of
R4729 T5314 T5312 arg1Of or,either
R4730 T5315 T5314 arg2Of C40,or
R4731 T5317 T5316 arg1Of activity,Luciferase
R4732 T5317 T5318 arg1Of activity,was
R4733 T5317 T5319 arg2Of activity,determined
R4734 T5319 T5318 arg2Of determined,was
R4735 T5320 T5319 arg3Of using,determined
R4736 T5322 T5320 arg2Of luminometer,using
R4737 T5322 T5321 arg1Of luminometer,a
R4738 T5322 T5323 arg1Of luminometer,(
R4739 T5326 T5323 arg2Of Luminometer,(
R4740 T5326 T5324 arg1Of Luminometer,Monolight
R4741 T5326 T5325 arg1Of Luminometer,2010
R4742 T5326 T5327 arg1Of Luminometer,","
R4743 T5329 T5327 arg2Of Arbor,","
R4744 T5329 T5328 arg1Of Arbor,Ann
R4745 T5329 T5330 arg1Of Arbor,","
R4746 T5331 T5330 arg2Of MI,","
R4747 T5332 T5323 arg3Of ),(
R4845 T5493 T5492 arg1Of Blot,Western
R4846 T5494 T5493 arg1Of Analysis,Blot
R4847 T5496 T5495 arg1Of cells,THP-1
R4848 T5496 T5497 arg1Of cells,were
R4849 T5496 T5498 arg2Of cells,stimulated
R4850 T5498 T5497 arg2Of stimulated,were
R4851 T5498 T5499 arg1Of stimulated,for
R4852 T5501 T5499 arg2Of lengths,for
R4853 T5501 T5500 arg1Of lengths,various
R4854 T5501 T5502 arg1Of lengths,of
R4855 T5503 T5502 arg2Of time,of
R4856 T5503 T5504 arg1Of time,with
R4857 T5505 T5506 arg1Of 0.1,mg/ml
R4858 T5507 T5505 arg1Of C10,0.1
R4859 T5507 T5508 arg1Of C10,or
R4860 T5508 T5511 arg1Of or,or
R4861 T5509 T5508 arg2Of C40,or
R4862 T5511 T5504 arg2Of or,with
R4863 T5511 T5510 arg1Of or,","
R4864 T5514 T5511 arg2Of LPS,or
R4865 T5514 T5512 arg1Of LPS,10
R4866 T5514 T5513 arg1Of LPS,µg/ml
R4867 T5515 T5516 arg1Of Cells,were
R4868 T5515 T5518 arg2Of Cells,pelleted
R4869 T5515 T5520 arg2Of Cells,washed
R4870 T5515 T5522 arg2Of Cells,homogenised
R4871 T5518 T5519 arg1Of pelleted,","
R4872 T5519 T5521 arg1Of ",",and
R4873 T5520 T5519 arg2Of washed,","
R4874 T5521 T5516 arg2Of and,were
R4875 T5521 T5517 arg1Of and,then
R4876 T5522 T5521 arg2Of homogenised,and
R4877 T5522 T5523 arg1Of homogenised,in
R4878 T5525 T5523 arg2Of buffer,in
R4879 T5525 T5524 arg1Of buffer,lysis
R4880 T5525 T5526 arg1Of buffer,(
R4881 T5525 T5551 arg1Of buffer,on
R4882 T5529 T5526 arg2Of Hepes,(
R4883 T5529 T5527 arg1Of Hepes,10
R4884 T5529 T5528 arg1Of Hepes,mM
R4885 T5529 T5530 arg1Of Hepes,","
R4886 T5531 T5532 arg1Of pH,7.9
R4887 T5531 T5533 arg1Of pH,","
R4888 T5533 T5537 arg1Of ",",","
R4889 T5534 T5535 arg1Of 150,mM
R4890 T5536 T5533 arg2Of NaCl,","
R4891 T5536 T5534 arg1Of NaCl,150
R4892 T5537 T5541 arg1Of ",",","
R4893 T5538 T5539 arg1Of 1,mM
R4894 T5540 T5537 arg2Of EDTA,","
R4895 T5540 T5538 arg1Of EDTA,1
R4896 T5541 T5546 arg1Of ",",and
R4897 T5542 T5543 arg1Of 0.6,%
R4898 T5544 T5541 arg2Of NP-40,","
R4899 T5544 T5542 arg1Of NP-40,0.6
R4900 T5546 T5530 arg2Of and,","
R4901 T5546 T5545 arg1Of and,","
R4902 T5547 T5548 arg1Of 0.5,mM
R4903 T5549 T5546 arg2Of PMSF,and
R4904 T5549 T5547 arg1Of PMSF,0.5
R4905 T5550 T5526 arg3Of ),(
R4906 T5552 T5551 arg2Of ice,on
R4907 T5553 T5554 arg1Of Homogenates,were
R4908 T5553 T5555 arg2Of Homogenates,sonicated
R4909 T5553 T5557 arg2Of Homogenates,centrifuged
R4910 T5555 T5556 arg1Of sonicated,","
R4911 T5556 T5554 arg2Of ",",were
R4912 T5556 T5561 modOf ",",to
R4913 T5557 T5556 arg2Of centrifuged,","
R4914 T5557 T5558 arg1Of centrifuged,at
R4915 T5560 T5558 arg2Of rpm,at
R4916 T5560 T5559 arg1Of rpm,"10,000"
R4917 T5562 T5561 arg1Of remove,to
R4918 T5564 T5563 arg1Of debris,cellular
R4919 T5564 T5566 arg1Of debris,and
R4920 T5566 T5562 arg2Of and,remove
R4921 T5566 T5565 arg1Of and,","
R4922 T5566 T5568 arg2Of and,collected
R4923 T5567 T5566 arg2Of supernatant,and
R4924 T5570 T5569 arg1Of concentration,Protein
R4925 T5570 T5571 arg1Of concentration,was
R4926 T5570 T5572 arg2Of concentration,determined
R4927 T5572 T5571 arg2Of determined,was
R4928 T5573 T5572 arg3Of using,determined
R4929 T5577 T5573 arg2Of Assay,using
R4930 T5577 T5574 arg1Of Assay,the
R4931 T5577 T5575 arg1Of Assay,DC
R4932 T5577 T5576 arg1Of Assay,Protein
R4933 T5577 T5578 arg1Of Assay,(
R4934 T5579 T5578 arg2Of Bio-Rad,(
R4935 T5580 T5578 arg3Of ),(
R4936 T5581 T5582 arg1Of Proteins,in
R4937 T5581 T5590 arg1Of Proteins,were
R4938 T5581 T5591 arg2Of Proteins,resolved
R4939 T5581 T5600 arg2Of Proteins,transferred
R4940 T5583 T5582 arg2Of samples,in
R4941 T5583 T5584 arg1Of samples,(
R4942 T5588 T5584 arg2Of proteins,(
R4943 T5588 T5585 arg1Of proteins,15
R4944 T5588 T5586 arg1Of proteins,µg
R4945 T5588 T5587 arg1Of proteins,total
R4946 T5589 T5584 arg3Of ),(
R4947 T5591 T5592 arg1Of resolved,in
R4948 T5591 T5599 arg1Of resolved,and
R4949 T5595 T5596 arg1Of 12,%
R4950 T5598 T5592 arg2Of gel,in
R4951 T5598 T5593 arg1Of gel,a
R4952 T5598 T5594 arg1Of gel,denaturing
R4953 T5598 T5595 arg1Of gel,12
R4954 T5598 T5597 arg1Of gel,polyacrylamide
R4955 T5599 T5590 arg2Of and,were
R4956 T5600 T5599 arg2Of transferred,and
R4957 T5600 T5601 arg1Of transferred,to
R4958 T5604 T5601 arg2Of membrane,to
R4959 T5604 T5602 arg1Of membrane,a
R4960 T5604 T5603 arg1Of membrane,nitrocellulose
R4961 T5606 T5605 arg1Of protein,I-κBα
R4962 T5606 T5607 arg1Of protein,was
R4963 T5606 T5608 arg2Of protein,detected
R4964 T5606 T5609 arg1Of protein,using
R4965 T5608 T5607 arg2Of detected,was
R4966 T5609 T5608 arg3Of using,detected
R4967 T5609 T5621 modOf using,followed
R4968 T5613 T5609 arg2Of antibody,using
R4969 T5613 T5610 arg1Of antibody,a
R4970 T5613 T5611 arg1Of antibody,rabbit
R4971 T5613 T5612 arg1Of antibody,polyclonal
R4972 T5613 T5614 arg1Of antibody,(
R4973 T5617 T5614 arg2Of Biotechnology,(
R4974 T5617 T5615 arg1Of Biotechnology,Santa
R4975 T5617 T5616 arg1Of Biotechnology,Cruz
R4976 T5617 T5618 arg1Of Biotechnology,","
R4977 T5619 T5618 arg2Of CA,","
R4978 T5620 T5614 arg3Of ),(
R4979 T5628 T5621 arg1Of antibody,followed
R4980 T5628 T5622 arg2Of antibody,by
R4981 T5628 T5623 arg1Of antibody,a
R4982 T5628 T5624 arg1Of antibody,horseradish
R4983 T5628 T5625 arg1Of antibody,peroxidase-coupled
R4984 T5628 T5626 arg1Of antibody,goat
R4985 T5628 T5627 arg1Of antibody,polyclonal
R4986 T5628 T5629 arg1Of antibody,against
R4987 T5631 T5629 arg2Of Ig,against
R4988 T5631 T5630 arg1Of Ig,rabbit
R4989 T5631 T5632 arg1Of Ig,(
R4990 T5634 T5632 arg2Of Laboratories,(
R4991 T5634 T5633 arg1Of Laboratories,Caltag
R4992 T5635 T5632 arg3Of ),(
R4993 T5639 T5638 arg1Of bands,IκB
R4994 T5639 T5640 arg1Of bands,were
R4995 T5639 T5641 arg2Of bands,revealed
R4996 T5641 T5636 arg1Of revealed,Finally
R4997 T5641 T5637 arg1Of revealed,","
R4998 T5641 T5640 arg2Of revealed,were
R4999 T5641 T5642 modOf revealed,using
R5000 T5641 T5647 arg1Of revealed,(
R5001 T5641 T5657 arg1Of revealed,according
R5002 T5646 T5642 arg2Of system,using
R5003 T5646 T5643 arg1Of system,the
R5004 T5646 T5644 arg1Of system,ECL™
R5005 T5646 T5645 arg1Of system,detection
R5006 T5650 T5647 arg2Of Biotech,(
R5007 T5650 T5648 arg1Of Biotech,Amersham
R5008 T5650 T5649 arg1Of Biotech,Pharmacia
R5009 T5650 T5651 arg1Of Biotech,","
R5010 T5653 T5651 arg2Of Ullis,","
R5011 T5653 T5652 arg1Of Ullis,Les
R5012 T5653 T5654 arg1Of Ullis,","
R5013 T5655 T5654 arg2Of France,","
R5014 T5656 T5647 arg3Of ),(
R5015 T5658 T5657 arg2Of to,according
R5016 T5660 T5659 arg1Of manufacturers,the
R5017 T5660 T5661 arg2Of manufacturers,'
R5018 T5662 T5658 arg2Of instruction,to
R5019 T5662 T5661 arg1Of instruction,'
R5020 T5663 T5664 arg1Of Antibody,to
R5021 T5663 T5670 arg1Of Antibody,was
R5022 T5665 T5664 arg2Of α-Tubulin,to
R5023 T5665 T5666 arg1Of α-Tubulin,(
R5024 T5668 T5666 arg2Of Cruz,(
R5025 T5668 T5667 arg1Of Cruz,Santa
R5026 T5669 T5666 arg3Of ),(
R5027 T5670 T5672 arg1Of was,as
R5028 T5671 T5670 arg2Of use,was
R5029 T5673 T5672 arg2Of loading,as
R5030 T5674 T5673 arg2Of control,loading
R5031 T5677 T5675 arg2Of NF-κB,For
R5032 T5677 T5676 arg1Of NF-κB,nuclear
R5033 T5680 T5679 arg1Of cells,THP-1
R5034 T5680 T5681 arg1Of cells,were
R5035 T5680 T5682 arg2Of cells,stimulated
R5036 T5682 T5681 arg2Of stimulated,were
R5037 T5682 T5683 arg1Of stimulated,with
R5038 T5686 T5687 arg1Of C10,or
R5039 T5687 T5683 arg2Of or,with
R5040 T5687 T5684 arg1Of or,1
R5041 T5687 T5685 arg1Of or,mg/ml
R5042 T5687 T5689 arg1Of or,for
R5043 T5688 T5687 arg2Of C40,or
R5044 T5691 T5690 arg1Of minutes,30
R5045 T5691 T5692 arg1Of minutes,at
R5046 T5691 T5695 arg1Of minutes,were
R5047 T5691 T5697 arg2Of minutes,pelleted
R5048 T5694 T5692 arg2Of Cells,at
R5049 T5694 T5693 arg1Of Cells,37°C.
R5050 T5697 T5675 arg1Of pelleted,For
R5051 T5697 T5678 arg1Of pelleted,","
R5052 T5697 T5681 modOf pelleted,were
R5053 T5697 T5695 arg2Of pelleted,were
R5054 T5697 T5696 arg1Of pelleted,then
R5055 T5697 T5698 arg1Of pelleted,and
R5056 T5699 T5697 arg3Of nuclei,pelleted
R5057 T5699 T5700 arg2Of nuclei,separated
R5058 T5700 T5701 arg1Of separated,as
R5059 T5702 T5701 arg2Of described,as
R5060 T5702 T5703 arg1Of described,[
R5061 T5704 T5703 arg2Of 31,[
R5062 T5705 T5703 arg3Of ],[
R5063 T5706 T5707 arg1Of Nuclei,were
R5064 T5706 T5708 arg2Of Nuclei,washed
R5065 T5706 T5710 arg2Of Nuclei,homogenized
R5066 T5706 T5719 arg2Of Nuclei,heated
R5067 T5708 T5709 arg1Of washed,and
R5068 T5709 T5707 arg2Of and,were
R5069 T5709 T5726 arg1Of and,in
R5070 T5710 T5711 arg1Of homogenized,directly
R5071 T5710 T5712 arg1Of homogenized,in
R5072 T5710 T5718 arg1Of homogenized,and
R5073 T5715 T5714 arg2Of Laemli,(
R5074 T5716 T5714 arg3Of ),(
R5075 T5717 T5712 arg2Of buffer,in
R5076 T5717 T5713 arg1Of buffer,loading
R5077 T5717 T5714 arg1Of buffer,(
R5078 T5718 T5709 arg2Of and,and
R5079 T5719 T5718 arg2Of heated,and
R5080 T5719 T5720 arg1Of heated,for
R5081 T5722 T5720 arg2Of minutes,for
R5082 T5722 T5721 arg1Of minutes,5
R5083 T5722 T5723 arg1Of minutes,at
R5084 T5725 T5723 arg2Of Proteins,at
R5085 T5725 T5724 arg1Of Proteins,100°C.
R5086 T5727 T5728 arg1Of samples,were
R5087 T5727 T5729 arg2Of samples,resolved
R5088 T5727 T5738 arg2Of samples,transferred
R5089 T5729 T5730 arg1Of resolved,in
R5090 T5729 T5737 arg1Of resolved,and
R5091 T5733 T5734 arg1Of 8,%
R5092 T5736 T5730 arg2Of gel,in
R5093 T5736 T5731 arg1Of gel,a
R5094 T5736 T5732 arg1Of gel,denaturing
R5095 T5736 T5733 arg1Of gel,8
R5096 T5736 T5735 arg1Of gel,polyacrylamide
R5097 T5737 T5726 arg2Of and,in
R5098 T5737 T5728 arg2Of and,were
R5099 T5738 T5737 arg2Of transferred,and
R5100 T5738 T5739 arg1Of transferred,to
R5101 T5744 T5743 arg2Of PVDF,(
R5102 T5745 T5743 arg3Of ),(
R5103 T5746 T5739 arg2Of membrane,to
R5104 T5746 T5740 arg1Of membrane,a
R5105 T5746 T5741 arg1Of membrane,polyvinylidine
R5106 T5746 T5742 arg1Of membrane,fluoride
R5107 T5746 T5743 arg1Of membrane,(
R5108 T5746 T5747 arg1Of membrane,(
R5109 T5748 T5747 arg2Of Immobilon-P,(
R5110 T5748 T5749 arg1Of Immobilon-P,;
R5111 T5748 T5753 arg1Of Immobilon-P,","
R5112 T5748 T5754 arg1Of Immobilon-P,MA
R5113 T5750 T5751 arg1Of Millipore,","
R5114 T5751 T5749 arg2Of ",",;
R5115 T5752 T5751 arg2Of Bedford,","
R5116 T5755 T5747 arg3Of ),(
R5117 T5756 T5757 arg1Of Membranes,were
R5118 T5756 T5758 arg2Of Membranes,incubated
R5119 T5758 T5757 arg2Of incubated,were
R5120 T5758 T5759 arg1Of incubated,in
R5121 T5760 T5759 arg2Of blocking,in
R5122 T5761 T5760 arg2Of buffer,blocking
R5123 T5761 T5762 arg1Of buffer,(
R5124 T5761 T5770 arg1Of buffer,for
R5125 T5763 T5764 arg1Of 1,%
R5126 T5765 T5762 arg2Of BSA,(
R5127 T5765 T5763 arg1Of BSA,1
R5128 T5765 T5766 arg1Of BSA,","
R5129 T5765 T5767 arg1Of BSA,in
R5130 T5768 T5767 arg2Of PBS,in
R5131 T5769 T5762 arg3Of ),(
R5132 T5772 T5770 arg2Of hours,for
R5133 T5772 T5771 arg1Of hours,two
R5134 T5772 T5773 arg1Of hours,at
R5135 T5775 T5773 arg2Of temperature,at
R5136 T5775 T5774 arg1Of temperature,room
R5137 T5776 T5777 arg1Of Membranes,were
R5138 T5776 T5779 arg2Of Membranes,probed
R5139 T5779 T5777 arg2Of probed,were
R5140 T5779 T5778 arg1Of probed,subsequently
R5141 T5779 T5780 arg1Of probed,with
R5142 T5779 T5787 arg1Of probed,","
R5143 T5779 T5788 arg1Of probed,overnight
R5144 T5783 T5780 arg2Of antibody,with
R5145 T5783 T5781 arg1Of antibody,the
R5146 T5783 T5782 arg1Of antibody,corresponding
R5147 T5783 T5784 arg1Of antibody,in
R5148 T5785 T5784 arg2Of blocking,in
R5149 T5786 T5785 arg2Of buffer,blocking
R5150 T5794 T5789 arg1Of subunit,Rabbit
R5151 T5794 T5790 arg1Of subunit,polyclonal
R5152 T5794 T5791 arg1Of subunit,antibody
R5153 T5794 T5792 arg1Of subunit,anti-NF-κB
R5154 T5794 T5793 arg1Of subunit,p50
R5155 T5794 T5795 arg1Of subunit,(
R5156 T5794 T5799 arg1Of subunit,or
R5157 T5797 T5795 arg2Of sc-114,(
R5158 T5797 T5796 arg1Of sc-114,#
R5159 T5798 T5795 arg3Of ),(
R5160 T5799 T5807 arg1Of or,from
R5161 T5799 T5811 arg1Of or,were
R5162 T5799 T5812 arg2Of or,used
R5163 T5802 T5799 arg2Of subunit,or
R5164 T5802 T5800 arg1Of subunit,anti-NF-κB
R5165 T5802 T5801 arg1Of subunit,p65
R5166 T5802 T5803 arg1Of subunit,(
R5167 T5805 T5803 arg2Of sc-109,(
R5168 T5805 T5804 arg1Of sc-109,#
R5169 T5806 T5803 arg3Of ),(
R5170 T5810 T5807 arg2Of Biotechnology,from
R5171 T5810 T5808 arg1Of Biotechnology,Santa
R5172 T5810 T5809 arg1Of Biotechnology,Cruz
R5173 T5812 T5811 arg2Of used,were
R5174 T5813 T5814 arg1Of Membranes,were
R5175 T5813 T5815 arg2Of Membranes,washed
R5176 T5813 T5832 arg2Of Membranes,incubated
R5177 T5815 T5817 arg1Of washed,times
R5178 T5815 T5818 arg1Of washed,in
R5179 T5815 T5820 arg1Of washed,with
R5180 T5815 T5824 arg1Of washed,20
R5181 T5815 T5827 arg1Of washed,minutes
R5182 T5815 T5829 arg1Of washed,time
R5183 T5815 T5831 arg1Of washed,and
R5184 T5817 T5816 arg1Of times,six
R5185 T5819 T5818 arg2Of PBS,in
R5186 T5822 T5820 arg2Of %,with
R5187 T5822 T5821 arg1Of %,0.05
R5188 T5824 T5823 arg1Of 20,Tween
R5189 T5824 T5825 arg1Of 20,","
R5190 T5827 T5825 arg2Of minutes,","
R5191 T5827 T5826 arg1Of minutes,5
R5192 T5829 T5828 arg1Of time,each
R5193 T5831 T5814 arg2Of and,were
R5194 T5831 T5830 arg1Of and,","
R5195 T5832 T5831 arg2Of incubated,and
R5196 T5832 T5833 arg1Of incubated,with
R5197 T5832 T5861 arg1Of incubated,for
R5198 T5833 T5859 arg1Of with,in
R5199 T5836 T5834 arg1Of dilution,a
R5200 T5836 T5835 arg1Of dilution,1/3000
R5201 T5836 T5837 arg1Of dilution,of
R5202 T5839 T5837 arg2Of F,of
R5203 T5839 T5838 arg1Of F,HRP-conjugated
R5204 T5841 T5840 arg2Of ab,(
R5205 T5841 T5842 arg1Of ab,'
R5206 T5843 T5840 arg3Of ),(
R5207 T5847 T5833 arg2Of IgG,with
R5208 T5847 T5836 arg1Of IgG,dilution
R5209 T5847 T5840 arg1Of IgG,(
R5210 T5847 T5844 arg1Of IgG,2
R5211 T5847 T5845 arg1Of IgG,goat
R5212 T5847 T5846 arg1Of IgG,anti-rabbit
R5213 T5847 T5848 arg1Of IgG,in
R5214 T5849 T5850 arg1Of 5,%
R5215 T5853 T5849 arg1Of milk,5
R5216 T5853 T5851 arg1Of milk,nonfat
R5217 T5853 T5852 arg1Of milk,dry
R5218 T5853 T5854 arg1Of milk,and
R5219 T5854 T5848 arg2Of and,in
R5220 T5856 T5855 arg1Of %,0.05
R5221 T5858 T5854 arg2Of 20,and
R5222 T5858 T5856 arg1Of 20,%
R5223 T5858 T5857 arg1Of 20,Tween
R5224 T5860 T5859 arg2Of PBS,in
R5225 T5863 T5861 arg2Of hour,for
R5226 T5863 T5862 arg1Of hour,1
R5227 T5863 T5864 arg1Of hour,at
R5228 T5866 T5864 arg2Of temperature,at
R5229 T5866 T5865 arg1Of temperature,room
R5230 T5868 T5867 arg2Of washing,After
R5231 T5868 T5872 arg1Of washing,in
R5232 T5868 T5874 arg1Of washing,with
R5233 T5870 T5869 arg1Of more,six
R5234 T5871 T5868 arg2Of times,washing
R5235 T5871 T5870 arg1Of times,more
R5236 T5873 T5872 arg2Of PBS,in
R5237 T5876 T5875 arg1Of %,0.05
R5238 T5878 T5874 arg2Of 20,with
R5239 T5878 T5876 arg1Of 20,%
R5240 T5878 T5877 arg1Of 20,Tween
R5241 T5881 T5868 arg1Of proteins,washing
R5242 T5881 T5880 arg1Of proteins,antibody-reactive
R5243 T5881 T5882 arg1Of proteins,were
R5244 T5881 T5883 arg2Of proteins,detected
R5245 T5883 T5867 arg1Of detected,After
R5246 T5883 T5879 arg1Of detected,","
R5247 T5883 T5882 arg2Of detected,were
R5248 T5884 T5883 arg3Of using,detected
R5249 T5884 T5888 arg1Of using,(
R5250 T5884 T5897 arg1Of using,according
R5251 T5887 T5884 arg2Of substrate,using
R5252 T5887 T5885 arg1Of substrate,a
R5253 T5887 T5886 arg1Of substrate,chemiluminescence
R5254 T5889 T5890 arg1Of SuperSignal,;
R5255 T5890 T5888 arg2Of ;,(
R5256 T5890 T5894 arg1Of ;,","
R5257 T5891 T5892 arg1Of Pierce,","
R5258 T5892 T5890 arg2Of ",",;
R5259 T5893 T5892 arg2Of Rockford,","
R5260 T5895 T5894 arg2Of IL,","
R5261 T5896 T5888 arg3Of ),(
R5262 T5898 T5897 arg2Of to,according
R5263 T5900 T5899 arg1Of manufacturer,the
R5264 T5900 T5901 arg2Of manufacturer,'s
R5265 T5902 T5898 arg2Of instructions,to
R5266 T5902 T5901 arg1Of instructions,'s
R5267 T5904 T5903 arg1Of confirm,To
R5268 T5907 T5906 arg1Of amounts,equivalent
R5269 T5907 T5908 arg1Of amounts,of
R5270 T5907 T5910 arg1Of amounts,were
R5271 T5907 T5911 arg2Of amounts,loaded
R5272 T5909 T5908 arg2Of protein,of
R5273 T5911 T5904 arg2Of loaded,confirm
R5274 T5911 T5905 arg1Of loaded,that
R5275 T5911 T5910 arg2Of loaded,were
R5276 T5911 T5912 arg1Of loaded,in
R5277 T5914 T5912 arg2Of line,in
R5278 T5914 T5913 arg1Of line,each
R5279 T5916 T5904 arg1Of membranes,confirm
R5280 T5916 T5917 arg1Of membranes,were
R5281 T5916 T5920 arg1Of membranes,blotted
R5282 T5917 T5903 modOf were,To
R5283 T5917 T5915 arg1Of were,","
R5284 T5917 T5918 arg1Of were,also
R5285 T5917 T5923 arg1Of were,as
R5286 T5920 T5917 arg2Of blotted,were
R5287 T5920 T5919 arg1Of blotted,Western
R5288 T5920 T5921 arg1Of blotted,for
R5289 T5922 T5921 arg2Of ERK,for
R5290 T5924 T5923 arg2Of described,as
R5291 T5924 T5925 arg1Of described,[
R5292 T5926 T5925 arg2Of 32,[
R5293 T5927 T5925 arg3Of ],[
R5753 T6465 T6466 arg1Of Analysis,of
R5754 T6465 T6469 arg1Of Analysis,by
R5755 T6468 T6466 arg2Of Activation,of
R5756 T6468 T6467 arg1Of Activation,NF-κB
R5757 T6471 T6469 arg2Of Cytometry,by
R5758 T6471 T6470 arg1Of Cytometry,Flow
R5759 T6473 T6472 arg1Of activation,Nuclear
R5760 T6473 T6474 arg1Of activation,of
R5761 T6473 T6476 arg1Of activation,by
R5762 T6473 T6479 arg1Of activation,was
R5763 T6473 T6480 arg2Of activation,performed
R5764 T6475 T6474 arg2Of NF−κΒ,of
R5765 T6478 T6476 arg2Of cytometry,by
R5766 T6478 T6477 arg1Of cytometry,flow
R5767 T6480 T6479 arg2Of performed,was
R5768 T6480 T6481 arg1Of performed,as
R5769 T6482 T6481 arg2Of described,as
R5770 T6482 T6483 arg1Of described,[
R5771 T6484 T6483 arg2Of 31,[
R5772 T6485 T6483 arg3Of ],[
R5800 T6521 T6520 arg1Of Analysis,Statistical
R5801 T6523 T6522 arg1Of results,The
R5802 T6523 T6524 arg1Of results,were
R5803 T6523 T6525 arg2Of results,expressed
R5804 T6525 T6524 arg2Of expressed,were
R5805 T6525 T6526 arg1Of expressed,as
R5806 T6529 T6527 arg1Of value,the
R5807 T6529 T6528 arg1Of value,mean
R5808 T6529 T6530 arg1Of value,±
R5809 T6530 T6526 arg2Of ±,as
R5810 T6531 T6530 arg2Of S.E.M.,±
R5811 T6531 T6532 arg1Of S.E.M.,of
R5812 T6534 T6532 arg2Of experiments,of
R5813 T6534 T6533 arg1Of experiments,individual
R5814 T6537 T6535 arg1Of significance,The
R5815 T6537 T6536 arg1Of significance,statistical
R5816 T6537 T6538 arg1Of significance,of
R5817 T6537 T6544 arg1Of significance,was
R5818 T6537 T6545 arg2Of significance,assessed
R5819 T6540 T6538 arg2Of differences,of
R5820 T6540 T6539 arg1Of differences,the
R5821 T6540 T6541 arg1Of differences,between
R5822 T6543 T6541 arg2Of values,between
R5823 T6543 T6542 arg1Of values,mean
R5824 T6545 T6544 arg2Of assessed,was
R5825 T6550 T6551 arg1Of t-test,and
R5826 T6551 T6545 arg1Of and,assessed
R5827 T6551 T6546 arg2Of and,by
R5828 T6551 T6547 arg1Of and,the
R5829 T6551 T6548 arg1Of and,Student
R5830 T6551 T6549 arg1Of and,'s
R5831 T6551 T6553 arg1Of and,of
R5832 T6552 T6551 arg2Of analysis,and
R5833 T6554 T6553 arg2Of variance,of
R5834 T6554 T6555 arg1Of variance,(
R5835 T6556 T6555 arg2Of ANOVA,(
R5836 T6557 T6555 arg3Of ),(
R5877 T6627 T6623 arg2Of Inflammation,Degraded
R5878 T6627 T6624 arg1Of Inflammation,CGN
R5879 T6627 T6625 arg1Of Inflammation,Induce
R5880 T6627 T6626 arg1Of Inflammation,Colonic
R5881 T6629 T6628 arg1Of rats,All
R5882 T6629 T6630 arg1Of rats,developed
R5883 T6630 T6632 arg1Of developed,during
R5884 T6630 T6636 arg1Of developed,and
R5885 T6631 T6630 arg2Of diarrhea,developed
R5886 T6635 T6632 arg2Of administration,during
R5887 T6635 T6633 arg2Of administration,degraded
R5888 T6635 T6634 arg1Of administration,carrageenan
R5889 T6638 T6637 arg1Of evidence,gross
R5890 T6638 T6639 arg1Of evidence,of
R5891 T6638 T6641 arg1Of evidence,was
R5892 T6638 T6643 arg2Of evidence,detected
R5893 T6640 T6639 arg2Of blood,of
R5894 T6643 T6636 arg2Of detected,and
R5895 T6643 T6641 arg2Of detected,was
R5896 T6643 T6642 arg1Of detected,frequently
R5897 T6643 T6644 arg1Of detected,in
R5898 T6646 T6644 arg2Of stools,in
R5899 T6646 T6645 arg1Of stools,the
R5900 T6648 T6647 arg1Of length,Colon
R5901 T6648 T6650 arg2Of length,decreased
R5902 T6650 T6649 arg1Of decreased,dramatically
R5903 T6650 T6651 arg1Of decreased,in
R5904 T6650 T6655 arg1Of decreased,with
R5905 T6654 T6651 arg2Of rats,in
R5906 T6654 T6652 arg1Of rats,all
R5907 T6654 T6653 arg2Of rats,treated
R5908 T6658 T6657 arg1Of pronounced,more
R5909 T6659 T6655 arg2Of effect,with
R5910 T6659 T6656 arg1Of effect,a
R5911 T6659 T6658 arg1Of effect,pronounced
R5912 T6659 T6660 arg1Of effect,being
R5913 T6659 T6661 arg2Of effect,observed
R5914 T6661 T6660 arg2Of observed,being
R5915 T6661 T6662 arg1Of observed,in
R5916 T6667 T6666 arg1Of treated,dCGN
R5917 T6668 T6662 arg2Of group,in
R5918 T6668 T6663 arg1Of group,the
R5919 T6668 T6664 arg1Of group,40
R5920 T6668 T6665 arg1Of group,kDa
R5921 T6668 T6667 arg1Of group,treated
R5922 T6668 T6669 arg1Of group,(
R5923 T6671 T6669 arg2Of 1A,(
R5924 T6671 T6670 arg1Of 1A,Fig.
R5925 T6672 T6669 arg3Of ),(
R5926 T6676 T6675 arg2Of exposure,prolonged
R5927 T6676 T6677 arg1Of exposure,to
R5928 T6676 T6681 arg1Of exposure,resulted
R5929 T6680 T6677 arg2Of dCGN,to
R5930 T6680 T6678 arg1Of dCGN,40
R5931 T6680 T6679 arg1Of dCGN,kDa
R5932 T6681 T6673 arg1Of resulted,Furthermore
R5933 T6681 T6674 arg1Of resulted,","
R5934 T6681 T6682 arg1Of resulted,in
R5935 T6681 T6690 arg1Of resulted,(
R5936 T6684 T6685 arg1Of macroscopic,and
R5937 T6686 T6685 arg2Of histological,and
R5938 T6687 T6682 arg2Of scores,in
R5939 T6687 T6683 arg1Of scores,high
R5940 T6687 T6684 arg1Of scores,macroscopic
R5941 T6687 T6686 arg1Of scores,histological
R5942 T6687 T6688 arg1Of scores,of
R5943 T6689 T6688 arg2Of inflammation,of
R5944 T6692 T6690 arg2Of 1B,(
R5945 T6692 T6691 arg1Of 1B,Fig.
R5946 T6692 T6693 arg1Of 1B,","
R5947 T6694 T6693 arg2Of C,","
R5948 T6695 T6690 arg3Of ),(
R5949 T6699 T6696 arg1Of activity,Only
R5950 T6699 T6697 arg1Of activity,weak
R5951 T6699 T6698 arg1Of activity,myeloperoxidase
R5952 T6699 T6700 arg1Of activity,was
R5953 T6699 T6701 arg2Of activity,detected
R5954 T6701 T6700 arg2Of detected,was
R5955 T6701 T6702 arg1Of detected,in
R5956 T6701 T6712 arg1Of detected,","
R5957 T6701 T6713 modOf detected,indicating
R5958 T6704 T6705 arg1Of control,and
R5959 T6706 T6705 arg2Of dCGN-treated,and
R5960 T6707 T6702 arg2Of groups,in
R5961 T6707 T6703 arg1Of groups,both
R5962 T6707 T6704 arg1Of groups,control
R5963 T6707 T6706 arg1Of groups,dCGN-treated
R5964 T6707 T6708 arg1Of groups,(
R5965 T6710 T6708 arg2Of 1D,(
R5966 T6710 T6709 arg1Of 1D,Fig.
R5967 T6711 T6708 arg3Of ),(
R5968 T6715 T6716 arg1Of granulocytes,did
R5969 T6715 T6718 arg1Of granulocytes,play
R5970 T6718 T6713 arg2Of play,indicating
R5971 T6718 T6714 arg1Of play,that
R5972 T6718 T6716 arg2Of play,did
R5973 T6718 T6717 arg1Of play,not
R5974 T6718 T6722 arg1Of play,in
R5975 T6718 T6725 arg1Of play,at
R5976 T6721 T6718 arg2Of role,play
R5977 T6721 T6719 arg1Of role,a
R5978 T6721 T6720 arg1Of role,major
R5979 T6724 T6722 arg2Of inflammation,in
R5980 T6724 T6723 arg1Of inflammation,the
R5981 T6727 T6725 arg2Of stage,at
R5982 T6727 T6726 arg1Of stage,that
R5983 T6729 T6728 arg1Of examination,Histological
R5984 T6729 T6730 arg1Of examination,revealed
R5985 T6732 T6730 arg2Of degrees,revealed
R5986 T6732 T6731 arg1Of degrees,various
R5987 T6732 T6733 arg1Of degrees,of
R5988 T6735 T6733 arg2Of inflammation,of
R5989 T6735 T6734 arg1Of inflammation,mucosal
R5990 T6736 T6737 arg2Of Rats,treated
R5991 T6736 T6742 arg1Of Rats,showed
R5992 T6737 T6738 arg1Of treated,with
R5993 T6741 T6738 arg2Of dCGN,with
R5994 T6741 T6739 arg1Of dCGN,10
R5995 T6741 T6740 arg1Of dCGN,kDa
R5996 T6743 T6744 arg1Of edema,","
R5997 T6744 T6747 arg1Of ",",and
R5998 T6746 T6744 arg2Of atrophy,","
R5999 T6746 T6745 arg1Of atrophy,epithelium
R6000 T6747 T6742 arg2Of and,showed
R6001 T6750 T6747 arg2Of infiltration,and
R6002 T6750 T6748 arg1Of infiltration,slight
R6003 T6750 T6749 arg1Of infiltration,lymphocyte
R6004 T6750 T6751 arg1Of infiltration,(
R6005 T6752 T6751 arg2Of data,(
R6006 T6752 T6754 arg2Of data,shown
R6007 T6754 T6753 arg1Of shown,not
R6008 T6755 T6751 arg3Of ),(
R6009 T6757 T6756 arg1Of symptoms,These
R6010 T6757 T6758 arg1Of symptoms,were
R6011 T6757 T6760 arg1Of symptoms,absent
R6012 T6760 T6758 arg2Of absent,were
R6013 T6760 T6759 arg1Of absent,totally
R6014 T6760 T6761 arg1Of absent,in
R6015 T6763 T6761 arg2Of colon,in
R6016 T6763 T6762 arg1Of colon,the
R6017 T6763 T6764 arg1Of colon,of
R6018 T6766 T6764 arg2Of rats,of
R6019 T6766 T6765 arg1Of rats,control
R6020 T6766 T6767 arg1Of rats,(
R6021 T6769 T6767 arg2Of 1E,(
R6022 T6769 T6768 arg1Of 1E,Fig.
R6023 T6770 T6767 arg3Of ),(
R6024 T6772 T6771 arg1Of severe,More
R6025 T6774 T6772 arg1Of injuries,severe
R6026 T6774 T6773 arg1Of injuries,mucosal
R6027 T6774 T6775 arg1Of injuries,including
R6028 T6774 T6789 arg1Of injuries,were
R6029 T6774 T6790 arg2Of injuries,observed
R6030 T6776 T6777 arg1Of ulceration,","
R6031 T6777 T6780 arg1Of ",",","
R6032 T6779 T6777 arg2Of epithelium,","
R6033 T6779 T6778 arg1Of epithelium,hyperplastic
R6034 T6780 T6783 arg1Of ",",and
R6035 T6782 T6780 arg2Of distortion,","
R6036 T6782 T6781 arg1Of distortion,crypt
R6037 T6783 T6775 arg2Of and,including
R6038 T6787 T6783 arg2Of infiltration,and
R6039 T6787 T6784 arg1Of infiltration,a
R6040 T6787 T6785 arg1Of infiltration,strong
R6041 T6787 T6786 arg1Of infiltration,macrophage
R6042 T6790 T6788 arg1Of observed,","
R6043 T6790 T6789 arg2Of observed,were
R6044 T6790 T6791 arg1Of observed,in
R6045 T6796 T6791 arg2Of rats,in
R6046 T6796 T6792 arg1Of rats,the
R6047 T6796 T6793 arg1Of rats,40
R6048 T6796 T6794 arg1Of rats,kDa
R6049 T6796 T6795 arg1Of rats,dCGN-treated
R6050 T6796 T6797 arg1Of rats,(
R6051 T6799 T6797 arg2Of 1F,(
R6052 T6799 T6798 arg1Of 1F,Fig.
R6053 T6800 T6797 arg3Of ),(
R6054 T6803 T6801 arg1Of polysaccharides,No
R6055 T6803 T6802 arg2Of polysaccharides,sulphated
R6056 T6803 T6804 arg1Of polysaccharides,were
R6057 T6803 T6805 arg2Of polysaccharides,detected
R6058 T6805 T6804 arg2Of detected,were
R6059 T6809 T6805 arg1Of staining,detected
R6060 T6809 T6806 arg2Of staining,by
R6061 T6809 T6807 arg1Of staining,toluidine
R6062 T6809 T6808 arg1Of staining,blue
R6063 T6809 T6810 arg1Of staining,of
R6064 T6812 T6810 arg2Of mucosa,of
R6065 T6812 T6811 arg1Of mucosa,colon
R6066 T6812 T6813 arg1Of mucosa,from
R6067 T6814 T6813 arg2Of rats,from
R6068 T6814 T6815 arg2Of rats,treated
R6069 T6815 T6816 arg1Of treated,with
R6070 T6819 T6820 arg1Of 10,or
R6071 T6821 T6820 arg2Of 40,or
R6072 T6823 T6816 arg2Of dCGN,with
R6073 T6823 T6817 arg1Of dCGN,either
R6074 T6823 T6818 arg1Of dCGN,the
R6075 T6823 T6819 arg1Of dCGN,10
R6076 T6823 T6821 arg1Of dCGN,40
R6077 T6823 T6822 arg1Of dCGN,kDa
R6078 T6823 T6824 arg1Of dCGN,(
R6079 T6826 T6824 arg2Of shown,(
R6080 T6826 T6825 arg1Of shown,not
R6081 T6827 T6824 arg3Of ),(
R6082 T6829 T6830 arg1Of we,can
R6083 T6829 T6832 arg1Of we,exclude
R6084 T6832 T6828 arg2Of exclude,Although
R6085 T6832 T6830 arg2Of exclude,can
R6086 T6832 T6831 arg1Of exclude,not
R6087 T6833 T6832 arg2Of that,exclude
R6088 T6835 T6834 arg1Of mat,dCGN
R6089 T6835 T6837 arg1Of mat,have
R6090 T6835 T6838 arg1Of mat,retained
R6091 T6838 T6833 arg1Of retained,that
R6092 T6838 T6836 arg1Of retained,not
R6093 T6838 T6837 arg2Of retained,have
R6094 T6838 T6839 arg1Of retained,in
R6095 T6838 T6842 arg1Of retained,during
R6096 T6841 T6839 arg2Of section,in
R6097 T6841 T6840 arg1Of section,the
R6098 T6845 T6842 arg2Of procedure,during
R6099 T6845 T6843 arg1Of procedure,the
R6100 T6845 T6844 arg1Of procedure,histology
R6101 T6847 T6848 arg1Of this,indicates
R6102 T6848 T6828 arg1Of indicates,Although
R6103 T6848 T6846 arg1Of indicates,","
R6104 T6851 T6850 arg1Of polymers,these
R6105 T6851 T6852 arg1Of polymers,may
R6106 T6851 T6854 arg1Of polymers,have
R6107 T6851 T6855 arg1Of polymers,been
R6108 T6851 T6856 arg2Of polymers,phagocytosed
R6109 T6856 T6848 arg2Of phagocytosed,indicates
R6110 T6856 T6849 arg1Of phagocytosed,that
R6111 T6856 T6852 arg2Of phagocytosed,may
R6112 T6856 T6853 arg1Of phagocytosed,not
R6113 T6856 T6854 arg2Of phagocytosed,have
R6114 T6856 T6855 arg2Of phagocytosed,been
R6441 T7289 T7286 arg2Of Production,Degraded
R6442 T7289 T7287 arg1Of Production,CGN
R6443 T7289 T7288 arg1Of Production,"inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced-TNF-α"
R6444 T7289 T7290 arg1Of Production,by
R6445 T7289 T7292 arg1Of Production,In
R6446 T7291 T7290 arg2Of Monocytes,by
R6447 T7293 T7292 arg2Of Vitro,In
R13350 T15572 T15571 arg2Of CGN,Degraded
R13351 T15572 T15573 arg1Of CGN,induced
R13352 T15575 T15573 arg2Of inflammation,induced
R13353 T15575 T15574 arg1Of inflammation,colon
R13354 T15575 T15576 arg1Of inflammation,in
R13355 T15577 T15576 arg2Of rats,in
R13356 T15578 T15579 arg1Of Histograms,showing
R13357 T15578 T15592 arg1Of Histograms,;
R13358 T15581 T15579 arg2Of effect,showing
R13359 T15581 T15580 arg1Of effect,the
R13360 T15581 T15582 arg1Of effect,of
R13361 T15581 T15585 arg1Of effect,on
R13362 T15584 T15582 arg2Of CGN,of
R13363 T15584 T15583 arg2Of CGN,degraded
R13364 T15585 T15586 arg1Of on,:
R13365 T15588 T15585 arg2Of length,on
R13366 T15588 T15587 arg1Of length,colon
R13367 T15588 T15589 arg1Of length,(
R13368 T15590 T15589 arg2Of A,(
R13369 T15591 T15589 arg3Of ),(
R13370 T15593 T15594 arg1Of macroscopic,(
R13371 T15593 T15597 arg1Of macroscopic,and
R13372 T15595 T15594 arg2Of B,(
R13373 T15596 T15594 arg3Of ),(
R13374 T15597 T15592 arg2Of and,;
R13375 T15600 T15599 arg2Of C,(
R13376 T15601 T15599 arg3Of ),(
R13377 T15603 T15598 arg1Of score,histological
R13378 T15603 T15599 arg1Of score,(
R13379 T15603 T15602 arg1Of score,inflammation
R13380 T15603 T15604 arg1Of score,of
R13381 T15603 T15606 arg1Of score,;
R13382 T15605 T15604 arg2Of colon,of
R13383 T15606 T15597 arg2Of ;,and
R13384 T15609 T15608 arg2Of MPO,(
R13385 T15610 T15608 arg3Of ),(
R13386 T15611 T15606 arg2Of activity,;
R13387 T15611 T15607 arg1Of activity,Myeloperoxidase
R13388 T15611 T15608 arg1Of activity,(
R13389 T15611 T15612 arg1Of activity,(
R13390 T15613 T15612 arg2Of D,(
R13391 T15614 T15612 arg3Of ),(
R13392 T15616 T15615 arg1Of rats,Control
R13393 T15616 T15617 arg1Of rats,(
R13394 T15616 T15621 arg1Of rats,;
R13395 T15619 T15617 arg2Of bars,(
R13396 T15619 T15618 arg1Of bars,white
R13397 T15620 T15617 arg3Of ),(
R13398 T15621 T15624 arg1Of ;,degraded
R13399 T15623 T15621 arg2Of kDa,;
R13400 T15623 T15622 arg1Of kDa,10
R13401 T15624 T15631 arg1Of degraded,;
R13402 T15626 T15624 arg2Of rats,degraded
R13403 T15626 T15625 arg1Of rats,CGN-treated
R13404 T15626 T15627 arg1Of rats,(
R13405 T15629 T15627 arg2Of bars,(
R13406 T15629 T15628 arg1Of bars,grey
R13407 T15630 T15627 arg3Of ),(
R13408 T15633 T15632 arg1Of kDa,40
R13409 T15633 T15634 arg1Of kDa,degraded
R13410 T15634 T15631 arg2Of degraded,;
R13411 T15636 T15634 arg2Of rats,degraded
R13412 T15636 T15635 arg1Of rats,CGN-treated
R13413 T15636 T15637 arg1Of rats,(
R13414 T15639 T15637 arg2Of bars,(
R13415 T15639 T15638 arg1Of bars,black
R13416 T15640 T15637 arg3Of ),(
R13417 T15642 T15634 arg3Of *,degraded
R13418 T15642 T15641 arg1Of *,.
R13419 T15642 T15643 arg1Of *,p
R13420 T15642 T15649 arg1Of *,*
R13421 T15644 T15645 arg1Of <0.0,5 fr
R13422 T15648 T15645 arg2Of .,5 fr
R13423 T15648 T15646 arg1Of .,om contr
R13424 T15648 T15647 arg1Of .,ol
R13425 T15649 T15644 arg1Of *,<0.0
R13426 T15649 T15650 arg1Of *,* p<
R13427 T15649 T15651 arg1Of *,0.01
R13428 T15652 T15651 arg2Of from co,0.01
R13429 T15654 T15653 arg1Of analysis,Histological
R13430 T15654 T15655 arg1Of analysis,of
R13431 T15654 T15657 arg1Of analysis,from
R13432 T15654 T15665 arg1Of analysis,from
R13433 T15656 T15655 arg2Of colon,of
R13434 T15657 T15664 arg1Of from,and
R13435 T15659 T15657 arg2Of rats,from
R13436 T15659 T15658 arg1Of rats,control
R13437 T15659 T15660 arg1Of rats,(
R13438 T15661 T15660 arg2Of E,(
R13439 T15662 T15660 arg3Of ),(
R13440 T15664 T15663 arg1Of and,","
R13441 T15665 T15664 arg2Of from,and
R13442 T15669 T15665 arg2Of rats,from
R13443 T15669 T15666 arg1Of rats,40
R13444 T15669 T15667 arg1Of rats,kDa
R13445 T15669 T15668 arg1Of rats,dCGN-treated
R13446 T15669 T15670 arg1Of rats,(
R13447 T15671 T15670 arg2Of F,(
R13448 T15672 T15670 arg3Of ),(
R1782 T2071 T2070 arg2Of carrageenan,degraded
R1783 T2071 T2072 arg1Of carrageenan,with
R1785 T2073 T2074 arg1Of low,","
R1793 T2082 T2072 arg2Of and,with

bionlp-st-ge-2016-spacy-parsed

Id Subject Object Predicate Lexical cue
T467 0-8 JJ denotes Degraded
T468 9-12 NNP denotes CGN
T469 13-22 VBD denotes inhibited
T470 23-28 CD denotes THP-1
T471 29-33 NN denotes cell
T472 34-47 NN denotes proliferation
T473 48-50 IN denotes in
T474 51-56 NN denotes vitro
T475 56-57 , denotes ,
T476 58-67 VBG denotes arresting
T477 68-71 DT denotes the
T478 72-77 NNS denotes cells
T479 78-80 IN denotes in
T480 81-83 CD denotes G1
T481 84-89 NN denotes phase
T482 89-90 . denotes .
T483 91-93 IN denotes In
T484 94-102 NN denotes addition
T485 102-103 , denotes ,
T486 104-108 NNP denotes dCGN
T487 109-118 VBD denotes increased
T488 119-125 NNP denotes ICAM-1
T489 126-136 NN denotes expression
T490 137-139 IN denotes in
T491 140-144 DT denotes both
T492 145-148 NNP denotes PBM
T493 149-152 CC denotes and
T494 153-158 NNP denotes THP-1
T495 159-164 NNS denotes cells
T496 165-169 IN denotes with
T497 170-171 DT denotes a
T498 172-177 JJ denotes major
T499 178-184 NN denotes effect
T500 185-189 VBN denotes seen
T501 190-195 IN denotes after
T502 196-198 CD denotes 40
T503 199-202 NNP denotes kDa
T504 203-207 NNP denotes dCGN
T505 208-216 NN denotes exposure
T506 216-217 . denotes .
T507 218-222 RB denotes Also
T508 222-223 , denotes ,
T509 224-228 NNP denotes dCGN
T510 229-239 VBD denotes stimulated
T511 240-248 NN denotes monocyte
T512 249-260 NN denotes aggregation
T513 261-263 IN denotes in
T514 264-269 NN denotes vitro
T515 270-274 WDT denotes that
T516 275-278 VBD denotes was
T517 279-288 VBN denotes prevented
T518 289-291 IN denotes by
T519 292-302 NN denotes incubation
T520 303-307 IN denotes with
T521 308-319 JJ denotes anti-ICAM-1
T522 320-328 NN denotes antibody
T523 328-329 . denotes .
T524 330-337 RB denotes Finally
T525 337-338 , denotes ,
T526 339-343 NNP denotes dCGN
T527 344-354 VBD denotes stimulated
T528 355-360 JJ denotes TNF-α
T529 361-371 NN denotes expression
T530 372-375 CC denotes and
T531 376-385 NN denotes secretion
T532 386-388 IN denotes by
T533 389-393 DT denotes both
T534 394-397 NNP denotes PBM
T535 398-401 CC denotes and
T536 402-407 NNP denotes THP-1
T537 408-413 NNS denotes cells
T538 413-414 . denotes .
T539 415-418 PDT denotes All
T540 419-424 DT denotes these
T541 425-432 NNS denotes effects
T542 433-437 VBD denotes were
T543 438-444 VBN denotes linked
T544 445-447 TO denotes to
T545 448-453 NNP denotes NF-κB
T546 454-464 NN denotes activation
T547 464-465 . denotes .
T548 466-471 DT denotes These
T549 472-476 NNS denotes data
T550 477-485 RB denotes strongly
T551 486-493 VBP denotes suggest
T552 494-498 IN denotes that
T553 499-502 DT denotes the
T554 503-511 JJ denotes degraded
T555 512-517 NNS denotes forms
T556 518-520 IN denotes of
T557 521-524 NNP denotes CGN
T558 525-529 VBP denotes have
T559 530-531 DT denotes a
T560 532-542 JJ denotes pronounced
T561 543-549 NN denotes effect
T562 550-552 IN denotes on
T563 553-562 NNS denotes monocytes
T564 562-563 , denotes ,
T565 564-578 NN denotes characteristic
T566 579-581 IN denotes of
T567 582-584 DT denotes an
T568 585-597 JJ denotes inflammatory
T569 598-607 NN denotes phenotype
T570 607-608 . denotes .
T1344 623-634 NNP denotes Carrageenan
T1345 635-636 -LRB- denotes (
T1346 636-639 NNP denotes CGN
T1347 639-640 -RRB- denotes )
T1348 641-643 VBZ denotes is
T1349 644-645 DT denotes a
T1350 646-650 JJ denotes high
T1351 651-660 JJ denotes molecular
T1352 661-667 NN denotes weight
T1353 668-677 VBN denotes sulphated
T1354 678-692 NN denotes polysaccharide
T1355 693-694 -LRB- denotes (
T1356 694-695 FW denotes >
T1357 695-698 CD denotes 200
T1358 699-702 NNP denotes kDa
T1359 702-703 -RRB- denotes )
T1360 704-711 VBN denotes derived
T1361 712-716 IN denotes from
T1362 717-720 JJ denotes red
T1363 721-726 NN denotes algae
T1364 727-728 -LRB- denotes (
T1365 728-740 NNP denotes Rhodophyceae
T1366 740-741 -RRB- denotes )
T1367 741-742 . denotes .
T1368 743-748 CD denotes Three
T1369 749-753 JJ denotes main
T1370 754-759 NNS denotes forms
T1371 760-762 IN denotes of
T1372 763-766 NNP denotes CGN
T1373 767-771 VBP denotes have
T1374 772-776 VBN denotes been
T1375 777-787 VBN denotes identified
T1376 787-788 : denotes :
T1377 789-794 NN denotes kappa
T1378 794-795 , denotes ,
T1379 796-800 NN denotes iota
T1380 800-801 , denotes ,
T1381 802-805 CC denotes and
T1382 806-812 NN denotes lambda
T1383 812-813 . denotes .
T1384 814-818 PRP denotes They
T1385 819-825 VBP denotes differ
T1386 826-830 IN denotes from
T1387 831-835 DT denotes each
T1388 836-841 JJ denotes other
T1389 842-844 IN denotes in
T1390 845-855 NN denotes sulphation
T1391 856-862 NN denotes degree
T1392 863-866 CC denotes and
T1393 867-877 NN denotes solubility
T1394 878-879 NN denotes [
T1395 879-880 CD denotes 1
T1396 880-881 NNP denotes ]
T1397 881-882 , denotes ,
T1398 883-884 NNP denotes [
T1399 884-885 CD denotes 2
T1400 885-886 NNP denotes ]
T1401 886-887 . denotes .
T1402 888-894 JJ denotes Native
T1403 895-898 NNP denotes CGN
T1404 899-901 VBZ denotes is
T1405 902-909 VBN denotes thought
T1406 910-912 TO denotes to
T1407 913-915 VB denotes be
T1408 916-924 JJ denotes harmless
T1409 925-928 CC denotes and
T1410 929-931 VBZ denotes is
T1411 932-938 RB denotes widely
T1412 939-943 VBN denotes used
T1413 944-946 IN denotes as
T1414 947-948 DT denotes a
T1415 949-953 NN denotes food
T1416 954-962 JJ denotes additive
T1417 963-965 TO denotes to
T1418 966-973 VB denotes improve
T1419 974-981 NN denotes texture
T1420 981-982 . denotes .
T1421 983-985 PRP denotes It
T1422 986-988 VBZ denotes is
T1423 989-993 RB denotes also
T1424 994-998 VBN denotes used
T1425 999-1001 IN denotes in
T1426 1002-1011 NNS denotes cosmetics
T1427 1012-1015 CC denotes and
T1428 1016-1031 NNS denotes pharmaceuticals
T1429 1031-1032 . denotes .
T1430 1033-1040 RB denotes However
T1431 1040-1041 , denotes ,
T1432 1042-1046 NN denotes acid
T1433 1047-1056 NN denotes treatment
T1434 1057-1059 IN denotes at
T1435 1060-1064 JJ denotes high
T1436 1065-1076 NN denotes temperature
T1437 1077-1078 -LRB- denotes (
T1438 1078-1080 CD denotes 80
T1439 1080-1081 CD denotes °
T1440 1081-1082 NNP denotes C
T1441 1082-1083 -RRB- denotes )
T1442 1084-1092 VBZ denotes triggers
T1443 1093-1096 NNP denotes CGN
T1444 1097-1107 NN denotes hydrolysis
T1445 1108-1110 TO denotes to
T1446 1111-1116 JJR denotes lower
T1447 1117-1126 JJ denotes molecular
T1448 1127-1133 NN denotes weight
T1449 1134-1135 -LRB- denotes (
T1450 1135-1136 FW denotes <
T1451 1136-1138 CD denotes 50
T1452 1139-1142 NNP denotes kDa
T1453 1142-1143 -RRB- denotes )
T1454 1144-1153 VBZ denotes compounds
T1455 1154-1159 VBN denotes known
T1456 1160-1162 IN denotes as
T1457 1163-1173 NN denotes poligeenan
T1458 1174-1176 CC denotes or
T1459 1177-1185 JJ denotes degraded
T1460 1186-1189 NNP denotes CGN
T1461 1190-1191 -LRB- denotes (
T1462 1191-1195 NNP denotes dCGN
T1463 1195-1196 -RRB- denotes )
T1464 1196-1197 . denotes .
T1465 1198-1203 DT denotes These
T1466 1204-1209 NNS denotes dCGNs
T1467 1210-1216 VBP denotes induce
T1468 1217-1229 NN denotes inflammation
T1469 1230-1233 CC denotes and
T1470 1234-1238 VBP denotes have
T1471 1239-1243 VBN denotes been
T1472 1244-1250 RB denotes widely
T1473 1251-1255 VBN denotes used
T1474 1256-1258 IN denotes as
T1475 1259-1265 NNS denotes models
T1476 1266-1268 IN denotes of
T1477 1269-1276 NN denotes colitis
T1478 1277-1279 IN denotes in
T1479 1280-1287 JJ denotes several
T1480 1288-1295 NNS denotes species
T1481 1295-1296 , denotes ,
T1482 1297-1306 VBG denotes including
T1483 1307-1311 NNS denotes rats
T1484 1312-1313 JJ denotes [
T1485 1313-1314 CD denotes 3
T1486 1314-1315 NNP denotes ]
T1487 1315-1316 , denotes ,
T1488 1317-1324 VBZ denotes rabbits
T1489 1325-1326 NNP denotes [
T1490 1326-1327 CD denotes 4
T1491 1327-1328 NNP denotes ]
T1492 1329-1332 CC denotes and
T1493 1333-1339 NN denotes guinea
T1494 1340-1344 NNS denotes pigs
T1495 1345-1346 VBP denotes [
T1496 1346-1347 CD denotes 5
T1497 1347-1348 NNP denotes ]
T1498 1348-1349 . denotes .
T1499 1350-1353 DT denotes The
T1500 1354-1358 NN denotes role
T1501 1359-1361 IN denotes of
T1502 1362-1366 NNP denotes dCGN
T1503 1367-1369 IN denotes as
T1504 1370-1371 DT denotes a
T1505 1372-1387 JJ denotes tumor-promoting
T1506 1388-1394 NN denotes factor
T1507 1395-1402 VBZ denotes remains
T1508 1403-1416 JJ denotes controversial
T1509 1417-1418 NN denotes [
T1510 1418-1419 CD denotes 4
T1511 1419-1420 NNP denotes ]
T1512 1420-1421 , denotes ,
T1513 1422-1423 NNP denotes [
T1514 1423-1424 CD denotes 6
T1515 1424-1425 NNP denotes ]
T1516 1426-1427 NN denotes [
T1517 1427-1428 CD denotes 8
T1518 1428-1429 NNP denotes ]
T1519 1429-1430 . denotes .
T1520 1431-1439 IN denotes Although
T1521 1440-1443 DT denotes the
T1522 1444-1450 JJ denotes native
T1523 1451-1455 NN denotes form
T1524 1456-1458 VBZ denotes is
T1525 1459-1466 VBN denotes thought
T1526 1467-1469 TO denotes to
T1527 1470-1472 VB denotes be
T1528 1473-1481 JJ denotes harmless
T1529 1482-1485 IN denotes for
T1530 1486-1491 JJ denotes human
T1531 1492-1503 NN denotes consumption
T1532 1503-1504 , denotes ,
T1533 1505-1510 JJ denotes small
T1534 1511-1518 NNS denotes amounts
T1535 1519-1521 IN denotes of
T1536 1522-1526 NNP denotes dCGN
T1537 1527-1530 VBP denotes are
T1538 1531-1539 RB denotes probably
T1539 1540-1548 VBN denotes produced
T1540 1549-1551 IN denotes by
T1541 1552-1556 NN denotes acid
T1542 1557-1567 NN denotes hydrolysis
T1543 1568-1574 IN denotes during
T1544 1575-1582 JJ denotes gastric
T1545 1583-1592 NN denotes digestion
T1546 1593-1594 NN denotes [
T1547 1594-1595 CD denotes 9
T1548 1595-1596 NNP denotes ]
T1549 1596-1597 , denotes ,
T1550 1598-1599 NNP denotes [
T1551 1599-1601 CD denotes 10
T1552 1601-1602 NNP denotes ]
T1553 1603-1605 CC denotes or
T1554 1606-1617 NN denotes interaction
T1555 1618-1622 IN denotes with
T1556 1623-1633 JJ denotes intestinal
T1557 1634-1642 NNS denotes bacteria
T1558 1643-1644 NNP denotes [
T1559 1644-1646 CD denotes 11
T1560 1646-1647 NNP denotes ]
T1561 1647-1648 , denotes ,
T1562 1649-1650 NNP denotes [
T1563 1650-1652 CD denotes 12
T1564 1652-1653 NNP denotes ]
T1565 1653-1654 . denotes .
T1566 1655-1662 IN denotes Whereas
T1567 1663-1666 DT denotes the
T1568 1667-1674 NNS denotes effects
T1569 1675-1677 IN denotes of
T1570 1678-1684 JJ denotes native
T1571 1685-1688 CC denotes and
T1572 1689-1693 NNP denotes dCGN
T1573 1694-1696 IN denotes on
T1574 1697-1707 JJ denotes intestinal
T1575 1708-1720 NN denotes inflammation
T1576 1721-1725 VBP denotes have
T1577 1726-1730 VBN denotes been
T1578 1731-1742 RB denotes extensively
T1579 1743-1751 VBN denotes analyzed
T1580 1752-1754 IN denotes in
T1581 1755-1761 NN denotes animal
T1582 1762-1768 NNS denotes models
T1583 1768-1769 , denotes ,
T1584 1770-1774 RB denotes only
T1585 1775-1778 JJ denotes few
T1586 1779-1786 NNS denotes studies
T1587 1787-1791 VBP denotes have
T1588 1792-1796 VBN denotes been
T1589 1797-1806 VBN denotes conducted
T1590 1807-1812 VBG denotes using
T1591 1813-1818 JJ denotes human
T1592 1819-1823 NN denotes cell
T1593 1824-1829 NNS denotes lines
T1594 1829-1830 . denotes .
T1595 1831-1837 JJ denotes Recent
T1596 1838-1845 NNS denotes studies
T1597 1846-1850 VBP denotes have
T1598 1851-1856 VBN denotes shown
T1599 1857-1858 DT denotes a
T1600 1859-1863 NN denotes link
T1601 1864-1871 IN denotes between
T1602 1872-1880 NN denotes exposure
T1603 1881-1883 TO denotes to
T1604 1884-1890 JJ denotes native
T1605 1891-1895 NN denotes form
T1606 1896-1899 NNP denotes CGN
T1607 1900-1903 CC denotes and
T1608 1904-1908 JJ denotes IL-8
T1609 1909-1919 NN denotes production
T1610 1920-1922 IN denotes by
T1611 1923-1926 DT denotes the
T1612 1927-1932 JJ denotes human
T1613 1933-1943 JJ denotes intestinal
T1614 1944-1954 JJ denotes epithelial
T1615 1955-1959 NN denotes cell
T1616 1960-1964 NN denotes line
T1617 1964-1965 , denotes ,
T1618 1966-1972 NNP denotes NCM460
T1619 1972-1973 , denotes ,
T1620 1974-1977 IN denotes via
T1621 1978-1985 NNP denotes Nuclear
T1622 1986-1995 NNP denotes Factor-κB
T1623 1996-1997 -LRB- denotes (
T1624 1997-2002 NNP denotes NF-κB
T1625 2002-2003 -RRB- denotes )
T1626 2004-2014 NN denotes activation
T1627 2015-2016 NNP denotes [
T1628 2016-2018 CD denotes 13
T1629 2018-2019 NNP denotes ]
T1630 2019-2020 , denotes ,
T1631 2021-2022 NNP denotes [
T1632 2022-2024 CD denotes 14
T1633 2024-2025 NNP denotes ]
T1634 2025-2026 . denotes .
T1635 2027-2032 NN denotes NF-κB
T1636 2033-2035 VBZ denotes is
T1637 2036-2037 DT denotes a
T1638 2038-2051 NN denotes transcription
T1639 2052-2058 NN denotes factor
T1640 2059-2063 WDT denotes that
T1641 2064-2073 VBZ denotes regulates
T1642 2074-2077 DT denotes the
T1643 2078-2088 NN denotes expression
T1644 2089-2091 IN denotes of
T1645 2092-2097 NNS denotes genes
T1646 2098-2108 VBN denotes associated
T1647 2109-2113 IN denotes with
T1648 2114-2126 NN denotes inflammation
T1649 2127-2128 NNP denotes [
T1650 2128-2130 CD denotes 15
T1651 2130-2131 NNP denotes ]
T1652 2131-2132 , denotes ,
T1653 2133-2134 NNP denotes [
T1654 2134-2136 CD denotes 16
T1655 2136-2137 NNP denotes ]
T1656 2137-2138 . denotes .
T1657 2139-2149 NNP denotes Macrophage
T1658 2150-2162 NN denotes infiltration
T1659 2163-2166 CC denotes and
T1660 2167-2179 NN denotes accumulation
T1661 2180-2182 VBZ denotes is
T1662 2183-2184 DT denotes a
T1663 2185-2191 JJ denotes common
T1664 2192-2206 NN denotes characteristic
T1665 2207-2209 IN denotes of
T1666 2210-2220 JJ denotes intestinal
T1667 2221-2229 NNS denotes diseases
T1668 2230-2231 NNP denotes [
T1669 2231-2233 CD denotes 17
T1670 2233-2234 NNP denotes ]
T1671 2234-2235 . denotes .
T1672 2236-2247 NNS denotes Macrophages
T1673 2248-2257 VBP denotes represent
T1674 2258-2260 CD denotes 10
T1675 2260-2261 NN denotes %
T1676 2262-2264 IN denotes of
T1677 2265-2270 JJ denotes total
T1678 2271-2277 NN denotes lamina
T1679 2278-2285 NN denotes propria
T1680 2286-2291 NNS denotes cells
T1681 2291-2292 , denotes ,
T1682 2293-2300 VBP denotes secrete
T1683 2301-2302 DT denotes a
T1684 2303-2307 JJ denotes wide
T1685 2308-2313 NN denotes range
T1686 2314-2316 IN denotes of
T1687 2317-2329 RB denotes biologically
T1688 2330-2336 JJ denotes active
T1689 2337-2346 NNS denotes compounds
T1690 2347-2350 CC denotes and
T1691 2351-2358 VB denotes express
T1692 2359-2372 NN denotes cell-adhesion
T1693 2373-2382 NNS denotes molecules
T1694 2382-2383 . denotes .
T1695 2384-2387 DT denotes The
T1696 2388-2394 JJ denotes immune
T1697 2395-2399 NN denotes cell
T1698 2400-2408 NN denotes response
T1699 2409-2411 TO denotes to
T1700 2412-2414 DT denotes an
T1701 2415-2427 JJ denotes inflammatory
T1702 2428-2436 NN denotes stimulus
T1703 2437-2442 VBZ denotes seems
T1704 2443-2445 TO denotes to
T1705 2446-2448 VB denotes be
T1706 2449-2458 VBN denotes amplified
T1707 2459-2461 CC denotes or
T1708 2462-2470 RB denotes directly
T1709 2471-2480 VBN denotes generated
T1710 2481-2483 IN denotes by
T1711 2484-2489 NNS denotes cells
T1712 2490-2497 VBN denotes exposed
T1713 2498-2500 TO denotes to
T1714 2501-2510 VBN denotes sulphated
T1715 2511-2526 NNS denotes polysaccharides
T1716 2527-2531 JJ denotes such
T1717 2532-2534 IN denotes as
T1718 2535-2547 NNS denotes carrageenans
T1719 2547-2548 . denotes .
T1720 2549-2555 RB denotes Indeed
T1721 2555-2556 , denotes ,
T1722 2557-2569 NN denotes inflammation
T1723 2570-2577 VBN denotes induced
T1724 2578-2580 IN denotes by
T1725 2581-2585 NNP denotes dCGN
T1726 2586-2589 VBD denotes was
T1727 2590-2600 VBN denotes associated
T1728 2601-2605 IN denotes with
T1729 2606-2617 NN denotes recruitment
T1730 2618-2620 IN denotes of
T1731 2621-2632 NNS denotes macrophages
T1732 2633-2635 TO denotes to
T1733 2636-2648 NN denotes inflammation
T1734 2649-2654 NNS denotes sites
T1735 2655-2656 NNP denotes [
T1736 2656-2658 CD denotes 18
T1737 2658-2659 NNP denotes ]
T1738 2659-2660 , denotes ,
T1739 2661-2662 NNP denotes [
T1740 2662-2664 CD denotes 19
T1741 2664-2665 NNP denotes ]
T1742 2665-2666 . denotes .
T1743 2667-2671 RB denotes Also
T1744 2671-2672 , denotes ,
T1745 2673-2685 NN denotes inflammation
T1746 2686-2693 VBN denotes induced
T1747 2694-2696 IN denotes by
T1748 2697-2704 NNP denotes Dextran
T1749 2705-2713 NNP denotes Sulphate
T1750 2714-2720 NNP denotes Sodium
T1751 2721-2722 -LRB- denotes (
T1752 2722-2725 NNP denotes DSS
T1753 2725-2726 -RRB- denotes )
T1754 2726-2727 , denotes ,
T1755 2728-2735 DT denotes another
T1756 2736-2745 VBN denotes sulphated
T1757 2746-2754 NN denotes compound
T1758 2754-2755 , denotes ,
T1759 2756-2759 VBD denotes was
T1760 2760-2768 RB denotes directly
T1761 2769-2779 VBN denotes associated
T1762 2780-2784 IN denotes with
T1763 2785-2796 NNS denotes macrophages
T1764 2797-2808 NN denotes recruitment
T1765 2809-2810 NNP denotes [
T1766 2810-2812 CD denotes 20
T1767 2812-2813 NNP denotes ]
T1768 2813-2814 , denotes ,
T1769 2815-2820 IN denotes since
T1770 2821-2824 NNP denotes DSS
T1771 2825-2830 RB denotes still
T1772 2831-2839 VBD denotes provoked
T1773 2840-2852 NN denotes inflammation
T1774 2853-2858 IN denotes after
T1775 2859-2871 NN denotes T-lymphocyte
T1776 2872-2875 CC denotes and
T1777 2876-2878 NNP denotes NK
T1778 2879-2883 NN denotes cell
T1779 2884-2893 NN denotes depletion
T1780 2894-2895 NNP denotes [
T1781 2895-2897 CD denotes 20
T1782 2897-2898 NNP denotes ]
T1783 2898-2899 . denotes .
T1784 2900-2908 IN denotes Although
T1785 2909-2921 NN denotes inflammation
T1786 2922-2925 MD denotes can
T1787 2926-2928 VB denotes be
T1788 2929-2936 VBN denotes induced
T1789 2937-2939 IN denotes by
T1790 2940-2944 NNP denotes dCGN
T1791 2944-2945 , denotes ,
T1792 2946-2951 EX denotes there
T1793 2952-2955 VBP denotes are
T1794 2956-2958 DT denotes no
T1795 2959-2963 NNS denotes data
T1796 2964-2966 IN denotes on
T1797 2967-2972 JJ denotes human
T1798 2973-2981 NN denotes monocyte
T1799 2982-2991 NNS denotes responses
T1800 2992-2994 TO denotes to
T1801 2995-2999 NNP denotes dCGN
T1802 3000-3008 NN denotes exposure
T1803 3008-3009 . denotes .
T1804 3010-3019 RB denotes Therefore
T1805 3019-3020 , denotes ,
T1806 3021-3023 TO denotes to
T1807 3024-3035 VB denotes investigate
T1808 3036-3039 DT denotes the
T1809 3040-3047 NNS denotes effects
T1810 3048-3050 IN denotes of
T1811 3051-3055 NNP denotes dCGN
T1812 3056-3058 IN denotes on
T1813 3059-3064 JJ denotes human
T1814 3065-3074 NNS denotes monocytes
T1815 3074-3075 , denotes ,
T1816 3076-3082 JJ denotes normal
T1817 3083-3093 NNP denotes Peripheral
T1818 3094-3099 NNP denotes Blood
T1819 3100-3109 NNP denotes Monocytes
T1820 3110-3111 -LRB- denotes (
T1821 3111-3114 NNP denotes PBM
T1822 3114-3115 -RRB- denotes )
T1823 3116-3119 CC denotes and
T1824 3120-3127 JJ denotes tumoral
T1825 3128-3147 NN denotes monocyte/macrophage
T1826 3148-3153 CD denotes THP-1
T1827 3154-3159 NNS denotes cells
T1828 3160-3164 VBD denotes were
T1829 3165-3172 VBN denotes exposed
T1830 3173-3175 TO denotes to
T1831 3176-3178 CD denotes 10
T1832 3179-3182 NN denotes kDa
T1833 3183-3186 CC denotes and
T1834 3187-3189 CD denotes 40
T1835 3190-3193 NNP denotes kDa
T1836 3194-3198 NNP denotes dCGN
T1837 3198-3199 . denotes .
T1838 3200-3202 PRP denotes We
T1839 3203-3208 VBD denotes found
T1840 3209-3213 IN denotes that
T1841 3214-3218 NNP denotes dCGN
T1842 3219-3228 VBD denotes inhibited
T1843 3229-3234 CD denotes THP-1
T1844 3235-3239 NN denotes cell
T1845 3240-3253 NN denotes proliferation
T1846 3254-3256 IN denotes in
T1847 3257-3262 NN denotes vitro
T1848 3262-3263 , denotes ,
T1849 3264-3273 VBD denotes increased
T1850 3274-3280 NNP denotes ICAM-1
T1851 3281-3291 NN denotes expression
T1852 3291-3292 , denotes ,
T1853 3293-3303 VBN denotes stimulated
T1854 3304-3320 JJ denotes ICAM-1-dependent
T1855 3321-3329 NN denotes monocyte
T1856 3330-3341 NN denotes aggregation
T1857 3341-3342 , denotes ,
T1858 3343-3346 CC denotes and
T1859 3347-3357 VBD denotes stimulated
T1860 3358-3363 NNP denotes TNF-α
T1861 3364-3374 NN denotes expression
T1862 3375-3378 CC denotes and
T1863 3379-3388 NN denotes secretion
T1864 3388-3389 . denotes .
T1865 3390-3395 DT denotes These
T1866 3396-3405 NNS denotes responses
T1867 3406-3410 VBD denotes were
T1868 3411-3415 RBR denotes more
T1869 3416-3426 JJ denotes pronounced
T1870 3427-3432 IN denotes after
T1871 3433-3435 CD denotes 40
T1872 3436-3439 NNP denotes kDa
T1873 3440-3444 NNP denotes dCGN
T1874 3445-3453 NN denotes exposure
T1875 3454-3457 CC denotes and
T1876 3458-3462 VBD denotes were
T1877 3463-3469 VBN denotes linked
T1878 3470-3472 TO denotes to
T1879 3473-3478 NNP denotes NF-κB
T1880 3479-3489 NN denotes activation
T1881 3489-3490 . denotes .
T1882 3491-3493 IN denotes In
T1883 3494-3502 NN denotes addition
T1884 3502-3503 , denotes ,
T1885 3504-3507 DT denotes the
T1886 3508-3510 CD denotes 40
T1887 3511-3514 NNP denotes kDa
T1888 3515-3519 NNP denotes dCGN
T1889 3519-3520 , denotes ,
T1890 3521-3524 CC denotes but
T1891 3525-3528 RB denotes not
T1892 3529-3532 DT denotes the
T1893 3533-3535 CD denotes 10
T1894 3536-3539 NNP denotes kDa
T1895 3540-3544 NNP denotes dCGN
T1896 3545-3552 VBD denotes induced
T1897 3553-3555 IN denotes in
T1898 3556-3560 NN denotes vivo
T1899 3561-3568 NNS denotes colitis
T1900 3569-3571 IN denotes as
T1901 3572-3577 VBN denotes shown
T1902 3578-3580 IN denotes by
T1903 3581-3584 DT denotes the
T1904 3585-3597 JJ denotes inflammatory
T1905 3598-3606 NN denotes response
T1906 3607-3609 IN denotes in
T1907 3610-3613 DT denotes the
T1908 3614-3617 NN denotes rat
T1909 3618-3623 NN denotes colon
T1910 3623-3624 . denotes .
T1911 3625-3630 DT denotes These
T1912 3631-3638 NNS denotes results
T1913 3639-3646 VBP denotes suggest
T1914 3647-3651 IN denotes that
T1915 3652-3655 DT denotes the
T1916 3656-3664 JJ denotes degraded
T1917 3665-3670 NNS denotes forms
T1918 3671-3673 IN denotes of
T1919 3674-3677 NNP denotes CGN
T1920 3678-3682 VBP denotes have
T1921 3683-3685 DT denotes an
T1922 3686-3695 JJ denotes important
T1923 3696-3702 NN denotes effect
T1924 3703-3705 IN denotes on
T1925 3706-3715 NNS denotes monocytes
T1926 3716-3725 VBG denotes resulting
T1927 3726-3728 IN denotes in
T1928 3729-3731 DT denotes an
T1929 3732-3744 JJ denotes inflammatory
T1930 3745-3754 NN denotes phenotype
T1931 3754-3755 . denotes .
T2416 3780-3791 NNP denotes Preparation
T2417 3792-3794 IN denotes of
T2418 3795-3803 NNP denotes Degraded
T2419 3804-3815 NNP denotes Carrageenan
T2420 3816-3819 CD denotes Two
T2421 3820-3832 NNS denotes preparations
T2422 3833-3835 IN denotes of
T2423 3836-3844 JJ denotes degraded
T2424 3845-3856 NN denotes carrageenan
T2425 3857-3861 IN denotes with
T2426 3862-3865 JJ denotes low
T2427 3865-3866 , denotes ,
T2428 3867-3868 -LRB- denotes (
T2429 3868-3869 FW denotes ∼
T2430 3869-3871 CD denotes 10
T2431 3872-3875 NN denotes kDa
T2432 3875-3876 : denotes ;
T2433 3877-3880 NNP denotes C10
T2434 3880-3881 -RRB- denotes )
T2435 3881-3882 , denotes ,
T2436 3883-3886 CC denotes and
T2437 3887-3893 NN denotes medium
T2438 3893-3894 , denotes ,
T2439 3895-3896 -LRB- denotes (
T2440 3896-3897 FW denotes ∼
T2441 3897-3899 CD denotes 40
T2442 3900-3903 NN denotes kDa
T2443 3903-3904 : denotes ;
T2444 3905-3908 NNP denotes C40
T2445 3908-3909 -RRB- denotes )
T2446 3910-3919 JJ denotes molecular
T2447 3920-3926 NN denotes weight
T2448 3927-3931 VBD denotes were
T2449 3932-3940 VBN denotes prepared
T2450 3941-3945 IN denotes from
T2451 3946-3952 JJ denotes native
T2452 3953-3969 JJ denotes iota-carrageenan
T2453 3970-3979 VBN denotes extracted
T2454 3980-3984 IN denotes from
T2455 3985-3992 NNP denotes Euchema
T2456 3993-4001 NN denotes spinosum
T2457 4002-4003 -LRB- denotes (
T2458 4003-4013 RB denotes generously
T2459 4014-4022 VBN denotes provided
T2460 4023-4025 IN denotes by
T2461 4026-4032 NNP denotes Sanofi
T2462 4033-4043 NNPS denotes Biosystems
T2463 4044-4052 NNP denotes Industry
T2464 4052-4053 , denotes ,
T2465 4054-4074 NNP denotes Boulogne-Billancourt
T2466 4074-4075 , denotes ,
T2467 4076-4082 NNP denotes France
T2468 4082-4083 -RRB- denotes )
T2469 4083-4084 . denotes .
T2470 4085-4091 JJ denotes Native
T2471 4092-4103 NN denotes carrageenan
T2472 4104-4107 VBD denotes was
T2473 4108-4117 VBN denotes dissolved
T2474 4118-4120 IN denotes in
T2475 4121-4130 JJ denotes distilled
T2476 4131-4136 NN denotes water
T2477 4137-4138 -LRB- denotes (
T2478 4138-4139 CD denotes 5
T2479 4139-4140 NN denotes %
T2480 4141-4144 NN denotes w/v
T2481 4144-4145 -RRB- denotes )
T2482 4146-4151 IN denotes under
T2483 4152-4160 JJ denotes vigorous
T2484 4161-4169 VBG denotes stirring
T2485 4170-4173 CC denotes and
T2486 4174-4180 VBN denotes heated
T2487 4181-4183 TO denotes to
T2488 4184-4186 CD denotes 60
T2489 4186-4187 CD denotes °
T2490 4187-4189 NNP denotes C.
T2491 4190-4194 RB denotes Then
T2492 4194-4195 , denotes ,
T2493 4196-4199 DT denotes the
T2494 4200-4211 NN denotes carrageenan
T2495 4212-4220 NN denotes solution
T2496 4221-4224 VBD denotes was
T2497 4225-4234 VBN denotes submitted
T2498 4235-4237 TO denotes to
T2499 4238-4241 CD denotes two
T2500 4242-4251 JJ denotes different
T2501 4252-4262 NNS denotes treatments
T2502 4263-4265 TO denotes to
T2503 4266-4272 VB denotes obtain
T2504 4273-4277 DT denotes both
T2505 4278-4281 JJ denotes low
T2506 4282-4285 CC denotes and
T2507 4286-4292 JJ denotes medium
T2508 4293-4302 JJ denotes molecular
T2509 4303-4309 NN denotes weight
T2510 4310-4319 NNS denotes fractions
T2511 4319-4320 . denotes .
T2512 4321-4328 RB denotes Briefly
T2513 4328-4329 , denotes ,
T2514 4330-4333 IN denotes for
T2515 4334-4337 DT denotes the
T2516 4338-4341 JJ denotes low
T2517 4342-4351 JJ denotes molecular
T2518 4352-4358 NN denotes weight
T2519 4359-4367 NN denotes fraction
T2520 4367-4368 , denotes ,
T2521 4369-4380 NN denotes carrageenan
T2522 4381-4389 NN denotes solution
T2523 4390-4393 VBD denotes was
T2524 4394-4404 VBN denotes hydrolyzed
T2525 4405-4409 IN denotes with
T2526 4410-4413 CD denotes 0.3
T2527 4413-4414 NN denotes %
T2528 4415-4416 -LRB- denotes (
T2529 4416-4419 FW denotes v/v
T2530 4419-4420 -RRB- denotes )
T2531 4421-4433 VBD denotes concentrated
T2532 4434-4443 JJ denotes sulphuric
T2533 4444-4448 NN denotes acid
T2534 4449-4452 IN denotes for
T2535 4453-4455 CD denotes 15
T2536 4456-4459 NN denotes min
T2537 4460-4462 IN denotes at
T2538 4463-4465 CD denotes 80
T2539 4465-4466 CD denotes °
T2540 4466-4467 NNP denotes C
T2541 4467-4468 . denotes .
T2542 4469-4474 IN denotes After
T2543 4475-4489 NN denotes neutralization
T2544 4490-4494 IN denotes with
T2545 4495-4499 NNP denotes NaOH
T2546 4500-4502 NNP denotes 4N
T2547 4502-4503 , denotes ,
T2548 4504-4507 DT denotes the
T2549 4508-4516 NN denotes solution
T2550 4517-4520 VBD denotes was
T2551 4521-4526 JJ denotes ultra
T2552 4527-4535 VBN denotes filtered
T2553 4536-4543 IN denotes through
T2554 4544-4545 DT denotes a
T2555 4546-4552 JJ denotes hollow
T2556 4553-4558 NN denotes fibre
T2557 4559-4568 NN denotes cartridge
T2558 4569-4573 IN denotes with
T2559 4574-4576 NNP denotes MW
T2560 4577-4584 NN denotes cut-off
T2561 4585-4586 CD denotes 5
T2562 4587-4590 NNP denotes kDa
T2563 4590-4591 , denotes ,
T2564 4592-4593 -LRB- denotes (
T2565 4593-4599 NNP denotes Amicon
T2566 4600-4603 NNP denotes Inc
T2567 4603-4604 , denotes ,
T2568 4605-4612 NNP denotes Beverly
T2569 4612-4613 , denotes ,
T2570 4614-4617 NNP denotes USA
T2571 4617-4618 -RRB- denotes )
T2572 4618-4619 . denotes .
T2573 4620-4623 IN denotes For
T2574 4624-4627 DT denotes the
T2575 4628-4634 NN denotes medium
T2576 4635-4644 JJ denotes molecular
T2577 4645-4651 NN denotes weight
T2578 4652-4660 NN denotes fraction
T2579 4660-4661 , denotes ,
T2580 4662-4665 DT denotes the
T2581 4666-4677 NN denotes carrageenan
T2582 4678-4686 NN denotes solution
T2583 4687-4690 VBD denotes was
T2584 4691-4701 VBN denotes hydrolyzed
T2585 4702-4706 IN denotes with
T2586 4707-4710 CD denotes 0.3
T2587 4710-4711 NN denotes %
T2588 4712-4713 -LRB- denotes (
T2589 4713-4716 FW denotes v/v
T2590 4716-4717 -RRB- denotes )
T2591 4718-4730 VBD denotes concentrated
T2592 4731-4740 JJ denotes sulphuric
T2593 4741-4745 NN denotes acid
T2594 4746-4749 IN denotes for
T2595 4750-4752 CD denotes 30
T2596 4753-4756 NN denotes min
T2597 4757-4759 IN denotes at
T2598 4760-4762 CD denotes 60
T2599 4762-4763 CD denotes °
T2600 4763-4764 NNP denotes C
T2601 4764-4765 . denotes .
T2602 4766-4771 IN denotes After
T2603 4772-4786 NN denotes neutralization
T2604 4786-4787 , denotes ,
T2605 4788-4791 DT denotes the
T2606 4792-4803 NN denotes supernatant
T2607 4804-4807 VBD denotes was
T2608 4808-4813 JJ denotes ultra
T2609 4814-4822 VBN denotes filtered
T2610 4823-4824 -LRB- denotes (
T2611 4824-4826 NNP denotes MW
T2612 4827-4834 NN denotes cut-off
T2613 4835-4838 CD denotes 100
T2614 4839-4842 NNP denotes kDa
T2615 4842-4843 -RRB- denotes )
T2616 4843-4844 . denotes .
T2617 4845-4848 DT denotes The
T2618 4849-4857 NN denotes filtrate
T2619 4858-4861 VBD denotes was
T2620 4862-4871 VBN denotes submitted
T2621 4872-4874 TO denotes to
T2622 4875-4876 DT denotes a
T2623 4877-4883 JJ denotes second
T2624 4884-4889 NN denotes ultra
T2625 4890-4900 NN denotes filtration
T2626 4901-4902 -LRB- denotes (
T2627 4902-4904 NNP denotes MW
T2628 4905-4912 NN denotes cut-off
T2629 4913-4914 CD denotes 5
T2630 4915-4918 NNP denotes kDa
T2631 4918-4919 -RRB- denotes )
T2632 4919-4920 . denotes .
T2633 4921-4925 DT denotes Both
T2634 4926-4938 NNS denotes preparations
T2635 4939-4941 IN denotes of
T2636 4942-4946 NNP denotes dCGN
T2637 4947-4951 VBD denotes were
T2638 4952-4964 VBN denotes precipitated
T2639 4965-4969 IN denotes with
T2640 4970-4971 CD denotes 4
T2641 4972-4979 NNS denotes volumes
T2642 4980-4982 IN denotes of
T2643 4983-4985 CD denotes 95
T2644 4985-4986 NN denotes %
T2645 4987-4994 NN denotes ethanol
T2646 4994-4995 , denotes ,
T2647 4996-5001 VBD denotes dried
T2648 5002-5004 IN denotes at
T2649 5005-5009 NN denotes room
T2650 5010-5021 NN denotes temperature
T2651 5022-5025 CC denotes and
T2652 5026-5032 NN denotes ground
T2653 5033-5035 TO denotes to
T2654 5036-5041 JJ denotes small
T2655 5042-5051 NNS denotes particles
T2656 5052-5053 -LRB- denotes (
T2657 5053-5054 CD denotes 1
T2658 5055-5057 NN denotes mm
T2659 5058-5060 IN denotes in
T2660 5061-5069 NN denotes diameter
T2661 5069-5070 -RRB- denotes )
T2662 5070-5071 . denotes .
T2663 5072-5077 VBG denotes Using
T2664 5078-5092 NN denotes gel-permeation
T2665 5093-5107 NN denotes chromatography
T2666 5108-5110 IN denotes in
T2667 5111-5122 NN denotes combination
T2668 5123-5127 IN denotes with
T2669 5128-5133 JJ denotes light
T2670 5134-5144 VBG denotes scattering
T2671 5145-5157 NNS denotes measurements
T2672 5158-5159 -LRB- denotes (
T2673 5159-5162 VB denotes see
T2674 5163-5169 NNP denotes Viebke
T2675 5170-5172 FW denotes et
T2676 5173-5176 FW denotes al.
T2677 5177-5178 NNP denotes [
T2678 5178-5180 CD denotes 21
T2679 5180-5181 NNP denotes ]
T2680 5181-5182 -RRB- denotes )
T2681 5182-5183 , denotes ,
T2682 5184-5186 PRP denotes it
T2683 5187-5190 VBD denotes was
T2684 5191-5200 VBN denotes confirmed
T2685 5201-5205 IN denotes that
T2686 5206-5209 DT denotes the
T2687 5210-5213 JJ denotes low
T2688 5214-5222 NN denotes fraction
T2689 5223-5226 VBD denotes had
T2690 5227-5229 DT denotes an
T2691 5230-5237 JJ denotes average
T2692 5238-5247 JJ denotes molecular
T2693 5248-5254 NN denotes weight
T2694 5255-5257 IN denotes of
T2695 5258-5260 CD denotes 10
T2696 5261-5264 NN denotes kDa
T2697 5264-5265 , denotes ,
T2698 5266-5269 CC denotes and
T2699 5270-5273 DT denotes the
T2700 5274-5280 NN denotes medium
T2701 5281-5289 NN denotes fraction
T2702 5290-5292 IN denotes of
T2703 5293-5295 CD denotes 40
T2704 5296-5299 NN denotes kDa
T2705 5299-5300 . denotes .
T2706 5301-5304 DT denotes The
T2707 5305-5313 NN denotes sulphate
T2708 5314-5321 NN denotes content
T2709 5322-5324 IN denotes of
T2710 5325-5340 NNS denotes polysaccharides
T2711 5341-5343 IN denotes in
T2712 5344-5348 DT denotes both
T2713 5349-5358 NNS denotes fractions
T2714 5359-5362 VBD denotes was
T2715 5363-5371 VBN denotes measured
T2716 5372-5381 VBG denotes following
T2717 5382-5385 DT denotes the
T2718 5386-5392 NN denotes method
T2719 5393-5395 IN denotes of
T2720 5396-5404 NNP denotes Quemener
T2721 5405-5407 FW denotes et
T2722 5408-5411 FW denotes al.
T2723 5412-5413 NNP denotes [
T2724 5413-5415 CD denotes 22
T2725 5415-5416 NNP denotes ]
T2726 5416-5417 . denotes .
T2727 5418-5425 RB denotes Finally
T2728 5425-5426 , denotes ,
T2729 5427-5430 DT denotes the
T2730 5431-5438 NN denotes absence
T2731 5439-5441 IN denotes of
T2732 5442-5456 NN denotes polysaccharide
T2733 5457-5466 NN denotes structure
T2734 5467-5480 NNS denotes modifications
T2735 5481-5483 IN denotes in
T2736 5484-5487 DT denotes the
T2737 5488-5491 CD denotes two
T2738 5492-5501 NNS denotes fractions
T2739 5502-5505 VBD denotes was
T2740 5506-5515 VBN denotes confirmed
T2741 5516-5521 VBG denotes using
T2742 5522-5528 JJ denotes 2H-NMR
T2743 5529-5541 NN denotes spectroscopy
T2744 5541-5542 . denotes .
T2745 5543-5546 DT denotes The
T2746 5547-5554 NN denotes absence
T2747 5555-5557 IN denotes of
T2748 5558-5561 NNP denotes LPS
T2749 5562-5575 NN denotes contamination
T2750 5576-5578 IN denotes in
T2751 5579-5582 DT denotes the
T2752 5583-5586 CD denotes two
T2753 5587-5596 NNS denotes fractions
T2754 5597-5600 VBD denotes was
T2755 5601-5610 VBN denotes confirmed
T2756 5611-5616 VBG denotes using
T2757 5617-5620 DT denotes the
T2758 5621-5629 JJ denotes e-Toxate
T2759 5629-5630 NN denotes ®
T2760 5631-5634 NN denotes kit
T2761 5635-5636 -LRB- denotes (
T2762 5636-5641 NNP denotes Sigma
T2763 5641-5642 , denotes ,
T2764 5643-5645 NNP denotes St
T2765 5646-5653 NNP denotes Quentin
T2766 5654-5663 NNP denotes Fallavier
T2767 5663-5664 , denotes ,
T2768 5665-5671 NNP denotes France
T2769 5671-5672 -RRB- denotes )
T2770 5672-5673 . denotes .
T2771 5674-5680 IN denotes Before
T2772 5681-5684 NN denotes use
T2773 5685-5687 IN denotes in
T2774 5688-5692 NN denotes cell
T2775 5693-5700 NN denotes culture
T2776 5700-5701 , denotes ,
T2777 5702-5705 DT denotes the
T2778 5706-5709 CD denotes two
T2779 5710-5719 NNS denotes fractions
T2780 5720-5724 VBD denotes were
T2781 5725-5734 VBN denotes dissolved
T2782 5735-5737 IN denotes in
T2783 5738-5746 JJ denotes complete
T2784 5747-5753 NN denotes medium
T2785 5754-5760 IN denotes during
T2786 5761-5763 CD denotes 30
T2787 5764-5767 NN denotes min
T2788 5768-5770 IN denotes at
T2789 5771-5773 CD denotes 56
T2790 5773-5774 CD denotes °
T2791 5774-5776 NNP denotes C.
T2945 5778-5785 NNS denotes Animals
T2946 5785-5786 , denotes ,
T2947 5787-5796 NNPS denotes Chemicals
T2948 5797-5800 CC denotes and
T2949 5801-5805 NNP denotes Diet
T2950 5806-5810 NNP denotes Male
T2951 5811-5817 NNP denotes Wistar
T2952 5818-5822 NNS denotes rats
T2953 5823-5824 -LRB- denotes (
T2954 5824-5827 CD denotes 150
T2955 5828-5829 JJ denotes g
T2956 5830-5837 JJ denotes average
T2957 5838-5844 NN denotes weight
T2958 5844-5845 -RRB- denotes )
T2959 5846-5850 VBD denotes were
T2960 5851-5857 VBN denotes housed
T2961 5858-5863 IN denotes under
T2962 5864-5872 JJ denotes standard
T2963 5873-5883 NNS denotes conditions
T2964 5884-5887 CC denotes and
T2965 5888-5891 VBN denotes fed
T2966 5892-5894 NN denotes ad
T2967 5895-5902 NN denotes libitum
T2968 5903-5907 IN denotes with
T2969 5908-5916 JJ denotes standard
T2970 5917-5923 NN denotes rodent
T2971 5924-5934 NN denotes laboratory
T2972 5935-5939 NN denotes chow
T2973 5939-5940 . denotes .
T2974 5941-5949 JJ denotes Degraded
T2975 5950-5967 NNS denotes iota-carrageenans
T2976 5968-5972 VBD denotes were
T2977 5973-5985 VBN denotes administered
T2978 5986-5988 IN denotes in
T2979 5989-5992 DT denotes the
T2980 5993-6001 NN denotes drinking
T2981 6002-6007 NN denotes water
T2982 6008-6009 -LRB- denotes (
T2983 6009-6010 CD denotes 5
T2984 6010-6011 NN denotes %
T2985 6012-6015 NN denotes w/v
T2986 6015-6016 -RRB- denotes )
T2987 6017-6020 IN denotes for
T2988 6021-6023 CD denotes 55
T2989 6024-6028 NNS denotes days
T2990 6029-6031 TO denotes to
T2991 6032-6033 CD denotes 2
T2992 6034-6040 NNS denotes groups
T2993 6041-6043 IN denotes of
T2994 6044-6047 CD denotes six
T2995 6048-6055 NNS denotes animals
T2996 6056-6060 DT denotes each
T2997 6060-6061 . denotes .
T2998 6062-6065 DT denotes The
T2999 6066-6071 JJ denotes first
T3000 6072-6077 NN denotes group
T3001 6078-6086 VBD denotes received
T3002 6087-6090 DT denotes the
T3003 6091-6094 JJ denotes low
T3004 6095-6104 JJ denotes molecular
T3005 6105-6111 NN denotes weight
T3006 6112-6123 NN denotes carrageenan
T3007 6124-6125 -LRB- denotes (
T3008 6125-6127 CD denotes 10
T3009 6128-6131 NNP denotes kDa
T3010 6132-6136 NNP denotes dCGN
T3011 6136-6137 -RRB- denotes )
T3012 6138-6141 CC denotes and
T3013 6142-6145 DT denotes the
T3014 6146-6152 JJ denotes second
T3015 6153-6161 VBD denotes received
T3016 6162-6165 DT denotes the
T3017 6166-6172 NN denotes medium
T3018 6173-6182 JJ denotes molecular
T3019 6183-6189 NN denotes weight
T3020 6190-6201 NN denotes carrageenan
T3021 6202-6203 -LRB- denotes (
T3022 6203-6205 CD denotes 40
T3023 6206-6209 NNP denotes kDa
T3024 6210-6214 NNP denotes dCGN
T3025 6214-6215 -RRB- denotes )
T3026 6215-6216 . denotes .
T3027 6217-6219 DT denotes An
T3028 6220-6230 JJ denotes additional
T3029 6231-6236 NN denotes group
T3030 6237-6239 IN denotes of
T3031 6240-6244 CD denotes four
T3032 6245-6249 NNS denotes rats
T3033 6250-6254 VBD denotes were
T3034 6255-6265 VBN denotes maintained
T3035 6266-6268 IN denotes on
T3036 6269-6276 JJ denotes regular
T3037 6277-6280 VBP denotes tap
T3038 6281-6286 NN denotes water
T3039 6287-6288 -LRB- denotes (
T3040 6288-6295 NN denotes control
T3041 6296-6301 NN denotes group
T3042 6301-6302 -RRB- denotes )
T3043 6302-6303 . denotes .
T3044 6304-6306 TO denotes To
T3045 6307-6315 VB denotes increase
T3046 6316-6328 NN denotes palatability
T3047 6329-6332 CD denotes 0.2
T3048 6332-6333 NN denotes %
T3049 6334-6341 NN denotes sucrose
T3050 6342-6345 VBD denotes was
T3051 6346-6351 VBN denotes added
T3052 6352-6354 TO denotes to
T3053 6355-6358 DT denotes the
T3054 6359-6367 NN denotes drinking
T3055 6368-6373 NN denotes water
T3056 6374-6376 IN denotes of
T3057 6377-6380 DT denotes all
T3058 6381-6387 NNS denotes groups
T3059 6388-6389 -LRB- denotes (
T3060 6389-6392 NNP denotes Van
T3061 6393-6396 NN denotes der
T3062 6397-6402 NNP denotes Waaji
T3063 6403-6405 FW denotes et
T3064 6406-6409 FW denotes al.
T3065 6409-6410 , denotes ,
T3066 6411-6412 NNP denotes [
T3067 6412-6414 CD denotes 23
T3068 6414-6415 NNP denotes ]
T3069 6415-6416 -RRB- denotes )
T3070 6416-6417 . denotes .
T3071 6418-6423 JJ denotes Fresh
T3072 6424-6435 NN denotes carrageenan
T3073 6436-6445 NNS denotes solutions
T3074 6446-6450 VBD denotes were
T3075 6451-6459 JJ denotes prepared
T3076 6460-6465 JJ denotes daily
T3077 6465-6466 . denotes .
T3368 6468-6478 NN denotes Evaluation
T3369 6479-6481 IN denotes of
T3370 6482-6489 NNP denotes Colitis
T3371 6490-6494 NNP denotes Body
T3372 6495-6501 NN denotes weight
T3373 6501-6502 , denotes ,
T3374 6503-6509 NN denotes liquid
T3375 6510-6513 CC denotes and
T3376 6514-6518 NN denotes food
T3377 6519-6530 NN denotes consumption
T3378 6530-6531 , denotes ,
T3379 6532-6540 NN denotes diarrhea
T3380 6541-6544 CC denotes and
T3381 6545-6551 JJ denotes rectal
T3382 6552-6560 NN denotes bleeding
T3383 6561-6562 -LRB- denotes (
T3384 6562-6570 VBN denotes detected
T3385 6571-6573 IN denotes by
T3386 6574-6577 NN denotes eye
T3387 6578-6588 NN denotes inspection
T3388 6588-6589 -RRB- denotes )
T3389 6590-6594 VBD denotes were
T3390 6595-6603 VBN denotes recorded
T3391 6604-6614 IN denotes throughout
T3392 6615-6618 DT denotes the
T3393 6619-6626 NN denotes feeding
T3394 6627-6633 NN denotes period
T3395 6633-6634 . denotes .
T3396 6635-6640 IN denotes After
T3397 6641-6643 CD denotes 55
T3398 6644-6648 NNS denotes days
T3399 6648-6649 , denotes ,
T3400 6650-6657 NNS denotes animals
T3401 6658-6662 VBD denotes were
T3402 6663-6673 VBN denotes sacrificed
T3403 6674-6676 IN denotes by
T3404 6677-6685 JJ denotes cervical
T3405 6686-6697 NN denotes dislocation
T3406 6697-6698 . denotes .
T3407 6699-6702 DT denotes The
T3408 6703-6709 NN denotes length
T3409 6710-6712 IN denotes of
T3410 6713-6716 DT denotes the
T3411 6717-6722 NN denotes colon
T3412 6723-6726 VBD denotes was
T3413 6727-6735 VBN denotes measured
T3414 6736-6738 IN denotes as
T3415 6739-6748 VBN denotes described
T3416 6749-6751 IN denotes by
T3417 6752-6760 NNP denotes Okayashu
T3418 6761-6763 FW denotes et
T3419 6764-6767 FW denotes al.
T3420 6768-6769 NNP denotes [
T3421 6769-6771 CD denotes 24
T3422 6771-6772 NNP denotes ]
T3423 6772-6773 . denotes .
T3424 6774-6778 RB denotes Then
T3425 6778-6779 , denotes ,
T3426 6780-6784 DT denotes each
T3427 6785-6790 NN denotes colon
T3428 6791-6794 VBD denotes was
T3429 6795-6802 VBN denotes ligated
T3430 6803-6805 IN denotes in
T3431 6806-6814 NNS denotes sections
T3432 6815-6817 IN denotes of
T3433 6818-6819 CD denotes 2
T3434 6820-6822 NN denotes cm
T3435 6823-6826 CC denotes and
T3436 6827-6828 CD denotes 1
T3437 6829-6831 TO denotes to
T3438 6832-6833 CD denotes 2
T3439 6834-6836 NN denotes ml
T3440 6837-6839 IN denotes of
T3441 6840-6842 CD denotes 10
T3442 6842-6843 NN denotes %
T3443 6844-6852 NN denotes formalin
T3444 6853-6856 VBD denotes was
T3445 6857-6864 VBN denotes infused
T3446 6865-6869 IN denotes into
T3447 6870-6873 DT denotes the
T3448 6874-6884 JJ denotes intestinal
T3449 6885-6890 NNS denotes lumen
T3450 6890-6891 . denotes .
T3451 6892-6895 DT denotes The
T3452 6896-6906 RB denotes moderately
T3453 6907-6916 JJ denotes distended
T3454 6917-6924 NN denotes segment
T3455 6925-6928 VBD denotes was
T3456 6929-6938 VBN denotes sectioned
T3457 6939-6942 CC denotes and
T3458 6943-6948 VBN denotes fixed
T3459 6949-6951 IN denotes in
T3460 6952-6954 CD denotes 10
T3461 6954-6955 NN denotes %
T3462 6956-6964 NN denotes formalin
T3463 6964-6965 . denotes .
T3464 6966-6969 DT denotes The
T3465 6970-6979 JJ denotes following
T3466 6980-6983 NN denotes day
T3467 6983-6984 , denotes ,
T3468 6985-6988 DT denotes the
T3469 6989-6999 JJ denotes intestinal
T3470 7000-7007 NN denotes content
T3471 7008-7011 VBD denotes was
T3472 7012-7019 VBN denotes removed
T3473 7020-7022 IN denotes by
T3474 7023-7032 VBG denotes vortexing
T3475 7032-7033 . denotes .
T3476 7034-7037 DT denotes The
T3477 7038-7043 VBN denotes fixed
T3478 7044-7051 NN denotes segment
T3479 7052-7055 VBD denotes was
T3480 7056-7060 VBN denotes kept
T3481 7061-7063 IN denotes in
T3482 7064-7066 CD denotes 10
T3483 7066-7067 NN denotes %
T3484 7068-7076 NN denotes formalin
T3485 7077-7079 IN denotes at
T3486 7080-7081 CD denotes 4
T3487 7081-7082 CD denotes °
T3488 7082-7083 NNP denotes C
T3489 7084-7089 IN denotes until
T3490 7090-7093 DT denotes the
T3491 7094-7102 NN denotes paraffin
T3492 7103-7112 NN denotes embedding
T3493 7113-7122 NN denotes procedure
T3494 7122-7123 . denotes .
T3495 7124-7126 TO denotes To
T3496 7127-7135 VB denotes evaluate
T3497 7136-7139 DT denotes the
T3498 7140-7146 NN denotes degree
T3499 7147-7149 IN denotes of
T3500 7150-7162 NN denotes inflammation
T3501 7162-7163 , denotes ,
T3502 7164-7168 DT denotes this
T3503 7169-7176 NN denotes segment
T3504 7177-7179 IN denotes of
T3505 7180-7185 NN denotes colon
T3506 7186-7189 VBD denotes was
T3507 7190-7196 VBN denotes opened
T3508 7197-7211 RB denotes longitudinally
T3509 7212-7215 CC denotes and
T3510 7216-7227 JJ denotes macroscopic
T3511 7228-7231 CC denotes and
T3512 7232-7244 JJ denotes histological
T3513 7245-7251 NNS denotes scores
T3514 7252-7254 IN denotes of
T3515 7255-7267 NN denotes inflammation
T3516 7268-7272 VBD denotes were
T3517 7273-7281 VBN denotes recorded
T3518 7282-7284 IN denotes as
T3519 7285-7295 RB denotes previously
T3520 7296-7305 VBN denotes described
T3521 7306-7307 NNP denotes [
T3522 7307-7309 CD denotes 25
T3523 7309-7310 NNP denotes ]
T3524 7310-7311 , denotes ,
T3525 7312-7313 NNP denotes [
T3526 7313-7315 CD denotes 26
T3527 7315-7316 NNP denotes ]
T3528 7316-7317 . denotes .
T3529 7318-7321 DT denotes The
T3530 7322-7331 NN denotes toluidine
T3531 7332-7336 JJ denotes blue
T3532 7337-7345 NN denotes staining
T3533 7346-7349 VBD denotes was
T3534 7350-7354 VBN denotes used
T3535 7355-7358 IN denotes for
T3536 7359-7373 NN denotes identification
T3537 7374-7376 IN denotes of
T3538 7377-7386 VBN denotes sulphated
T3539 7387-7402 NNS denotes polysaccharides
T3540 7403-7405 IN denotes in
T3541 7406-7409 DT denotes the
T3542 7410-7420 JJ denotes intestinal
T3543 7421-7427 NN denotes mucosa
T3544 7427-7428 . denotes .
T3545 7429-7431 IN denotes On
T3546 7432-7435 DT denotes the
T3547 7436-7439 NN denotes day
T3548 7440-7442 IN denotes of
T3549 7443-7452 NN denotes sacrifice
T3550 7452-7453 , denotes ,
T3551 7454-7455 DT denotes a
T3552 7456-7461 JJ denotes fresh
T3553 7462-7468 NN denotes sample
T3554 7469-7471 IN denotes of
T3555 7472-7476 DT denotes each
T3556 7477-7482 NN denotes colon
T3557 7483-7484 -LRB- denotes (
T3558 7484-7486 CD denotes 50
T3559 7487-7489 NN denotes mg
T3560 7489-7490 -RRB- denotes )
T3561 7491-7494 VBD denotes was
T3562 7495-7504 VBN denotes collected
T3563 7505-7508 IN denotes for
T3564 7509-7524 NN denotes myeloperoxidase
T3565 7525-7526 -LRB- denotes (
T3566 7526-7529 NNP denotes MPO
T3567 7529-7530 -RRB- denotes )
T3568 7531-7536 JJ denotes assay
T3569 7537-7546 VBG denotes according
T3570 7547-7549 TO denotes to
T3571 7550-7557 NNP denotes Krawisz
T3572 7558-7560 FW denotes et
T3573 7561-7564 FW denotes al.
T3574 7564-7565 , denotes ,
T3575 7566-7567 NNP denotes [
T3576 7567-7569 CD denotes 27
T3577 7569-7570 NNP denotes ]
T3578 7570-7571 . denotes .
T3579 7572-7575 DT denotes The
T3580 7576-7581 NN denotes level
T3581 7582-7584 IN denotes of
T3582 7585-7588 NNP denotes MPO
T3583 7588-7589 , denotes ,
T3584 7590-7596 RB denotes mainly
T3585 7597-7606 VBN denotes expressed
T3586 7607-7609 IN denotes by
T3587 7610-7621 NNS denotes neutrophils
T3588 7621-7622 , denotes ,
T3589 7623-7632 VBZ denotes indicates
T3590 7633-7636 DT denotes the
T3591 7637-7641 NN denotes rate
T3592 7642-7644 IN denotes of
T3593 7645-7656 NN denotes recruitment
T3594 7657-7659 IN denotes of
T3595 7660-7671 NNS denotes neutrophils
T3596 7672-7674 TO denotes to
T3597 7675-7678 DT denotes the
T3598 7679-7689 JJ denotes intestinal
T3599 7690-7696 NN denotes mucosa
T3600 7696-7697 . denotes .
T3601 7698-7701 CD denotes One
T3602 7702-7706 NN denotes unit
T3603 7707-7709 IN denotes of
T3604 7710-7713 NNP denotes MPO
T3605 7714-7722 NN denotes activity
T3606 7723-7734 VBZ denotes corresponds
T3607 7735-7737 TO denotes to
T3608 7738-7741 DT denotes the
T3609 7742-7753 NN denotes degradation
T3610 7754-7756 IN denotes of
T3611 7757-7758 CD denotes 1
T3612 7759-7763 NN denotes µmol
T3613 7764-7766 IN denotes of
T3614 7767-7775 NN denotes peroxide
T3615 7776-7779 IN denotes per
T3616 7780-7786 NN denotes minute
T3617 7787-7789 IN denotes at
T3618 7790-7792 CD denotes 25
T3619 7792-7793 CD denotes °
T3620 7793-7795 NNP denotes C.
T3816 7797-7801 NNP denotes Cell
T3817 7802-7809 NNP denotes Culture
T3818 7810-7813 NNP denotes All
T3819 7814-7820 NN denotes tissue
T3820 7821-7828 NN denotes culture
T3821 7829-7837 NNS denotes reagents
T3822 7838-7842 VBD denotes were
T3823 7843-7847 IN denotes from
T3824 7848-7858 NNP denotes Invitrogen
T3825 7859-7860 -LRB- denotes (
T3826 7860-7865 NNP denotes Cergy
T3827 7866-7874 NNP denotes Pontoise
T3828 7874-7875 , denotes ,
T3829 7876-7882 NNP denotes France
T3830 7882-7883 -RRB- denotes )
T3831 7883-7884 . denotes .
T3832 7885-7890 CD denotes THP-1
T3833 7891-7896 JJ denotes human
T3834 7897-7906 JJ denotes monocytic
T3835 7907-7912 NNS denotes cells
T3836 7913-7917 VBD denotes were
T3837 7918-7928 VBN denotes maintained
T3838 7929-7931 IN denotes in
T3839 7932-7941 NN denotes RPMI-1640
T3840 7942-7954 VBN denotes supplemented
T3841 7955-7959 IN denotes with
T3842 7960-7962 CD denotes 10
T3843 7962-7963 NN denotes %
T3844 7964-7967 NNP denotes FCS
T3845 7967-7968 , denotes ,
T3846 7969-7970 CD denotes 2
T3847 7971-7973 NNP denotes mM
T3848 7974-7975 NNP denotes L
T3849 7976-7977 : denotes -
T3850 7977-7986 NN denotes glutamine
T3851 7986-7987 , denotes ,
T3852 7988-7990 CD denotes 50
T3853 7991-7995 NN denotes U/ml
T3854 7996-8006 NN denotes penicillin
T3855 8007-8010 CC denotes and
T3856 8011-8013 CD denotes 50
T3857 8014-8019 JJ denotes mg/ml
T3858 8020-8032 NN denotes streptomycin
T3859 8033-8035 IN denotes at
T3860 8036-8038 CD denotes 37
T3861 8038-8039 CD denotes °
T3862 8039-8040 NNP denotes C
T3863 8041-8043 IN denotes in
T3864 8044-8045 DT denotes a
T3865 8046-8047 CD denotes 5
T3866 8047-8048 NN denotes %
T3867 8049-8052 CD denotes CO2
T3868 8053-8062 NN denotes incubator
T3869 8062-8063 . denotes .
T3870 8064-8069 JJ denotes Human
T3871 8070-8080 JJ denotes peripheral
T3872 8081-8086 NN denotes blood
T3873 8087-8098 NN denotes mononuclear
T3874 8099-8104 NNS denotes cells
T3875 8105-8109 VBD denotes were
T3876 8110-8118 VBN denotes obtained
T3877 8119-8123 IN denotes from
T3878 8124-8135 VBN denotes heparinized
T3879 8136-8141 NN denotes blood
T3880 8142-8144 IN denotes by
T3881 8145-8159 JJ denotes Ficoll-Hypaque
T3882 8160-8167 NN denotes density
T3883 8168-8176 NN denotes gradient
T3884 8176-8177 . denotes .
T3885 8178-8187 NNS denotes Monocytes
T3886 8188-8192 VBD denotes were
T3887 8193-8197 RB denotes then
T3888 8198-8206 VBN denotes isolated
T3889 8207-8209 IN denotes by
T3890 8210-8219 NN denotes adherence
T3891 8220-8222 TO denotes to
T3892 8223-8230 NN denotes culture
T3893 8231-8237 NNS denotes flasks
T3894 8238-8240 IN denotes as
T3895 8241-8250 VBN denotes described
T3896 8251-8252 NNP denotes [
T3897 8252-8254 CD denotes 28
T3898 8254-8255 NNP denotes ]
T3899 8255-8256 . denotes .
T3900 8257-8260 IN denotes For
T3901 8261-8265 NN denotes cell
T3902 8266-8277 NN denotes aggregation
T3903 8277-8278 , denotes ,
T3904 8279-8288 NNS denotes monocytes
T3905 8289-8293 VBD denotes were
T3906 8294-8302 VBN denotes cultured
T3907 8303-8305 IN denotes in
T3908 8306-8309 DT denotes the
T3909 8310-8318 NN denotes presence
T3910 8319-8321 CC denotes or
T3911 8322-8329 NN denotes absence
T3912 8330-8332 IN denotes of
T3913 8333-8336 CD denotes C10
T3914 8337-8339 CC denotes or
T3915 8340-8343 CD denotes C40
T3916 8344-8347 IN denotes for
T3917 8348-8350 CD denotes 72
T3918 8351-8353 NN denotes h.
T3919 8354-8358 NNP denotes Cell
T3920 8359-8367 NNS denotes colonies
T3921 8368-8372 VBD denotes were
T3922 8373-8382 VBN denotes monitored
T3923 8383-8388 IN denotes under
T3924 8389-8391 DT denotes an
T3925 8392-8400 JJ denotes inverted
T3926 8401-8406 NN denotes phase
T3927 8407-8415 NN denotes contrast
T3928 8416-8426 NN denotes microscope
T3929 8427-8434 VBN denotes coupled
T3930 8435-8442 IN denotes through
T3931 8443-8444 DT denotes a
T3932 8445-8450 NN denotes video
T3933 8451-8457 NN denotes camera
T3934 8458-8460 TO denotes to
T3935 8461-8462 DT denotes a
T3936 8463-8471 NN denotes computer
T3937 8471-8472 . denotes .
T3938 8473-8475 IN denotes In
T3939 8476-8480 DT denotes some
T3940 8481-8486 NNS denotes wells
T3941 8486-8487 , denotes ,
T3942 8488-8500 VBG denotes neutralizing
T3943 8501-8511 JJ denotes monoclonal
T3944 8512-8520 NN denotes antibody
T3945 8521-8523 TO denotes to
T3946 8524-8530 NNP denotes ICAM-1
T3947 8531-8532 -LRB- denotes (
T3948 8532-8535 CD denotes 2.5
T3949 8536-8538 NN denotes µg
T3950 8538-8539 NN denotes /
T3951 8539-8541 NN denotes ml
T3952 8541-8542 -RRB- denotes )
T3953 8543-8544 -LRB- denotes (
T3954 8544-8548 NNP denotes Tebu
T3955 8548-8549 , denotes ,
T3956 8550-8552 NNP denotes Le
T3957 8553-8559 NNP denotes Perray
T3958 8560-8562 IN denotes en
T3959 8563-8571 NNP denotes Yvelines
T3960 8571-8572 , denotes ,
T3961 8573-8579 NNP denotes France
T3962 8579-8580 -RRB- denotes )
T3963 8581-8584 VBD denotes was
T3964 8585-8590 VBN denotes added
T3965 8590-8591 . denotes .
T4100 8593-8597 NNP denotes Cell
T4101 8598-8603 NNP denotes Cycle
T4102 8604-8612 NNP denotes Analysis
T4103 8613-8618 NNP denotes THP-1
T4104 8619-8624 NNS denotes cells
T4105 8625-8627 IN denotes in
T4106 8628-8639 JJ denotes exponential
T4107 8640-8646 NN denotes growth
T4108 8647-8652 NN denotes phase
T4109 8653-8657 VBD denotes were
T4110 8658-8665 VBN denotes exposed
T4111 8666-8668 TO denotes to
T4112 8669-8677 VB denotes complete
T4113 8678-8684 NN denotes medium
T4114 8685-8687 IN denotes in
T4115 8688-8691 DT denotes the
T4116 8692-8700 NN denotes presence
T4117 8701-8703 CC denotes or
T4118 8704-8711 NN denotes absence
T4119 8712-8714 IN denotes of
T4120 8715-8727 NNS denotes carrageenans
T4121 8728-8731 IN denotes for
T4122 8732-8734 CD denotes 24
T4123 8735-8736 NN denotes h
T4124 8737-8743 IN denotes before
T4125 8744-8749 VBG denotes being
T4126 8750-8757 VBN denotes stained
T4127 8758-8762 IN denotes with
T4128 8763-8772 NN denotes propidium
T4129 8773-8779 NN denotes iodide
T4130 8780-8785 VBG denotes using
T4131 8786-8789 DT denotes the
T4132 8790-8798 NNP denotes DNA-Prep
T4133 8799-8806 NNP denotes Coulter
T4134 8807-8810 NN denotes kit
T4135 8811-8820 VBG denotes according
T4136 8821-8823 TO denotes to
T4137 8824-8827 DT denotes the
T4138 8828-8840 NN denotes manufacturer
T4139 8840-8842 POS denotes 's
T4140 8843-8854 NN denotes instruction
T4141 8855-8856 -LRB- denotes (
T4142 8856-8871 NNP denotes Beckman-Coulter
T4143 8871-8872 , denotes ,
T4144 8873-8883 NNP denotes Villepinte
T4145 8883-8884 , denotes ,
T4146 8885-8891 NNP denotes France
T4147 8891-8892 -RRB- denotes )
T4148 8892-8893 . denotes .
T4149 8894-8898 NNP denotes Cell
T4150 8899-8902 NNP denotes DNA
T4151 8903-8910 NN denotes content
T4152 8911-8914 VBD denotes was
T4153 8915-8919 RB denotes then
T4154 8920-8928 VBN denotes analyzed
T4155 8929-8931 IN denotes by
T4156 8932-8936 NN denotes flow
T4157 8937-8946 NN denotes cytometry
T4158 8947-8952 VBG denotes using
T4159 8953-8955 DT denotes an
T4160 8956-8961 NNP denotes EPICS
T4161 8962-8965 NNP denotes XL2
T4162 8966-8967 -LRB- denotes (
T4163 8967-8982 NNP denotes Beckman-Coulter
T4164 8982-8983 -RRB- denotes )
T4165 8983-8984 . denotes .
T4166 8985-8988 JJ denotes Raw
T4167 8989-8993 NNS denotes data
T4168 8994-8997 IN denotes for
T4169 8998-9001 DT denotes the
T4170 9002-9014 NN denotes distribution
T4171 9015-9017 IN denotes of
T4172 9018-9021 NNP denotes DNA
T4173 9022-9029 NN denotes content
T4174 9030-9032 IN denotes of
T4175 9033-9039 CD denotes 30,000
T4176 9040-9045 NNS denotes cells
T4177 9046-9055 VBN denotes retrieved
T4178 9056-9060 IN denotes from
T4179 9061-9064 DT denotes the
T4180 9065-9074 NN denotes cytometer
T4181 9075-9079 VBD denotes were
T4182 9080-9089 VBN denotes expressed
T4183 9090-9092 IN denotes as
T4184 9093-9096 DT denotes the
T4185 9097-9107 NN denotes percentage
T4186 9108-9110 IN denotes of
T4187 9111-9116 CD denotes G0/G1
T4188 9117-9124 IN denotes through
T4189 9125-9129 NNP denotes G2/M
T4190 9130-9141 NNS denotes populations
T4191 9141-9142 . denotes .
T4192 9143-9153 NNP denotes Multicycle
T4193 9154-9156 NNP denotes AV
T4194 9157-9165 NN denotes software
T4195 9166-9167 -LRB- denotes (
T4196 9167-9174 NNP denotes Phoenix
T4197 9175-9179 NNP denotes Flow
T4198 9180-9187 NNPS denotes Systems
T4199 9187-9188 , denotes ,
T4200 9189-9192 NNP denotes San
T4201 9193-9198 NNP denotes Diego
T4202 9198-9199 , denotes ,
T4203 9200-9202 NNP denotes CA
T4204 9202-9203 -RRB- denotes )
T4205 9204-9207 VBD denotes was
T4206 9208-9212 VBN denotes used
T4207 9213-9215 TO denotes to
T4208 9216-9224 VB denotes generate
T4209 9225-9228 NNP denotes DNA
T4210 9229-9236 JJ denotes content
T4211 9237-9246 NN denotes frequency
T4212 9247-9257 NNS denotes histograms
T4213 9258-9261 CC denotes and
T4214 9262-9272 VB denotes facilitate
T4215 9273-9277 NNS denotes data
T4216 9278-9286 NN denotes analysis
T4217 9286-9287 . denotes .
T4382 9289-9293 NNP denotes Cell
T4383 9294-9301 NNP denotes Surface
T4384 9302-9309 NNP denotes Antigen
T4385 9310-9320 NNP denotes Expression
T4386 9321-9329 NNP denotes Analysis
T4387 9330-9340 NNP denotes Peripheral
T4388 9341-9346 NNP denotes Blood
T4389 9347-9356 NNPS denotes Monocytes
T4390 9357-9359 CC denotes or
T4391 9360-9365 CD denotes THP-1
T4392 9366-9371 NNS denotes cells
T4393 9372-9376 VBD denotes were
T4394 9377-9384 VBN denotes exposed
T4395 9385-9387 TO denotes to
T4396 9388-9396 VB denotes complete
T4397 9397-9403 NN denotes medium
T4398 9404-9406 IN denotes in
T4399 9407-9410 DT denotes the
T4400 9411-9419 NN denotes presence
T4401 9420-9422 CC denotes or
T4402 9423-9430 NN denotes absence
T4403 9431-9433 IN denotes of
T4404 9434-9445 NN denotes carrageenan
T4405 9446-9449 IN denotes for
T4406 9450-9452 CD denotes 36
T4407 9453-9454 NN denotes h
T4408 9454-9455 . denotes .
T4409 9456-9461 IN denotes After
T4410 9462-9465 CD denotes two
T4411 9466-9472 NNS denotes washes
T4412 9473-9475 IN denotes in
T4413 9476-9479 NNP denotes PBS
T4414 9480-9487 IN denotes without
T4415 9488-9491 NNP denotes Ca2
T4416 9491-9492 NNP denotes +
T4417 9493-9496 CC denotes and
T4418 9497-9500 NNP denotes Mg2
T4419 9500-9501 NNP denotes +
T4420 9501-9502 , denotes ,
T4421 9503-9508 NNS denotes cells
T4422 9509-9513 VBD denotes were
T4423 9514-9523 VBN denotes incubated
T4424 9524-9526 IN denotes in
T4425 9527-9530 NNP denotes PBS
T4426 9531-9541 VBG denotes containing
T4427 9542-9545 CD denotes 0.1
T4428 9545-9546 NN denotes %
T4429 9547-9554 NN denotes gelatin
T4430 9555-9558 CC denotes and
T4431 9559-9560 CD denotes 8
T4432 9560-9561 NN denotes %
T4433 9562-9564 NNP denotes AB
T4434 9565-9570 JJ denotes human
T4435 9571-9576 NN denotes serum
T4436 9577-9579 TO denotes to
T4437 9580-9587 VB denotes prevent
T4438 9588-9595 JJ denotes binding
T4439 9596-9598 TO denotes to
T4440 9599-9601 VB denotes Fc
T4441 9602-9611 NNS denotes receptors
T4442 9611-9612 . denotes .
T4443 9613-9617 RB denotes Then
T4444 9617-9618 , denotes ,
T4445 9619-9620 CD denotes 5
T4446 9620-9621 NN denotes ×
T4447 9621-9624 CD denotes 105
T4448 9625-9630 NNS denotes cells
T4449 9631-9635 VBD denotes were
T4450 9636-9645 VBN denotes incubated
T4451 9646-9650 IN denotes with
T4452 9651-9658 JJ denotes primary
T4453 9659-9669 NNS denotes antibodies
T4454 9670-9672 IN denotes at
T4455 9673-9674 CD denotes 4
T4456 9674-9675 CD denotes °
T4457 9675-9676 NNP denotes C
T4458 9677-9680 IN denotes for
T4459 9681-9683 CD denotes 30
T4460 9684-9687 NN denotes min
T4461 9687-9688 . denotes .
T4462 9689-9692 CD denotes Two
T4463 9693-9698 JJ denotes other
T4464 9699-9705 NNS denotes washes
T4465 9706-9708 IN denotes in
T4466 9709-9712 NNP denotes PBS
T4467 9713-9721 VBD denotes preceded
T4468 9722-9732 NN denotes incubation
T4469 9733-9737 IN denotes with
T4470 9738-9753 JJ denotes FITC-conjugated
T4471 9754-9758 NN denotes goat
T4472 9759-9767 NN denotes antibody
T4473 9768-9778 JJ denotes anti-mouse
T4474 9779-9782 NNP denotes IgG
T4475 9783-9790 VBD denotes diluted
T4476 9791-9797 CD denotes 1/1000
T4477 9798-9800 IN denotes at
T4478 9801-9802 CD denotes 4
T4479 9802-9803 CD denotes °
T4480 9803-9804 NNP denotes C
T4481 9805-9808 IN denotes for
T4482 9809-9811 CD denotes 30
T4483 9812-9815 NN denotes min
T4484 9816-9817 -LRB- denotes (
T4485 9817-9821 NNP denotes Tebu
T4486 9821-9822 -RRB- denotes )
T4487 9822-9823 . denotes .
T4488 9824-9829 IN denotes After
T4489 9830-9833 CD denotes two
T4490 9834-9844 JJ denotes additional
T4491 9845-9851 NNS denotes washes
T4492 9851-9852 , denotes ,
T4493 9853-9861 NN denotes analysis
T4494 9862-9864 IN denotes of
T4495 9865-9872 JJ denotes stained
T4496 9873-9878 NNS denotes cells
T4497 9879-9882 VBD denotes was
T4498 9883-9892 VBN denotes performed
T4499 9893-9895 IN denotes on
T4500 9896-9898 DT denotes an
T4501 9899-9904 NNP denotes EPICS
T4502 9905-9908 NNP denotes XL2
T4503 9909-9910 -LRB- denotes (
T4504 9910-9925 NNP denotes Beckman-Coulter
T4505 9925-9926 -RRB- denotes )
T4506 9926-9927 . denotes .
T4507 9928-9931 DT denotes The
T4508 9932-9936 NN denotes cell
T4509 9937-9947 NN denotes population
T4510 9948-9951 VBD denotes was
T4511 9952-9957 VBN denotes gated
T4512 9958-9967 VBG denotes according
T4513 9968-9970 TO denotes to
T4514 9971-9974 PRP$ denotes its
T4515 9975-9982 JJ denotes forward
T4516 9983-9986 CC denotes and
T4517 9987-9997 JJ denotes wide-angle
T4518 9998-10003 JJ denotes light
T4519 10004-10014 NN denotes scattering
T4520 10014-10015 . denotes .
T4521 10016-10020 NNS denotes Data
T4522 10021-10025 VBD denotes were
T4523 10026-10035 VBN denotes expressed
T4524 10036-10038 IN denotes as
T4525 10039-10043 JJ denotes mean
T4526 10044-10052 JJ denotes relative
T4527 10053-10065 NN denotes fluorescence
T4528 10066-10075 NN denotes intensity
T4529 10076-10077 -LRB- denotes (
T4530 10077-10080 NNP denotes MFI
T4531 10080-10081 -RRB- denotes )
T4532 10082-10084 IN denotes of
T4533 10085-10089 CD denotes 3000
T4534 10090-10095 NNS denotes cells
T4535 10095-10096 . denotes .
T4648 10098-10101 NNP denotes TNF
T4649 10102-10110 NNP denotes Activity
T4650 10111-10119 NNP denotes Bioassay
T4651 10120-10129 NNPS denotes Monocytes
T4652 10130-10132 CC denotes or
T4653 10133-10138 CD denotes THP-1
T4654 10139-10144 NNS denotes cells
T4655 10145-10149 VBD denotes were
T4656 10150-10158 VBN denotes cultured
T4657 10159-10163 IN denotes with
T4658 10164-10166 CC denotes or
T4659 10167-10174 IN denotes without
T4660 10175-10184 JJ denotes different
T4661 10185-10199 NNS denotes concentrations
T4662 10200-10202 IN denotes of
T4663 10203-10207 NNS denotes CGNs
T4664 10208-10210 CC denotes or
T4665 10211-10214 NNP denotes LPS
T4666 10215-10216 -LRB- denotes (
T4667 10216-10226 NNP denotes Salmonella
T4668 10227-10234 NN denotes typhosa
T4669 10234-10235 , denotes ,
T4670 10236-10241 NNP denotes Sigma
T4671 10241-10242 -RRB- denotes )
T4672 10243-10246 IN denotes for
T4673 10247-10249 CD denotes 24
T4674 10250-10251 NN denotes h
T4675 10252-10254 CC denotes or
T4676 10255-10258 DT denotes the
T4677 10259-10268 JJ denotes indicated
T4678 10269-10273 NN denotes time
T4679 10273-10274 . denotes .
T4680 10275-10287 RB denotes Biologically
T4681 10288-10294 JJ denotes active
T4682 10295-10300 NNP denotes TNF-α
T4683 10300-10301 NN denotes /
T4684 10301-10302 NN denotes β
T4685 10303-10305 IN denotes in
T4686 10306-10312 NN denotes tissue
T4687 10313-10320 NN denotes culture
T4688 10321-10332 NN denotes supernatant
T4689 10333-10336 VBD denotes was
T4690 10337-10345 VBN denotes measured
T4691 10346-10351 VBG denotes using
T4692 10352-10355 DT denotes the
T4693 10356-10360 NNP denotes WEHI
T4694 10361-10364 CD denotes 164
T4695 10365-10370 NN denotes clone
T4696 10371-10378 JJ denotes 13-cell
T4697 10379-10386 VBG denotes killing
T4698 10387-10392 NN denotes assay
T4699 10393-10394 NNP denotes [
T4700 10394-10396 CD denotes 29
T4701 10396-10397 NNP denotes ]
T4702 10397-10398 . denotes .
T4703 10399-10402 NNP denotes TNF
T4704 10403-10417 NNS denotes concentrations
T4705 10418-10421 VBP denotes are
T4706 10422-10431 VBN denotes expressed
T4707 10432-10434 IN denotes as
T4708 10435-10440 NN denotes pg/ml
T4709 10440-10441 . denotes .
T4951 10443-10449 NNP denotes RT-PCR
T4952 10450-10458 NNP denotes Analysis
T4953 10459-10464 NNP denotes Total
T4954 10465-10468 NNP denotes RNA
T4955 10469-10473 IN denotes from
T4956 10474-10483 NNS denotes monocytes
T4957 10484-10487 VBD denotes was
T4958 10488-10496 VBN denotes isolated
T4959 10497-10502 VBG denotes using
T4960 10503-10509 NNP denotes TRIzol
T4961 10510-10517 NNP denotes Reagent
T4962 10517-10518 NNP denotes ™
T4963 10519-10520 -LRB- denotes (
T4964 10520-10530 NNP denotes Invitrogen
T4965 10530-10531 -RRB- denotes )
T4966 10531-10532 . denotes .
T4967 10533-10537 NNP denotes cDNA
T4968 10538-10541 VBD denotes was
T4969 10542-10551 VBN denotes generated
T4970 10552-10554 IN denotes on
T4971 10555-10556 CD denotes 1
T4972 10557-10559 NN denotes µg
T4973 10560-10562 IN denotes of
T4974 10563-10568 JJ denotes total
T4975 10569-10572 NNP denotes RNA
T4976 10573-10575 IN denotes in
T4977 10576-10577 DT denotes a
T4978 10578-10586 NN denotes reaction
T4979 10587-10593 NN denotes volume
T4980 10594-10596 IN denotes of
T4981 10597-10599 CD denotes 20
T4982 10600-10602 NN denotes µl
T4983 10602-10603 , denotes ,
T4984 10604-10609 VBG denotes using
T4985 10610-10615 NNP denotes M-MLV
T4986 10616-10623 VB denotes reverse
T4987 10624-10637 NN denotes transcriptase
T4988 10638-10639 -LRB- denotes (
T4989 10639-10649 NNP denotes Invitrogen
T4990 10649-10650 -RRB- denotes )
T4991 10650-10651 . denotes .
T4992 10652-10655 NNP denotes PCR
T4993 10656-10659 VBD denotes was
T4994 10660-10664 VBN denotes done
T4995 10665-10667 IN denotes in
T4996 10668-10671 DT denotes the
T4997 10672-10678 JJ denotes linear
T4998 10679-10684 NN denotes range
T4999 10685-10687 IN denotes of
T5000 10688-10701 NN denotes amplification
T5001 10702-10703 -LRB- denotes (
T5002 10703-10713 VBN denotes determined
T5003 10714-10717 IN denotes for
T5004 10718-10722 DT denotes each
T5005 10723-10729 NN denotes primer
T5006 10730-10739 NN denotes pair-cDNA
T5007 10740-10751 NN denotes combination
T5008 10751-10752 -RRB- denotes )
T5009 10752-10753 . denotes .
T5010 10754-10762 NNP denotes Standard
T5011 10763-10766 NNP denotes PCR
T5012 10767-10776 NNS denotes reactions
T5013 10777-10781 VBD denotes were
T5014 10782-10791 VBN denotes performed
T5015 10792-10796 IN denotes with
T5016 10797-10798 CD denotes 1
T5017 10799-10801 NN denotes µl
T5018 10802-10804 IN denotes of
T5019 10805-10808 DT denotes the
T5020 10809-10813 NNP denotes cDNA
T5021 10814-10822 NN denotes solution
T5022 10822-10823 , denotes ,
T5023 10824-10826 CD denotes 50
T5024 10827-10829 NN denotes µM
T5025 10830-10832 IN denotes of
T5026 10833-10837 DT denotes each
T5027 10838-10844 NN denotes primer
T5028 10845-10853 NN denotes solution
T5029 10853-10854 , denotes ,
T5030 10855-10857 CD denotes 10
T5031 10858-10860 NN denotes mM
T5032 10861-10863 IN denotes of
T5033 10864-10868 DT denotes each
T5034 10869-10873 NNP denotes dNTP
T5035 10873-10874 , denotes ,
T5036 10875-10877 CD denotes 25
T5037 10878-10880 NNP denotes mM
T5038 10881-10886 NNP denotes MgCl2
T5039 10886-10887 , denotes ,
T5040 10888-10891 NNP denotes 10X
T5041 10892-10900 NNP denotes Goldstar
T5042 10901-10904 NNP denotes DNA
T5043 10905-10915 NN denotes polymerase
T5044 10916-10924 NN denotes reaction
T5045 10925-10931 NN denotes buffer
T5046 10931-10932 , denotes ,
T5047 10933-10936 CC denotes and
T5048 10937-10940 CD denotes 0.5
T5049 10941-10946 NNS denotes units
T5050 10947-10949 IN denotes of
T5051 10950-10958 NNP denotes Goldstar
T5052 10959-10962 NNP denotes DNA
T5053 10963-10973 NN denotes polymerase
T5054 10974-10975 -LRB- denotes (
T5055 10975-10985 NNP denotes Eurogentec
T5056 10985-10986 , denotes ,
T5057 10987-10994 NNP denotes Seraing
T5058 10994-10995 , denotes ,
T5059 10996-11003 NNP denotes Belgium
T5060 11003-11004 -RRB- denotes )
T5061 11004-11005 . denotes .
T5062 11006-11011 NNP denotes First
T5063 11012-11015 NNP denotes PCR
T5064 11016-11021 NN denotes cycle
T5065 11022-11031 VBD denotes consisted
T5066 11032-11034 IN denotes of
T5067 11035-11036 CD denotes 1
T5068 11037-11040 NN denotes min
T5069 11041-11043 IN denotes at
T5070 11044-11046 CD denotes 92
T5071 11046-11047 CD denotes °
T5072 11047-11048 NNP denotes C
T5073 11048-11049 , denotes ,
T5074 11050-11051 CD denotes 1
T5075 11052-11055 NN denotes min
T5076 11056-11058 IN denotes at
T5077 11059-11061 CD denotes 58
T5078 11061-11062 CD denotes °
T5079 11062-11063 NNP denotes C
T5080 11064-11067 CC denotes and
T5081 11068-11069 CD denotes 1
T5082 11070-11073 NN denotes min
T5083 11074-11076 IN denotes at
T5084 11077-11079 CD denotes 72
T5085 11079-11080 CD denotes °
T5086 11080-11081 NNP denotes C
T5087 11081-11082 : denotes ;
T5088 11083-11087 RB denotes then
T5089 11088-11092 DT denotes each
T5090 11093-11096 NNP denotes PCR
T5091 11097-11102 NN denotes cycle
T5092 11103-11112 VBD denotes consisted
T5093 11113-11115 IN denotes of
T5094 11116-11118 CD denotes 40
T5095 11119-11122 NN denotes sec
T5096 11123-11125 IN denotes at
T5097 11126-11128 CD denotes 92
T5098 11128-11129 CD denotes °
T5099 11129-11130 NNP denotes C
T5100 11130-11131 , denotes ,
T5101 11132-11134 CD denotes 40
T5102 11135-11138 NN denotes sec
T5103 11139-11141 IN denotes at
T5104 11142-11144 CD denotes 58
T5105 11144-11145 CD denotes °
T5106 11145-11146 NNP denotes C
T5107 11147-11150 CC denotes and
T5108 11151-11153 CD denotes 50
T5109 11154-11157 NN denotes sec
T5110 11158-11160 IN denotes at
T5111 11161-11163 CD denotes 72
T5112 11163-11164 CD denotes °
T5113 11164-11166 NNP denotes C.
T5114 11167-11171 NNP denotes cDNA
T5115 11172-11175 IN denotes for
T5116 11176-11183 NN denotes β-actin
T5117 11184-11187 VBD denotes was
T5118 11188-11197 VBN denotes amplified
T5119 11198-11201 IN denotes for
T5120 11202-11204 CD denotes 28
T5121 11205-11211 NNS denotes cycles
T5122 11212-11217 VBG denotes using
T5123 11218-11221 DT denotes the
T5124 11222-11228 NNS denotes oligos
T5125 11228-11229 : denotes :
T5126 11230-11235 NN denotes sense
T5127 11236-11237 CD denotes 5
T5128 11237-11238 SYM denotes ′
T5129 11238-11239 : denotes -
T5130 11239-11260 NN denotes GGCATCGTGATGGACTCCG-3
T5131 11260-11261 NN denotes ′
T5132 11262-11265 CC denotes and
T5133 11266-11275 JJ denotes antisense
T5134 11276-11277 CD denotes 5
T5135 11277-11278 CD denotes ′
T5136 11278-11299 NNP denotes GCTGGAAGGTGGACAGCGA-3
T5137 11299-11300 NN denotes ′
T5138 11300-11301 . denotes .
T5139 11302-11306 NNP denotes cDNA
T5140 11307-11310 IN denotes for
T5141 11311-11316 NNP denotes TNF-α
T5142 11317-11320 VBD denotes was
T5143 11321-11330 VBN denotes amplified
T5144 11331-11334 IN denotes for
T5145 11335-11337 CD denotes 35
T5146 11338-11344 NNS denotes cycles
T5147 11345-11350 VBG denotes using
T5148 11351-11354 DT denotes the
T5149 11355-11361 NNS denotes oligos
T5150 11361-11362 : denotes :
T5151 11363-11368 NN denotes sense
T5152 11369-11370 CD denotes 5
T5153 11370-11371 SYM denotes ′
T5154 11371-11372 : denotes -
T5155 11372-11394 NN denotes AAGCCTGTAGCCCATGTTGT-3
T5156 11394-11395 NN denotes ′
T5157 11396-11399 CC denotes and
T5158 11400-11409 JJ denotes antisense
T5159 11410-11411 CD denotes 5
T5160 11411-11412 SYM denotes ′
T5161 11412-11413 : denotes -
T5162 11413-11435 NN denotes CAGATAGATGGGCTCATACC-3
T5163 11435-11436 NN denotes ′
T5164 11436-11437 . denotes .
T5165 11438-11442 NNP denotes cDNA
T5166 11443-11446 IN denotes for
T5167 11447-11453 NNP denotes ICAM-1
T5168 11454-11457 VBD denotes was
T5169 11458-11467 VBN denotes amplified
T5170 11468-11471 IN denotes for
T5171 11472-11474 CD denotes 35
T5172 11475-11481 NNS denotes cycles
T5173 11482-11487 VBG denotes using
T5174 11488-11491 DT denotes the
T5175 11492-11498 NNS denotes oligos
T5176 11499-11504 VBP denotes sense
T5177 11505-11506 CD denotes 5
T5178 11506-11507 SYM denotes ′
T5179 11507-11508 : denotes -
T5180 11508-11532 NN denotes GTAGCAGCCGCAGTCATAATGG-3
T5181 11532-11533 NN denotes ′
T5182 11534-11537 CC denotes and
T5183 11538-11547 JJ denotes antisense
T5184 11548-11549 CD denotes 5
T5185 11549-11550 SYM denotes ′
T5186 11550-11551 : denotes -
T5187 11551-11552 DT denotes A
T5188 11553-11576 JJ denotes TGCTGTTGTATCTGACTGAGG-3
T5189 11576-11577 NN denotes ′
T5190 11577-11578 . denotes .
T5335 11580-11585 NNP denotes NF-kB
T5336 11586-11599 NNP denotes Transcription
T5337 11600-11608 NNP denotes Reporter
T5338 11609-11613 NNP denotes Gene
T5339 11614-11619 NNP denotes Assay
T5340 11620-11623 NNP denotes The
T5341 11624-11631 VBD denotes plasmid
T5342 11632-11641 NNP denotes 3XMHC-luc
T5343 11642-11643 -LRB- denotes (
T5344 11643-11644 DT denotes a
T5345 11645-11653 JJ denotes generous
T5346 11654-11658 NN denotes gift
T5347 11659-11663 IN denotes from
T5348 11664-11668 NNP denotes Drs.
T5349 11669-11671 NNP denotes J.
T5350 11672-11680 NNP denotes Westwick
T5351 11681-11684 CC denotes and
T5352 11685-11689 NNP denotes D.A.
T5353 11690-11697 NNP denotes Brenner
T5354 11697-11698 , denotes ,
T5355 11699-11709 NNP denotes University
T5356 11710-11712 IN denotes of
T5357 11713-11718 NNP denotes North
T5358 11719-11727 NNP denotes Carolina
T5359 11727-11728 , denotes ,
T5360 11729-11735 NNP denotes Chapel
T5361 11736-11740 NNP denotes Hill
T5362 11740-11741 -RRB- denotes )
T5363 11742-11750 VBZ denotes contains
T5364 11751-11756 CD denotes three
T5365 11757-11763 NNS denotes copies
T5366 11764-11766 IN denotes of
T5367 11767-11783 JJ denotes NF-κB-responsive
T5368 11784-11791 NN denotes element
T5369 11792-11796 IN denotes from
T5370 11797-11800 DT denotes the
T5371 11801-11804 NNP denotes MHC
T5372 11805-11810 NN denotes class
T5373 11811-11812 PRP denotes I
T5374 11813-11818 VBZ denotes locus
T5375 11818-11819 , denotes ,
T5376 11820-11826 VBN denotes placed
T5377 11827-11835 RB denotes upstream
T5378 11836-11838 IN denotes of
T5379 11839-11842 DT denotes the
T5380 11843-11853 NN denotes luciferase
T5381 11854-11858 NN denotes gene
T5382 11858-11859 . denotes .
T5383 11860-11865 JJ denotes Human
T5384 11866-11875 JJ denotes monocytic
T5385 11876-11881 NN denotes THP-1
T5386 11882-11887 NNS denotes cells
T5387 11888-11892 VBD denotes were
T5388 11893-11904 RB denotes transiently
T5389 11905-11916 VBN denotes transfected
T5390 11917-11919 IN denotes as
T5391 11920-11930 RB denotes previously
T5392 11931-11940 VBN denotes described
T5393 11941-11942 NNP denotes [
T5394 11942-11944 CD denotes 30
T5395 11944-11945 NNP denotes ]
T5396 11945-11946 , denotes ,
T5397 11947-11950 CC denotes and
T5398 11951-11955 RB denotes then
T5399 11956-11964 VBD denotes cultured
T5400 11965-11968 IN denotes for
T5401 11969-11970 CD denotes 4
T5402 11971-11972 NN denotes h
T5403 11973-11978 RB denotes alone
T5404 11979-11981 CC denotes or
T5405 11982-11986 IN denotes with
T5406 11987-11997 VBG denotes increasing
T5407 11998-12011 NN denotes concentration
T5408 12012-12014 IN denotes of
T5409 12015-12021 DT denotes either
T5410 12022-12025 NNP denotes C10
T5411 12026-12028 CC denotes or
T5412 12029-12032 NNP denotes C40
T5413 12032-12033 . denotes .
T5414 12034-12044 JJ denotes Luciferase
T5415 12045-12053 NN denotes activity
T5416 12054-12057 VBD denotes was
T5417 12058-12068 VBN denotes determined
T5418 12069-12074 VBG denotes using
T5419 12075-12076 DT denotes a
T5420 12077-12088 NN denotes luminometer
T5421 12089-12090 -LRB- denotes (
T5422 12090-12099 NNP denotes Monolight
T5423 12100-12104 CD denotes 2010
T5424 12105-12116 NNP denotes Luminometer
T5425 12116-12117 , denotes ,
T5426 12118-12121 NNP denotes Ann
T5427 12122-12127 NNP denotes Arbor
T5428 12127-12128 , denotes ,
T5429 12129-12131 NNP denotes MI
T5430 12131-12132 -RRB- denotes )
T5431 12132-12133 . denotes .
T5933 12135-12142 JJ denotes Western
T5934 12143-12147 NNP denotes Blot
T5935 12148-12156 NNP denotes Analysis
T5936 12157-12162 NNP denotes THP-1
T5937 12163-12168 NNS denotes cells
T5938 12169-12173 VBD denotes were
T5939 12174-12184 VBN denotes stimulated
T5940 12185-12188 IN denotes for
T5941 12189-12196 JJ denotes various
T5942 12197-12204 NNS denotes lengths
T5943 12205-12207 IN denotes of
T5944 12208-12212 NN denotes time
T5945 12213-12217 IN denotes with
T5946 12218-12221 CD denotes 0.1
T5947 12222-12227 NN denotes mg/ml
T5948 12228-12231 CD denotes C10
T5949 12232-12234 CC denotes or
T5950 12235-12238 CD denotes C40
T5951 12238-12239 , denotes ,
T5952 12240-12242 CC denotes or
T5953 12243-12245 CD denotes 10
T5954 12246-12248 NN denotes µg
T5955 12248-12249 NN denotes /
T5956 12249-12251 NN denotes ml
T5957 12252-12255 NNP denotes LPS
T5958 12255-12256 . denotes .
T5959 12257-12262 NNS denotes Cells
T5960 12263-12267 VBD denotes were
T5961 12268-12272 RB denotes then
T5962 12273-12281 VBN denotes pelleted
T5963 12281-12282 , denotes ,
T5964 12283-12289 VBN denotes washed
T5965 12290-12293 CC denotes and
T5966 12294-12305 VBN denotes homogenised
T5967 12306-12308 IN denotes in
T5968 12309-12314 NN denotes lysis
T5969 12315-12321 NN denotes buffer
T5970 12322-12323 -LRB- denotes (
T5971 12323-12325 CD denotes 10
T5972 12326-12328 NNP denotes mM
T5973 12329-12334 NNP denotes Hepes
T5974 12334-12335 , denotes ,
T5975 12336-12338 NNP denotes pH
T5976 12339-12342 CD denotes 7.9
T5977 12342-12343 , denotes ,
T5978 12344-12347 CD denotes 150
T5979 12348-12350 NNP denotes mM
T5980 12351-12355 NNP denotes NaCl
T5981 12355-12356 , denotes ,
T5982 12357-12358 CD denotes 1
T5983 12359-12361 NNP denotes mM
T5984 12362-12366 NNP denotes EDTA
T5985 12366-12367 , denotes ,
T5986 12368-12371 CD denotes 0.6
T5987 12371-12372 NN denotes %
T5988 12373-12378 NN denotes NP-40
T5989 12378-12379 , denotes ,
T5990 12380-12383 CC denotes and
T5991 12384-12387 CD denotes 0.5
T5992 12388-12390 NNP denotes mM
T5993 12391-12395 NNP denotes PMSF
T5994 12395-12396 -RRB- denotes )
T5995 12397-12399 IN denotes on
T5996 12400-12403 NN denotes ice
T5997 12403-12404 . denotes .
T5998 12405-12416 NNS denotes Homogenates
T5999 12417-12421 VBD denotes were
T6000 12422-12431 VBN denotes sonicated
T6001 12431-12432 , denotes ,
T6002 12433-12444 VBN denotes centrifuged
T6003 12445-12447 IN denotes at
T6004 12448-12454 CD denotes 10,000
T6005 12455-12458 NN denotes rpm
T6006 12459-12461 TO denotes to
T6007 12462-12468 VB denotes remove
T6008 12469-12477 JJ denotes cellular
T6009 12478-12484 NN denotes debris
T6010 12484-12485 , denotes ,
T6011 12486-12489 CC denotes and
T6012 12490-12501 JJ denotes supernatant
T6013 12502-12511 VBN denotes collected
T6014 12511-12512 . denotes .
T6015 12513-12520 NN denotes Protein
T6016 12521-12534 NN denotes concentration
T6017 12535-12538 VBD denotes was
T6018 12539-12549 VBN denotes determined
T6019 12550-12555 VBG denotes using
T6020 12556-12559 DT denotes the
T6021 12560-12562 NNP denotes DC
T6022 12563-12570 NNP denotes Protein
T6023 12571-12576 NNP denotes Assay
T6024 12577-12578 -LRB- denotes (
T6025 12578-12585 NNP denotes Bio-Rad
T6026 12585-12586 -RRB- denotes )
T6027 12586-12587 . denotes .
T6028 12588-12596 NNPS denotes Proteins
T6029 12597-12599 IN denotes in
T6030 12600-12607 NNS denotes samples
T6031 12608-12609 -LRB- denotes (
T6032 12609-12611 CD denotes 15
T6033 12612-12614 NN denotes µg
T6034 12615-12620 JJ denotes total
T6035 12621-12629 NNS denotes proteins
T6036 12629-12630 -RRB- denotes )
T6037 12631-12635 VBD denotes were
T6038 12636-12644 VBN denotes resolved
T6039 12645-12647 IN denotes in
T6040 12648-12649 DT denotes a
T6041 12650-12660 VBG denotes denaturing
T6042 12661-12663 CD denotes 12
T6043 12663-12664 NN denotes %
T6044 12665-12679 NN denotes polyacrylamide
T6045 12680-12683 NN denotes gel
T6046 12684-12687 CC denotes and
T6047 12688-12699 VBN denotes transferred
T6048 12700-12702 TO denotes to
T6049 12703-12704 DT denotes a
T6050 12705-12719 JJ denotes nitrocellulose
T6051 12720-12728 NN denotes membrane
T6052 12728-12729 . denotes .
T6053 12730-12735 JJ denotes I-κBα
T6054 12736-12743 NN denotes protein
T6055 12744-12747 VBD denotes was
T6056 12748-12756 VBN denotes detected
T6057 12757-12762 VBG denotes using
T6058 12763-12764 DT denotes a
T6059 12765-12771 NN denotes rabbit
T6060 12772-12782 NN denotes polyclonal
T6061 12783-12791 NN denotes antibody
T6062 12792-12793 -LRB- denotes (
T6063 12793-12798 NNP denotes Santa
T6064 12799-12803 NNP denotes Cruz
T6065 12804-12817 NNP denotes Biotechnology
T6066 12817-12818 , denotes ,
T6067 12819-12821 NNP denotes CA
T6068 12821-12822 -RRB- denotes )
T6069 12823-12831 VBN denotes followed
T6070 12832-12834 IN denotes by
T6071 12835-12836 DT denotes a
T6072 12837-12848 JJ denotes horseradish
T6073 12849-12867 JJ denotes peroxidase-coupled
T6074 12868-12872 NN denotes goat
T6075 12873-12883 NN denotes polyclonal
T6076 12884-12892 NN denotes antibody
T6077 12893-12900 IN denotes against
T6078 12901-12907 NN denotes rabbit
T6079 12908-12910 NNP denotes Ig
T6080 12911-12912 -LRB- denotes (
T6081 12912-12918 NNP denotes Caltag
T6082 12919-12931 NNPS denotes Laboratories
T6083 12931-12932 -RRB- denotes )
T6084 12932-12933 . denotes .
T6085 12934-12941 RB denotes Finally
T6086 12941-12942 , denotes ,
T6087 12943-12946 NNP denotes IκB
T6088 12947-12952 NNS denotes bands
T6089 12953-12957 VBD denotes were
T6090 12958-12966 VBN denotes revealed
T6091 12967-12972 VBG denotes using
T6092 12973-12976 DT denotes the
T6093 12977-12980 NNP denotes ECL
T6094 12980-12981 NN denotes ™
T6095 12982-12991 NN denotes detection
T6096 12992-12998 NN denotes system
T6097 12999-13000 -LRB- denotes (
T6098 13000-13008 NNP denotes Amersham
T6099 13009-13018 NNP denotes Pharmacia
T6100 13019-13026 NNP denotes Biotech
T6101 13026-13027 , denotes ,
T6102 13028-13031 NNP denotes Les
T6103 13032-13037 NNP denotes Ullis
T6104 13037-13038 , denotes ,
T6105 13039-13045 NNP denotes France
T6106 13045-13046 -RRB- denotes )
T6107 13047-13056 VBG denotes according
T6108 13057-13059 TO denotes to
T6109 13060-13063 DT denotes the
T6110 13064-13077 NNS denotes manufacturers
T6111 13077-13078 POS denotes '
T6112 13079-13090 NN denotes instruction
T6113 13090-13091 . denotes .
T6114 13092-13100 NN denotes Antibody
T6115 13101-13103 TO denotes to
T6116 13104-13113 NNP denotes α-Tubulin
T6117 13114-13115 -LRB- denotes (
T6118 13115-13120 NNP denotes Santa
T6119 13121-13125 NNP denotes Cruz
T6120 13125-13126 -RRB- denotes )
T6121 13127-13130 VBD denotes was
T6122 13131-13134 NN denotes use
T6123 13135-13137 IN denotes as
T6124 13138-13145 VBG denotes loading
T6125 13146-13153 NN denotes control
T6126 13153-13154 . denotes .
T6127 13155-13158 IN denotes For
T6128 13159-13166 JJ denotes nuclear
T6129 13167-13172 NNP denotes NF-κB
T6130 13172-13173 , denotes ,
T6131 13174-13179 NNP denotes THP-1
T6132 13180-13185 NNS denotes cells
T6133 13186-13190 VBD denotes were
T6134 13191-13201 VBN denotes stimulated
T6135 13202-13206 IN denotes with
T6136 13207-13208 CD denotes 1
T6137 13209-13214 NN denotes mg/ml
T6138 13215-13218 CD denotes C10
T6139 13219-13221 CC denotes or
T6140 13222-13225 CD denotes C40
T6141 13226-13229 IN denotes for
T6142 13230-13232 CD denotes 30
T6143 13233-13240 NNS denotes minutes
T6144 13241-13243 IN denotes at
T6145 13244-13246 CD denotes 37
T6146 13246-13247 NN denotes °
T6147 13247-13249 NNP denotes C.
T6148 13250-13255 NNP denotes Cells
T6149 13256-13260 VBD denotes were
T6150 13261-13265 RB denotes then
T6151 13266-13274 VBN denotes pelleted
T6152 13275-13278 CC denotes and
T6153 13279-13285 JJ denotes nuclei
T6154 13286-13295 VBN denotes separated
T6155 13296-13298 IN denotes as
T6156 13299-13308 VBN denotes described
T6157 13309-13310 NNP denotes [
T6158 13310-13312 CD denotes 31
T6159 13312-13313 NNP denotes ]
T6160 13313-13314 . denotes .
T6161 13315-13321 NNP denotes Nuclei
T6162 13322-13326 VBD denotes were
T6163 13327-13333 VBN denotes washed
T6164 13334-13337 CC denotes and
T6165 13338-13349 VBN denotes homogenized
T6166 13350-13358 RB denotes directly
T6167 13359-13361 IN denotes in
T6168 13362-13369 VBG denotes loading
T6169 13370-13371 -LRB- denotes (
T6170 13371-13377 NNP denotes Laemli
T6171 13377-13378 -RRB- denotes )
T6172 13379-13385 NN denotes buffer
T6173 13386-13389 CC denotes and
T6174 13390-13396 VBN denotes heated
T6175 13397-13400 IN denotes for
T6176 13401-13402 CD denotes 5
T6177 13403-13410 NNS denotes minutes
T6178 13411-13413 IN denotes at
T6179 13414-13417 CD denotes 100
T6180 13417-13418 CD denotes °
T6181 13418-13420 NNP denotes C.
T6182 13421-13429 NNPS denotes Proteins
T6183 13430-13432 IN denotes in
T6184 13433-13440 NNS denotes samples
T6185 13441-13445 VBD denotes were
T6186 13446-13454 VBN denotes resolved
T6187 13455-13457 IN denotes in
T6188 13458-13459 DT denotes a
T6189 13460-13470 NN denotes denaturing
T6190 13471-13472 CD denotes 8
T6191 13472-13473 NN denotes %
T6192 13474-13488 NN denotes polyacrylamide
T6193 13489-13492 NN denotes gel
T6194 13493-13496 CC denotes and
T6195 13497-13508 VBN denotes transferred
T6196 13509-13511 TO denotes to
T6197 13512-13513 DT denotes a
T6198 13514-13528 NN denotes polyvinylidine
T6199 13529-13537 NN denotes fluoride
T6200 13538-13539 -LRB- denotes (
T6201 13539-13543 NNP denotes PVDF
T6202 13543-13544 -RRB- denotes )
T6203 13545-13553 NN denotes membrane
T6204 13554-13555 -LRB- denotes (
T6205 13555-13566 JJ denotes Immobilon-P
T6206 13566-13567 : denotes ;
T6207 13568-13577 NNP denotes Millipore
T6208 13577-13578 , denotes ,
T6209 13579-13586 NNP denotes Bedford
T6210 13586-13587 , denotes ,
T6211 13588-13590 NNP denotes MA
T6212 13590-13591 -RRB- denotes )
T6213 13591-13592 . denotes .
T6214 13593-13602 NNS denotes Membranes
T6215 13603-13607 VBD denotes were
T6216 13608-13617 VBN denotes incubated
T6217 13618-13620 IN denotes in
T6218 13621-13629 VBG denotes blocking
T6219 13630-13636 NN denotes buffer
T6220 13637-13638 -LRB- denotes (
T6221 13638-13639 CD denotes 1
T6222 13639-13640 NN denotes %
T6223 13641-13644 NNP denotes BSA
T6224 13644-13645 , denotes ,
T6225 13646-13648 IN denotes in
T6226 13649-13652 NNP denotes PBS
T6227 13652-13653 -RRB- denotes )
T6228 13654-13657 IN denotes for
T6229 13658-13661 CD denotes two
T6230 13662-13667 NNS denotes hours
T6231 13668-13670 IN denotes at
T6232 13671-13675 NN denotes room
T6233 13676-13687 NN denotes temperature
T6234 13687-13688 . denotes .
T6235 13689-13698 NNS denotes Membranes
T6236 13699-13703 VBD denotes were
T6237 13704-13716 RB denotes subsequently
T6238 13717-13723 VBN denotes probed
T6239 13724-13728 IN denotes with
T6240 13729-13732 DT denotes the
T6241 13733-13746 JJ denotes corresponding
T6242 13747-13755 NN denotes antibody
T6243 13756-13758 IN denotes in
T6244 13759-13767 VBG denotes blocking
T6245 13768-13774 NN denotes buffer
T6246 13774-13775 , denotes ,
T6247 13776-13785 JJ denotes overnight
T6248 13785-13786 . denotes .
T6249 13787-13793 NN denotes Rabbit
T6250 13794-13804 NN denotes polyclonal
T6251 13805-13813 NN denotes antibody
T6252 13814-13824 JJ denotes anti-NF-κB
T6253 13825-13828 JJ denotes p50
T6254 13829-13836 NN denotes subunit
T6255 13837-13838 -LRB- denotes (
T6256 13838-13839 # denotes #
T6257 13840-13846 CD denotes sc-114
T6258 13846-13847 -RRB- denotes )
T6259 13848-13850 CC denotes or
T6260 13851-13861 JJ denotes anti-NF-κB
T6261 13862-13865 NNS denotes p65
T6262 13866-13873 VBN denotes subunit
T6263 13874-13875 -LRB- denotes (
T6264 13875-13876 # denotes #
T6265 13877-13883 CD denotes sc-109
T6266 13883-13884 -RRB- denotes )
T6267 13885-13889 IN denotes from
T6268 13890-13895 NNP denotes Santa
T6269 13896-13900 NNP denotes Cruz
T6270 13901-13914 NNP denotes Biotechnology
T6271 13915-13919 VBD denotes were
T6272 13920-13924 VBN denotes used
T6273 13924-13925 . denotes .
T6274 13926-13935 NNS denotes Membranes
T6275 13936-13940 VBD denotes were
T6276 13941-13947 VBN denotes washed
T6277 13948-13951 CD denotes six
T6278 13952-13957 NNS denotes times
T6279 13958-13960 IN denotes in
T6280 13961-13964 NNP denotes PBS
T6281 13965-13969 IN denotes with
T6282 13970-13974 CD denotes 0.05
T6283 13974-13975 NN denotes %
T6284 13976-13981 CD denotes Tween
T6285 13982-13984 CD denotes 20
T6286 13984-13985 , denotes ,
T6287 13986-13987 CD denotes 5
T6288 13988-13995 NNS denotes minutes
T6289 13996-14000 DT denotes each
T6290 14001-14005 NN denotes time
T6291 14005-14006 , denotes ,
T6292 14007-14010 CC denotes and
T6293 14011-14020 VBN denotes incubated
T6294 14021-14025 IN denotes with
T6295 14026-14027 DT denotes a
T6296 14028-14034 CD denotes 1/3000
T6297 14035-14043 NN denotes dilution
T6298 14044-14046 IN denotes of
T6299 14047-14061 JJ denotes HRP-conjugated
T6300 14062-14063 NN denotes F
T6301 14063-14064 -LRB- denotes (
T6302 14064-14066 NN denotes ab
T6303 14066-14067 '' denotes '
T6304 14067-14068 -RRB- denotes )
T6305 14068-14069 CD denotes 2
T6306 14070-14074 NN denotes goat
T6307 14075-14086 JJ denotes anti-rabbit
T6308 14087-14090 NNP denotes IgG
T6309 14091-14093 IN denotes in
T6310 14094-14095 CD denotes 5
T6311 14095-14096 NN denotes %
T6312 14097-14103 JJ denotes nonfat
T6313 14104-14107 JJ denotes dry
T6314 14108-14112 NN denotes milk
T6315 14113-14116 CC denotes and
T6316 14117-14121 CD denotes 0.05
T6317 14121-14122 NN denotes %
T6318 14123-14128 CD denotes Tween
T6319 14129-14131 CD denotes 20
T6320 14132-14134 IN denotes in
T6321 14135-14138 NNP denotes PBS
T6322 14139-14142 IN denotes for
T6323 14143-14144 CD denotes 1
T6324 14145-14149 NN denotes hour
T6325 14150-14152 IN denotes at
T6326 14153-14157 NN denotes room
T6327 14158-14169 NN denotes temperature
T6328 14169-14170 . denotes .
T6329 14171-14176 IN denotes After
T6330 14177-14184 VBG denotes washing
T6331 14185-14188 CD denotes six
T6332 14189-14193 JJR denotes more
T6333 14194-14199 NNS denotes times
T6334 14200-14202 IN denotes in
T6335 14203-14206 NNP denotes PBS
T6336 14207-14211 IN denotes with
T6337 14212-14216 CD denotes 0.05
T6338 14216-14217 NN denotes %
T6339 14218-14223 CD denotes Tween
T6340 14224-14226 CD denotes 20
T6341 14226-14227 , denotes ,
T6342 14228-14245 JJ denotes antibody-reactive
T6343 14246-14254 NNS denotes proteins
T6344 14255-14259 VBD denotes were
T6345 14260-14268 VBN denotes detected
T6346 14269-14274 VBG denotes using
T6347 14275-14276 DT denotes a
T6348 14277-14294 NN denotes chemiluminescence
T6349 14295-14304 NN denotes substrate
T6350 14305-14306 -LRB- denotes (
T6351 14306-14317 NNP denotes SuperSignal
T6352 14317-14318 : denotes ;
T6353 14319-14325 NNP denotes Pierce
T6354 14325-14326 , denotes ,
T6355 14327-14335 NNP denotes Rockford
T6356 14335-14336 , denotes ,
T6357 14337-14339 NNP denotes IL
T6358 14339-14340 -RRB- denotes )
T6359 14341-14350 VBG denotes according
T6360 14351-14353 TO denotes to
T6361 14354-14357 DT denotes the
T6362 14358-14370 NN denotes manufacturer
T6363 14370-14372 POS denotes 's
T6364 14373-14385 NNS denotes instructions
T6365 14385-14386 . denotes .
T6366 14387-14389 TO denotes To
T6367 14390-14397 VB denotes confirm
T6368 14398-14402 DT denotes that
T6369 14403-14413 JJ denotes equivalent
T6370 14414-14421 NNS denotes amounts
T6371 14422-14424 IN denotes of
T6372 14425-14432 NN denotes protein
T6373 14433-14437 VBD denotes were
T6374 14438-14444 VBN denotes loaded
T6375 14445-14447 IN denotes in
T6376 14448-14452 DT denotes each
T6377 14453-14457 NN denotes line
T6378 14457-14458 , denotes ,
T6379 14459-14468 NNS denotes membranes
T6380 14469-14473 VBD denotes were
T6381 14474-14478 RB denotes also
T6382 14479-14486 JJ denotes Western
T6383 14487-14494 VBN denotes blotted
T6384 14495-14498 IN denotes for
T6385 14499-14502 NNP denotes ERK
T6386 14503-14505 IN denotes as
T6387 14506-14515 VBN denotes described
T6388 14516-14517 NNP denotes [
T6389 14517-14519 CD denotes 32
T6390 14519-14520 NNP denotes ]
T6391 14520-14521 . denotes .
T6486 14523-14531 NN denotes Analysis
T6487 14532-14534 IN denotes of
T6488 14535-14540 NNP denotes NF-κB
T6489 14541-14551 NNP denotes Activation
T6490 14552-14554 IN denotes by
T6491 14555-14559 NNP denotes Flow
T6492 14560-14569 NNP denotes Cytometry
T6493 14570-14577 NNP denotes Nuclear
T6494 14578-14588 NN denotes activation
T6495 14589-14591 IN denotes of
T6496 14592-14594 NNP denotes NF
T6497 14594-14595 NNP denotes −
T6498 14595-14597 NNP denotes κΒ
T6499 14598-14600 IN denotes by
T6500 14601-14605 NN denotes flow
T6501 14606-14615 NN denotes cytometry
T6502 14616-14619 VBD denotes was
T6503 14620-14629 VBN denotes performed
T6504 14630-14632 IN denotes as
T6505 14633-14642 VBN denotes described
T6506 14643-14644 NNP denotes [
T6507 14644-14646 CD denotes 31
T6508 14646-14647 NNP denotes ]
T6509 14647-14648 . denotes .
T6558 14650-14661 NNP denotes Statistical
T6559 14662-14670 NNP denotes Analysis
T6560 14671-14674 NNP denotes The
T6561 14675-14682 NNS denotes results
T6562 14683-14687 VBD denotes were
T6563 14688-14697 VBN denotes expressed
T6564 14698-14700 IN denotes as
T6565 14701-14704 DT denotes the
T6566 14705-14709 JJ denotes mean
T6567 14710-14715 NN denotes value
T6568 14716-14717 NN denotes ±
T6569 14718-14724 NNP denotes S.E.M.
T6570 14725-14727 IN denotes of
T6571 14728-14738 JJ denotes individual
T6572 14739-14750 NNS denotes experiments
T6573 14750-14751 . denotes .
T6574 14752-14755 DT denotes The
T6575 14756-14767 JJ denotes statistical
T6576 14768-14780 NN denotes significance
T6577 14781-14783 IN denotes of
T6578 14784-14787 DT denotes the
T6579 14788-14799 NNS denotes differences
T6580 14800-14807 IN denotes between
T6581 14808-14812 JJ denotes mean
T6582 14813-14819 NNS denotes values
T6583 14820-14823 VBD denotes was
T6584 14824-14832 VBN denotes assessed
T6585 14833-14835 IN denotes by
T6586 14836-14839 DT denotes the
T6587 14840-14847 NN denotes Student
T6588 14847-14849 POS denotes 's
T6589 14850-14856 JJS denotes t-test
T6590 14857-14860 CC denotes and
T6591 14861-14869 NN denotes analysis
T6592 14870-14872 IN denotes of
T6593 14873-14881 NN denotes variance
T6594 14882-14883 -LRB- denotes (
T6595 14883-14888 NNP denotes ANOVA
T6596 14888-14889 -RRB- denotes )
T6597 14889-14890 . denotes .
T6857 14901-14909 JJ denotes Degraded
T6858 14910-14913 NNP denotes CGN
T6859 14914-14920 NNP denotes Induce
T6860 14921-14928 NNP denotes Colonic
T6861 14929-14941 NNP denotes Inflammation
T6862 14942-14945 NNP denotes All
T6863 14946-14950 NNS denotes rats
T6864 14951-14960 VBD denotes developed
T6865 14961-14969 NN denotes diarrhea
T6866 14970-14976 IN denotes during
T6867 14977-14985 JJ denotes degraded
T6868 14986-14997 NN denotes carrageenan
T6869 14998-15012 NN denotes administration
T6870 15013-15016 CC denotes and
T6871 15017-15022 JJ denotes gross
T6872 15023-15031 NN denotes evidence
T6873 15032-15034 IN denotes of
T6874 15035-15040 NN denotes blood
T6875 15041-15044 VBD denotes was
T6876 15045-15055 RB denotes frequently
T6877 15056-15064 VBN denotes detected
T6878 15065-15067 IN denotes in
T6879 15068-15071 DT denotes the
T6880 15072-15078 NNS denotes stools
T6881 15078-15079 . denotes .
T6882 15080-15085 NN denotes Colon
T6883 15086-15092 NN denotes length
T6884 15093-15105 RB denotes dramatically
T6885 15106-15115 VBD denotes decreased
T6886 15116-15118 IN denotes in
T6887 15119-15122 DT denotes all
T6888 15123-15130 VBN denotes treated
T6889 15131-15135 NNS denotes rats
T6890 15136-15140 IN denotes with
T6891 15141-15142 DT denotes a
T6892 15143-15147 RBR denotes more
T6893 15148-15158 JJ denotes pronounced
T6894 15159-15165 NN denotes effect
T6895 15166-15171 VBG denotes being
T6896 15172-15180 VBN denotes observed
T6897 15181-15183 IN denotes in
T6898 15184-15187 DT denotes the
T6899 15188-15190 CD denotes 40
T6900 15191-15194 NNP denotes kDa
T6901 15195-15199 NNP denotes dCGN
T6902 15200-15207 VBD denotes treated
T6903 15208-15213 NN denotes group
T6904 15214-15215 -LRB- denotes (
T6905 15215-15219 NNP denotes Fig.
T6906 15220-15222 NNP denotes 1A
T6907 15222-15223 -RRB- denotes )
T6908 15223-15224 . denotes .
T6909 15225-15236 RB denotes Furthermore
T6910 15236-15237 , denotes ,
T6911 15238-15247 VBN denotes prolonged
T6912 15248-15256 NN denotes exposure
T6913 15257-15259 TO denotes to
T6914 15260-15262 CD denotes 40
T6915 15263-15266 NNP denotes kDa
T6916 15267-15271 NNP denotes dCGN
T6917 15272-15280 VBD denotes resulted
T6918 15281-15283 IN denotes in
T6919 15284-15288 JJ denotes high
T6920 15289-15300 JJ denotes macroscopic
T6921 15301-15304 CC denotes and
T6922 15305-15317 JJ denotes histological
T6923 15318-15324 NNS denotes scores
T6924 15325-15327 IN denotes of
T6925 15328-15340 NN denotes inflammation
T6926 15341-15342 -LRB- denotes (
T6927 15342-15346 NNP denotes Fig.
T6928 15347-15349 NNP denotes 1B
T6929 15349-15350 , denotes ,
T6930 15351-15352 NNP denotes C
T6931 15352-15353 -RRB- denotes )
T6932 15353-15354 . denotes .
T6933 15355-15359 RB denotes Only
T6934 15360-15364 JJ denotes weak
T6935 15365-15380 NN denotes myeloperoxidase
T6936 15381-15389 NN denotes activity
T6937 15390-15393 VBD denotes was
T6938 15394-15402 VBN denotes detected
T6939 15403-15405 IN denotes in
T6940 15406-15410 DT denotes both
T6941 15411-15418 NN denotes control
T6942 15419-15422 CC denotes and
T6943 15423-15435 JJ denotes dCGN-treated
T6944 15436-15442 NNS denotes groups
T6945 15443-15444 -LRB- denotes (
T6946 15444-15448 NNP denotes Fig.
T6947 15449-15451 NNP denotes 1D
T6948 15451-15452 -RRB- denotes )
T6949 15452-15453 , denotes ,
T6950 15454-15464 VBG denotes indicating
T6951 15465-15469 IN denotes that
T6952 15470-15482 NNS denotes granulocytes
T6953 15483-15486 VBD denotes did
T6954 15487-15490 RB denotes not
T6955 15491-15495 VB denotes play
T6956 15496-15497 DT denotes a
T6957 15498-15503 JJ denotes major
T6958 15504-15508 NN denotes role
T6959 15509-15511 IN denotes in
T6960 15512-15515 DT denotes the
T6961 15516-15528 NN denotes inflammation
T6962 15529-15531 IN denotes at
T6963 15532-15536 DT denotes that
T6964 15537-15542 NN denotes stage
T6965 15542-15543 . denotes .
T6966 15544-15556 JJ denotes Histological
T6967 15557-15568 NN denotes examination
T6968 15569-15577 VBD denotes revealed
T6969 15578-15585 JJ denotes various
T6970 15586-15593 NNS denotes degrees
T6971 15594-15596 IN denotes of
T6972 15597-15604 JJ denotes mucosal
T6973 15605-15617 NN denotes inflammation
T6974 15617-15618 . denotes .
T6975 15619-15623 NNS denotes Rats
T6976 15624-15631 VBN denotes treated
T6977 15632-15636 IN denotes with
T6978 15637-15639 CD denotes 10
T6979 15640-15643 NNP denotes kDa
T6980 15644-15648 NNP denotes dCGN
T6981 15649-15655 VBD denotes showed
T6982 15656-15661 NN denotes edema
T6983 15661-15662 , denotes ,
T6984 15663-15673 NN denotes epithelium
T6985 15674-15681 NN denotes atrophy
T6986 15682-15685 CC denotes and
T6987 15686-15692 JJ denotes slight
T6988 15693-15703 NN denotes lymphocyte
T6989 15704-15716 NN denotes infiltration
T6990 15717-15718 -LRB- denotes (
T6991 15718-15722 NNS denotes data
T6992 15723-15726 RB denotes not
T6993 15727-15732 VBN denotes shown
T6994 15732-15733 -RRB- denotes )
T6995 15733-15734 . denotes .
T6996 15735-15740 DT denotes These
T6997 15741-15749 NNS denotes symptoms
T6998 15750-15754 VBD denotes were
T6999 15755-15762 RB denotes totally
T7000 15763-15769 JJ denotes absent
T7001 15770-15772 IN denotes in
T7002 15773-15776 DT denotes the
T7003 15777-15782 NN denotes colon
T7004 15783-15785 IN denotes of
T7005 15786-15793 NN denotes control
T7006 15794-15798 NNS denotes rats
T7007 15799-15800 -LRB- denotes (
T7008 15800-15804 NNP denotes Fig.
T7009 15805-15807 NNP denotes 1E
T7010 15807-15808 -RRB- denotes )
T7011 15808-15809 . denotes .
T7012 15810-15814 RBR denotes More
T7013 15815-15821 JJ denotes severe
T7014 15822-15829 JJ denotes mucosal
T7015 15830-15838 NNS denotes injuries
T7016 15839-15848 VBG denotes including
T7017 15849-15859 NN denotes ulceration
T7018 15859-15860 , denotes ,
T7019 15861-15873 JJ denotes hyperplastic
T7020 15874-15884 NN denotes epithelium
T7021 15884-15885 , denotes ,
T7022 15886-15891 JJ denotes crypt
T7023 15892-15902 NN denotes distortion
T7024 15903-15906 CC denotes and
T7025 15907-15908 DT denotes a
T7026 15909-15915 JJ denotes strong
T7027 15916-15926 NN denotes macrophage
T7028 15927-15939 NN denotes infiltration
T7029 15939-15940 , denotes ,
T7030 15941-15945 VBD denotes were
T7031 15946-15954 VBN denotes observed
T7032 15955-15957 IN denotes in
T7033 15958-15961 DT denotes the
T7034 15962-15964 CD denotes 40
T7035 15965-15968 NNP denotes kDa
T7036 15969-15981 NNP denotes dCGN-treated
T7037 15982-15986 NNS denotes rats
T7038 15987-15988 -LRB- denotes (
T7039 15988-15992 NNP denotes Fig.
T7040 15993-15995 NNP denotes 1F
T7041 15995-15996 -RRB- denotes )
T7042 15996-15997 . denotes .
T7043 15998-16000 DT denotes No
T7044 16001-16010 VBN denotes sulphated
T7045 16011-16026 NNS denotes polysaccharides
T7046 16027-16031 VBD denotes were
T7047 16032-16040 VBN denotes detected
T7048 16041-16043 IN denotes by
T7049 16044-16053 NN denotes toluidine
T7050 16054-16058 JJ denotes blue
T7051 16059-16067 NN denotes staining
T7052 16068-16070 IN denotes of
T7053 16071-16076 NN denotes colon
T7054 16077-16083 NN denotes mucosa
T7055 16084-16088 IN denotes from
T7056 16089-16093 NNS denotes rats
T7057 16094-16101 VBN denotes treated
T7058 16102-16106 IN denotes with
T7059 16107-16113 CC denotes either
T7060 16114-16117 DT denotes the
T7061 16118-16120 CD denotes 10
T7062 16121-16123 CC denotes or
T7063 16124-16126 CD denotes 40
T7064 16127-16130 NNP denotes kDa
T7065 16131-16135 NNP denotes dCGN
T7066 16136-16137 -LRB- denotes (
T7067 16137-16140 RB denotes not
T7068 16141-16146 VBN denotes shown
T7069 16146-16147 -RRB- denotes )
T7070 16147-16148 . denotes .
T7071 16149-16157 IN denotes Although
T7072 16158-16160 PRP denotes we
T7073 16161-16164 MD denotes can
T7074 16164-16167 RB denotes not
T7075 16168-16175 VB denotes exclude
T7076 16176-16180 DT denotes that
T7077 16181-16185 NNP denotes dCGN
T7078 16186-16189 NN denotes mat
T7079 16190-16193 RB denotes not
T7080 16194-16198 VB denotes have
T7081 16199-16207 VBN denotes retained
T7082 16208-16210 IN denotes in
T7083 16211-16214 DT denotes the
T7084 16215-16222 NN denotes section
T7085 16223-16229 IN denotes during
T7086 16230-16233 DT denotes the
T7087 16234-16243 NN denotes histology
T7088 16244-16253 NN denotes procedure
T7089 16253-16254 , denotes ,
T7090 16255-16259 DT denotes this
T7091 16260-16269 VBZ denotes indicates
T7092 16270-16274 IN denotes that
T7093 16275-16280 DT denotes these
T7094 16281-16289 NNS denotes polymers
T7095 16290-16293 MD denotes may
T7096 16294-16297 RB denotes not
T7097 16298-16302 VB denotes have
T7098 16303-16307 VBN denotes been
T7099 16308-16320 VBN denotes phagocytosed
T7100 16320-16321 . denotes .
T7657 0-8 JJ denotes Degraded
T7658 9-12 NNP denotes CGN
T7659 13-16872 NNP denotes inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced-TNF-α
T7660 16873-16883 NN denotes Production
T7661 16884-16886 IN denotes by
T7662 16887-16896 NNP denotes Monocytes
T7663 16897-16899 IN denotes In
T7664 16900-16905 NNP denotes Vitro
T15673 16366-16374 JJ denotes Degraded
T15674 16375-16378 NNP denotes CGN
T15675 16379-16386 VBD denotes induced
T15676 16387-16392 NN denotes colon
T15677 16393-16405 NN denotes inflammation
T15678 16406-16408 IN denotes in
T15679 16409-16413 NNS denotes rats
T15680 16413-16414 . denotes .
T15681 16415-16425 NNP denotes Histograms
T15682 16426-16433 VBG denotes showing
T15683 16434-16437 DT denotes the
T15684 16438-16444 NN denotes effect
T15685 16445-16447 IN denotes of
T15686 16448-16456 JJ denotes degraded
T15687 16457-16460 NNP denotes CGN
T15688 16461-16463 IN denotes on
T15689 16463-16464 : denotes :
T15690 16465-16470 NN denotes colon
T15691 16471-16477 NN denotes length
T15692 16478-16479 -LRB- denotes (
T15693 16479-16480 NNP denotes A
T15694 16480-16481 -RRB- denotes )
T15695 16481-16482 : denotes ;
T15696 16483-16494 JJ denotes macroscopic
T15697 16495-16496 -LRB- denotes (
T15698 16496-16497 NNP denotes B
T15699 16497-16498 -RRB- denotes )
T15700 16499-16502 CC denotes and
T15701 16503-16515 JJ denotes histological
T15702 16516-16517 -LRB- denotes (
T15703 16517-16518 NNP denotes C
T15704 16518-16519 -RRB- denotes )
T15705 16520-16532 NN denotes inflammation
T15706 16533-16538 NN denotes score
T15707 16539-16541 IN denotes of
T15708 16542-16547 NN denotes colon
T15709 16547-16548 : denotes ;
T15710 16549-16564 NNP denotes Myeloperoxidase
T15711 16565-16566 -LRB- denotes (
T15712 16566-16569 NNP denotes MPO
T15713 16569-16570 -RRB- denotes )
T15714 16571-16579 NN denotes activity
T15715 16580-16581 -LRB- denotes (
T15716 16581-16582 NNP denotes D
T15717 16582-16583 -RRB- denotes )
T15718 16583-16584 . denotes .
T15719 16585-16592 NNP denotes Control
T15720 16593-16597 NNS denotes rats
T15721 16598-16599 -LRB- denotes (
T15722 16599-16604 JJ denotes white
T15723 16605-16609 NNS denotes bars
T15724 16609-16610 -RRB- denotes )
T15725 16610-16611 : denotes ;
T15726 16612-16614 CD denotes 10
T15727 16615-16618 NN denotes kDa
T15728 16619-16627 JJ denotes degraded
T15729 16628-16639 JJ denotes CGN-treated
T15730 16640-16644 NNS denotes rats
T15731 16645-16646 -LRB- denotes (
T15732 16646-16650 NN denotes grey
T15733 16651-16655 NNS denotes bars
T15734 16655-16656 -RRB- denotes )
T15735 16656-16657 : denotes ;
T15736 16658-16660 CD denotes 40
T15737 16661-16664 NN denotes kDa
T15738 16665-16673 JJ denotes degraded
T15739 16674-16685 JJ denotes CGN-treated
T15740 16686-16690 NNS denotes rats
T15741 16691-16692 -LRB- denotes (
T15742 16692-16697 JJ denotes black
T15743 16698-16702 NNS denotes bars
T15744 16702-16703 -RRB- denotes )
T15745 16703-16704 . denotes .
T15746 16705-16706 SYM denotes *
T15747 16707-16708 NN denotes p
T15748 16708-16709 NN denotes <
T15749 16709-16713 CD denotes 0.05
T15750 16714-16718 IN denotes from
T15751 16719-16726 NN denotes control
T15752 16726-16727 . denotes .
T15753 16728-16730 SYM denotes **
T15754 16731-16732 FW denotes p
T15755 16732-16733 FW denotes <
T15756 16733-16737 CD denotes 0.01
T15757 16738-16742 IN denotes from
T15758 16743-16750 NN denotes control
T15759 16750-16751 . denotes .
T15760 16752-16764 JJ denotes Histological
T15761 16765-16773 NN denotes analysis
T15762 16774-16776 IN denotes of
T15763 16777-16782 NN denotes colon
T15764 16783-16787 IN denotes from
T15765 16788-16795 NN denotes control
T15766 16796-16800 NNS denotes rats
T15767 16801-16802 -LRB- denotes (
T15768 16802-16803 NNP denotes E
T15769 16803-16804 -RRB- denotes )
T15770 16804-16805 , denotes ,
T15771 16806-16809 CC denotes and
T15772 16810-16814 IN denotes from
T15773 16815-16817 CD denotes 40
T15774 16818-16821 NNP denotes kDa
T15775 16822-16834 NNP denotes dCGN-treated
T15776 16835-16839 NNS denotes rats
T15777 16840-16841 -LRB- denotes (
T15778 16841-16842 NN denotes F
T15779 16842-16843 -RRB- denotes )
T15780 16843-16844 . denotes .
R396 T467 T468 amod Degraded,CGN
R397 T468 T469 nsubj CGN,inhibited
R398 T469 T469 ROOT inhibited,inhibited
R399 T470 T472 nummod THP-1,proliferation
R400 T471 T472 compound cell,proliferation
R401 T472 T469 dobj proliferation,inhibited
R402 T473 T469 prep in,inhibited
R403 T474 T473 pobj vitro,in
R404 T475 T469 punct ",",inhibited
R405 T476 T469 advcl arresting,inhibited
R406 T477 T478 det the,cells
R407 T478 T476 dobj cells,arresting
R408 T479 T476 prep in,arresting
R409 T480 T481 nummod G1,phase
R410 T481 T479 pobj phase,in
R411 T482 T469 punct .,inhibited
R412 T483 T487 prep In,increased
R413 T484 T483 pobj addition,In
R414 T485 T487 punct ",",increased
R415 T486 T487 nsubj dCGN,increased
R416 T487 T487 ROOT increased,increased
R417 T488 T489 compound ICAM-1,expression
R418 T489 T487 dobj expression,increased
R419 T490 T487 prep in,increased
R420 T491 T492 preconj both,PBM
R421 T492 T490 pobj PBM,in
R422 T493 T492 cc and,PBM
R423 T494 T492 conj THP-1,PBM
R424 T495 T487 conj cells,increased
R425 T496 T495 prep with,cells
R426 T497 T499 det a,effect
R427 T498 T499 amod major,effect
R428 T499 T496 pobj effect,with
R429 T500 T499 acl seen,effect
R430 T501 T500 prep after,seen
R431 T502 T505 nummod 40,exposure
R432 T503 T504 compound kDa,dCGN
R433 T504 T505 compound dCGN,exposure
R434 T505 T501 pobj exposure,after
R435 T506 T487 punct .,increased
R436 T507 T510 advmod Also,stimulated
R437 T508 T510 punct ",",stimulated
R438 T509 T510 nsubj dCGN,stimulated
R439 T510 T510 ROOT stimulated,stimulated
R440 T511 T512 compound monocyte,aggregation
R441 T512 T510 dobj aggregation,stimulated
R442 T513 T510 prep in,stimulated
R443 T514 T513 pobj vitro,in
R444 T515 T517 nsubjpass that,prevented
R445 T516 T517 auxpass was,prevented
R446 T517 T510 conj prevented,stimulated
R447 T518 T517 agent by,prevented
R448 T519 T518 pobj incubation,by
R449 T520 T517 prep with,prevented
R450 T521 T522 amod anti-ICAM-1,antibody
R451 T522 T520 pobj antibody,with
R452 T523 T510 punct .,stimulated
R453 T524 T527 advmod Finally,stimulated
R454 T525 T527 punct ",",stimulated
R455 T526 T527 nsubj dCGN,stimulated
R456 T527 T527 ROOT stimulated,stimulated
R457 T528 T529 amod TNF-α,expression
R458 T529 T527 dobj expression,stimulated
R459 T530 T529 cc and,expression
R460 T531 T529 conj secretion,expression
R461 T532 T527 prep by,stimulated
R462 T533 T534 preconj both,PBM
R463 T534 T537 nmod PBM,cells
R464 T535 T534 cc and,PBM
R465 T536 T534 conj THP-1,PBM
R466 T537 T532 pobj cells,by
R467 T538 T527 punct .,stimulated
R468 T539 T541 predet All,effects
R469 T540 T541 det these,effects
R470 T541 T543 nsubjpass effects,linked
R471 T542 T543 auxpass were,linked
R472 T543 T543 ROOT linked,linked
R473 T544 T543 prep to,linked
R474 T545 T546 compound NF-κB,activation
R475 T546 T544 pobj activation,to
R476 T547 T543 punct .,linked
R477 T548 T549 det These,data
R478 T549 T551 nsubj data,suggest
R479 T550 T551 advmod strongly,suggest
R480 T551 T551 ROOT suggest,suggest
R481 T552 T558 mark that,have
R482 T553 T555 det the,forms
R483 T554 T555 amod degraded,forms
R484 T555 T558 nsubj forms,have
R485 T556 T555 prep of,forms
R486 T557 T556 pobj CGN,of
R487 T558 T551 ccomp have,suggest
R488 T559 T561 det a,effect
R489 T560 T561 amod pronounced,effect
R490 T561 T558 dobj effect,have
R491 T562 T561 prep on,effect
R492 T563 T562 pobj monocytes,on
R493 T564 T563 punct ",",monocytes
R494 T565 T561 conj characteristic,effect
R495 T566 T565 prep of,characteristic
R496 T567 T569 det an,phenotype
R497 T568 T569 amod inflammatory,phenotype
R498 T569 T566 pobj phenotype,of
R499 T570 T551 punct .,suggest
R1155 T1344 T1348 nsubj Carrageenan,is
R1156 T1345 T1346 punct (,CGN
R1157 T1346 T1344 appos CGN,Carrageenan
R1158 T1347 T1344 punct ),Carrageenan
R1159 T1348 T1348 ROOT is,is
R1160 T1349 T1354 det a,polysaccharide
R1161 T1350 T1352 amod high,weight
R1162 T1351 T1352 amod molecular,weight
R1163 T1352 T1354 nmod weight,polysaccharide
R1164 T1353 T1354 amod sulphated,polysaccharide
R1165 T1354 T1348 attr polysaccharide,is
R1166 T1355 T1356 punct (,>
R1167 T1356 T1354 appos >,polysaccharide
R1168 T1357 T1358 nummod 200,kDa
R1169 T1358 T1354 appos kDa,polysaccharide
R1170 T1359 T1358 punct ),kDa
R1171 T1360 T1354 acl derived,polysaccharide
R1172 T1361 T1360 prep from,derived
R1173 T1362 T1363 amod red,algae
R1174 T1363 T1361 pobj algae,from
R1175 T1364 T1363 punct (,algae
R1176 T1365 T1363 appos Rhodophyceae,algae
R1177 T1366 T1363 punct ),algae
R1178 T1367 T1348 punct .,is
R1179 T1368 T1370 nummod Three,forms
R1180 T1369 T1370 amod main,forms
R1181 T1370 T1375 nsubjpass forms,identified
R1182 T1371 T1370 prep of,forms
R1183 T1372 T1371 pobj CGN,of
R1184 T1373 T1375 aux have,identified
R1185 T1374 T1375 auxpass been,identified
R1186 T1375 T1375 ROOT identified,identified
R1187 T1376 T1375 punct :,identified
R1188 T1377 T1375 npadvmod kappa,identified
R1189 T1378 T1377 punct ",",kappa
R1190 T1379 T1377 conj iota,kappa
R1191 T1380 T1379 punct ",",iota
R1192 T1381 T1379 cc and,iota
R1193 T1382 T1379 conj lambda,iota
R1194 T1383 T1375 punct .,identified
R1195 T1384 T1385 nsubj They,differ
R1196 T1385 T1385 ROOT differ,differ
R1197 T1386 T1385 prep from,differ
R1198 T1387 T1388 det each,other
R1199 T1388 T1386 pobj other,from
R1200 T1389 T1385 prep in,differ
R1201 T1390 T1391 compound sulphation,degree
R1202 T1391 T1389 pobj degree,in
R1203 T1392 T1391 cc and,degree
R1204 T1393 T1394 compound solubility,[
R1205 T1394 T1385 conj [,differ
R1206 T1395 T1396 nummod 1,]
R1207 T1396 T1394 conj ],[
R1208 T1397 T1396 punct ",",]
R1209 T1398 T1396 conj [,]
R1210 T1399 T1400 nummod 2,]
R1211 T1400 T1396 conj ],]
R1212 T1401 T1385 punct .,differ
R1213 T1402 T1403 amod Native,CGN
R1214 T1403 T1405 nsubjpass CGN,thought
R1215 T1404 T1405 auxpass is,thought
R1216 T1405 T1405 ROOT thought,thought
R1217 T1406 T1407 aux to,be
R1218 T1407 T1405 xcomp be,thought
R1219 T1408 T1407 acomp harmless,be
R1220 T1409 T1405 cc and,thought
R1221 T1410 T1412 auxpass is,used
R1222 T1411 T1412 advmod widely,used
R1223 T1412 T1405 conj used,thought
R1224 T1413 T1412 prep as,used
R1225 T1414 T1416 det a,additive
R1226 T1415 T1416 compound food,additive
R1227 T1416 T1413 pobj additive,as
R1228 T1417 T1418 aux to,improve
R1229 T1418 T1416 relcl improve,additive
R1230 T1419 T1418 dobj texture,improve
R1231 T1420 T1405 punct .,thought
R1232 T1421 T1424 nsubjpass It,used
R1233 T1422 T1424 auxpass is,used
R1234 T1423 T1424 advmod also,used
R1235 T1424 T1424 ROOT used,used
R1236 T1425 T1424 prep in,used
R1237 T1426 T1425 pobj cosmetics,in
R1238 T1427 T1426 cc and,cosmetics
R1239 T1428 T1426 conj pharmaceuticals,cosmetics
R1240 T1429 T1424 punct .,used
R1241 T1430 T1442 advmod However,triggers
R1242 T1431 T1442 punct ",",triggers
R1243 T1432 T1433 compound acid,treatment
R1244 T1433 T1442 nsubj treatment,triggers
R1245 T1434 T1433 prep at,treatment
R1246 T1435 T1436 amod high,temperature
R1247 T1436 T1434 pobj temperature,at
R1248 T1437 T1436 punct (,temperature
R1249 T1438 T1440 nummod 80,C
R1250 T1439 T1440 nummod °,C
R1251 T1440 T1433 npadvmod C,treatment
R1252 T1441 T1433 punct ),treatment
R1253 T1442 T1442 ROOT triggers,triggers
R1254 T1443 T1444 compound CGN,hydrolysis
R1255 T1444 T1442 dobj hydrolysis,triggers
R1256 T1445 T1446 aux to,lower
R1257 T1446 T1448 amod lower,weight
R1258 T1447 T1448 amod molecular,weight
R1259 T1448 T1444 appos weight,hydrolysis
R1260 T1449 T1450 punct (,<
R1261 T1450 T1452 nmod <,kDa
R1262 T1451 T1452 nummod 50,kDa
R1263 T1452 T1448 appos kDa,weight
R1264 T1453 T1448 punct ),weight
R1265 T1454 T1442 conj compounds,triggers
R1266 T1455 T1454 acl known,compounds
R1267 T1456 T1455 prep as,known
R1268 T1457 T1456 pobj poligeenan,as
R1269 T1458 T1457 cc or,poligeenan
R1270 T1459 T1460 amod degraded,CGN
R1271 T1460 T1457 conj CGN,poligeenan
R1272 T1461 T1460 punct (,CGN
R1273 T1462 T1460 appos dCGN,CGN
R1274 T1463 T1460 punct ),CGN
R1275 T1464 T1442 punct .,triggers
R1276 T1465 T1466 det These,dCGNs
R1277 T1466 T1467 nsubj dCGNs,induce
R1278 T1467 T1467 ROOT induce,induce
R1279 T1468 T1467 dobj inflammation,induce
R1280 T1469 T1467 cc and,induce
R1281 T1470 T1473 aux have,used
R1282 T1471 T1473 auxpass been,used
R1283 T1472 T1473 advmod widely,used
R1284 T1473 T1467 conj used,induce
R1285 T1474 T1473 prep as,used
R1286 T1475 T1474 pobj models,as
R1287 T1476 T1475 prep of,models
R1288 T1477 T1476 pobj colitis,of
R1289 T1478 T1475 prep in,models
R1290 T1479 T1480 amod several,species
R1291 T1480 T1478 pobj species,in
R1292 T1481 T1480 punct ",",species
R1293 T1482 T1480 prep including,species
R1294 T1483 T1484 nsubj rats,[
R1295 T1484 T1486 nmod [,]
R1296 T1485 T1486 nummod 3,]
R1297 T1486 T1482 pobj ],including
R1298 T1487 T1486 punct ",",]
R1299 T1488 T1486 conj rabbits,]
R1300 T1489 T1491 nmod [,]
R1301 T1490 T1491 nummod 4,]
R1302 T1491 T1494 nmod ],pigs
R1303 T1492 T1491 cc and,]
R1304 T1493 T1494 compound guinea,pigs
R1305 T1494 T1495 nsubj pigs,[
R1306 T1495 T1488 relcl [,rabbits
R1307 T1496 T1497 nummod 5,]
R1308 T1497 T1495 dobj ],[
R1309 T1498 T1467 punct .,induce
R1310 T1499 T1500 det The,role
R1311 T1500 T1507 nsubj role,remains
R1312 T1501 T1500 prep of,role
R1313 T1502 T1501 pobj dCGN,of
R1314 T1503 T1500 prep as,role
R1315 T1504 T1506 det a,factor
R1316 T1505 T1506 amod tumor-promoting,factor
R1317 T1506 T1503 pobj factor,as
R1318 T1507 T1507 ROOT remains,remains
R1319 T1508 T1507 acomp controversial,remains
R1320 T1509 T1518 nmod [,]
R1321 T1510 T1509 nummod 4,[
R1322 T1511 T1509 conj ],[
R1323 T1512 T1511 punct ",",]
R1324 T1513 T1515 quantmod [,]
R1325 T1514 T1515 nummod 6,]
R1326 T1515 T1509 appos ],[
R1327 T1516 T1518 nmod [,]
R1328 T1517 T1518 nummod 8,]
R1329 T1518 T1507 npadvmod ],remains
R1330 T1519 T1507 punct .,remains
R1331 T1520 T1525 mark Although,thought
R1332 T1521 T1523 det the,form
R1333 T1522 T1523 amod native,form
R1334 T1523 T1525 nsubjpass form,thought
R1335 T1524 T1525 auxpass is,thought
R1336 T1525 T1539 advcl thought,produced
R1337 T1526 T1527 aux to,be
R1338 T1527 T1525 xcomp be,thought
R1339 T1528 T1527 acomp harmless,be
R1340 T1529 T1527 prep for,be
R1341 T1530 T1531 amod human,consumption
R1342 T1531 T1529 pobj consumption,for
R1343 T1532 T1539 punct ",",produced
R1344 T1533 T1534 amod small,amounts
R1345 T1534 T1539 nsubjpass amounts,produced
R1346 T1535 T1534 prep of,amounts
R1347 T1536 T1535 pobj dCGN,of
R1348 T1537 T1539 auxpass are,produced
R1349 T1538 T1539 advmod probably,produced
R1350 T1539 T1539 ROOT produced,produced
R1351 T1540 T1539 agent by,produced
R1352 T1541 T1542 compound acid,hydrolysis
R1353 T1542 T1540 pobj hydrolysis,by
R1354 T1543 T1539 prep during,produced
R1355 T1544 T1545 amod gastric,digestion
R1356 T1545 T1543 pobj digestion,during
R1357 T1546 T1548 nmod [,]
R1358 T1547 T1548 nummod 9,]
R1359 T1548 T1545 appos ],digestion
R1360 T1549 T1548 punct ",",]
R1361 T1550 T1552 nmod [,]
R1362 T1551 T1552 nummod 10,]
R1363 T1552 T1548 conj ],]
R1364 T1553 T1552 cc or,]
R1365 T1554 T1552 conj interaction,]
R1366 T1555 T1554 prep with,interaction
R1367 T1556 T1557 amod intestinal,bacteria
R1368 T1557 T1555 pobj bacteria,with
R1369 T1558 T1560 compound [,]
R1370 T1559 T1560 compound 11,]
R1371 T1560 T1557 appos ],bacteria
R1372 T1561 T1560 punct ",",]
R1373 T1562 T1564 nmod [,]
R1374 T1563 T1564 nummod 12,]
R1375 T1564 T1560 appos ],]
R1376 T1565 T1539 punct .,produced
R1377 T1566 T1579 mark Whereas,analyzed
R1378 T1567 T1568 det the,effects
R1379 T1568 T1579 nsubjpass effects,analyzed
R1380 T1569 T1568 prep of,effects
R1381 T1570 T1573 quantmod native,on
R1382 T1571 T1570 cc and,native
R1383 T1572 T1570 conj dCGN,native
R1384 T1573 T1568 prep on,effects
R1385 T1574 T1575 amod intestinal,inflammation
R1386 T1575 T1573 pobj inflammation,on
R1387 T1576 T1579 aux have,analyzed
R1388 T1577 T1579 auxpass been,analyzed
R1389 T1578 T1579 advmod extensively,analyzed
R1390 T1579 T1589 advcl analyzed,conducted
R1391 T1580 T1579 prep in,analyzed
R1392 T1581 T1582 compound animal,models
R1393 T1582 T1580 pobj models,in
R1394 T1583 T1589 punct ",",conducted
R1395 T1584 T1586 advmod only,studies
R1396 T1585 T1586 amod few,studies
R1397 T1586 T1589 nsubjpass studies,conducted
R1398 T1587 T1589 aux have,conducted
R1399 T1588 T1589 auxpass been,conducted
R1400 T1589 T1589 ROOT conducted,conducted
R1401 T1590 T1589 advcl using,conducted
R1402 T1591 T1593 amod human,lines
R1403 T1592 T1593 compound cell,lines
R1404 T1593 T1590 dobj lines,using
R1405 T1594 T1589 punct .,conducted
R1406 T1595 T1596 amod Recent,studies
R1407 T1596 T1598 nsubj studies,shown
R1408 T1597 T1598 aux have,shown
R1409 T1598 T1598 ROOT shown,shown
R1410 T1599 T1600 det a,link
R1411 T1600 T1598 dobj link,shown
R1412 T1601 T1600 prep between,link
R1413 T1602 T1606 compound exposure,CGN
R1414 T1603 T1604 aux to,native
R1415 T1604 T1605 amod native,form
R1416 T1605 T1606 compound form,CGN
R1417 T1606 T1601 pobj CGN,between
R1418 T1607 T1606 cc and,CGN
R1419 T1608 T1609 amod IL-8,production
R1420 T1609 T1606 conj production,CGN
R1421 T1610 T1609 prep by,production
R1422 T1611 T1616 det the,line
R1423 T1612 T1615 amod human,cell
R1424 T1613 T1615 amod intestinal,cell
R1425 T1614 T1615 compound epithelial,cell
R1426 T1615 T1616 compound cell,line
R1427 T1616 T1610 pobj line,by
R1428 T1617 T1616 punct ",",line
R1429 T1618 T1616 appos NCM460,line
R1430 T1619 T1598 punct ",",shown
R1431 T1620 T1598 prep via,shown
R1432 T1621 T1622 compound Nuclear,Factor-κB
R1433 T1622 T1620 pobj Factor-κB,via
R1434 T1623 T1622 punct (,Factor-κB
R1435 T1624 T1622 appos NF-κB,Factor-κB
R1436 T1625 T1622 punct ),Factor-κB
R1437 T1626 T1627 compound activation,[
R1438 T1627 T1629 nmod [,]
R1439 T1628 T1629 nummod 13,]
R1440 T1629 T1598 dobj ],shown
R1441 T1630 T1629 punct ",",]
R1442 T1631 T1629 conj [,]
R1443 T1632 T1633 nummod 14,]
R1444 T1633 T1629 conj ],]
R1445 T1634 T1598 punct .,shown
R1446 T1635 T1636 nsubj NF-κB,is
R1447 T1636 T1636 ROOT is,is
R1448 T1637 T1639 det a,factor
R1449 T1638 T1639 compound transcription,factor
R1450 T1639 T1636 attr factor,is
R1451 T1640 T1641 nsubj that,regulates
R1452 T1641 T1639 relcl regulates,factor
R1453 T1642 T1643 det the,expression
R1454 T1643 T1641 dobj expression,regulates
R1455 T1644 T1643 prep of,expression
R1456 T1645 T1644 pobj genes,of
R1457 T1646 T1645 acl associated,genes
R1458 T1647 T1646 prep with,associated
R1459 T1648 T1647 pobj inflammation,with
R1460 T1649 T1651 nmod [,]
R1461 T1650 T1651 nummod 15,]
R1462 T1651 T1643 appos ],expression
R1463 T1652 T1651 punct ",",]
R1464 T1653 T1655 nmod [,]
R1465 T1654 T1655 nummod 16,]
R1466 T1655 T1651 appos ],]
R1467 T1656 T1636 punct .,is
R1468 T1657 T1658 compound Macrophage,infiltration
R1469 T1658 T1661 nsubj infiltration,is
R1470 T1659 T1658 cc and,infiltration
R1471 T1660 T1658 conj accumulation,infiltration
R1472 T1661 T1661 ROOT is,is
R1473 T1662 T1664 det a,characteristic
R1488 T1677 T1680 amod total,cells
R1489 T1678 T1680 compound lamina,cells
R1490 T1679 T1680 compound propria,cells
R1491 T1680 T1676 pobj cells,of
R1492 T1681 T1673 punct ",",represent
R1493 T1682 T1673 conj secrete,represent
R1494 T1683 T1685 det a,range
R1495 T1684 T1685 amod wide,range
R1496 T1685 T1682 dobj range,secrete
R1497 T1686 T1685 prep of,range
R1498 T1687 T1688 advmod biologically,active
R1499 T1688 T1689 amod active,compounds
R1500 T1689 T1686 pobj compounds,of
R1501 T1690 T1682 cc and,secrete
R1502 T1691 T1682 conj express,secrete
R1503 T1692 T1693 amod cell-adhesion,molecules
R1504 T1693 T1691 dobj molecules,express
R1505 T1694 T1673 punct .,represent
R1506 T1695 T1698 det The,response
R1507 T1696 T1698 amod immune,response
R1508 T1697 T1698 compound cell,response
R1509 T1698 T1703 nsubj response,seems
R1510 T1699 T1698 prep to,response
R1511 T1700 T1702 det an,stimulus
R1512 T1701 T1702 amod inflammatory,stimulus
R1513 T1702 T1699 pobj stimulus,to
R1514 T1703 T1703 ROOT seems,seems
R1515 T1704 T1706 aux to,amplified
R1516 T1705 T1706 auxpass be,amplified
R1517 T1706 T1703 xcomp amplified,seems
R1518 T1707 T1706 cc or,amplified
R1519 T1708 T1709 advmod directly,generated
R1520 T1709 T1706 conj generated,amplified
R1521 T1710 T1709 agent by,generated
R1522 T1711 T1710 pobj cells,by
R1523 T1712 T1711 acl exposed,cells
R1524 T1713 T1712 prep to,exposed
R1525 T1714 T1715 amod sulphated,polysaccharides
R1526 T1715 T1713 pobj polysaccharides,to
R1527 T1716 T1717 amod such,as
R1528 T1717 T1715 prep as,polysaccharides
R1529 T1718 T1717 pobj carrageenans,as
R1530 T1719 T1703 punct .,seems
R1531 T1720 T1727 advmod Indeed,associated
R1532 T1721 T1727 punct ",",associated
R1533 T1722 T1727 nsubjpass inflammation,associated
R1534 T1723 T1722 acl induced,inflammation
R1535 T1724 T1723 agent by,induced
R1536 T1725 T1724 pobj dCGN,by
R1537 T1726 T1727 auxpass was,associated
R1538 T1727 T1727 ROOT associated,associated
R1539 T1728 T1727 prep with,associated
R1540 T1729 T1728 pobj recruitment,with
R1541 T1730 T1729 prep of,recruitment
R1542 T1731 T1730 pobj macrophages,of
R1543 T1732 T1727 prep to,associated
R1544 T1733 T1734 compound inflammation,sites
R1545 T1734 T1732 pobj sites,to
R1546 T1735 T1737 nmod [,]
R1547 T1736 T1737 nummod 18,]
R1548 T1737 T1734 appos ],sites
R1549 T1738 T1737 punct ",",]
R1550 T1739 T1741 nmod [,]
R1551 T1740 T1741 nummod 19,]
R1552 T1741 T1737 conj ],]
R1553 T1742 T1727 punct .,associated
R1554 T1743 T1761 advmod Also,associated
R1555 T1744 T1761 punct ",",associated
R1556 T1745 T1761 nsubjpass inflammation,associated
R1557 T1746 T1745 acl induced,inflammation
R1558 T1747 T1746 agent by,induced
R1559 T1748 T1750 compound Dextran,Sodium
R1560 T1749 T1750 compound Sulphate,Sodium
R1561 T1750 T1761 nsubjpass Sodium,associated
R1562 T1751 T1752 punct (,DSS
R1563 T1752 T1750 appos DSS,Sodium
R1564 T1753 T1750 punct ),Sodium
R1565 T1754 T1750 punct ",",Sodium
R1566 T1755 T1757 det another,compound
R1567 T1756 T1757 amod sulphated,compound
R1568 T1757 T1750 appos compound,Sodium
R1569 T1758 T1761 punct ",",associated
R1570 T1759 T1761 auxpass was,associated
R1571 T1760 T1761 advmod directly,associated
R1572 T1761 T1761 ROOT associated,associated
R1573 T1762 T1761 prep with,associated
R1574 T1763 T1764 compound macrophages,recruitment
R1575 T1764 T1762 pobj recruitment,with
R1576 T1765 T1767 nmod [,]
R1577 T1766 T1767 nummod 20,]
R1578 T1767 T1764 appos ],recruitment
R1579 T1768 T1761 punct ",",associated
R1580 T1769 T1772 mark since,provoked
R1581 T1770 T1772 nsubj DSS,provoked
R1582 T1771 T1772 advmod still,provoked
R1583 T1772 T1761 advcl provoked,associated
R1584 T1773 T1772 dobj inflammation,provoked
R1585 T1774 T1772 prep after,provoked
R1586 T1775 T1774 pobj T-lymphocyte,after
R1587 T1776 T1775 cc and,T-lymphocyte
R1588 T1777 T1779 compound NK,depletion
R1589 T1778 T1779 compound cell,depletion
R1590 T1779 T1775 conj depletion,T-lymphocyte
R1591 T1780 T1782 nsubj [,]
R1592 T1781 T1782 nummod 20,]
R1593 T1782 T1782 ROOT ],]
R1594 T1783 T1761 punct .,associated
R1595 T1784 T1788 mark Although,induced
R1596 T1785 T1788 nsubjpass inflammation,induced
R1597 T1786 T1788 aux can,induced
R1598 T1787 T1788 auxpass be,induced
R1599 T1788 T1793 advcl induced,are
R1600 T1789 T1788 agent by,induced
R1601 T1790 T1789 pobj dCGN,by
R1602 T1791 T1793 punct ",",are
R1603 T1792 T1793 expl there,are
R1604 T1793 T1793 ROOT are,are
R1605 T1794 T1795 det no,data
R1606 T1795 T1793 attr data,are
R1607 T1796 T1795 prep on,data
R1608 T1797 T1799 amod human,responses
R1609 T1798 T1799 compound monocyte,responses
R1610 T1799 T1796 pobj responses,on
R1611 T1800 T1799 prep to,responses
R1612 T1801 T1802 compound dCGN,exposure
R1613 T1802 T1800 pobj exposure,to
R1614 T1803 T1793 punct .,are
R1615 T1804 T1829 advmod Therefore,exposed
R1616 T1805 T1829 punct ",",exposed
R1617 T1806 T1807 aux to,investigate
R1618 T1807 T1829 advcl investigate,exposed
R1619 T1808 T1809 det the,effects
R1620 T1809 T1807 dobj effects,investigate
R1621 T1810 T1809 prep of,effects
R1622 T1811 T1810 pobj dCGN,of
R1623 T1812 T1809 prep on,effects
R1624 T1813 T1814 amod human,monocytes
R1625 T1814 T1812 pobj monocytes,on
R1626 T1815 T1814 punct ",",monocytes
R1627 T1816 T1819 amod normal,Monocytes
R1628 T1817 T1818 compound Peripheral,Blood
R1629 T1818 T1819 compound Blood,Monocytes
R1630 T1819 T1827 nmod Monocytes,cells
R1631 T1820 T1821 punct (,PBM
R1632 T1821 T1819 appos PBM,Monocytes
R1633 T1822 T1821 punct ),PBM
R1634 T1823 T1819 cc and,Monocytes
R1635 T1824 T1825 amod tumoral,monocyte/macrophage
R1636 T1825 T1819 conj monocyte/macrophage,Monocytes
R1637 T1826 T1827 nummod THP-1,cells
R1638 T1827 T1829 nsubjpass cells,exposed
R1639 T1828 T1829 auxpass were,exposed
R1640 T1829 T1829 ROOT exposed,exposed
R1641 T1830 T1829 prep to,exposed
R1642 T1831 T1832 nummod 10,kDa
R1643 T1832 T1830 pobj kDa,to
R1644 T1833 T1832 cc and,kDa
R1645 T1834 T1836 nummod 40,dCGN
R1646 T1835 T1836 compound kDa,dCGN
R1647 T1836 T1832 conj dCGN,kDa
R1648 T1837 T1829 punct .,exposed
R1649 T1838 T1839 nsubj We,found
R1650 T1839 T1839 ROOT found,found
R1651 T1840 T1842 mark that,inhibited
R1652 T1841 T1842 nsubj dCGN,inhibited
R1653 T1842 T1839 ccomp inhibited,found
R1654 T1843 T1845 nummod THP-1,proliferation
R1655 T1844 T1845 compound cell,proliferation
R1656 T1845 T1842 dobj proliferation,inhibited
R1657 T1846 T1842 prep in,inhibited
R1658 T1847 T1846 pobj vitro,in
R1659 T1848 T1842 punct ",",inhibited
R1660 T1849 T1851 amod increased,expression
R1661 T1850 T1851 compound ICAM-1,expression
R1662 T1851 T1839 dobj expression,found
R1663 T1852 T1851 punct ",",expression
R1664 T1853 T1851 acl stimulated,expression
R1665 T1854 T1856 amod ICAM-1-dependent,aggregation
R1666 T1855 T1856 compound monocyte,aggregation
R1667 T1856 T1853 dobj aggregation,stimulated
R1668 T1857 T1839 punct ",",found
R1669 T1858 T1839 cc and,found
R1670 T1859 T1839 conj stimulated,found
R1671 T1860 T1861 compound TNF-α,expression
R1672 T1861 T1859 dobj expression,stimulated
R1673 T1862 T1861 cc and,expression
R1674 T1863 T1861 conj secretion,expression
R1675 T1864 T1839 punct .,found
R1676 T1865 T1866 det These,responses
R1677 T1866 T1867 nsubj responses,were
R1678 T1867 T1867 ROOT were,were
R1679 T1868 T1869 advmod more,pronounced
R1680 T1869 T1867 acomp pronounced,were
R1681 T1870 T1869 prep after,pronounced
R1682 T1871 T1874 nummod 40,exposure
R1683 T1872 T1874 compound kDa,exposure
R1684 T1873 T1874 compound dCGN,exposure
R1685 T1874 T1870 pobj exposure,after
R1686 T1875 T1867 cc and,were
R1687 T1876 T1877 auxpass were,linked
R1688 T1877 T1867 conj linked,were
R1689 T1878 T1877 prep to,linked
R1690 T1879 T1880 compound NF-κB,activation
R1691 T1880 T1878 pobj activation,to
R1692 T1881 T1867 punct .,were
R1693 T1882 T1896 prep In,induced
R1694 T1883 T1882 pobj addition,In
R1695 T1884 T1896 punct ",",induced
R1696 T1885 T1888 det the,dCGN
R1697 T1886 T1888 nummod 40,dCGN
R1698 T1887 T1888 compound kDa,dCGN
R1699 T1888 T1896 nsubj dCGN,induced
R1700 T1889 T1888 punct ",",dCGN
R1701 T1890 T1896 cc but,induced
R1702 T1891 T1896 neg not,induced
R1703 T1892 T1895 det the,dCGN
R1704 T1893 T1895 nummod 10,dCGN
R1705 T1894 T1895 compound kDa,dCGN
R1706 T1895 T1896 nsubj dCGN,induced
R1707 T1896 T1896 ROOT induced,induced
R1708 T1897 T1896 prep in,induced
R1709 T1898 T1899 compound vivo,colitis
R1710 T1899 T1897 pobj colitis,in
R1711 T1900 T1901 mark as,shown
R1712 T1901 T1896 advcl shown,induced
R1713 T1902 T1901 agent by,shown
R1714 T1903 T1905 det the,response
R1715 T1904 T1905 amod inflammatory,response
R1716 T1905 T1902 pobj response,by
R1717 T1906 T1905 prep in,response
R1718 T1907 T1909 det the,colon
R1719 T1908 T1909 compound rat,colon
R1720 T1909 T1906 pobj colon,in
R1721 T1910 T1896 punct .,induced
R1722 T1911 T1912 det These,results
R1723 T1912 T1913 nsubj results,suggest
R1724 T1913 T1913 ROOT suggest,suggest
R1725 T1914 T1920 mark that,have
R1726 T1915 T1917 det the,forms
R1727 T1916 T1917 amod degraded,forms
R1728 T1917 T1920 nsubj forms,have
R1729 T1918 T1917 prep of,forms
R1730 T1919 T1918 pobj CGN,of
R1731 T1920 T1913 ccomp have,suggest
R1732 T1921 T1923 det an,effect
R1733 T1922 T1923 amod important,effect
R1734 T1923 T1920 dobj effect,have
R1735 T1924 T1923 prep on,effect
R1736 T1925 T1924 pobj monocytes,on
R1737 T1926 T1923 acl resulting,effect
R1738 T1927 T1926 prep in,resulting
R1739 T1928 T1930 det an,phenotype
R1740 T1929 T1930 amod inflammatory,phenotype
R1741 T1930 T1927 pobj phenotype,in
R1742 T1931 T1913 punct .,suggest
R2132 T2416 T2449 nsubjpass Preparation,prepared
R2133 T2417 T2416 prep of,Preparation
R2134 T2418 T2419 compound Degraded,Carrageenan
R2135 T2419 T2417 pobj Carrageenan,of
R2136 T2420 T2421 nummod Two,preparations
R2137 T2421 T2447 nmod preparations,weight
R2138 T2422 T2421 prep of,preparations
R2139 T2423 T2424 amod degraded,carrageenan
R2140 T2424 T2422 pobj carrageenan,of
R2141 T2425 T2424 prep with,carrageenan
R2142 T2426 T2425 pobj low,with
R2143 T2427 T2421 punct ",",preparations
R2144 T2428 T2429 punct (,∼
R2145 T2429 T2421 appos ∼,preparations
R2146 T2430 T2431 nummod 10,kDa
R2147 T2431 T2429 npadvmod kDa,∼
R2148 T2432 T2421 punct ;,preparations
R2149 T2433 T2421 appos C10,preparations
R2150 T2434 T2421 punct ),preparations
R2151 T2435 T2421 punct ",",preparations
R2152 T2436 T2421 cc and,preparations
R2180 T2464 T2463 punct ",",Industry
R2181 T2465 T2463 conj Boulogne-Billancourt,Industry
R2182 T2466 T2465 punct ",",Boulogne-Billancourt
R2183 T2467 T2465 conj France,Boulogne-Billancourt
R2184 T2468 T2452 punct ),iota-carrageenan
R2185 T2469 T2449 punct .,prepared
R2186 T2470 T2471 amod Native,carrageenan
R2187 T2471 T2473 nsubjpass carrageenan,dissolved
R2188 T2472 T2473 auxpass was,dissolved
R2189 T2473 T2473 ROOT dissolved,dissolved
R2190 T2474 T2473 prep in,dissolved
R2191 T2475 T2476 amod distilled,water
R2192 T2476 T2474 pobj water,in
R2193 T2477 T2480 punct (,w/v
R2194 T2478 T2479 nummod 5,%
R2195 T2479 T2480 compound %,w/v
R2196 T2480 T2473 parataxis w/v,dissolved
R2197 T2481 T2480 punct ),w/v
R2198 T2482 T2473 prep under,dissolved
R2199 T2483 T2484 amod vigorous,stirring
R2200 T2484 T2482 pobj stirring,under
R2201 T2485 T2484 cc and,stirring
R2202 T2486 T2484 conj heated,stirring
R2203 T2487 T2486 prep to,heated
R2204 T2488 T2489 nummod 60,°
R2232 T2516 T2518 amod low,weight
R2233 T2517 T2518 amod molecular,weight
R2234 T2518 T2519 compound weight,fraction
R2235 T2519 T2514 pobj fraction,for
R2236 T2520 T2524 punct ",",hydrolyzed
R2237 T2521 T2522 compound carrageenan,solution
R2238 T2522 T2524 nsubjpass solution,hydrolyzed
R2239 T2523 T2524 auxpass was,hydrolyzed
R2240 T2524 T2524 ROOT hydrolyzed,hydrolyzed
R2241 T2525 T2524 prep with,hydrolyzed
R2242 T2526 T2527 nummod 0.3,%
R2243 T2527 T2525 pobj %,with
R2244 T2528 T2529 punct (,v/v
R2245 T2529 T2527 appos v/v,%
R2246 T2530 T2524 punct ),hydrolyzed
R2247 T2531 T2533 nmod concentrated,acid
R2248 T2532 T2533 compound sulphuric,acid
R2249 T2533 T2524 dobj acid,hydrolyzed
R2250 T2534 T2524 prep for,hydrolyzed
R2251 T2535 T2536 nummod 15,min
R2252 T2536 T2534 pobj min,for
R2253 T2537 T2524 prep at,hydrolyzed
R2254 T2538 T2540 nummod 80,C
R2255 T2539 T2540 nummod °,C
R2256 T2540 T2537 pobj C,at
R2257 T2541 T2524 punct .,hydrolyzed
R2258 T2542 T2552 prep After,filtered
R2259 T2543 T2542 pobj neutralization,After
R2260 T2544 T2543 prep with,neutralization
R2261 T2545 T2546 compound NaOH,4N
R2262 T2546 T2544 pobj 4N,with
R2263 T2547 T2552 punct ",",filtered
R2264 T2548 T2549 det the,solution
R2265 T2549 T2552 nsubjpass solution,filtered
R2266 T2550 T2552 auxpass was,filtered
R2267 T2551 T2552 advmod ultra,filtered
R2268 T2552 T2552 ROOT filtered,filtered
R2269 T2553 T2552 prep through,filtered
R2270 T2554 T2557 det a,cartridge
R2271 T2555 T2557 amod hollow,cartridge
R2272 T2556 T2557 compound fibre,cartridge
R2273 T2557 T2553 pobj cartridge,through
R2274 T2558 T2557 prep with,cartridge
R2275 T2559 T2562 nmod MW,kDa
R2276 T2560 T2562 nmod cut-off,kDa
R2277 T2561 T2562 nummod 5,kDa
R2278 T2562 T2558 pobj kDa,with
R2279 T2563 T2562 punct ",",kDa
R2280 T2564 T2566 punct (,Inc
R2281 T2565 T2566 compound Amicon,Inc
R2282 T2566 T2562 conj Inc,kDa
R2283 T2567 T2566 punct ",",Inc
R2284 T2568 T2566 conj Beverly,Inc
R2285 T2569 T2568 punct ",",Beverly
R2286 T2570 T2568 conj USA,Beverly
R2287 T2571 T2566 punct ),Inc
R2288 T2572 T2552 punct .,filtered
R2289 T2573 T2584 prep For,hydrolyzed
R2290 T2574 T2578 det the,fraction
R2291 T2575 T2578 compound medium,fraction
R2292 T2576 T2577 amod molecular,weight
R2293 T2577 T2578 compound weight,fraction
R2294 T2578 T2573 pobj fraction,For
R2295 T2579 T2584 punct ",",hydrolyzed
R2296 T2580 T2582 det the,solution
R2297 T2581 T2582 compound carrageenan,solution
R2298 T2582 T2584 nsubjpass solution,hydrolyzed
R2299 T2583 T2584 auxpass was,hydrolyzed
R2300 T2584 T2584 ROOT hydrolyzed,hydrolyzed
R2301 T2585 T2584 prep with,hydrolyzed
R2302 T2586 T2587 nummod 0.3,%
R2303 T2587 T2585 pobj %,with
R2304 T2588 T2589 punct (,v/v
R2305 T2589 T2587 appos v/v,%
R2306 T2590 T2584 punct ),hydrolyzed
R2307 T2591 T2591 ROOT concentrated,concentrated
R2308 T2592 T2593 amod sulphuric,acid
R2309 T2593 T2591 dobj acid,concentrated
R2310 T2594 T2591 prep for,concentrated
R2311 T2595 T2596 nummod 30,min
R2312 T2596 T2594 pobj min,for
R2313 T2597 T2591 prep at,concentrated
R2314 T2598 T2599 nummod 60,°
R2315 T2599 T2597 pobj °,at
R2316 T2600 T2591 npadvmod C,concentrated
R2317 T2601 T2584 punct .,hydrolyzed
R2318 T2602 T2609 prep After,filtered
R2319 T2603 T2602 pobj neutralization,After
R2320 T2604 T2609 punct ",",filtered
R2321 T2605 T2606 det the,supernatant
R2322 T2606 T2609 nsubjpass supernatant,filtered
R2323 T2607 T2609 auxpass was,filtered
R2324 T2608 T2609 advmod ultra,filtered
R2325 T2609 T2609 ROOT filtered,filtered
R2326 T2610 T2614 punct (,kDa
R2327 T2611 T2614 prt MW,kDa
R2328 T2612 T2614 nmod cut-off,kDa
R2329 T2613 T2614 nummod 100,kDa
R2330 T2614 T2609 dobj kDa,filtered
R2331 T2615 T2614 punct ),kDa
R2332 T2616 T2609 punct .,filtered
R2333 T2617 T2618 det The,filtrate
R2334 T2618 T2620 nsubjpass filtrate,submitted
R2335 T2619 T2620 auxpass was,submitted
R2336 T2620 T2620 ROOT submitted,submitted
R2337 T2621 T2620 prep to,submitted
R2338 T2622 T2625 det a,filtration
R2339 T2623 T2625 amod second,filtration
R2340 T2624 T2625 compound ultra,filtration
R2341 T2625 T2621 pobj filtration,to
R2342 T2626 T2630 punct (,kDa
R2343 T2627 T2630 nmod MW,kDa
R2344 T2628 T2630 punct cut-off,kDa
R2345 T2629 T2628 nummod 5,cut-off
R2346 T2630 T2625 appos kDa,filtration
R2347 T2631 T2630 punct ),kDa
R2348 T2632 T2620 punct .,submitted
R2349 T2633 T2634 det Both,preparations
R2350 T2634 T2638 nsubjpass preparations,precipitated
R2351 T2635 T2634 prep of,preparations
R2352 T2636 T2635 pobj dCGN,of
R2353 T2637 T2638 auxpass were,precipitated
R2354 T2638 T2638 ROOT precipitated,precipitated
R2355 T2639 T2638 prep with,precipitated
R2356 T2640 T2641 nummod 4,volumes
R2357 T2641 T2639 pobj volumes,with
R2358 T2642 T2641 prep of,volumes
R2359 T2643 T2644 nummod 95,%
R2360 T2644 T2645 compound %,ethanol
R2361 T2645 T2642 pobj ethanol,of
R2362 T2646 T2638 punct ",",precipitated
R2363 T2647 T2638 conj dried,precipitated
R2364 T2648 T2647 prep at,dried
R2365 T2649 T2650 compound room,temperature
R2366 T2650 T2648 pobj temperature,at
R2367 T2651 T2650 cc and,temperature
R2368 T2652 T2650 conj ground,temperature
R2369 T2653 T2647 prep to,dried
R2370 T2654 T2655 amod small,particles
R2371 T2655 T2653 pobj particles,to
R2372 T2656 T2658 punct (,mm
R2373 T2657 T2658 nummod 1,mm
R2374 T2658 T2655 parataxis mm,particles
R2375 T2659 T2658 prep in,mm
R2376 T2660 T2659 pobj diameter,in
R2377 T2661 T2658 punct ),mm
R2378 T2662 T2638 punct .,precipitated
R2379 T2663 T2663 ROOT Using,Using
R2380 T2664 T2665 compound gel-permeation,chromatography
R2381 T2665 T2663 dobj chromatography,Using
R2382 T2666 T2665 prep in,chromatography
R2383 T2667 T2666 pobj combination,in
R2384 T2668 T2663 prep with,Using
R2385 T2669 T2671 amod light,measurements
R2386 T2670 T2671 compound scattering,measurements
R2387 T2671 T2668 pobj measurements,with
R2388 T2672 T2673 punct (,see
R2389 T2673 T2663 parataxis see,Using
R2390 T2674 T2684 nsubj Viebke,confirmed
R2391 T2675 T2676 compound et,al.
R2392 T2676 T2684 prep al.,confirmed
R2393 T2677 T2676 appos [,al.
R2394 T2678 T2679 nummod 21,]
R2395 T2679 T2677 appos ],[
R2396 T2680 T2677 punct ),[
R2397 T2681 T2684 punct ",",confirmed
R2398 T2682 T2684 nsubjpass it,confirmed
R2399 T2683 T2684 auxpass was,confirmed
R2400 T2684 T2673 ccomp confirmed,see
R2401 T2685 T2689 mark that,had
R2402 T2686 T2688 det the,fraction
R2403 T2687 T2688 amod low,fraction
R2404 T2688 T2689 nsubj fraction,had
R2405 T2689 T2684 ccomp had,confirmed
R2406 T2690 T2693 det an,weight
R2407 T2691 T2693 amod average,weight
R2408 T2692 T2693 amod molecular,weight
R2409 T2693 T2689 dobj weight,had
R2410 T2694 T2693 prep of,weight
R2411 T2695 T2696 nummod 10,kDa
R2412 T2696 T2694 pobj kDa,of
R2413 T2697 T2689 punct ",",had
R2414 T2698 T2689 cc and,had
R2415 T2699 T2701 det the,fraction
R2416 T2700 T2701 compound medium,fraction
R2417 T2701 T2689 conj fraction,had
R2418 T2702 T2701 prep of,fraction
R2419 T2703 T2704 nummod 40,kDa
R2420 T2704 T2702 pobj kDa,of
R2421 T2705 T2673 punct .,see
R2422 T2706 T2708 det The,content
R2423 T2707 T2708 compound sulphate,content
R2424 T2708 T2715 nsubjpass content,measured
R2425 T2709 T2708 prep of,content
R2426 T2710 T2709 pobj polysaccharides,of
R2427 T2711 T2710 prep in,polysaccharides
R2428 T2712 T2713 det both,fractions
R2429 T2713 T2711 pobj fractions,in
R2430 T2714 T2715 auxpass was,measured
R2431 T2715 T2715 ROOT measured,measured
R2432 T2716 T2715 prep following,measured
R2433 T2717 T2718 det the,method
R2434 T2718 T2716 pobj method,following
R2435 T2719 T2718 prep of,method
R2436 T2720 T2723 compound Quemener,[
R2437 T2721 T2722 compound et,al.
R2438 T2722 T2723 compound al.,[
R2439 T2723 T2719 pobj [,of
R2440 T2724 T2725 nummod 22,]
R2441 T2725 T2718 npadvmod ],method
R2442 T2726 T2715 punct .,measured
R2443 T2727 T2740 advmod Finally,confirmed
R2444 T2728 T2740 punct ",",confirmed
R2445 T2729 T2730 det the,absence
R2446 T2730 T2740 nsubjpass absence,confirmed
R2447 T2731 T2730 prep of,absence
R2448 T2732 T2733 compound polysaccharide,structure
R2449 T2733 T2734 compound structure,modifications
R2450 T2734 T2731 pobj modifications,of
R2451 T2735 T2734 prep in,modifications
R2452 T2736 T2738 det the,fractions
R2453 T2737 T2738 nummod two,fractions
R2454 T2738 T2735 pobj fractions,in
R2455 T2739 T2740 auxpass was,confirmed
R2456 T2740 T2740 ROOT confirmed,confirmed
R2457 T2741 T2740 advcl using,confirmed
R2458 T2742 T2743 amod 2H-NMR,spectroscopy
R2459 T2743 T2741 dobj spectroscopy,using
R2460 T2744 T2740 punct .,confirmed
R2461 T2745 T2746 det The,absence
R2462 T2746 T2755 nsubjpass absence,confirmed
R2463 T2747 T2746 prep of,absence
R2464 T2748 T2749 compound LPS,contamination
R2465 T2749 T2747 pobj contamination,of
R2466 T2750 T2749 prep in,contamination
R2467 T2751 T2753 det the,fractions
R2468 T2752 T2753 nummod two,fractions
R2469 T2753 T2750 pobj fractions,in
R2470 T2754 T2755 auxpass was,confirmed
R2471 T2755 T2755 ROOT confirmed,confirmed
R2472 T2756 T2755 advcl using,confirmed
R2473 T2757 T2760 det the,kit
R2474 T2758 T2760 amod e-Toxate,kit
R2475 T2759 T2760 compound ®,kit
R2476 T2760 T2756 dobj kit,using
R2477 T2761 T2760 punct (,kit
R2478 T2762 T2760 appos Sigma,kit
R2479 T2763 T2762 punct ",",Sigma
R2480 T2764 T2766 compound St,Fallavier
R2481 T2765 T2766 compound Quentin,Fallavier
R2482 T2766 T2762 conj Fallavier,Sigma
R2483 T2767 T2766 punct ",",Fallavier
R2484 T2768 T2766 conj France,Fallavier
R2485 T2769 T2766 punct ),Fallavier
R2486 T2770 T2755 punct .,confirmed
R2487 T2771 T2781 prep Before,dissolved
R2488 T2772 T2771 pobj use,Before
R2489 T2773 T2772 prep in,use
R2490 T2774 T2775 compound cell,culture
R2491 T2775 T2773 pobj culture,in
R2492 T2776 T2781 punct ",",dissolved
R2647 T2959 T2960 auxpass were,housed
R2648 T2960 T2960 ROOT housed,housed
R2649 T2961 T2960 prep under,housed
R2650 T2962 T2963 amod standard,conditions
R2651 T2963 T2961 pobj conditions,under
R2652 T2964 T2960 cc and,housed
R2653 T2965 T2960 conj fed,housed
R2654 T2966 T2967 compound ad,libitum
R2655 T2967 T2965 dobj libitum,fed
R2656 T2968 T2967 prep with,libitum
R2657 T2969 T2972 amod standard,chow
R2658 T2970 T2972 compound rodent,chow
R2659 T2971 T2972 compound laboratory,chow
R2660 T2972 T2968 pobj chow,with
R2661 T2973 T2960 punct .,housed
R2662 T2974 T2975 amod Degraded,iota-carrageenans
R2663 T2975 T2977 nsubjpass iota-carrageenans,administered
R2664 T2976 T2977 auxpass were,administered
R2665 T2977 T2977 ROOT administered,administered
R2666 T2978 T2977 prep in,administered
R2667 T2979 T2981 det the,water
R2669 T2981 T2978 pobj water,in
R2730 T3042 T3038 punct ),water
R2731 T3043 T3034 punct .,maintained
R2732 T3044 T3045 aux To,increase
R2733 T3045 T3051 advcl increase,added
R2734 T3046 T3049 amod palatability,sucrose
R2735 T3047 T3048 nummod 0.2,%
R2736 T3048 T3049 compound %,sucrose
R2737 T3049 T3051 nsubjpass sucrose,added
R2738 T3050 T3051 auxpass was,added
R2739 T3051 T3051 ROOT added,added
R2740 T3052 T3051 prep to,added
R2741 T3053 T3055 det the,water
R2742 T3054 T3055 compound drinking,water
R2743 T3055 T3052 pobj water,to
R2744 T3056 T3055 prep of,water
R2745 T3057 T3058 det all,groups
R2746 T3058 T3056 pobj groups,of
R2747 T3059 T3062 punct (,Waaji
R2748 T3060 T3062 compound Van,Waaji
R2749 T3061 T3062 compound der,Waaji
R2750 T3062 T3064 compound Waaji,al.
R2751 T3063 T3064 compound et,al.
R2752 T3064 T3055 appos al.,water
R2753 T3065 T3055 punct ",",water
R2754 T3066 T3055 appos [,water
R2755 T3067 T3068 nummod 23,]
R2756 T3068 T3066 appos ],[
R2757 T3069 T3066 punct ),[
R2758 T3070 T3051 punct .,added
R2759 T3071 T3073 amod Fresh,solutions
R2760 T3072 T3073 compound carrageenan,solutions
R2761 T3073 T3075 nsubjpass solutions,prepared
R2762 T3074 T3075 auxpass were,prepared
R2763 T3075 T3075 ROOT prepared,prepared
R2764 T3076 T3075 advmod daily,prepared
R2765 T3077 T3075 punct .,prepared
R3012 T3368 T3390 nsubjpass Evaluation,recorded
R3013 T3369 T3368 prep of,Evaluation
R3014 T3370 T3371 compound Colitis,Body
R3015 T3371 T3372 compound Body,weight
R3016 T3372 T3369 pobj weight,of
R3017 T3373 T3372 punct ",",weight
R3018 T3374 T3372 conj liquid,weight
R3019 T3375 T3374 cc and,liquid
R3020 T3376 T3377 compound food,consumption
R3021 T3377 T3374 conj consumption,liquid
R3022 T3378 T3377 punct ",",consumption
R3023 T3379 T3377 conj diarrhea,consumption
R3024 T3380 T3379 cc and,diarrhea
R3025 T3381 T3382 amod rectal,bleeding
R3026 T3382 T3379 conj bleeding,diarrhea
R3027 T3383 T3382 punct (,bleeding
R3028 T3384 T3382 acl detected,bleeding
R3029 T3385 T3384 agent by,detected
R3030 T3386 T3387 compound eye,inspection
R3031 T3387 T3385 pobj inspection,by
R3032 T3388 T3368 punct ),Evaluation
R3033 T3389 T3390 auxpass were,recorded
R3034 T3390 T3390 ROOT recorded,recorded
R3035 T3391 T3390 prep throughout,recorded
R3036 T3392 T3394 det the,period
R3037 T3393 T3394 compound feeding,period
R3038 T3394 T3391 pobj period,throughout
R3039 T3395 T3390 punct .,recorded
R3040 T3396 T3402 prep After,sacrificed
R3041 T3397 T3398 nummod 55,days
R3042 T3398 T3396 pobj days,After
R3043 T3399 T3402 punct ",",sacrificed
R3044 T3400 T3402 nsubjpass animals,sacrificed
R3045 T3401 T3402 auxpass were,sacrificed
R3046 T3402 T3402 ROOT sacrificed,sacrificed
R3047 T3403 T3402 agent by,sacrificed
R3048 T3404 T3405 amod cervical,dislocation
R3049 T3405 T3403 pobj dislocation,by
R3050 T3406 T3402 punct .,sacrificed
R3051 T3407 T3408 det The,length
R3052 T3408 T3413 nsubjpass length,measured
R3053 T3409 T3408 prep of,length
R3054 T3410 T3411 det the,colon
R3055 T3411 T3409 pobj colon,of
R3056 T3412 T3413 auxpass was,measured
R3057 T3413 T3413 ROOT measured,measured
R3058 T3414 T3415 mark as,described
R3059 T3415 T3413 advcl described,measured
R3060 T3416 T3415 agent by,described
R3061 T3417 T3416 pobj Okayashu,by
R3062 T3418 T3422 nmod et,]
R3063 T3419 T3422 nmod al.,]
R3064 T3420 T3422 nmod [,]
R3065 T3421 T3422 nummod 24,]
R3066 T3422 T3415 npadvmod ],described
R3067 T3423 T3413 punct .,measured
R3068 T3424 T3429 advmod Then,ligated
R3069 T3425 T3429 punct ",",ligated
R3070 T3426 T3427 det each,colon
R3071 T3427 T3429 nsubjpass colon,ligated
R3072 T3428 T3429 auxpass was,ligated
R3073 T3429 T3429 ROOT ligated,ligated
R3074 T3430 T3429 prep in,ligated
R3075 T3431 T3430 pobj sections,in
R3076 T3432 T3431 prep of,sections
R3077 T3433 T3434 nummod 2,cm
R3078 T3434 T3432 pobj cm,of
R3079 T3435 T3429 cc and,ligated
R3080 T3436 T3438 quantmod 1,2
R3081 T3437 T3438 quantmod to,2
R3082 T3438 T3439 nummod 2,ml
R3083 T3439 T3445 nsubjpass ml,infused
R3084 T3440 T3439 prep of,ml
R3085 T3441 T3442 nummod 10,%
R3086 T3442 T3443 compound %,formalin
R3087 T3443 T3440 pobj formalin,of
R3088 T3444 T3445 auxpass was,infused
R3089 T3445 T3429 conj infused,ligated
R3090 T3446 T3445 prep into,infused
R3091 T3447 T3449 det the,lumen
R3092 T3448 T3449 amod intestinal,lumen
R3093 T3449 T3446 pobj lumen,into
R3094 T3450 T3445 punct .,infused
R3095 T3451 T3454 det The,segment
R3096 T3452 T3453 advmod moderately,distended
R3097 T3453 T3454 amod distended,segment
R3098 T3454 T3455 nsubj segment,was
R3099 T3455 T3455 ROOT was,was
R3100 T3456 T3455 attr sectioned,was
R3101 T3457 T3456 cc and,sectioned
R3102 T3458 T3456 conj fixed,sectioned
R3103 T3459 T3458 prep in,fixed
R3104 T3460 T3461 nummod 10,%
R3105 T3461 T3462 compound %,formalin
R3106 T3462 T3459 pobj formalin,in
R3107 T3463 T3455 punct .,was
R3108 T3464 T3466 det The,day
R3109 T3465 T3466 amod following,day
R3110 T3466 T3472 npadvmod day,removed
R3111 T3467 T3472 punct ",",removed
R3112 T3468 T3470 det the,content
R3113 T3469 T3470 amod intestinal,content
R3114 T3470 T3472 nsubjpass content,removed
R3115 T3471 T3472 auxpass was,removed
R3116 T3472 T3472 ROOT removed,removed
R3117 T3473 T3472 agent by,removed
R3118 T3474 T3473 pobj vortexing,by
R3119 T3475 T3472 punct .,removed
R3120 T3476 T3478 det The,segment
R3121 T3477 T3478 amod fixed,segment
R3122 T3478 T3480 nsubjpass segment,kept
R3123 T3479 T3480 auxpass was,kept
R3124 T3480 T3480 ROOT kept,kept
R3125 T3481 T3480 oprd in,kept
R3126 T3482 T3483 nummod 10,%
R3127 T3483 T3484 compound %,formalin
R3128 T3484 T3481 pobj formalin,in
R3129 T3485 T3480 prep at,kept
R3130 T3486 T3487 nummod 4,°
R3131 T3487 T3488 nummod °,C
R3132 T3488 T3485 pobj C,at
R3133 T3489 T3480 prep until,kept
R3134 T3490 T3493 det the,procedure
R3135 T3491 T3492 compound paraffin,embedding
R3136 T3492 T3493 compound embedding,procedure
R3137 T3493 T3489 pobj procedure,until
R3138 T3494 T3480 punct .,kept
R3139 T3495 T3496 aux To,evaluate
R3140 T3496 T3507 advcl evaluate,opened
R3141 T3497 T3498 det the,degree
R3142 T3498 T3496 dobj degree,evaluate
R3143 T3499 T3498 prep of,degree
R3144 T3500 T3499 pobj inflammation,of
R3145 T3501 T3507 punct ",",opened
R3146 T3502 T3503 det this,segment
R3161 T3517 T3507 conj recorded,opened
R3162 T3518 T3520 mark as,described
R3163 T3519 T3520 advmod previously,described
R3164 T3520 T3517 advcl described,recorded
R3165 T3521 T3523 nmod [,]
R3166 T3522 T3523 nummod 25,]
R3167 T3523 T3520 dobj ],described
R3168 T3524 T3523 punct ",",]
R3169 T3525 T3527 nmod [,]
R3170 T3526 T3527 nummod 26,]
R3171 T3527 T3523 appos ],]
R3172 T3528 T3517 punct .,recorded
R3173 T3529 T3532 det The,staining
R3174 T3530 T3532 nmod toluidine,staining
R3175 T3531 T3532 amod blue,staining
R3176 T3532 T3534 nsubjpass staining,used
R3177 T3533 T3534 auxpass was,used
R3178 T3534 T3534 ROOT used,used
R3179 T3535 T3534 prep for,used
R3180 T3536 T3535 pobj identification,for
R3181 T3537 T3536 prep of,identification
R3182 T3538 T3539 amod sulphated,polysaccharides
R3183 T3539 T3537 pobj polysaccharides,of
R3184 T3540 T3539 prep in,polysaccharides
R3185 T3541 T3543 det the,mucosa
R3186 T3542 T3543 amod intestinal,mucosa
R3187 T3543 T3540 pobj mucosa,in
R3188 T3544 T3534 punct .,used
R3189 T3545 T3562 prep On,collected
R3190 T3546 T3547 det the,day
R3191 T3547 T3545 pobj day,On
R3192 T3548 T3547 prep of,day
R3193 T3549 T3548 pobj sacrifice,of
R3194 T3550 T3562 punct ",",collected
R3195 T3551 T3553 det a,sample
R3196 T3552 T3553 amod fresh,sample
R3197 T3553 T3562 nsubjpass sample,collected
R3198 T3554 T3553 prep of,sample
R3199 T3555 T3556 det each,colon
R3200 T3556 T3554 pobj colon,of
R3201 T3557 T3556 punct (,colon
R3202 T3558 T3559 nummod 50,mg
R3203 T3559 T3556 appos mg,colon
R3204 T3560 T3556 punct ),colon
R3205 T3561 T3562 auxpass was,collected
R3206 T3562 T3562 ROOT collected,collected
R3207 T3563 T3562 prep for,collected
R3208 T3564 T3563 pobj myeloperoxidase,for
R3209 T3565 T3566 punct (,MPO
R3210 T3566 T3564 appos MPO,myeloperoxidase
R3211 T3567 T3566 punct ),MPO
R3212 T3568 T3569 amod assay,according
R3213 T3569 T3562 prep according,collected
R3214 T3570 T3569 prep to,according
R3215 T3571 T3573 compound Krawisz,al.
R3216 T3572 T3573 nmod et,al.
R3217 T3573 T3570 pobj al.,to
R3218 T3574 T3573 punct ",",al.
R3219 T3575 T3577 nmod [,]
R3220 T3576 T3577 nummod 27,]
R3221 T3577 T3573 appos ],al.
R3222 T3578 T3562 punct .,collected
R3223 T3579 T3580 det The,level
R3224 T3580 T3589 nsubj level,indicates
R3234 T3590 T3591 det the,rate
R3235 T3591 T3589 dobj rate,indicates
R3236 T3592 T3591 prep of,rate
R3237 T3593 T3592 pobj recruitment,of
R3238 T3594 T3593 prep of,recruitment
R3239 T3595 T3594 pobj neutrophils,of
R3240 T3596 T3589 prep to,indicates
R3241 T3597 T3599 det the,mucosa
R3242 T3598 T3599 amod intestinal,mucosa
R3243 T3599 T3596 pobj mucosa,to
R3244 T3600 T3589 punct .,indicates
R3245 T3601 T3602 nummod One,unit
R3246 T3602 T3606 nsubj unit,corresponds
R3247 T3603 T3602 prep of,unit
R3248 T3604 T3605 compound MPO,activity
R3249 T3605 T3603 pobj activity,of
R3250 T3606 T3606 ROOT corresponds,corresponds
R3251 T3607 T3606 prep to,corresponds
R3252 T3608 T3609 det the,degradation
R3253 T3609 T3607 pobj degradation,to
R3254 T3610 T3609 prep of,degradation
R3255 T3611 T3612 nummod 1,µmol
R3256 T3612 T3610 pobj µmol,of
R3257 T3613 T3612 prep of,µmol
R3258 T3614 T3613 pobj peroxide,of
R3259 T3615 T3614 prep per,peroxide
R3260 T3616 T3615 pobj minute,per
R3261 T3617 T3609 prep at,degradation
R3262 T3618 T3619 nummod 25,°
R3263 T3619 T3620 nummod °,C.
R3264 T3620 T3617 pobj C.,at
R3413 T3816 T3818 compound Cell,All
R3414 T3817 T3818 compound Culture,All
R3415 T3818 T3821 det All,reagents
R3416 T3819 T3820 compound tissue,culture
R3417 T3820 T3821 compound culture,reagents
R3418 T3821 T3822 nsubj reagents,were
R3419 T3822 T3822 ROOT were,were
R3420 T3823 T3822 prep from,were
R3421 T3824 T3823 pobj Invitrogen,from
R3422 T3825 T3824 punct (,Invitrogen
R3423 T3826 T3827 compound Cergy,Pontoise
R3424 T3827 T3824 appos Pontoise,Invitrogen
R3425 T3828 T3827 punct ",",Pontoise
R3426 T3829 T3827 npadvmod France,Pontoise
R3427 T3830 T3824 punct ),Invitrogen
R3428 T3831 T3822 punct .,were
R3429 T3832 T3835 nummod THP-1,cells
R3430 T3833 T3835 amod human,cells
R3431 T3834 T3835 amod monocytic,cells
R3432 T3835 T3837 nsubjpass cells,maintained
R3433 T3836 T3837 auxpass were,maintained
R3434 T3837 T3837 ROOT maintained,maintained
R3435 T3838 T3837 prep in,maintained
R3436 T3839 T3838 pobj RPMI-1640,in
R3437 T3840 T3837 conj supplemented,maintained
R3438 T3841 T3840 prep with,supplemented
R3439 T3842 T3843 nummod 10,%
R3440 T3843 T3844 compound %,FCS
R3441 T3844 T3841 pobj FCS,with
R3442 T3845 T3844 punct ",",FCS
R3443 T3846 T3850 nummod 2,glutamine
R3444 T3847 T3850 compound mM,glutamine
R3445 T3848 T3850 compound L,glutamine
R3446 T3849 T3850 punct -,glutamine
R3447 T3850 T3844 appos glutamine,FCS
R3448 T3851 T3850 punct ",",glutamine
R3449 T3852 T3854 nummod 50,penicillin
R3450 T3853 T3854 compound U/ml,penicillin
R3451 T3854 T3862 nsubj penicillin,C
R3452 T3855 T3854 cc and,penicillin
R3453 T3856 T3857 nummod 50,mg/ml
R3454 T3857 T3858 compound mg/ml,streptomycin
R3455 T3858 T3854 conj streptomycin,penicillin
R3456 T3859 T3854 prep at,penicillin
R3457 T3860 T3861 nummod 37,°
R3458 T3861 T3859 pobj °,at
R3459 T3862 T3844 conj C,FCS
R3460 T3863 T3837 prep in,maintained
R3461 T3864 T3868 det a,incubator
R3462 T3865 T3866 nummod 5,%
R3463 T3866 T3868 nmod %,incubator
R3464 T3867 T3868 nummod CO2,incubator
R3465 T3868 T3863 pobj incubator,in
R3466 T3869 T3837 punct .,maintained
R3467 T3870 T3874 amod Human,cells
R3468 T3871 T3874 amod peripheral,cells
R3469 T3872 T3874 compound blood,cells
R3470 T3873 T3874 compound mononuclear,cells
R3471 T3874 T3876 nsubjpass cells,obtained
R3472 T3875 T3876 auxpass were,obtained
R3473 T3876 T3876 ROOT obtained,obtained
R3474 T3877 T3876 prep from,obtained
R3475 T3878 T3879 amod heparinized,blood
R3476 T3879 T3877 pobj blood,from
R3477 T3880 T3876 agent by,obtained
R3478 T3881 T3883 amod Ficoll-Hypaque,gradient
R3479 T3882 T3883 compound density,gradient
R3480 T3883 T3880 pobj gradient,by
R3481 T3884 T3876 punct .,obtained
R3482 T3885 T3888 nsubjpass Monocytes,isolated
R3483 T3886 T3888 auxpass were,isolated
R3484 T3887 T3888 advmod then,isolated
R3485 T3888 T3888 ROOT isolated,isolated
R3486 T3889 T3888 agent by,isolated
R3487 T3890 T3889 pobj adherence,by
R3488 T3891 T3890 prep to,adherence
R3489 T3892 T3893 compound culture,flasks
R3490 T3893 T3891 pobj flasks,to
R3491 T3894 T3895 mark as,described
R3492 T3895 T3888 advcl described,isolated
R3493 T3896 T3898 compound [,]
R3494 T3897 T3898 compound 28,]
R3495 T3898 T3895 dobj ],described
R3496 T3899 T3888 punct .,isolated
R3497 T3900 T3906 prep For,cultured
R3498 T3901 T3902 compound cell,aggregation
R3499 T3902 T3900 pobj aggregation,For
R3500 T3903 T3906 punct ",",cultured
R3501 T3904 T3906 nsubjpass monocytes,cultured
R3502 T3905 T3906 auxpass were,cultured
R3503 T3906 T3906 ROOT cultured,cultured
R3504 T3907 T3906 prep in,cultured
R3505 T3908 T3909 det the,presence
R3506 T3909 T3907 pobj presence,in
R3507 T3910 T3909 cc or,presence
R3508 T3911 T3909 conj absence,presence
R3509 T3912 T3911 prep of,absence
R3510 T3913 T3912 pobj C10,of
R3511 T3914 T3913 cc or,C10
R3512 T3915 T3913 conj C40,C10
R3513 T3916 T3906 prep for,cultured
R3514 T3917 T3920 nummod 72,colonies
R3515 T3918 T3920 compound h.,colonies
R3516 T3919 T3920 compound Cell,colonies
R3517 T3920 T3916 pobj colonies,for
R3518 T3921 T3922 auxpass were,monitored
R3519 T3922 T3906 conj monitored,cultured
R3520 T3923 T3922 prep under,monitored
R3521 T3924 T3928 det an,microscope
R3522 T3925 T3926 amod inverted,phase
R3523 T3926 T3928 compound phase,microscope
R3524 T3927 T3928 compound contrast,microscope
R3525 T3928 T3923 pobj microscope,under
R3526 T3929 T3928 acl coupled,microscope
R3527 T3930 T3929 prep through,coupled
R3528 T3931 T3933 det a,camera
R3529 T3932 T3933 compound video,camera
R3530 T3933 T3930 pobj camera,through
R3531 T3934 T3929 prep to,coupled
R3532 T3935 T3936 det a,computer
R3533 T3936 T3934 pobj computer,to
R3534 T3937 T3906 punct .,cultured
R3535 T3938 T3964 prep In,added
R3536 T3939 T3940 det some,wells
R3537 T3940 T3938 pobj wells,In
R3538 T3941 T3964 punct ",",added
R3539 T3942 T3964 csubj neutralizing,added
R3540 T3943 T3944 amod monoclonal,antibody
R3541 T3944 T3942 dobj antibody,neutralizing
R3542 T3945 T3942 prep to,neutralizing
R3543 T3946 T3945 pobj ICAM-1,to
R3544 T3947 T3951 punct (,ml
R3545 T3948 T3949 nummod 2.5,µg
R3546 T3949 T3951 nmod µg,ml
R3547 T3950 T3951 compound /,ml
R3548 T3951 T3946 appos ml,ICAM-1
R3549 T3952 T3946 punct ),ICAM-1
R3550 T3953 T3964 punct (,added
R3551 T3954 T3964 dep Tebu,added
R3552 T3955 T3954 punct ",",Tebu
R3553 T3956 T3957 compound Le,Perray
R3554 T3957 T3964 nsubjpass Perray,added
R3555 T3958 T3957 prep en,Perray
R3556 T3959 T3958 pobj Yvelines,en
R3557 T3960 T3959 punct ",",Yvelines
R3558 T3961 T3959 appos France,Yvelines
R3559 T3962 T3959 punct ),Yvelines
R3560 T3963 T3964 auxpass was,added
R3561 T3964 T3964 ROOT added,added
R3562 T3965 T3964 punct .,added
R3680 T4100 T4102 compound Cell,Analysis
R3681 T4101 T4102 compound Cycle,Analysis
R3682 T4102 T4104 compound Analysis,cells
R3683 T4103 T4104 compound THP-1,cells
R3684 T4104 T4110 nsubjpass cells,exposed
R3685 T4105 T4104 prep in,cells
R3686 T4106 T4108 amod exponential,phase
R3687 T4107 T4108 compound growth,phase
R3688 T4108 T4105 pobj phase,in
R3689 T4109 T4110 auxpass were,exposed
R3690 T4110 T4110 ROOT exposed,exposed
R3691 T4111 T4112 aux to,complete
R3692 T4112 T4110 xcomp complete,exposed
R3693 T4113 T4112 dobj medium,complete
R3694 T4114 T4112 prep in,complete
R3695 T4115 T4116 det the,presence
R3696 T4116 T4114 pobj presence,in
R3697 T4117 T4116 cc or,presence
R3698 T4118 T4116 conj absence,presence
R3699 T4119 T4118 prep of,absence
R3700 T4120 T4119 pobj carrageenans,of
R3701 T4121 T4112 prep for,complete
R3702 T4122 T4123 nummod 24,h
R3703 T4123 T4121 pobj h,for
R3704 T4124 T4112 prep before,complete
R3705 T4125 T4126 auxpass being,stained
R3706 T4126 T4124 pcomp stained,before
R3707 T4127 T4126 prep with,stained
R3708 T4128 T4129 compound propidium,iodide
R3709 T4129 T4127 pobj iodide,with
R3710 T4130 T4124 pcomp using,before
R3711 T4131 T4134 det the,kit
R3712 T4132 T4133 compound DNA-Prep,Coulter
R3713 T4133 T4134 compound Coulter,kit
R3714 T4134 T4130 dobj kit,using
R3715 T4135 T4110 prep according,exposed
R3716 T4136 T4135 prep to,according
R3717 T4137 T4138 det the,manufacturer
R3718 T4138 T4140 poss manufacturer,instruction
R3719 T4139 T4138 case 's,manufacturer
R3720 T4140 T4136 pobj instruction,to
R3721 T4141 T4142 punct (,Beckman-Coulter
R3722 T4142 T4140 appos Beckman-Coulter,instruction
R3723 T4143 T4142 punct ",",Beckman-Coulter
R3724 T4144 T4142 conj Villepinte,Beckman-Coulter
R3725 T4145 T4144 punct ",",Villepinte
R3726 T4146 T4144 conj France,Villepinte
R3727 T4147 T4110 punct ),exposed
R3728 T4148 T4110 punct .,exposed
R3729 T4149 T4150 compound Cell,DNA
R3730 T4150 T4151 compound DNA,content
R3731 T4151 T4154 nsubjpass content,analyzed
R3732 T4152 T4154 auxpass was,analyzed
R3733 T4153 T4154 advmod then,analyzed
R3734 T4154 T4154 ROOT analyzed,analyzed
R3735 T4155 T4154 agent by,analyzed
R3736 T4156 T4157 compound flow,cytometry
R3737 T4157 T4155 pobj cytometry,by
R3738 T4158 T4154 advcl using,analyzed
R3739 T4159 T4161 det an,XL2
R3740 T4160 T4161 compound EPICS,XL2
R3741 T4161 T4158 dobj XL2,using
R3742 T4162 T4163 punct (,Beckman-Coulter
R3743 T4163 T4161 appos Beckman-Coulter,XL2
R3744 T4164 T4161 punct ),XL2
R3745 T4165 T4154 punct .,analyzed
R3746 T4166 T4167 amod Raw,data
R3747 T4167 T4182 nsubjpass data,expressed
R3748 T4168 T4167 prep for,data
R3749 T4169 T4170 det the,distribution
R3750 T4170 T4168 pobj distribution,for
R3751 T4171 T4170 prep of,distribution
R3752 T4172 T4173 compound DNA,content
R3753 T4173 T4171 pobj content,of
R3754 T4174 T4173 prep of,content
R3755 T4175 T4176 nummod "30,000",cells
R3756 T4176 T4174 pobj cells,of
R3757 T4177 T4170 acl retrieved,distribution
R3758 T4178 T4177 prep from,retrieved
R3759 T4179 T4180 det the,cytometer
R3760 T4180 T4178 pobj cytometer,from
R3761 T4181 T4182 auxpass were,expressed
R3762 T4182 T4182 ROOT expressed,expressed
R3763 T4183 T4182 prep as,expressed
R3764 T4184 T4185 det the,percentage
R3765 T4185 T4183 pobj percentage,as
R3766 T4186 T4185 prep of,percentage
R3767 T4187 T4186 pobj G0/G1,of
R3768 T4188 T4182 prep through,expressed
R3769 T4189 T4190 compound G2/M,populations
R3770 T4190 T4188 pobj populations,through
R3771 T4191 T4182 punct .,expressed
R3772 T4192 T4194 amod Multicycle,software
R3773 T4193 T4194 compound AV,software
R3774 T4194 T4206 nsubjpass software,used
R3775 T4195 T4198 punct (,Systems
R3776 T4196 T4198 compound Phoenix,Systems
R3777 T4197 T4198 compound Flow,Systems
R3778 T4198 T4194 appos Systems,software
R3779 T4199 T4198 punct ",",Systems
R3780 T4200 T4201 compound San,Diego
R3781 T4201 T4198 npadvmod Diego,Systems
R3782 T4202 T4201 punct ",",Diego
R3783 T4203 T4201 conj CA,Diego
R3784 T4204 T4198 punct ),Systems
R3785 T4205 T4206 auxpass was,used
R3786 T4206 T4206 ROOT used,used
R3787 T4207 T4208 aux to,generate
R3788 T4208 T4206 xcomp generate,used
R3789 T4209 T4212 compound DNA,histograms
R3790 T4210 T4211 amod content,frequency
R3791 T4211 T4212 compound frequency,histograms
R3792 T4212 T4208 dobj histograms,generate
R3793 T4213 T4208 cc and,generate
R3794 T4214 T4208 conj facilitate,generate
R3795 T4215 T4216 compound data,analysis
R3796 T4216 T4214 dobj analysis,facilitate
R3797 T4217 T4206 punct .,used
R3936 T4382 T4384 compound Cell,Antigen
R3937 T4383 T4384 compound Surface,Antigen
R3938 T4384 T4389 compound Antigen,Monocytes
R3939 T4385 T4386 compound Expression,Analysis
R3953 T4399 T4400 det the,presence
R3954 T4400 T4398 pobj presence,in
R3955 T4401 T4400 cc or,presence
R3956 T4402 T4400 conj absence,presence
R3957 T4403 T4402 prep of,absence
R3958 T4404 T4403 pobj carrageenan,of
R3959 T4405 T4396 prep for,complete
R3960 T4406 T4407 nummod 36,h
R3961 T4407 T4405 pobj h,for
R3962 T4408 T4394 punct .,exposed
R3963 T4409 T4423 prep After,incubated
R3964 T4410 T4411 nummod two,washes
R3965 T4411 T4409 pobj washes,After
R3966 T4412 T4411 prep in,washes
R3967 T4413 T4412 pobj PBS,in
R3968 T4414 T4411 prep without,washes
R3969 T4415 T4416 compound Ca2,+
R3970 T4416 T4414 pobj +,without
R3971 T4417 T4416 cc and,+
R3972 T4418 T4419 compound Mg2,+
R3973 T4419 T4416 conj +,+
R3974 T4420 T4423 punct ",",incubated
R3975 T4421 T4423 nsubjpass cells,incubated
R3976 T4422 T4423 auxpass were,incubated
R3977 T4423 T4423 ROOT incubated,incubated
R3978 T4424 T4423 prep in,incubated
R3979 T4425 T4424 pobj PBS,in
R3980 T4426 T4423 advcl containing,incubated
R3981 T4427 T4428 nummod 0.1,%
R3982 T4428 T4429 compound %,gelatin
R3983 T4429 T4426 dobj gelatin,containing
R3984 T4430 T4429 cc and,gelatin
R3985 T4431 T4432 nummod 8,%
R3986 T4432 T4429 conj %,gelatin
R3987 T4433 T4435 nmod AB,serum
R3988 T4434 T4435 amod human,serum
R3989 T4435 T4429 conj serum,gelatin
R3990 T4436 T4437 aux to,prevent
R3991 T4437 T4435 relcl prevent,serum
R3992 T4438 T4437 advmod binding,prevent
R3993 T4439 T4438 prep to,binding
R3994 T4440 T4441 compound Fc,receptors
R3995 T4441 T4439 pobj receptors,to
R3996 T4442 T4423 punct .,incubated
R3997 T4443 T4450 advmod Then,incubated
R3998 T4444 T4450 punct ",",incubated
R3999 T4445 T4448 nummod 5,cells
R4000 T4446 T4448 nmod ×,cells
R4001 T4447 T4448 nummod 105,cells
R4002 T4448 T4450 nsubjpass cells,incubated
R4003 T4449 T4450 auxpass were,incubated
R4004 T4450 T4450 ROOT incubated,incubated
R4005 T4451 T4450 prep with,incubated
R4006 T4452 T4453 amod primary,antibodies
R4007 T4453 T4451 pobj antibodies,with
R4008 T4454 T4450 prep at,incubated
R4009 T4455 T4456 nummod 4,°
R4010 T4456 T4454 pobj °,at
R4011 T4457 T4450 conj C,incubated
R4012 T4458 T4457 prep for,C
R4013 T4459 T4460 nummod 30,min
R4014 T4460 T4458 pobj min,for
R4015 T4461 T4450 punct .,incubated
R4016 T4462 T4464 nummod Two,washes
R4017 T4463 T4464 amod other,washes
R4018 T4464 T4467 nsubj washes,preceded
R4019 T4465 T4464 prep in,washes
R4020 T4466 T4465 pobj PBS,in
R4021 T4467 T4467 ROOT preceded,preceded
R4022 T4468 T4467 dobj incubation,preceded
R4023 T4469 T4467 prep with,preceded
R4024 T4470 T4475 advmod FITC-conjugated,diluted
R4025 T4471 T4472 compound goat,antibody
R4026 T4472 T4475 nsubj antibody,diluted
R4027 T4473 T4474 compound anti-mouse,IgG
R4028 T4474 T4472 appos IgG,antibody
R4029 T4475 T4469 pcomp diluted,with
R4030 T4476 T4475 dobj 1/1000,diluted
R4031 T4477 T4475 prep at,diluted
R4032 T4478 T4479 nummod 4,°
R4033 T4479 T4477 pobj °,at
R4034 T4480 T4475 npadvmod C,diluted
R4035 T4481 T4475 prep for,diluted
R4036 T4482 T4483 nummod 30,min
R4037 T4483 T4481 pobj min,for
R4038 T4484 T4483 punct (,min
R4039 T4485 T4483 appos Tebu,min
R4040 T4486 T4483 punct ),min
R4041 T4487 T4467 punct .,preceded
R4042 T4488 T4498 mark After,performed
R4043 T4489 T4491 nummod two,washes
R4044 T4490 T4491 amod additional,washes
R4045 T4491 T4488 pobj washes,After
R4046 T4492 T4491 punct ",",washes
R4047 T4493 T4491 conj analysis,washes
R4048 T4494 T4493 prep of,analysis
R4049 T4495 T4496 amod stained,cells
R4050 T4496 T4494 pobj cells,of
R4051 T4497 T4498 auxpass was,performed
R4052 T4498 T4498 ROOT performed,performed
R4053 T4499 T4498 prep on,performed
R4054 T4500 T4502 det an,XL2
R4055 T4501 T4502 compound EPICS,XL2
R4056 T4502 T4499 pobj XL2,on
R4057 T4503 T4502 punct (,XL2
R4058 T4504 T4502 appos Beckman-Coulter,XL2
R4059 T4505 T4502 punct ),XL2
R4060 T4506 T4498 punct .,performed
R4061 T4507 T4509 det The,population
R4062 T4508 T4509 compound cell,population
R4063 T4509 T4511 nsubjpass population,gated
R4064 T4510 T4511 auxpass was,gated
R4065 T4511 T4511 ROOT gated,gated
R4066 T4512 T4511 prep according,gated
R4067 T4513 T4512 prep to,according
R4068 T4514 T4519 poss its,scattering
R4069 T4515 T4519 amod forward,scattering
R4070 T4516 T4515 cc and,forward
R4071 T4517 T4515 conj wide-angle,forward
R4072 T4518 T4519 amod light,scattering
R4073 T4519 T4513 pobj scattering,to
R4074 T4520 T4511 punct .,gated
R4075 T4521 T4523 nsubjpass Data,expressed
R4076 T4522 T4523 auxpass were,expressed
R4077 T4523 T4523 ROOT expressed,expressed
R4078 T4524 T4523 prep as,expressed
R4079 T4525 T4528 amod mean,intensity
R4080 T4526 T4528 amod relative,intensity
R4081 T4527 T4528 compound fluorescence,intensity
R4082 T4528 T4524 pobj intensity,as
R4083 T4529 T4530 punct (,MFI
R4084 T4530 T4528 appos MFI,intensity
R4085 T4531 T4530 punct ),MFI
R4086 T4532 T4528 prep of,intensity
R4087 T4533 T4534 nummod 3000,cells
R4088 T4534 T4532 pobj cells,of
R4089 T4535 T4523 punct .,expressed
R4150 T4648 T4651 compound TNF,Monocytes
R4151 T4649 T4651 compound Activity,Monocytes
R4152 T4650 T4651 compound Bioassay,Monocytes
R4153 T4651 T4656 nsubjpass Monocytes,cultured
R4154 T4652 T4651 cc or,Monocytes
R4155 T4653 T4654 nummod THP-1,cells
R4156 T4654 T4651 conj cells,Monocytes
R4157 T4655 T4656 auxpass were,cultured
R4158 T4656 T4656 ROOT cultured,cultured
R4159 T4657 T4656 prep with,cultured
R4160 T4658 T4657 cc or,with
R4161 T4659 T4657 conj without,with
R4162 T4660 T4661 amod different,concentrations
R4163 T4661 T4659 pobj concentrations,without
R4164 T4662 T4661 prep of,concentrations
R4165 T4663 T4662 pobj CGNs,of
R4166 T4664 T4663 cc or,CGNs
R4167 T4665 T4663 conj LPS,CGNs
R4168 T4666 T4668 punct (,typhosa
R4169 T4667 T4668 compound Salmonella,typhosa
R4170 T4668 T4665 appos typhosa,LPS
R4171 T4669 T4668 punct ",",typhosa
R4172 T4670 T4668 npadvmod Sigma,typhosa
R4173 T4671 T4668 punct ),typhosa
R4174 T4672 T4665 prep for,LPS
R4175 T4673 T4674 nummod 24,h
R4176 T4674 T4672 pobj h,for
R4177 T4675 T4674 cc or,h
R4178 T4676 T4678 det the,time
R4179 T4677 T4678 amod indicated,time
R4180 T4678 T4674 conj time,h
R4181 T4679 T4656 punct .,cultured
R4182 T4680 T4684 advmod Biologically,β
R4183 T4681 T4684 amod active,β
R4184 T4682 T4684 compound TNF-α,β
R4185 T4683 T4684 compound /,β
R4186 T4684 T4690 nsubjpass β,measured
R4187 T4685 T4684 prep in,β
R4188 T4686 T4687 compound tissue,culture
R4189 T4687 T4685 pobj culture,in
R4190 T4688 T4690 nsubjpass supernatant,measured
R4191 T4689 T4690 auxpass was,measured
R4192 T4690 T4690 ROOT measured,measured
R4193 T4691 T4690 advcl using,measured
R4194 T4692 T4698 det the,assay
R4195 T4693 T4696 nmod WEHI,13-cell
R4196 T4694 T4693 nummod 164,WEHI
R4197 T4695 T4696 compound clone,13-cell
R4198 T4696 T4698 compound 13-cell,assay
R4199 T4697 T4698 compound killing,assay
R4200 T4698 T4691 dobj assay,using
R4201 T4699 T4701 nmod [,]
R4202 T4700 T4701 nummod 29,]
R4203 T4701 T4698 appos ],assay
R4204 T4702 T4690 punct .,measured
R4205 T4703 T4704 compound TNF,concentrations
R4206 T4704 T4706 nsubjpass concentrations,expressed
R4207 T4705 T4706 auxpass are,expressed
R4208 T4706 T4706 ROOT expressed,expressed
R4209 T4707 T4706 prep as,expressed
R4210 T4708 T4707 pobj pg/ml,as
R4211 T4709 T4706 punct .,expressed
R4414 T4951 T4952 compound RT-PCR,Analysis
R4415 T4952 T4958 nsubjpass Analysis,isolated
R4416 T4953 T4954 compound Total,RNA
R4417 T4954 T4952 appos RNA,Analysis
R4418 T4955 T4954 prep from,RNA
R4419 T4956 T4955 pobj monocytes,from
R4420 T4957 T4958 auxpass was,isolated
R4421 T4958 T4958 ROOT isolated,isolated
R4422 T4959 T4958 advcl using,isolated
R4423 T4960 T4962 compound TRIzol,™
R4424 T4961 T4962 compound Reagent,™
R4425 T4962 T4959 dobj ™,using
R4426 T4963 T4964 punct (,Invitrogen
R4427 T4964 T4962 appos Invitrogen,™
R4428 T4965 T4962 punct ),™
R4429 T4966 T4958 punct .,isolated
R4430 T4967 T4969 nsubjpass cDNA,generated
R4431 T4968 T4969 auxpass was,generated
R4432 T4969 T4969 ROOT generated,generated
R4433 T4970 T4969 prep on,generated
R4434 T4971 T4972 nummod 1,µg
R4435 T4972 T4970 pobj µg,on
R4436 T4973 T4972 prep of,µg
R4437 T4974 T4975 amod total,RNA
R4438 T4975 T4973 pobj RNA,of
R4439 T4976 T4969 prep in,generated
R4440 T4977 T4979 det a,volume
R4441 T4978 T4979 compound reaction,volume
R4442 T4979 T4976 pobj volume,in
R4443 T4980 T4979 prep of,volume
R4444 T4981 T4982 nummod 20,µl
R4445 T4982 T4980 pobj µl,of
R4446 T4983 T4969 punct ",",generated
R4447 T4984 T4969 advcl using,generated
R4448 T4985 T4986 compound M-MLV,reverse
R4449 T4986 T4984 dobj reverse,using
R4450 T4987 T4986 npadvmod transcriptase,reverse
R4451 T4988 T4987 punct (,transcriptase
R4452 T4989 T4987 appos Invitrogen,transcriptase
R4453 T4990 T4987 punct ),transcriptase
R4454 T4991 T4969 punct .,generated
R4455 T4992 T4994 nsubjpass PCR,done
R4456 T4993 T4994 auxpass was,done
R4457 T4994 T4994 ROOT done,done
R4458 T4995 T4994 prep in,done
R4459 T4996 T4998 det the,range
R4460 T4997 T4998 amod linear,range
R4461 T4998 T4995 pobj range,in
R4462 T4999 T4998 prep of,range
R4463 T5000 T4999 pobj amplification,of
R4464 T5001 T4998 punct (,range
R4465 T5002 T4994 conj determined,done
R4466 T5003 T5002 prep for,determined
R4467 T5004 T5007 det each,combination
R4468 T5005 T5006 compound primer,pair-cDNA
R4469 T5006 T5007 compound pair-cDNA,combination
R4470 T5007 T5003 pobj combination,for
R4471 T5008 T5002 punct ),determined
R4472 T5009 T4994 punct .,done
R4473 T5010 T5012 amod Standard,reactions
R4474 T5011 T5012 compound PCR,reactions
R4475 T5012 T5014 nsubjpass reactions,performed
R4476 T5013 T5014 auxpass were,performed
R4477 T5014 T5014 ROOT performed,performed
R4478 T5015 T5014 prep with,performed
R4479 T5016 T5017 nummod 1,µl
R4480 T5017 T5015 pobj µl,with
R4481 T5018 T5017 prep of,µl
R4482 T5019 T5021 det the,solution
R4483 T5020 T5021 compound cDNA,solution
R4484 T5021 T5018 pobj solution,of
R4485 T5022 T5021 punct ",",solution
R4486 T5023 T5024 nummod 50,µM
R4487 T5024 T5021 appos µM,solution
R4488 T5025 T5024 prep of,µM
R4489 T5026 T5028 det each,solution
R4490 T5027 T5028 compound primer,solution
R4491 T5028 T5025 pobj solution,of
R4492 T5029 T5024 punct ",",µM
R4493 T5030 T5031 nummod 10,mM
R4494 T5031 T5024 appos mM,µM
R4495 T5032 T5031 prep of,mM
R4496 T5033 T5034 det each,dNTP
R4497 T5034 T5032 pobj dNTP,of
R4498 T5035 T5024 punct ",",µM
R4499 T5036 T5038 nummod 25,MgCl2
R4500 T5037 T5038 compound mM,MgCl2
R4501 T5038 T5024 appos MgCl2,µM
R4502 T5039 T5038 punct ",",MgCl2
R4503 T5040 T5042 compound 10X,DNA
R4504 T5041 T5042 compound Goldstar,DNA
R4505 T5042 T5045 compound DNA,buffer
R4506 T5043 T5045 compound polymerase,buffer
R4507 T5044 T5045 compound reaction,buffer
R4508 T5045 T5038 appos buffer,MgCl2
R4509 T5046 T5038 punct ",",MgCl2
R4510 T5047 T5038 cc and,MgCl2
R4511 T5048 T5049 nummod 0.5,units
R4512 T5049 T5024 appos units,µM
R4513 T5050 T5049 prep of,units
R4514 T5051 T5053 compound Goldstar,polymerase
R4515 T5052 T5053 compound DNA,polymerase
R4516 T5053 T5050 pobj polymerase,of
R4517 T5054 T5055 punct (,Eurogentec
R4518 T5055 T5053 appos Eurogentec,polymerase
R4519 T5056 T5055 punct ",",Eurogentec
R4520 T5057 T5055 conj Seraing,Eurogentec
R4521 T5058 T5057 punct ",",Seraing
R4522 T5059 T5057 conj Belgium,Seraing
R4523 T5060 T5024 punct ),µM
R4524 T5061 T5014 punct .,performed
R4525 T5062 T5064 amod First,cycle
R4526 T5063 T5064 compound PCR,cycle
R4527 T5064 T5065 nsubj cycle,consisted
R4528 T5065 T5092 ccomp consisted,consisted
R4529 T5066 T5065 prep of,consisted
R4530 T5067 T5068 nummod 1,min
R4531 T5068 T5066 pobj min,of
R4532 T5069 T5065 prep at,consisted
R4533 T5070 T5071 nummod 92,°
R4534 T5071 T5069 pobj °,at
R4535 T5072 T5065 npadvmod C,consisted
R4536 T5073 T5072 punct ",",C
R4537 T5074 T5075 nummod 1,min
R4538 T5075 T5072 appos min,C
R4539 T5076 T5075 prep at,min
R4540 T5077 T5078 nummod 58,°
R4541 T5078 T5079 nummod °,C
R4542 T5079 T5076 pobj C,at
R4543 T5080 T5075 cc and,min
R4544 T5081 T5082 nummod 1,min
R4545 T5082 T5075 conj min,min
R4546 T5083 T5082 prep at,min
R4547 T5084 T5085 nummod 72,°
R4548 T5085 T5086 nummod °,C
R4549 T5086 T5083 pobj C,at
R4550 T5087 T5092 punct ;,consisted
R4551 T5088 T5092 advmod then,consisted
R4552 T5089 T5091 det each,cycle
R4553 T5090 T5091 compound PCR,cycle
R4554 T5091 T5092 nsubj cycle,consisted
R4555 T5092 T5092 ROOT consisted,consisted
R4556 T5093 T5092 prep of,consisted
R4557 T5094 T5095 nummod 40,sec
R4558 T5095 T5093 pobj sec,of
R4559 T5096 T5092 prep at,consisted
R4560 T5097 T5098 nummod 92,°
R4561 T5098 T5096 pobj °,at
R4562 T5099 T5092 dobj C,consisted
R4563 T5100 T5099 punct ",",C
R4564 T5101 T5102 nummod 40,sec
R4565 T5102 T5099 appos sec,C
R4566 T5103 T5102 prep at,sec
R4567 T5104 T5105 nummod 58,°
R4568 T5105 T5106 nummod °,C
R4569 T5106 T5103 pobj C,at
R4570 T5107 T5102 cc and,sec
R4571 T5108 T5109 nummod 50,sec
R4572 T5109 T5102 conj sec,sec
R4573 T5110 T5109 prep at,sec
R4574 T5111 T5112 nummod 72,°
R4575 T5112 T5114 nummod °,cDNA
R4576 T5113 T5114 compound C.,cDNA
R4577 T5114 T5110 pobj cDNA,at
R4578 T5115 T5109 prep for,sec
R4579 T5116 T5115 pobj β-actin,for
R4580 T5117 T5118 auxpass was,amplified
R4581 T5118 T5092 conj amplified,consisted
R4582 T5119 T5118 prep for,amplified
R4583 T5120 T5121 nummod 28,cycles
R4584 T5121 T5119 pobj cycles,for
R4585 T5122 T5118 advcl using,amplified
R4586 T5123 T5124 det the,oligos
R4587 T5124 T5122 dobj oligos,using
R4588 T5125 T5124 punct :,oligos
R4589 T5126 T5124 appos sense,oligos
R4590 T5127 T5131 nummod 5,′
R4591 T5128 T5130 punct ′,GGCATCGTGATGGACTCCG-3
R4592 T5129 T5130 punct -,GGCATCGTGATGGACTCCG-3
R4593 T5130 T5131 compound GGCATCGTGATGGACTCCG-3,′
R4594 T5131 T5124 appos ′,oligos
R4595 T5132 T5131 cc and,′
R4596 T5133 T5131 conj antisense,′
R4597 T5134 T5133 nummod 5,antisense
R4598 T5135 T5137 nummod ′,′
R4599 T5136 T5137 compound GCTGGAAGGTGGACAGCGA-3,′
R4600 T5137 T5133 appos ′,antisense
R4601 T5138 T5092 punct .,consisted
R4602 T5139 T5143 nsubjpass cDNA,amplified
R4603 T5140 T5139 prep for,cDNA
R4604 T5141 T5140 pobj TNF-α,for
R4605 T5142 T5143 auxpass was,amplified
R4606 T5143 T5143 ROOT amplified,amplified
R4607 T5144 T5143 prep for,amplified
R4608 T5145 T5146 nummod 35,cycles
R4609 T5146 T5144 pobj cycles,for
R4610 T5147 T5143 advcl using,amplified
R4611 T5148 T5149 det the,oligos
R4612 T5149 T5147 dobj oligos,using
R4613 T5150 T5149 punct :,oligos
R4614 T5151 T5149 appos sense,oligos
R4615 T5152 T5155 nummod 5,AAGCCTGTAGCCCATGTTGT-3
R4616 T5153 T5155 punct ′,AAGCCTGTAGCCCATGTTGT-3
R4617 T5154 T5155 punct -,AAGCCTGTAGCCCATGTTGT-3
R4618 T5155 T5156 compound AAGCCTGTAGCCCATGTTGT-3,′
R4619 T5156 T5149 appos ′,oligos
R4620 T5157 T5156 cc and,′
R4621 T5158 T5163 nmod antisense,′
R4622 T5159 T5158 nummod 5,antisense
R4623 T5160 T5162 punct ′,CAGATAGATGGGCTCATACC-3
R4624 T5161 T5162 punct -,CAGATAGATGGGCTCATACC-3
R4637 T5174 T5176 det the,sense
R4638 T5175 T5176 nsubj oligos,sense
R4639 T5176 T5173 dobj sense,using
R4640 T5177 T5181 nummod 5,′
R4641 T5178 T5180 punct ′,GTAGCAGCCGCAGTCATAATGG-3
R4642 T5179 T5180 punct -,GTAGCAGCCGCAGTCATAATGG-3
R4643 T5180 T5181 compound GTAGCAGCCGCAGTCATAATGG-3,′
R4644 T5181 T5176 appos ′,sense
R4645 T5182 T5181 cc and,′
R4646 T5183 T5181 conj antisense,′
R4647 T5184 T5183 nummod 5,antisense
R4648 T5185 T5189 punct ′,′
R4649 T5186 T5189 punct -,′
R4650 T5187 T5189 det A,′
R4651 T5188 T5189 amod TGCTGTTGTATCTGACTGAGG-3,′
R4652 T5189 T5181 appos ′,′
R4653 T5190 T5169 punct .,amplified
R4748 T5335 T5337 compound NF-kB,Reporter
R4749 T5336 T5337 compound Transcription,Reporter
R4750 T5337 T5363 nsubj Reporter,contains
R4751 T5338 T5339 compound Gene,Assay
R4752 T5339 T5340 compound Assay,The
R4753 T5340 T5342 det The,3XMHC-luc
R4754 T5341 T5342 compound plasmid,3XMHC-luc
R4755 T5342 T5363 nsubj 3XMHC-luc,contains
R4756 T5343 T5346 punct (,gift
R4757 T5344 T5346 det a,gift
R4758 T5345 T5346 amod generous,gift
R4759 T5346 T5342 appos gift,3XMHC-luc
R4760 T5347 T5346 prep from,gift
R4761 T5348 T5350 compound Drs.,Westwick
R4762 T5349 T5350 compound J.,Westwick
R4763 T5350 T5347 pobj Westwick,from
R4764 T5351 T5350 cc and,Westwick
R4765 T5352 T5353 compound D.A.,Brenner
R4766 T5353 T5350 conj Brenner,Westwick
R4767 T5354 T5353 punct ",",Brenner
R4768 T5355 T5353 appos University,Brenner
R4769 T5356 T5355 prep of,University
R4770 T5357 T5358 compound North,Carolina
R4771 T5358 T5356 pobj Carolina,of
R4772 T5359 T5353 punct ",",Brenner
R4773 T5360 T5361 compound Chapel,Hill
R4774 T5361 T5353 appos Hill,Brenner
R4775 T5362 T5353 punct ),Brenner
R4776 T5363 T5363 ROOT contains,contains
R4777 T5364 T5365 nummod three,copies
R4778 T5365 T5363 dobj copies,contains
R4779 T5366 T5365 prep of,copies
R4780 T5367 T5368 amod NF-κB-responsive,element
R4781 T5368 T5366 pobj element,of
R4782 T5369 T5368 prep from,element
R4783 T5370 T5372 det the,class
R4784 T5371 T5372 compound MHC,class
R4785 T5372 T5369 pobj class,from
R4786 T5373 T5374 compound I,locus
R4787 T5374 T5366 pobj locus,of
R4788 T5375 T5365 punct ",",copies
R4789 T5376 T5365 acl placed,copies
R4790 T5377 T5376 advmod upstream,placed
R4791 T5378 T5377 prep of,upstream
R4792 T5379 T5381 det the,gene
R4793 T5380 T5381 compound luciferase,gene
R4794 T5381 T5378 pobj gene,of
R4795 T5382 T5363 punct .,contains
R4796 T5383 T5386 amod Human,cells
R4797 T5384 T5386 amod monocytic,cells
R4798 T5385 T5386 compound THP-1,cells
R4799 T5386 T5389 nsubjpass cells,transfected
R4800 T5387 T5389 auxpass were,transfected
R4801 T5388 T5389 advmod transiently,transfected
R4802 T5389 T5389 ROOT transfected,transfected
R4803 T5390 T5392 mark as,described
R4804 T5391 T5392 advmod previously,described
R4805 T5392 T5389 advcl described,transfected
R4806 T5393 T5395 compound [,]
R4807 T5394 T5395 compound 30,]
R4808 T5395 T5392 dobj ],described
R4809 T5396 T5389 punct ",",transfected
R4810 T5397 T5389 cc and,transfected
R4811 T5398 T5399 advmod then,cultured
R4812 T5399 T5389 conj cultured,transfected
R4813 T5400 T5399 prep for,cultured
R4814 T5401 T5402 nummod 4,h
R4815 T5402 T5400 pobj h,for
R4816 T5403 T5402 advmod alone,h
R4817 T5404 T5400 cc or,for
R4818 T5405 T5400 conj with,for
R4819 T5406 T5407 amod increasing,concentration
R4820 T5407 T5405 pobj concentration,with
R4821 T5408 T5407 prep of,concentration
R4822 T5409 T5410 det either,C10
R4823 T5410 T5408 pobj C10,of
R4824 T5411 T5410 cc or,C10
R4825 T5412 T5410 conj C40,C10
R4826 T5413 T5389 punct .,transfected
R4827 T5414 T5415 amod Luciferase,activity
R4828 T5415 T5417 nsubjpass activity,determined
R4829 T5416 T5417 auxpass was,determined
R4830 T5417 T5417 ROOT determined,determined
R4831 T5418 T5417 advcl using,determined
R4832 T5419 T5420 det a,luminometer
R4833 T5420 T5418 dobj luminometer,using
R4834 T5421 T5420 punct (,luminometer
R4835 T5422 T5420 appos Monolight,luminometer
R4836 T5423 T5424 nummod 2010,Luminometer
R4837 T5424 T5424 ROOT Luminometer,Luminometer
R4838 T5425 T5424 punct ",",Luminometer
R4839 T5426 T5427 compound Ann,Arbor
R4840 T5427 T5424 conj Arbor,Luminometer
R4841 T5428 T5427 punct ",",Arbor
R4842 T5429 T5427 conj MI,Arbor
R4843 T5430 T5422 punct ),Monolight
R4844 T5431 T5417 punct .,determined
R5294 T5933 T5937 amod Western,cells
R5295 T5934 T5936 compound Blot,THP-1
R5296 T5935 T5936 compound Analysis,THP-1
R5297 T5936 T5937 compound THP-1,cells
R5298 T5937 T5939 nsubjpass cells,stimulated
R5299 T5938 T5939 auxpass were,stimulated
R5300 T5939 T5939 ROOT stimulated,stimulated
R5301 T5940 T5939 prep for,stimulated
R5302 T5941 T5942 amod various,lengths
R5303 T5942 T5940 pobj lengths,for
R5304 T5943 T5942 prep of,lengths
R5305 T5944 T5943 pobj time,of
R5306 T5945 T5942 prep with,lengths
R5307 T5946 T5947 nummod 0.1,mg/ml
R5308 T5947 T5945 pobj mg/ml,with
R5309 T5948 T5947 nummod C10,mg/ml
R5310 T5949 T5948 cc or,C10
R5311 T5950 T5948 conj C40,C10
R5312 T5951 T5950 punct ",",C40
R5313 T5952 T5950 cc or,C40
R5314 T5953 T5954 nummod 10,µg
R5315 T5954 T5957 compound µg,LPS
R5316 T5955 T5957 compound /,LPS
R5317 T5956 T5957 compound ml,LPS
R5318 T5957 T5950 conj LPS,C40
R5319 T5958 T5939 punct .,stimulated
R5320 T5959 T5962 nsubjpass Cells,pelleted
R5321 T5960 T5962 auxpass were,pelleted
R5322 T5961 T5962 advmod then,pelleted
R5323 T5962 T5962 ROOT pelleted,pelleted
R5324 T5963 T5962 punct ",",pelleted
R5325 T5964 T5962 conj washed,pelleted
R5326 T5965 T5964 cc and,washed
R5327 T5966 T5964 conj homogenised,washed
R5328 T5967 T5966 prep in,homogenised
R5329 T5968 T5969 compound lysis,buffer
R5330 T5969 T5967 pobj buffer,in
R5331 T5970 T5973 punct (,Hepes
R5332 T5971 T5973 nummod 10,Hepes
R5333 T5972 T5973 compound mM,Hepes
R5334 T5973 T5966 appos Hepes,homogenised
R5335 T5974 T5973 punct ",",Hepes
R5336 T5975 T5973 conj pH,Hepes
R5337 T5976 T5975 nummod 7.9,pH
R5338 T5977 T5975 punct ",",pH
R5339 T5978 T5980 nummod 150,NaCl
R5340 T5979 T5980 compound mM,NaCl
R5341 T5980 T5975 conj NaCl,pH
R5342 T5981 T5980 punct ",",NaCl
R5343 T5982 T5984 nummod 1,EDTA
R5344 T5983 T5984 compound mM,EDTA
R5345 T5984 T5980 conj EDTA,NaCl
R5346 T5985 T5984 punct ",",EDTA
R5347 T5986 T5987 nummod 0.6,%
R5348 T5987 T5988 compound %,NP-40
R5349 T5988 T5984 conj NP-40,EDTA
R5350 T5989 T5988 punct ",",NP-40
R5351 T5990 T5988 cc and,NP-40
R5352 T5991 T5993 nummod 0.5,PMSF
R5353 T5992 T5993 compound mM,PMSF
R5354 T5993 T5988 conj PMSF,NP-40
R5355 T5994 T5993 punct ),PMSF
R5356 T5995 T5988 prep on,NP-40
R5357 T5996 T5995 pobj ice,on
R5358 T5997 T5962 punct .,pelleted
R5359 T5998 T6000 nsubjpass Homogenates,sonicated
R5360 T5999 T6000 auxpass were,sonicated
R5361 T6000 T6000 ROOT sonicated,sonicated
R5362 T6001 T6000 punct ",",sonicated
R5363 T6002 T6000 conj centrifuged,sonicated
R5364 T6003 T6002 prep at,centrifuged
R5365 T6004 T6005 nummod "10,000",rpm
R5366 T6005 T6003 pobj rpm,at
R5367 T6006 T6007 aux to,remove
R5368 T6007 T6002 advcl remove,centrifuged
R5369 T6008 T6009 amod cellular,debris
R5370 T6009 T6007 dobj debris,remove
R5371 T6010 T6007 punct ",",remove
R5372 T6011 T6007 cc and,remove
R5373 T6012 T6013 amod supernatant,collected
R5374 T6013 T6002 conj collected,centrifuged
R5375 T6014 T6000 punct .,sonicated
R5376 T6015 T6016 compound Protein,concentration
R5377 T6016 T6018 nsubjpass concentration,determined
R5378 T6017 T6018 auxpass was,determined
R5379 T6018 T6018 ROOT determined,determined
R5380 T6019 T6018 advcl using,determined
R5381 T6020 T6023 det the,Assay
R5382 T6021 T6023 compound DC,Assay
R5383 T6022 T6023 compound Protein,Assay
R5384 T6023 T6019 dobj Assay,using
R5385 T6024 T6023 punct (,Assay
R5386 T6025 T6023 appos Bio-Rad,Assay
R5387 T6026 T6023 punct ),Assay
R5388 T6027 T6018 punct .,determined
R5389 T6028 T6038 nsubjpass Proteins,resolved
R5390 T6029 T6028 prep in,Proteins
R5391 T6030 T6029 pobj samples,in
R5392 T6031 T6030 punct (,samples
R5393 T6032 T6033 nummod 15,µg
R5394 T6033 T6038 nsubjpass µg,resolved
R5395 T6034 T6035 amod total,proteins
R5396 T6035 T6033 appos proteins,µg
R5397 T6036 T6033 punct ),µg
R5398 T6037 T6038 auxpass were,resolved
R5399 T6038 T6038 ROOT resolved,resolved
R5400 T6039 T6038 prep in,resolved
R5401 T6040 T6045 det a,gel
R5402 T6041 T6045 amod denaturing,gel
R5403 T6042 T6043 nummod 12,%
R5404 T6043 T6045 compound %,gel
R5405 T6044 T6045 compound polyacrylamide,gel
R5406 T6045 T6039 pobj gel,in
R5407 T6046 T6038 cc and,resolved
R5408 T6047 T6038 conj transferred,resolved
R5409 T6048 T6047 prep to,transferred
R5410 T6049 T6051 det a,membrane
R5411 T6050 T6051 amod nitrocellulose,membrane
R5412 T6051 T6048 pobj membrane,to
R5413 T6052 T6038 punct .,resolved
R5414 T6053 T6054 amod I-κBα,protein
R5415 T6054 T6056 nsubjpass protein,detected
R5416 T6055 T6056 auxpass was,detected
R5417 T6056 T6056 ROOT detected,detected
R5418 T6057 T6056 advcl using,detected
R5419 T6058 T6061 det a,antibody
R5420 T6059 T6061 nmod rabbit,antibody
R5421 T6060 T6061 compound polyclonal,antibody
R5422 T6061 T6057 dobj antibody,using
R5423 T6062 T6061 punct (,antibody
R5424 T6063 T6064 compound Santa,Cruz
R5425 T6064 T6065 compound Cruz,Biotechnology
R5426 T6065 T6061 appos Biotechnology,antibody
R5427 T6066 T6065 punct ",",Biotechnology
R5428 T6067 T6065 appos CA,Biotechnology
R5429 T6068 T6061 punct ),antibody
R5430 T6069 T6056 advcl followed,detected
R5431 T6070 T6069 agent by,followed
R5432 T6071 T6076 det a,antibody
R5433 T6072 T6076 amod horseradish,antibody
R5434 T6073 T6076 amod peroxidase-coupled,antibody
R5435 T6074 T6076 compound goat,antibody
R5436 T6075 T6076 compound polyclonal,antibody
R5437 T6076 T6070 pobj antibody,by
R5438 T6077 T6076 prep against,antibody
R5439 T6078 T6079 compound rabbit,Ig
R5440 T6079 T6077 pobj Ig,against
R5441 T6080 T6082 punct (,Laboratories
R5442 T6081 T6082 compound Caltag,Laboratories
R5443 T6082 T6079 appos Laboratories,Ig
R5444 T6083 T6082 punct ),Laboratories
R5445 T6084 T6056 punct .,detected
R5446 T6085 T6090 advmod Finally,revealed
R5447 T6086 T6090 punct ",",revealed
R5448 T6087 T6088 compound IκB,bands
R5449 T6088 T6090 nsubjpass bands,revealed
R5450 T6089 T6090 auxpass were,revealed
R5451 T6090 T6090 ROOT revealed,revealed
R5452 T6091 T6090 advcl using,revealed
R5453 T6092 T6096 det the,system
R5454 T6093 T6096 nmod ECL,system
R5455 T6094 T6096 compound ™,system
R5456 T6095 T6096 compound detection,system
R5457 T6096 T6091 dobj system,using
R5458 T6097 T6096 punct (,system
R5459 T6098 T6100 compound Amersham,Biotech
R5460 T6099 T6100 compound Pharmacia,Biotech
R5461 T6100 T6096 appos Biotech,system
R5462 T6101 T6096 punct ",",system
R5463 T6102 T6103 compound Les,Ullis
R5464 T6103 T6096 appos Ullis,system
R5465 T6104 T6103 punct ",",Ullis
R5466 T6105 T6103 appos France,Ullis
R5467 T6106 T6096 punct ),system
R5468 T6107 T6090 prep according,revealed
R5469 T6108 T6107 prep to,according
R5470 T6109 T6110 det the,manufacturers
R5471 T6110 T6112 poss manufacturers,instruction
R5472 T6111 T6110 case ',manufacturers
R5473 T6112 T6108 pobj instruction,to
R5474 T6113 T6090 punct .,revealed
R5475 T6114 T6121 nsubj Antibody,was
R5476 T6115 T6114 prep to,Antibody
R5477 T6116 T6115 pobj α-Tubulin,to
R5478 T6117 T6119 punct (,Cruz
R5479 T6118 T6119 compound Santa,Cruz
R5480 T6119 T6116 appos Cruz,α-Tubulin
R5481 T6120 T6116 punct ),α-Tubulin
R5482 T6121 T6121 ROOT was,was
R5483 T6122 T6121 attr use,was
R5484 T6123 T6122 prep as,use
R5485 T6124 T6123 pcomp loading,as
R5486 T6125 T6124 dobj control,loading
R5487 T6126 T6121 punct .,was
R5488 T6127 T6134 prep For,stimulated
R5489 T6128 T6129 amod nuclear,NF-κB
R5490 T6129 T6127 pobj NF-κB,For
R5491 T6130 T6134 punct ",",stimulated
R5492 T6131 T6132 compound THP-1,cells
R5493 T6132 T6134 nsubjpass cells,stimulated
R5494 T6133 T6134 auxpass were,stimulated
R5495 T6134 T6151 ccomp stimulated,pelleted
R5496 T6135 T6134 prep with,stimulated
R5497 T6136 T6137 nummod 1,mg/ml
R5498 T6137 T6135 pobj mg/ml,with
R5499 T6138 T6137 nummod C10,mg/ml
R5500 T6139 T6138 cc or,C10
R5501 T6140 T6138 conj C40,C10
R5502 T6141 T6140 prep for,C40
R5503 T6142 T6143 nummod 30,minutes
R5504 T6143 T6141 pobj minutes,for
R5505 T6144 T6143 prep at,minutes
R5506 T6145 T6146 nummod 37,°
R5507 T6146 T6148 compound °,Cells
R5508 T6147 T6148 compound C.,Cells
R5509 T6148 T6144 pobj Cells,at
R5510 T6149 T6151 auxpass were,pelleted
R5511 T6150 T6151 advmod then,pelleted
R5512 T6151 T6151 ROOT pelleted,pelleted
R5513 T6152 T6151 cc and,pelleted
R5514 T6153 T6151 conj nuclei,pelleted
R5515 T6154 T6151 conj separated,pelleted
R5516 T6155 T6156 mark as,described
R5517 T6156 T6151 advcl described,pelleted
R5518 T6157 T6159 nmod [,]
R5519 T6158 T6159 nummod 31,]
R5520 T6159 T6156 dobj ],described
R5521 T6160 T6151 punct .,pelleted
R5522 T6161 T6163 nsubjpass Nuclei,washed
R5523 T6162 T6163 auxpass were,washed
R5524 T6163 T6163 ROOT washed,washed
R5525 T6164 T6163 cc and,washed
R5526 T6165 T6163 conj homogenized,washed
R5527 T6166 T6165 advmod directly,homogenized
R5528 T6167 T6165 prep in,homogenized
R5529 T6168 T6167 pcomp loading,in
R5530 T6169 T6172 punct (,buffer
R5531 T6170 T6172 nmod Laemli,buffer
R5532 T6171 T6172 punct ),buffer
R5533 T6172 T6168 dobj buffer,loading
R5534 T6173 T6172 cc and,buffer
R5535 T6174 T6163 conj heated,washed
R5536 T6175 T6174 prep for,heated
R5537 T6176 T6177 nummod 5,minutes
R5538 T6177 T6175 pobj minutes,for
R5539 T6178 T6174 prep at,heated
R5540 T6179 T6182 nummod 100,Proteins
R5541 T6180 T6182 nummod °,Proteins
R5542 T6181 T6182 compound C.,Proteins
R5543 T6182 T6178 pobj Proteins,at
R5544 T6183 T6182 prep in,Proteins
R5545 T6184 T6183 pobj samples,in
R5546 T6185 T6186 auxpass were,resolved
R5547 T6186 T6163 conj resolved,washed
R5548 T6187 T6186 prep in,resolved
R5549 T6188 T6193 det a,gel
R5550 T6189 T6193 amod denaturing,gel
R5551 T6190 T6191 nummod 8,%
R5552 T6191 T6193 compound %,gel
R5553 T6192 T6193 compound polyacrylamide,gel
R5554 T6193 T6187 pobj gel,in
R5555 T6194 T6186 cc and,resolved
R5556 T6195 T6186 conj transferred,resolved
R5557 T6196 T6195 prep to,transferred
R5558 T6197 T6199 det a,fluoride
R5559 T6198 T6199 compound polyvinylidine,fluoride
R5560 T6199 T6196 pobj fluoride,to
R5561 T6200 T6199 punct (,fluoride
R5562 T6201 T6199 appos PVDF,fluoride
R5563 T6202 T6199 punct ),fluoride
R5564 T6203 T6195 dobj membrane,transferred
R5565 T6204 T6205 punct (,Immobilon-P
R5566 T6205 T6203 appos Immobilon-P,membrane
R5567 T6206 T6163 punct ;,washed
R5568 T6207 T6207 ROOT Millipore,Millipore
R5569 T6208 T6207 punct ",",Millipore
R5570 T6209 T6207 conj Bedford,Millipore
R5571 T6210 T6209 punct ",",Bedford
R5572 T6211 T6209 conj MA,Bedford
R5573 T6212 T6209 punct ),Bedford
R5574 T6213 T6207 punct .,Millipore
R5575 T6214 T6216 nsubjpass Membranes,incubated
R5576 T6215 T6216 auxpass were,incubated
R5577 T6216 T6216 ROOT incubated,incubated
R5578 T6217 T6216 prep in,incubated
R5579 T6218 T6217 pcomp blocking,in
R5580 T6219 T6218 dobj buffer,blocking
R5581 T6220 T6223 punct (,BSA
R5582 T6221 T6222 nummod 1,%
R5583 T6222 T6223 npadvmod %,BSA
R5584 T6223 T6218 dobj BSA,blocking
R5585 T6224 T6218 punct ",",blocking
R5586 T6225 T6224 oprd in,","
R5587 T6226 T6225 pobj PBS,in
R5588 T6227 T6225 punct ),in
R5589 T6228 T6216 prep for,incubated
R5590 T6229 T6230 nummod two,hours
R5591 T6230 T6228 pobj hours,for
R5592 T6231 T6230 prep at,hours
R5593 T6232 T6233 compound room,temperature
R5594 T6233 T6231 pobj temperature,at
R5595 T6234 T6216 punct .,incubated
R5596 T6235 T6238 nsubjpass Membranes,probed
R5597 T6236 T6238 auxpass were,probed
R5598 T6237 T6238 advmod subsequently,probed
R5599 T6238 T6238 ROOT probed,probed
R5600 T6239 T6238 prep with,probed
R5601 T6240 T6242 det the,antibody
R5602 T6241 T6242 amod corresponding,antibody
R5603 T6242 T6239 pobj antibody,with
R5604 T6243 T6238 prep in,probed
R5605 T6244 T6243 pcomp blocking,in
R5606 T6245 T6244 dobj buffer,blocking
R5607 T6246 T6247 punct ",",overnight
R5608 T6247 T6244 npadvmod overnight,blocking
R5609 T6248 T6238 punct .,probed
R5610 T6249 T6254 nmod Rabbit,subunit
R5611 T6250 T6254 amod polyclonal,subunit
R5612 T6251 T6254 nmod antibody,subunit
R5613 T6252 T6254 nmod anti-NF-κB,subunit
R5614 T6253 T6254 amod p50,subunit
R5615 T6254 T6272 nsubjpass subunit,used
R5616 T6255 T6254 punct (,subunit
R5617 T6256 T6257 nmod #,sc-114
R5618 T6257 T6272 nsubjpass sc-114,used
R5619 T6258 T6257 punct ),sc-114
R5620 T6259 T6257 cc or,sc-114
R5621 T6260 T6261 amod anti-NF-κB,p65
R5622 T6261 T6257 conj p65,sc-114
R5623 T6262 T6261 acl subunit,p65
R5624 T6263 T6261 punct (,p65
R5625 T6264 T6265 nmod #,sc-109
R5626 T6265 T6261 appos sc-109,p65
R5627 T6266 T6261 punct ),p65
R5628 T6267 T6261 prep from,p65
R5629 T6268 T6269 compound Santa,Cruz
R5630 T6269 T6270 compound Cruz,Biotechnology
R5631 T6270 T6267 pobj Biotechnology,from
R5632 T6271 T6272 auxpass were,used
R5633 T6272 T6272 ROOT used,used
R5634 T6273 T6272 punct .,used
R5635 T6274 T6276 nsubjpass Membranes,washed
R5636 T6275 T6276 auxpass were,washed
R5637 T6276 T6276 ROOT washed,washed
R5638 T6277 T6278 nummod six,times
R5639 T6278 T6276 npadvmod times,washed
R5640 T6279 T6276 prep in,washed
R5641 T6280 T6279 pobj PBS,in
R5642 T6281 T6276 prep with,washed
R5643 T6282 T6283 nummod 0.05,%
R5644 T6283 T6288 nummod %,minutes
R5645 T6284 T6288 nummod Tween,minutes
R5646 T6285 T6288 nummod 20,minutes
R5647 T6286 T6288 punct ",",minutes
R5648 T6287 T6288 nummod 5,minutes
R5649 T6288 T6281 pobj minutes,with
R5650 T6289 T6290 det each,time
R5651 T6290 T6276 npadvmod time,washed
R5652 T6291 T6276 punct ",",washed
R5653 T6292 T6276 cc and,washed
R5654 T6293 T6276 conj incubated,washed
R5655 T6294 T6293 prep with,incubated
R5656 T6295 T6297 det a,dilution
R5657 T6296 T6297 nummod 1/3000,dilution
R5658 T6297 T6294 pobj dilution,with
R5659 T6298 T6297 prep of,dilution
R5660 T6299 T6300 amod HRP-conjugated,F
R5661 T6300 T6298 pobj F,of
R5662 T6301 T6308 punct (,IgG
R5663 T6302 T6306 nmod ab,goat
R5664 T6303 T6302 case ',ab
R5665 T6304 T6302 punct ),ab
R5666 T6305 T6302 nummod 2,ab
R5667 T6306 T6308 nmod goat,IgG
R5668 T6307 T6308 compound anti-rabbit,IgG
R5669 T6308 T6314 nmod IgG,milk
R5670 T6309 T6308 prep in,IgG
R5671 T6310 T6311 nummod 5,%
R5672 T6311 T6314 nmod %,milk
R5673 T6312 T6314 amod nonfat,milk
R5674 T6313 T6314 amod dry,milk
R5675 T6314 T6297 appos milk,dilution
R5676 T6315 T6314 cc and,milk
R5677 T6316 T6317 nummod 0.05,%
R5678 T6317 T6314 conj %,milk
R5679 T6318 T6319 nummod Tween,20
R5680 T6319 T6322 dep 20,for
R5681 T6320 T6319 prep in,20
R5682 T6321 T6320 pobj PBS,in
R5683 T6322 T6293 conj for,incubated
R5684 T6323 T6324 nummod 1,hour
R5685 T6324 T6322 pobj hour,for
R5686 T6325 T6322 prep at,for
R5687 T6326 T6327 compound room,temperature
R5688 T6327 T6325 pobj temperature,at
R5689 T6328 T6276 punct .,washed
R5690 T6329 T6345 prep After,detected
R5691 T6330 T6329 pcomp washing,After
R5692 T6331 T6333 nummod six,times
R5693 T6332 T6331 amod more,six
R5694 T6333 T6330 dobj times,washing
R5695 T6334 T6330 prep in,washing
R5696 T6335 T6334 pobj PBS,in
R5697 T6336 T6330 prep with,washing
R5698 T6337 T6338 nummod 0.05,%
R5699 T6338 T6345 nsubj %,detected
R5700 T6339 T6340 nummod Tween,20
R5701 T6340 T6345 dep 20,detected
R5702 T6341 T6345 punct ",",detected
R5703 T6342 T6343 amod antibody-reactive,proteins
R5704 T6343 T6345 nsubjpass proteins,detected
R5705 T6344 T6345 auxpass were,detected
R5706 T6345 T6345 ROOT detected,detected
R5707 T6346 T6345 advcl using,detected
R5708 T6347 T6349 det a,substrate
R5709 T6348 T6349 compound chemiluminescence,substrate
R5710 T6349 T6346 dobj substrate,using
R5711 T6350 T6359 punct (,according
R5712 T6351 T6359 dep SuperSignal,according
R5713 T6352 T6351 punct ;,SuperSignal
R5714 T6353 T6351 conj Pierce,SuperSignal
R5715 T6354 T6353 punct ",",Pierce
R5716 T6355 T6353 conj Rockford,Pierce
R5717 T6356 T6355 punct ",",Rockford
R5718 T6357 T6355 conj IL,Rockford
R5719 T6358 T6359 punct ),according
R5720 T6359 T6345 prep according,detected
R5721 T6360 T6359 prep to,according
R5722 T6361 T6362 det the,manufacturer
R5723 T6362 T6364 poss manufacturer,instructions
R5724 T6363 T6362 case 's,manufacturer
R5725 T6364 T6360 pobj instructions,to
R5726 T6365 T6345 punct .,detected
R5727 T6366 T6367 aux To,confirm
R5728 T6367 T6380 advcl confirm,were
R5729 T6368 T6374 mark that,loaded
R5730 T6369 T6370 amod equivalent,amounts
R5731 T6370 T6374 nsubjpass amounts,loaded
R5732 T6371 T6370 prep of,amounts
R5733 T6372 T6371 pobj protein,of
R5734 T6373 T6374 auxpass were,loaded
R5735 T6374 T6367 ccomp loaded,confirm
R5736 T6375 T6374 prep in,loaded
R5737 T6376 T6377 det each,line
R5738 T6377 T6375 pobj line,in
R5739 T6378 T6380 punct ",",were
R5740 T6379 T6380 nsubj membranes,were
R5741 T6380 T6380 ROOT were,were
R5742 T6381 T6380 advmod also,were
R5743 T6382 T6383 amod Western,blotted
R5744 T6383 T6380 attr blotted,were
R5745 T6384 T6383 prep for,blotted
R5746 T6385 T6384 pobj ERK,for
R5747 T6386 T6387 mark as,described
R5748 T6387 T6380 advcl described,were
R5749 T6388 T6390 nmod [,]
R5750 T6389 T6390 nummod 32,]
R5751 T6390 T6387 dobj ],described
R5752 T6391 T6380 punct .,were
R5773 T6486 T6503 nsubjpass Analysis,performed
R5774 T6487 T6486 prep of,Analysis
R5775 T6488 T6489 compound NF-κB,Activation
R5776 T6489 T6487 pobj Activation,of
R5777 T6490 T6489 prep by,Activation
R5778 T6491 T6494 compound Flow,activation
R5779 T6492 T6494 compound Cytometry,activation
R5780 T6493 T6494 compound Nuclear,activation
R5781 T6494 T6490 pobj activation,by
R5782 T6495 T6494 prep of,activation
R5783 T6496 T6498 compound NF,κΒ
R5784 T6497 T6498 compound −,κΒ
R5785 T6498 T6495 pobj κΒ,of
R5786 T6499 T6486 prep by,Analysis
R5787 T6500 T6501 compound flow,cytometry
R5788 T6501 T6499 pobj cytometry,by
R5789 T6502 T6503 auxpass was,performed
R5790 T6503 T6503 ROOT performed,performed
R5791 T6504 T6505 mark as,described
R5792 T6505 T6503 advcl described,performed
R5793 T6506 T6508 nmod [,]
R5794 T6507 T6508 nummod 31,]
R5795 T6508 T6505 dobj ],described
R5796 T6509 T6503 punct .,performed
R5837 T6558 T6559 compound Statistical,Analysis
R5838 T6559 T6563 nsubjpass Analysis,expressed
R5839 T6560 T6561 det The,results
R5840 T6561 T6563 nsubjpass results,expressed
R5841 T6562 T6563 auxpass were,expressed
R5842 T6563 T6563 ROOT expressed,expressed
R5843 T6564 T6563 prep as,expressed
R5844 T6565 T6569 det the,S.E.M.
R5845 T6566 T6569 amod mean,S.E.M.
R5846 T6567 T6568 compound value,±
R5847 T6568 T6569 compound ±,S.E.M.
R5848 T6569 T6564 pobj S.E.M.,as
R5849 T6570 T6569 prep of,S.E.M.
R5850 T6571 T6572 amod individual,experiments
R5851 T6572 T6570 pobj experiments,of
R5852 T6573 T6563 punct .,expressed
R5853 T6574 T6576 det The,significance
R5854 T6575 T6576 amod statistical,significance
R5855 T6576 T6584 nsubjpass significance,assessed
R5856 T6577 T6576 prep of,significance
R5857 T6578 T6579 det the,differences
R5858 T6579 T6577 pobj differences,of
R5859 T6580 T6579 prep between,differences
R5860 T6581 T6582 amod mean,values
R5861 T6582 T6580 pobj values,between
R5862 T6583 T6584 auxpass was,assessed
R5863 T6584 T6584 ROOT assessed,assessed
R5864 T6585 T6584 agent by,assessed
R5865 T6586 T6587 det the,Student
R5866 T6587 T6591 poss Student,analysis
R5867 T6588 T6587 case 's,Student
R5868 T6589 T6591 amod t-test,analysis
R5869 T6590 T6589 cc and,t-test
R5870 T6591 T6585 pobj analysis,by
R5871 T6592 T6591 prep of,analysis
R5872 T6593 T6592 pobj variance,of
R5873 T6594 T6595 punct (,ANOVA
R5874 T6595 T6593 appos ANOVA,variance
R5875 T6596 T6584 punct ),assessed
R5876 T6597 T6584 punct .,assessed
R6115 T6857 T6859 amod Degraded,Induce
R6116 T6858 T6859 compound CGN,Induce
R6117 T6859 T6863 nmod Induce,rats
R6118 T6860 T6863 compound Colonic,rats
R6119 T6861 T6863 compound Inflammation,rats
R6120 T6862 T6863 compound All,rats
R6121 T6863 T6864 nsubj rats,developed
R6122 T6864 T6864 ROOT developed,developed
R6123 T6865 T6864 dobj diarrhea,developed
R6124 T6866 T6864 prep during,developed
R6125 T6867 T6869 amod degraded,administration
R6126 T6868 T6869 compound carrageenan,administration
R6127 T6869 T6866 pobj administration,during
R6128 T6870 T6869 cc and,administration
R6129 T6871 T6872 amod gross,evidence
R6130 T6872 T6869 conj evidence,administration
R6131 T6873 T6872 prep of,evidence
R6132 T6874 T6873 pobj blood,of
R6133 T6875 T6877 auxpass was,detected
R6134 T6876 T6877 advmod frequently,detected
R6135 T6877 T6864 conj detected,developed
R6136 T6878 T6877 prep in,detected
R6137 T6879 T6880 det the,stools
R6138 T6880 T6878 pobj stools,in
R6139 T6881 T6864 punct .,developed
R6140 T6882 T6883 compound Colon,length
R6141 T6883 T6885 nsubj length,decreased
R6142 T6884 T6885 advmod dramatically,decreased
R6143 T6885 T6885 ROOT decreased,decreased
R6144 T6886 T6885 prep in,decreased
R6145 T6887 T6889 det all,rats
R6146 T6888 T6889 amod treated,rats
R6147 T6889 T6886 pobj rats,in
R6148 T6890 T6889 prep with,rats
R6149 T6891 T6894 det a,effect
R6150 T6892 T6893 advmod more,pronounced
R6151 T6893 T6894 amod pronounced,effect
R6152 T6894 T6890 pobj effect,with
R6153 T6895 T6896 auxpass being,observed
R6154 T6896 T6894 acl observed,effect
R6155 T6897 T6896 prep in,observed
R6156 T6898 T6901 det the,dCGN
R6157 T6899 T6901 nummod 40,dCGN
R6158 T6900 T6901 compound kDa,dCGN
R6159 T6901 T6897 pobj dCGN,in
R6160 T6902 T6885 conj treated,decreased
R6161 T6903 T6902 dobj group,treated
R6162 T6904 T6906 punct (,1A
R6163 T6905 T6906 compound Fig.,1A
R6164 T6906 T6903 appos 1A,group
R6165 T6907 T6906 punct ),1A
R6166 T6908 T6885 punct .,decreased
R6167 T6909 T6917 advmod Furthermore,resulted
R6168 T6910 T6917 punct ",",resulted
R6169 T6911 T6912 amod prolonged,exposure
R6170 T6912 T6917 nsubj exposure,resulted
R6171 T6913 T6912 prep to,exposure
R6172 T6914 T6916 nummod 40,dCGN
R6173 T6915 T6916 compound kDa,dCGN
R6174 T6916 T6917 nsubj dCGN,resulted
R6175 T6917 T6917 ROOT resulted,resulted
R6176 T6918 T6917 prep in,resulted
R6177 T6919 T6923 amod high,scores
R6178 T6920 T6923 amod macroscopic,scores
R6179 T6921 T6920 cc and,macroscopic
R6180 T6922 T6920 conj histological,macroscopic
R6181 T6923 T6918 pobj scores,in
R6182 T6924 T6923 prep of,scores
R6183 T6925 T6924 pobj inflammation,of
R6184 T6926 T6930 punct (,C
R6185 T6927 T6928 compound Fig.,1B
R6186 T6928 T6930 dep 1B,C
R6187 T6929 T6930 punct ",",C
R6188 T6930 T6923 appos C,scores
R6189 T6931 T6923 punct ),scores
R6190 T6932 T6917 punct .,resulted
R6191 T6933 T6936 amod Only,activity
R6192 T6934 T6936 amod weak,activity
R6193 T6935 T6936 compound myeloperoxidase,activity
R6194 T6936 T6938 nsubjpass activity,detected
R6195 T6937 T6938 auxpass was,detected
R6196 T6938 T6938 ROOT detected,detected
R6197 T6939 T6938 prep in,detected
R6198 T6940 T6941 det both,control
R6199 T6941 T6939 pobj control,in
R6200 T6942 T6941 cc and,control
R6201 T6943 T6944 amod dCGN-treated,groups
R6202 T6944 T6941 conj groups,control
R6203 T6945 T6944 punct (,groups
R6204 T6946 T6947 compound Fig.,1D
R6205 T6947 T6944 appos 1D,groups
R6206 T6948 T6944 punct ),groups
R6207 T6949 T6938 punct ",",detected
R6208 T6950 T6938 advcl indicating,detected
R6209 T6951 T6955 mark that,play
R6210 T6952 T6955 nsubj granulocytes,play
R6211 T6953 T6955 aux did,play
R6212 T6954 T6955 neg not,play
R6213 T6955 T6950 ccomp play,indicating
R6214 T6956 T6958 det a,role
R6215 T6957 T6958 amod major,role
R6216 T6958 T6955 dobj role,play
R6217 T6959 T6955 prep in,play
R6218 T6960 T6961 det the,inflammation
R6219 T6961 T6959 pobj inflammation,in
R6220 T6962 T6955 prep at,play
R6221 T6963 T6964 det that,stage
R6222 T6964 T6962 pobj stage,at
R6223 T6965 T6938 punct .,detected
R6224 T6966 T6967 amod Histological,examination
R6225 T6967 T6968 nsubj examination,revealed
R6226 T6968 T6968 ROOT revealed,revealed
R6227 T6969 T6970 amod various,degrees
R6228 T6970 T6968 dobj degrees,revealed
R6229 T6971 T6970 prep of,degrees
R6230 T6972 T6973 amod mucosal,inflammation
R6231 T6973 T6971 pobj inflammation,of
R6232 T6974 T6968 punct .,revealed
R6233 T6975 T6976 nsubj Rats,treated
R6234 T6976 T6976 ROOT treated,treated
R6235 T6977 T6976 prep with,treated
R6236 T6978 T6980 nummod 10,dCGN
R6237 T6979 T6980 compound kDa,dCGN
R6238 T6980 T6977 pobj dCGN,with
R6239 T6981 T6976 conj showed,treated
R6240 T6982 T6981 dobj edema,showed
R6241 T6983 T6981 punct ",",showed
R6242 T6984 T6985 compound epithelium,atrophy
R6243 T6985 T6985 ROOT atrophy,atrophy
R6244 T6986 T6985 cc and,atrophy
R6245 T6987 T6989 amod slight,infiltration
R6246 T6988 T6989 compound lymphocyte,infiltration
R6247 T6989 T6985 conj infiltration,atrophy
R6248 T6990 T6993 punct (,shown
R6249 T6991 T6993 nsubjpass data,shown
R6250 T6992 T6993 neg not,shown
R6251 T6993 T6985 parataxis shown,atrophy
R6252 T6994 T6993 punct ),shown
R6253 T6995 T6976 punct .,treated
R6254 T6996 T6997 det These,symptoms
R6255 T6997 T6998 nsubj symptoms,were
R6256 T6998 T6998 ROOT were,were
R6257 T6999 T7000 advmod totally,absent
R6258 T7000 T6998 acomp absent,were
R6259 T7001 T7000 prep in,absent
R6260 T7002 T7003 det the,colon
R6261 T7003 T7001 pobj colon,in
R6262 T7004 T7003 prep of,colon
R6263 T7005 T7006 compound control,rats
R6264 T7006 T7004 pobj rats,of
R6265 T7007 T7003 punct (,colon
R6266 T7008 T7009 compound Fig.,1E
R6267 T7009 T7003 appos 1E,colon
R6268 T7010 T7003 punct ),colon
R6269 T7011 T6998 punct .,were
R6270 T7012 T7013 advmod More,severe
R6271 T7013 T7015 amod severe,injuries
R6272 T7014 T7015 amod mucosal,injuries
R6273 T7015 T7031 nsubjpass injuries,observed
R6274 T7016 T7015 prep including,injuries
R6275 T7017 T7016 pobj ulceration,including
R6276 T7018 T7017 punct ",",ulceration
R6277 T7019 T7020 amod hyperplastic,epithelium
R6278 T7020 T7017 conj epithelium,ulceration
R6279 T7021 T7020 punct ",",epithelium
R6280 T7022 T7023 compound crypt,distortion
R6281 T7023 T7020 conj distortion,epithelium
R6282 T7024 T7023 cc and,distortion
R6283 T7025 T7028 det a,infiltration
R6284 T7026 T7028 amod strong,infiltration
R6285 T7027 T7028 compound macrophage,infiltration
R6286 T7028 T7023 conj infiltration,distortion
R6287 T7029 T7031 punct ",",observed
R6288 T7030 T7031 auxpass were,observed
R6289 T7031 T7031 ROOT observed,observed
R6290 T7032 T7031 prep in,observed
R6291 T7033 T7037 det the,rats
R6292 T7034 T7037 nummod 40,rats
R6293 T7035 T7036 compound kDa,dCGN-treated
R6294 T7036 T7037 compound dCGN-treated,rats
R6295 T7037 T7032 pobj rats,in
R6296 T7038 T7031 punct (,observed
R6297 T7039 T7040 compound Fig.,1F
R6298 T7040 T7031 npadvmod 1F,observed
R6299 T7041 T7031 punct ),observed
R6300 T7042 T7031 punct .,observed
R6301 T7043 T7045 det No,polysaccharides
R6302 T7044 T7045 amod sulphated,polysaccharides
R6303 T7045 T7047 nsubjpass polysaccharides,detected
R6304 T7046 T7047 auxpass were,detected
R6305 T7047 T7047 ROOT detected,detected
R6306 T7048 T7047 agent by,detected
R6307 T7049 T7051 nmod toluidine,staining
R6308 T7050 T7051 amod blue,staining
R6309 T7051 T7048 pobj staining,by
R6310 T7052 T7051 prep of,staining
R6311 T7053 T7054 compound colon,mucosa
R6312 T7054 T7052 pobj mucosa,of
R6313 T7055 T7051 prep from,staining
R6314 T7056 T7055 pobj rats,from
R6315 T7057 T7056 acl treated,rats
R6316 T7058 T7057 prep with,treated
R6317 T7059 T7065 preconj either,dCGN
R6318 T7060 T7065 det the,dCGN
R6319 T7061 T7065 nummod 10,dCGN
R6320 T7062 T7061 cc or,10
R6321 T7063 T7061 conj 40,10
R6322 T7064 T7065 compound kDa,dCGN
R6323 T7065 T7058 pobj dCGN,with
R6324 T7066 T7068 punct (,shown
R6325 T7067 T7068 neg not,shown
R6326 T7068 T7065 parataxis shown,dCGN
R6327 T7069 T7068 punct ),shown
R6328 T7070 T7047 punct .,detected
R6329 T7071 T7075 mark Although,exclude
R6330 T7072 T7075 nsubj we,exclude
R6331 T7073 T7075 aux can,exclude
R6332 T7074 T7075 neg not,exclude
R6333 T7075 T7091 advcl exclude,indicates
R6334 T7076 T7078 det that,mat
R6335 T7077 T7078 compound dCGN,mat
R6336 T7078 T7075 dobj mat,exclude
R6337 T7079 T7081 neg not,retained
R6338 T7080 T7081 aux have,retained
R6339 T7081 T7078 acl retained,mat
R6340 T7082 T7081 prep in,retained
R6341 T7083 T7084 det the,section
R6342 T7084 T7082 pobj section,in
R6343 T7085 T7081 prep during,retained
R6344 T7086 T7088 det the,procedure
R6345 T7087 T7088 compound histology,procedure
R6346 T7088 T7085 pobj procedure,during
R6347 T7089 T7091 punct ",",indicates
R6348 T7090 T7091 nsubj this,indicates
R6349 T7091 T7091 ROOT indicates,indicates
R6350 T7092 T7099 mark that,phagocytosed
R6351 T7093 T7094 det these,polymers
R6352 T7094 T7099 nsubjpass polymers,phagocytosed
R6353 T7095 T7099 aux may,phagocytosed
R6354 T7096 T7099 neg not,phagocytosed
R6355 T7097 T7099 aux have,phagocytosed
R6356 T7098 T7099 auxpass been,phagocytosed
R6357 T7099 T7091 ccomp phagocytosed,indicates
R6358 T7100 T7091 punct .,indicates
R6775 T7657 T7660 amod Degraded,Production
R6776 T7658 T7659 compound CGN,"inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced-TNF-α"
R6777 T7659 T7660 compound "inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced-TNF-α",Production
R6778 T7660 T7660 ROOT Production,Production
R6779 T7661 T7660 prep by,Production
R6780 T7662 T7661 pobj Monocytes,by
R6781 T7663 T7663 ROOT In,In
R6782 T7664 T7663 pobj Vitro,In
R13449 T15673 T15674 amod Degraded,CGN
R13450 T15674 T15675 nsubj CGN,induced
R13451 T15675 T15675 ROOT induced,induced
R13452 T15676 T15677 compound colon,inflammation
R13453 T15677 T15675 dobj inflammation,induced
R13454 T15678 T15677 prep in,inflammation
R13455 T15679 T15678 pobj rats,in
R13456 T15680 T15675 punct .,induced
R13457 T15681 T15706 dep Histograms,score
R13458 T15682 T15681 acl showing,Histograms
R13459 T15683 T15684 det the,effect
R13460 T15684 T15682 dobj effect,showing
R13461 T15685 T15684 prep of,effect
R13462 T15686 T15687 amod degraded,CGN
R13463 T15687 T15685 pobj CGN,of
R13464 T15688 T15684 prep on,effect
R13465 T15689 T15688 punct :,on
R13466 T15690 T15691 compound colon,length
R13467 T15691 T15688 pobj length,on
R13468 T15692 T15693 punct (,A
R13469 T15693 T15691 appos A,length
R13470 T15694 T15693 punct ),A
R13471 T15695 T15706 punct ;,score
R13472 T15696 T15706 amod macroscopic,score
R13473 T15697 T15698 punct (,B
R13474 T15698 T15696 appos B,macroscopic
R13475 T15699 T15698 punct ),B
R13476 T15700 T15696 cc and,macroscopic
R13477 T15701 T15696 conj histological,macroscopic
R13478 T15702 T15701 punct (,histological
R13479 T15703 T15701 appos C,histological
R13480 T15704 T15701 punct ),histological
R13481 T15705 T15706 compound inflammation,score
R13482 T15706 T15706 ROOT score,score
R13483 T15707 T15706 prep of,score
R13484 T15708 T15707 pobj colon,of
R13485 T15709 T15706 punct ;,score
R13486 T15710 T15714 nmod Myeloperoxidase,activity
R13487 T15711 T15712 punct (,MPO
R13488 T15712 T15710 appos MPO,Myeloperoxidase
R13489 T15713 T15712 punct ),MPO
R13490 T15714 T15706 conj activity,score
R13491 T15715 T15716 punct (,D
R13492 T15716 T15714 appos D,activity
R13493 T15717 T15714 punct ),activity
R13494 T15718 T15706 punct .,score
R13495 T15719 T15720 compound Control,rats
R13496 T15720 T15720 ROOT rats,rats
R13497 T15721 T15723 punct (,bars
R13498 T15722 T15723 amod white,bars
R13499 T15723 T15720 appos bars,rats
R13500 T15724 T15723 punct ),bars
R13501 T15725 T15723 punct ;,bars
R13502 T15726 T15727 nummod 10,kDa
R13503 T15727 T15720 appos kDa,rats
R13504 T15728 T15730 amod degraded,rats
R13505 T15729 T15730 amod CGN-treated,rats
R13506 T15730 T15727 appos rats,kDa
R13507 T15731 T15733 punct (,bars
R13508 T15732 T15733 compound grey,bars
R13509 T15733 T15730 appos bars,rats
R13510 T15734 T15730 punct ),rats
R13511 T15735 T15727 punct ;,kDa
R13512 T15736 T15737 nummod 40,kDa
R13513 T15737 T15738 npadvmod kDa,degraded
R13514 T15738 T15740 amod degraded,rats
R13515 T15739 T15740 amod CGN-treated,rats
R13516 T15740 T15727 appos rats,kDa
R13517 T15741 T15743 punct (,bars
R13518 T15742 T15743 amod black,bars
R13519 T15743 T15740 appos bars,rats
R13520 T15744 T15740 punct ),rats
R13521 T15745 T15720 punct .,rats
R13522 T15746 T15748 punct *,<
R13523 T15747 T15748 compound p,<
R13524 T15748 T15748 ROOT <,<
R13525 T15749 T15748 nummod 0.05,<
R13526 T15750 T15748 prep from,<
R13527 T15751 T15750 pobj control,from
R13528 T15752 T15748 punct .,<
R13529 T15753 T15748 punct **,<
R13530 T15754 T15756 nmod p,0.01
R13531 T15755 T15756 nmod <,0.01
R13532 T15756 T15753 nummod 0.01,**
R13533 T15757 T15753 prep from,**
R13534 T15758 T15757 pobj control,from
R13535 T15759 T15753 punct .,**
R13536 T15760 T15761 amod Histological,analysis
R13537 T15761 T15761 ROOT analysis,analysis
R13538 T15762 T15761 prep of,analysis
R13539 T15763 T15762 pobj colon,of
R13540 T15764 T15761 prep from,analysis
R13541 T15765 T15766 compound control,rats
R13542 T15766 T15764 pobj rats,from
R13543 T15767 T15766 punct (,rats
R13544 T15768 T15766 appos E,rats
R13545 T15769 T15766 punct ),rats
R13546 T15770 T15761 punct ",",analysis
R13547 T15771 T15761 cc and,analysis
R13548 T15772 T15761 conj from,analysis
R13549 T15773 T15776 nummod 40,rats
R13550 T15774 T15775 compound kDa,dCGN-treated
R13551 T15775 T15776 compound dCGN-treated,rats
R13552 T15776 T15772 pobj rats,from
R13553 T15777 T15776 punct (,rats
R13554 T15778 T15776 appos F,rats
R13555 T15779 T15776 punct ),rats
R13556 T15780 T15761 punct .,analysis
R1474 T1663 T1664 amod common,characteristic
R1475 T1664 T1661 attr characteristic,is
R1476 T1665 T1664 prep of,characteristic
R1477 T1666 T1667 amod intestinal,diseases
R1478 T1667 T1665 pobj diseases,of
R1479 T1668 T1670 nmod [,]
R1480 T1669 T1670 nummod 17,]
R1481 T1670 T1664 appos ],characteristic
R1482 T1671 T1661 punct .,is
R1483 T1672 T1673 nsubj Macrophages,represent
R1484 T1673 T1673 ROOT represent,represent
R1485 T1674 T1675 nummod 10,%
R1486 T1675 T1673 dobj %,represent
R1487 T1676 T1675 prep of,%
R2153 T2437 T2421 conj medium,preparations
R2154 T2438 T2437 punct ",",medium
R2155 T2439 T2437 punct (,medium
R2156 T2440 T2437 appos ∼,medium
R2157 T2441 T2442 nummod 40,kDa
R2158 T2442 T2440 appos kDa,∼
R2159 T2443 T2437 punct ;,medium
R2160 T2444 T2421 appos C40,preparations
R2161 T2445 T2421 punct ),preparations
R2162 T2446 T2447 amod molecular,weight
R2163 T2447 T2416 conj weight,Preparation
R2164 T2448 T2449 auxpass were,prepared
R2165 T2449 T2449 ROOT prepared,prepared
R2166 T2450 T2449 prep from,prepared
R2167 T2451 T2452 amod native,iota-carrageenan
R2168 T2452 T2450 pobj iota-carrageenan,from
R2169 T2453 T2452 acl extracted,iota-carrageenan
R2170 T2454 T2453 prep from,extracted
R2171 T2455 T2456 compound Euchema,spinosum
R2172 T2456 T2454 pobj spinosum,from
R2173 T2457 T2459 punct (,provided
R2174 T2458 T2459 advmod generously,provided
R2175 T2459 T2452 acl provided,iota-carrageenan
R2176 T2460 T2459 agent by,provided
R2177 T2461 T2463 compound Sanofi,Industry
R2178 T2462 T2463 compound Biosystems,Industry
R2179 T2463 T2460 pobj Industry,by
R2205 T2489 T2487 pobj °,to
R2206 T2490 T2491 compound C.,Then
R2207 T2491 T2497 advmod Then,submitted
R2208 T2492 T2497 punct ",",submitted
R2209 T2493 T2495 det the,solution
R2210 T2494 T2495 compound carrageenan,solution
R2211 T2495 T2497 nsubjpass solution,submitted
R2212 T2496 T2497 auxpass was,submitted
R2213 T2497 T2497 ROOT submitted,submitted
R2214 T2498 T2497 prep to,submitted
R2215 T2499 T2501 nummod two,treatments
R2216 T2500 T2501 amod different,treatments
R2217 T2501 T2498 pobj treatments,to
R2218 T2502 T2503 aux to,obtain
R2219 T2503 T2497 xcomp obtain,submitted
R2220 T2504 T2505 advmod both,low
R2221 T2505 T2510 amod low,fractions
R2222 T2506 T2505 cc and,low
R2223 T2507 T2505 conj medium,low
R2224 T2508 T2509 amod molecular,weight
R2225 T2509 T2510 compound weight,fractions
R2226 T2510 T2503 dobj fractions,obtain
R2227 T2511 T2497 punct .,submitted
R2228 T2512 T2524 advmod Briefly,hydrolyzed
R2229 T2513 T2524 punct ",",hydrolyzed
R2230 T2514 T2524 prep for,hydrolyzed
R2231 T2515 T2519 det the,fraction
R2493 T2777 T2779 det the,fractions
R2494 T2778 T2779 nummod two,fractions
R2495 T2779 T2781 nsubjpass fractions,dissolved
R2496 T2780 T2781 auxpass were,dissolved
R2497 T2781 T2781 ROOT dissolved,dissolved
R2498 T2782 T2781 prep in,dissolved
R2499 T2783 T2784 amod complete,medium
R2500 T2784 T2782 pobj medium,in
R2501 T2785 T2781 prep during,dissolved
R2502 T2786 T2787 nummod 30,min
R2503 T2787 T2785 pobj min,during
R2504 T2788 T2787 prep at,min
R2505 T2789 T2790 nummod 56,°
R2506 T2790 T2791 nummod °,C.
R2507 T2791 T2788 pobj C.,at
R2633 T2945 T2960 nsubjpass Animals,housed
R2634 T2946 T2945 punct ",",Animals
R2635 T2947 T2952 nmod Chemicals,rats
R2636 T2948 T2947 cc and,Chemicals
R2637 T2949 T2947 conj Diet,Chemicals
R2638 T2950 T2951 compound Male,Wistar
R2639 T2951 T2947 conj Wistar,Chemicals
R2640 T2952 T2945 appos rats,Animals
R2641 T2953 T2952 punct (,rats
R2642 T2954 T2957 nummod 150,weight
R2643 T2955 T2957 amod g,weight
R2644 T2956 T2957 amod average,weight
R2645 T2957 T2952 appos weight,rats
R2646 T2958 T2952 punct ),rats
R2668 T2980 T2981 compound drinking,water
R2670 T2982 T2985 punct (,w/v
R2671 T2983 T2984 nummod 5,%
R2672 T2984 T2985 compound %,w/v
R2673 T2985 T2977 parataxis w/v,administered
R2674 T2986 T2985 punct ),w/v
R2675 T2987 T2977 prep for,administered
R2676 T2988 T2989 nummod 55,days
R2677 T2989 T2987 pobj days,for
R2678 T2990 T2989 prep to,days
R2679 T2991 T2992 nummod 2,groups
R2680 T2992 T2990 pobj groups,to
R2681 T2993 T2992 prep of,groups
R2682 T2994 T2995 nummod six,animals
R2683 T2995 T2993 pobj animals,of
R2684 T2996 T2992 npadvmod each,groups
R2685 T2997 T2977 punct .,administered
R2686 T2998 T3000 det The,group
R2687 T2999 T3000 amod first,group
R2688 T3000 T3001 nsubj group,received
R2689 T3001 T3001 ROOT received,received
R2690 T3002 T3006 det the,carrageenan
R2691 T3003 T3005 amod low,weight
R2692 T3004 T3005 amod molecular,weight
R2693 T3005 T3006 compound weight,carrageenan
R2694 T3006 T3001 dobj carrageenan,received
R2695 T3007 T3006 punct (,carrageenan
R2696 T3008 T3010 nummod 10,dCGN
R2697 T3009 T3010 compound kDa,dCGN
R2698 T3010 T3006 appos dCGN,carrageenan
R2699 T3011 T3010 punct ),dCGN
R2700 T3012 T3006 cc and,carrageenan
R2701 T3013 T3014 det the,second
R2702 T3014 T3015 nsubj second,received
R2703 T3015 T3001 conj received,received
R2704 T3016 T3020 det the,carrageenan
R2705 T3017 T3019 nmod medium,weight
R2706 T3018 T3019 amod molecular,weight
R2707 T3019 T3020 compound weight,carrageenan
R2708 T3020 T3015 dobj carrageenan,received
R2709 T3021 T3020 punct (,carrageenan
R2710 T3022 T3024 nummod 40,dCGN
R2711 T3023 T3024 compound kDa,dCGN
R2712 T3024 T3020 appos dCGN,carrageenan
R2713 T3025 T3024 punct ),dCGN
R2714 T3026 T3015 punct .,received
R2715 T3027 T3029 det An,group
R2716 T3028 T3029 amod additional,group
R2717 T3029 T3034 nsubjpass group,maintained
R2718 T3030 T3029 prep of,group
R2719 T3031 T3032 nummod four,rats
R2720 T3032 T3030 pobj rats,of
R2721 T3033 T3034 auxpass were,maintained
R2722 T3034 T3034 ROOT maintained,maintained
R2723 T3035 T3034 prep on,maintained
R2724 T3036 T3038 amod regular,water
R2725 T3037 T3038 compound tap,water
R2726 T3038 T3035 pobj water,on
R2727 T3039 T3041 punct (,group
R2728 T3040 T3041 compound control,group
R2729 T3041 T3038 appos group,water
R3147 T3503 T3507 nsubjpass segment,opened
R3148 T3504 T3503 prep of,segment
R3149 T3505 T3504 pobj colon,of
R3150 T3506 T3507 auxpass was,opened
R3151 T3507 T3507 ROOT opened,opened
R3152 T3508 T3507 advmod longitudinally,opened
R3153 T3509 T3508 cc and,longitudinally
R3154 T3510 T3508 conj macroscopic,longitudinally
R3155 T3511 T3510 cc and,macroscopic
R3156 T3512 T3510 conj histological,macroscopic
R3157 T3513 T3517 nsubjpass scores,recorded
R3158 T3514 T3513 prep of,scores
R3159 T3515 T3514 pobj inflammation,of
R3160 T3516 T3517 auxpass were,recorded
R3225 T3581 T3580 prep of,level
R3226 T3582 T3581 pobj MPO,of
R3227 T3583 T3580 punct ",",level
R3228 T3584 T3585 advmod mainly,expressed
R3229 T3585 T3580 acl expressed,level
R3230 T3586 T3585 agent by,expressed
R3231 T3587 T3586 pobj neutrophils,by
R3232 T3588 T3589 punct ",",indicates
R3233 T3589 T3589 ROOT indicates,indicates
R3940 T4386 T4389 compound Analysis,Monocytes
R3941 T4387 T4388 compound Peripheral,Blood
R3942 T4388 T4389 compound Blood,Monocytes
R3943 T4389 T4394 nsubjpass Monocytes,exposed
R3944 T4390 T4389 cc or,Monocytes
R3945 T4391 T4392 nummod THP-1,cells
R3946 T4392 T4389 conj cells,Monocytes
R3947 T4393 T4394 auxpass were,exposed
R3948 T4394 T4394 ROOT exposed,exposed
R3949 T4395 T4396 aux to,complete
R3950 T4396 T4394 xcomp complete,exposed
R3951 T4397 T4396 dobj medium,complete
R3952 T4398 T4396 prep in,complete
R4625 T5162 T5163 compound CAGATAGATGGGCTCATACC-3,′
R4626 T5163 T5156 conj ′,′
R4627 T5164 T5143 punct .,amplified
R4628 T5165 T5169 nsubjpass cDNA,amplified
R4629 T5166 T5169 prep for,amplified
R4630 T5167 T5166 pobj ICAM-1,for
R4631 T5168 T5169 auxpass was,amplified
R4632 T5169 T5169 ROOT amplified,amplified
R4633 T5170 T5169 prep for,amplified
R4634 T5171 T5172 nummod 35,cycles
R4635 T5172 T5170 pobj cycles,for
R4636 T5173 T5169 advcl using,amplified

UBERON-AE

Id Subject Object Predicate Lexical cue
T731 1623-1633 http://purl.obolibrary.org/obo/UBERON_0000160 denotes intestinal
T732 1697-1707 http://purl.obolibrary.org/obo/UBERON_0000160 denotes intestinal
T733 1933-1943 http://purl.obolibrary.org/obo/UBERON_0000160 denotes intestinal
T734 2210-2220 http://purl.obolibrary.org/obo/UBERON_0000160 denotes intestinal
T735 1731-1742 http://purl.obolibrary.org/obo/UBERON_2000106 denotes extensively
T736 1933-1954 http://purl.obolibrary.org/obo/UBERON_0001277 denotes intestinal epithelial
T737 2271-2277 http://purl.obolibrary.org/obo/UBERON_0000957 denotes lamina
T738 2271-2285 http://purl.obolibrary.org/obo/UBERON_0000030 denotes lamina propria
T739 3094-3099 http://purl.obolibrary.org/obo/UBERON_0000178 denotes Blood
T740 3618-3623 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T3100 6574-6577 http://purl.obolibrary.org/obo/UBERON_0000970 denotes eye
T3101 6717-6722 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T3102 6785-6790 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T3103 7180-7185 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T3104 7477-7482 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T3105 6874-6884 http://purl.obolibrary.org/obo/UBERON_0000160 denotes intestinal
T3665 7814-7820 http://purl.obolibrary.org/obo/UBERON_0000479 denotes tissue
T3666 8081-8086 http://purl.obolibrary.org/obo/UBERON_0000178 denotes blood
T3667 8136-8141 http://purl.obolibrary.org/obo/UBERON_0000178 denotes blood
T4232 9341-9346 http://purl.obolibrary.org/obo/UBERON_0000178 denotes Blood
T4233 9571-9576 http://purl.obolibrary.org/obo/UBERON_0001977 denotes serum
T4578 10306-10312 http://purl.obolibrary.org/obo/UBERON_0000479 denotes tissue
T5473 14108-14112 http://purl.obolibrary.org/obo/UBERON_0001913 denotes milk
T6603 14921-14928 http://purl.obolibrary.org/obo/UBERON_0001155 denotes Colonic
T6604 15777-15782 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T6605 16071-16076 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T6606 15035-15040 http://purl.obolibrary.org/obo/UBERON_0000178 denotes blood
T6607 15663-15673 http://purl.obolibrary.org/obo/UBERON_0000483 denotes epithelium
T6608 15874-15884 http://purl.obolibrary.org/obo/UBERON_0000483 denotes epithelium
T6609 16071-16083 http://purl.obolibrary.org/obo/UBERON_0000317 denotes colon mucosa
T6610 16077-16083 http://purl.obolibrary.org/obo/UBERON_0000344 denotes mucosa
T15563 16387-16392 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T15564 16465-16470 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T15565 16542-16547 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T15566 16777-16782 http://purl.obolibrary.org/obo/UBERON_0001155 denotes colon
T3106 6989-6999 http://purl.obolibrary.org/obo/UBERON_0000160 denotes intestinal
T3107 7410-7420 http://purl.obolibrary.org/obo/UBERON_0000160 denotes intestinal
T3108 7679-7689 http://purl.obolibrary.org/obo/UBERON_0000160 denotes intestinal
T3109 7410-7427 http://purl.obolibrary.org/obo/UBERON_0001242 denotes intestinal mucosa
T3110 7679-7696 http://purl.obolibrary.org/obo/UBERON_0001242 denotes intestinal mucosa
T3111 7421-7427 http://purl.obolibrary.org/obo/UBERON_0000344 denotes mucosa
T3112 7690-7696 http://purl.obolibrary.org/obo/UBERON_0000344 denotes mucosa

sentences

Id Subject Object Predicate Lexical cue
T78 0-90 Sentence denotes Degraded CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase.
T79 91-217 Sentence denotes In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure.
T80 218-329 Sentence denotes Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody.
T81 330-414 Sentence denotes Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells.
T82 415-465 Sentence denotes All these effects were linked to NF-κB activation.
T83 466-608 Sentence denotes These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype.
T753 623-742 Sentence denotes Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae).
T754 743-813 Sentence denotes Three main forms of CGN have been identified: kappa, iota, and lambda.
T755 814-887 Sentence denotes They differ from each other in sulphation degree and solubility [1], [2].
T756 888-982 Sentence denotes Native CGN is thought to be harmless and is widely used as a food additive to improve texture.
T757 983-1032 Sentence denotes It is also used in cosmetics and pharmaceuticals.
T758 1033-1197 Sentence denotes However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN).
T759 1198-1349 Sentence denotes These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5].
T760 1350-1430 Sentence denotes The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8].
T761 1431-1654 Sentence denotes Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12].
T762 1655-1830 Sentence denotes Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines.
T763 1831-2026 Sentence denotes Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14].
T764 2027-2138 Sentence denotes NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16].
T765 2139-2235 Sentence denotes Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17].
T766 2236-2383 Sentence denotes Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules.
T767 2384-2548 Sentence denotes The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans.
T768 2549-2666 Sentence denotes Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19].
T769 2667-2899 Sentence denotes Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20].
T770 2900-3009 Sentence denotes Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure.
T771 3010-3199 Sentence denotes Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN.
T772 3200-3389 Sentence denotes We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion.
T773 3390-3490 Sentence denotes These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation.
T774 3491-3624 Sentence denotes In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon.
T775 3625-3755 Sentence denotes These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype.
T2051 3780-3815 Sentence denotes Preparation of Degraded Carrageenan
T2052 3816-4084 Sentence denotes Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France).
T2053 4085-4320 Sentence denotes Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions.
T2054 4321-4619 Sentence denotes Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA).
T2055 4620-4844 Sentence denotes For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa).
T2056 4845-4920 Sentence denotes The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa).
T2057 4921-5071 Sentence denotes Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter).
T2058 5072-5300 Sentence denotes Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa.
T2059 5301-5417 Sentence denotes The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22].
T2060 5418-5542 Sentence denotes Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy.
T2061 5543-5673 Sentence denotes The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France).
T2062 5674-5776 Sentence denotes Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C.
T2810 5778-5805 Sentence denotes Animals, Chemicals and Diet
T2811 5806-5940 Sentence denotes Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow.
T2812 5941-6061 Sentence denotes Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each.
T2813 6062-6216 Sentence denotes The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN).
T2814 6217-6303 Sentence denotes An additional group of four rats were maintained on regular tap water (control group).
T2815 6304-6417 Sentence denotes To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]).
T2816 6418-6466 Sentence denotes Fresh carrageenan solutions were prepared daily.
T3117 6468-6489 Sentence denotes Evaluation of Colitis
T3118 6490-6634 Sentence denotes Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period.
T3119 6635-6698 Sentence denotes After 55 days, animals were sacrificed by cervical dislocation.
T3120 6699-6773 Sentence denotes The length of the colon was measured as described by Okayashu et al. [24].
T3121 6774-6891 Sentence denotes Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen.
T3122 6892-6965 Sentence denotes The moderately distended segment was sectioned and fixed in 10% formalin.
T3123 6966-7033 Sentence denotes The following day, the intestinal content was removed by vortexing.
T3124 7034-7123 Sentence denotes The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure.
T3125 7124-7317 Sentence denotes To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26].
T3126 7318-7428 Sentence denotes The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa.
T3127 7429-7571 Sentence denotes On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27].
T3128 7572-7697 Sentence denotes The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa.
T3129 7698-7795 Sentence denotes One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C.
T3668 7797-7809 Sentence denotes Cell Culture
T3669 7810-7884 Sentence denotes All tissue culture reagents were from Invitrogen (Cergy Pontoise, France).
T3670 7885-8063 Sentence denotes THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator.
T3671 8064-8177 Sentence denotes Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient.
T3672 8178-8256 Sentence denotes Monocytes were then isolated by adherence to culture flasks as described [28].
T3673 8257-8353 Sentence denotes For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h.
T3674 8354-8472 Sentence denotes Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer.
T3675 8473-8591 Sentence denotes In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added.
T3981 8593-8612 Sentence denotes Cell Cycle Analysis
T3982 8613-8893 Sentence denotes THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France).
T3983 8894-8984 Sentence denotes Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter).
T3984 8985-9142 Sentence denotes Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations.
T3985 9143-9287 Sentence denotes Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis.
T4234 9289-9329 Sentence denotes Cell Surface Antigen Expression Analysis
T4235 9330-9455 Sentence denotes Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h.
T4236 9456-9612 Sentence denotes After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors.
T4237 9613-9688 Sentence denotes Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min.
T4238 9689-9823 Sentence denotes Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu).
T4239 9824-9927 Sentence denotes After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter).
T4240 9928-10015 Sentence denotes The cell population was gated according to its forward and wide-angle light scattering.
T4241 10016-10096 Sentence denotes Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells.
T4583 10098-10119 Sentence denotes TNF Activity Bioassay
T4584 10120-10274 Sentence denotes Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time.
T4585 10275-10398 Sentence denotes Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29].
T4586 10399-10441 Sentence denotes TNF concentrations are expressed as pg/ml.
T4745 10443-10458 Sentence denotes RT-PCR Analysis
T4746 10459-10651 Sentence denotes Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen).
T4747 10652-10753 Sentence denotes PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination).
T4748 10754-11005 Sentence denotes Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium).
T4749 11006-11578 Sentence denotes First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′.
T5235 11580-11619 Sentence denotes NF-kB Transcription Reporter Gene Assay
T5236 11620-11859 Sentence denotes The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene.
T5237 11860-12033 Sentence denotes Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40.
T5238 12034-12133 Sentence denotes Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI).
T5475 12135-12156 Sentence denotes Western Blot Analysis
T5476 12157-12256 Sentence denotes THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS.
T5477 12257-12404 Sentence denotes Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice.
T5478 12405-12512 Sentence denotes Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected.
T5479 12513-12587 Sentence denotes Protein concentration was determined using the DC Protein Assay (Bio-Rad).
T5480 12588-12729 Sentence denotes Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane.
T5481 12730-12933 Sentence denotes I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories).
T5482 12934-13091 Sentence denotes Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction.
T5483 13092-13154 Sentence denotes Antibody to α-Tubulin (Santa Cruz) was use as loading control.
T5484 13155-13314 Sentence denotes For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31].
T5485 13315-13592 Sentence denotes Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA).
T5486 13593-13688 Sentence denotes Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature.
T5487 13689-13786 Sentence denotes Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight.
T5488 13787-13925 Sentence denotes Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used.
T5489 13926-14170 Sentence denotes Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature.
T5490 14171-14386 Sentence denotes After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions.
T5491 14387-14521 Sentence denotes To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32].
T6463 14523-14569 Sentence denotes Analysis of NF-κB Activation by Flow Cytometry
T6464 14570-14648 Sentence denotes Nuclear activation of NF−κΒ by flow cytometry was performed as described [31].
T6517 14650-14670 Sentence denotes Statistical Analysis
T6518 14671-14751 Sentence denotes The results were expressed as the mean value ± S.E.M. of individual experiments.
T6519 14752-14890 Sentence denotes The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA).
T6612 14901-14941 Sentence denotes Degraded CGN Induce Colonic Inflammation
T6613 14942-15079 Sentence denotes All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools.
T6614 15080-15224 Sentence denotes Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A).
T6615 15225-15354 Sentence denotes Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C).
T6616 15355-15543 Sentence denotes Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage.
T6617 15544-15618 Sentence denotes Histological examination revealed various degrees of mucosal inflammation.
T6618 15619-15734 Sentence denotes Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown).
T6619 15735-15809 Sentence denotes These symptoms were totally absent in the colon of control rats (Fig. 1E).
T6620 15810-15997 Sentence denotes More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F).
T6621 15998-16148 Sentence denotes No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown).
T6622 16149-16321 Sentence denotes Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed.
T7270 0-16905 Sentence denotes Degraded CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced-TNF-α Production by Monocytes In Vitro
T15567 16366-16414 Sentence denotes Degraded CGN induced colon inflammation in rats.
T15568 16415-16584 Sentence denotes Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D).
T15569 16585-16751 Sentence denotes Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control.
T15570 16752-16844 Sentence denotes Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F).
T9 0-90 Sentence denotes Degraded CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase.
T10 91-217 Sentence denotes In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure.
T11 218-329 Sentence denotes Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody.
T12 330-414 Sentence denotes Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells.
T13 415-465 Sentence denotes All these effects were linked to NF-κB activation.
T14 466-608 Sentence denotes These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype.
T15 610-622 Sentence denotes Introduction
T16 623-742 Sentence denotes Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae).
T17 743-813 Sentence denotes Three main forms of CGN have been identified: kappa, iota, and lambda.
T18 814-887 Sentence denotes They differ from each other in sulphation degree and solubility [1], [2].
T19 888-982 Sentence denotes Native CGN is thought to be harmless and is widely used as a food additive to improve texture.
T20 983-1032 Sentence denotes It is also used in cosmetics and pharmaceuticals.
T21 1033-1197 Sentence denotes However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN).
T22 1198-1349 Sentence denotes These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5].
T23 1350-1430 Sentence denotes The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8].
T24 1431-1654 Sentence denotes Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12].
T25 1655-1830 Sentence denotes Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines.
T26 1831-2026 Sentence denotes Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14].
T27 2027-2138 Sentence denotes NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16].
T28 2139-2235 Sentence denotes Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17].
T29 2236-2383 Sentence denotes Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules.
T30 2384-2548 Sentence denotes The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans.
T31 2549-2666 Sentence denotes Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19].
T32 2667-2899 Sentence denotes Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20].
T33 2900-3009 Sentence denotes Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure.
T34 3010-3199 Sentence denotes Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN.
T35 3200-3389 Sentence denotes We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion.
T36 3390-3490 Sentence denotes These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation.
T37 3491-3624 Sentence denotes In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon.
T38 3625-3755 Sentence denotes These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype.
T39 3757-3778 Sentence denotes Materials and Methods
T40 3780-3815 Sentence denotes Preparation of Degraded Carrageenan
T41 3816-4084 Sentence denotes Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France).
T42 4085-4189 Sentence denotes Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C.
T43 4190-4320 Sentence denotes Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions.
T44 4321-4468 Sentence denotes Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C.
T45 4469-4619 Sentence denotes After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA).
T46 4620-4765 Sentence denotes For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C.
T47 4766-4844 Sentence denotes After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa).
T48 4845-4920 Sentence denotes The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa).
T49 4921-5071 Sentence denotes Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter).
T50 5072-5300 Sentence denotes Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa.
T51 5301-5417 Sentence denotes The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22].
T52 5418-5542 Sentence denotes Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy.
T53 5543-5673 Sentence denotes The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France).
T54 5674-5776 Sentence denotes Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C.
T55 5778-5805 Sentence denotes Animals, Chemicals and Diet
T56 5806-5940 Sentence denotes Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow.
T57 5941-6061 Sentence denotes Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each.
T58 6062-6216 Sentence denotes The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN).
T59 6217-6303 Sentence denotes An additional group of four rats were maintained on regular tap water (control group).
T60 6304-6417 Sentence denotes To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]).
T61 6418-6466 Sentence denotes Fresh carrageenan solutions were prepared daily.
T62 6468-6489 Sentence denotes Evaluation of Colitis
T63 6490-6634 Sentence denotes Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period.
T64 6635-6698 Sentence denotes After 55 days, animals were sacrificed by cervical dislocation.
T65 6699-6773 Sentence denotes The length of the colon was measured as described by Okayashu et al. [24].
T66 6774-6891 Sentence denotes Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen.
T67 6892-6965 Sentence denotes The moderately distended segment was sectioned and fixed in 10% formalin.
T68 6966-7033 Sentence denotes The following day, the intestinal content was removed by vortexing.
T69 7034-7123 Sentence denotes The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure.
T70 7124-7317 Sentence denotes To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26].
T71 7318-7428 Sentence denotes The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa.
T72 7429-7571 Sentence denotes On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27].
T73 7572-7697 Sentence denotes The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa.
T74 7698-7795 Sentence denotes One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C.
T75 7797-7809 Sentence denotes Cell Culture
T76 7810-7884 Sentence denotes All tissue culture reagents were from Invitrogen (Cergy Pontoise, France).
T77 7885-8063 Sentence denotes THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator.
T78 8064-8177 Sentence denotes Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient.
T79 8178-8256 Sentence denotes Monocytes were then isolated by adherence to culture flasks as described [28].
T80 8257-8353 Sentence denotes For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h.
T81 8354-8472 Sentence denotes Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer.
T82 8473-8591 Sentence denotes In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added.
T83 8593-8612 Sentence denotes Cell Cycle Analysis
T84 8613-8893 Sentence denotes THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France).
T85 8894-8984 Sentence denotes Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter).
T86 8985-9142 Sentence denotes Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations.
T87 9143-9287 Sentence denotes Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis.
T88 9289-9329 Sentence denotes Cell Surface Antigen Expression Analysis
T89 9330-9455 Sentence denotes Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h.
T90 9456-9612 Sentence denotes After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors.
T91 9613-9688 Sentence denotes Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min.
T92 9689-9823 Sentence denotes Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu).
T93 9824-9927 Sentence denotes After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter).
T94 9928-10015 Sentence denotes The cell population was gated according to its forward and wide-angle light scattering.
T95 10016-10096 Sentence denotes Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells.
T96 10098-10119 Sentence denotes TNF Activity Bioassay
T97 10120-10274 Sentence denotes Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time.
T98 10275-10398 Sentence denotes Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29].
T99 10399-10441 Sentence denotes TNF concentrations are expressed as pg/ml.
T100 10443-10458 Sentence denotes RT-PCR Analysis
T101 10459-10651 Sentence denotes Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen).
T102 10652-10753 Sentence denotes PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination).
T103 10754-11005 Sentence denotes Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium).
T104 11006-11578 Sentence denotes First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′.
T105 11580-11619 Sentence denotes NF-kB Transcription Reporter Gene Assay
T106 11620-11668 Sentence denotes The plasmid 3XMHC-luc (a generous gift from Drs.
T107 11669-11671 Sentence denotes J.
T108 11672-11689 Sentence denotes Westwick and D.A.
T109 11690-11859 Sentence denotes Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene.
T110 11860-12033 Sentence denotes Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40.
T111 12034-12133 Sentence denotes Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI).
T112 12135-12156 Sentence denotes Western Blot Analysis
T113 12157-12256 Sentence denotes THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS.
T114 12257-12404 Sentence denotes Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice.
T115 12405-12512 Sentence denotes Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected.
T116 12513-12587 Sentence denotes Protein concentration was determined using the DC Protein Assay (Bio-Rad).
T117 12588-12729 Sentence denotes Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane.
T118 12730-12933 Sentence denotes I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories).
T119 12934-13091 Sentence denotes Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction.
T120 13092-13154 Sentence denotes Antibody to α-Tubulin (Santa Cruz) was use as loading control.
T121 13155-13249 Sentence denotes For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C.
T122 13250-13314 Sentence denotes Cells were then pelleted and nuclei separated as described [31].
T123 13315-13420 Sentence denotes Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C.
T124 13421-13592 Sentence denotes Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA).
T125 13593-13688 Sentence denotes Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature.
T126 13689-13786 Sentence denotes Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight.
T127 13787-13925 Sentence denotes Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used.
T128 13926-14170 Sentence denotes Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature.
T129 14171-14386 Sentence denotes After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions.
T130 14387-14521 Sentence denotes To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32].
T131 14523-14569 Sentence denotes Analysis of NF-κB Activation by Flow Cytometry
T132 14570-14648 Sentence denotes Nuclear activation of NF−κΒ by flow cytometry was performed as described [31].
T133 14650-14670 Sentence denotes Statistical Analysis
T134 14671-14751 Sentence denotes The results were expressed as the mean value ± S.E.M. of individual experiments.
T135 14752-14890 Sentence denotes The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA).
T136 14892-14899 Sentence denotes Results
T137 14901-14941 Sentence denotes Degraded CGN Induce Colonic Inflammation
T138 14942-15079 Sentence denotes All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools.
T139 15080-15224 Sentence denotes Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A).
T140 15225-15354 Sentence denotes Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C).
T141 15355-15543 Sentence denotes Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage.
T142 15544-15618 Sentence denotes Histological examination revealed various degrees of mucosal inflammation.
T143 15619-15734 Sentence denotes Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown).
T144 15735-15809 Sentence denotes These symptoms were totally absent in the colon of control rats (Fig. 1E).
T145 15810-15997 Sentence denotes More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F).
T146 15998-16148 Sentence denotes No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown).
T147 16149-16321 Sentence denotes Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed.
T148 16322-16414 Sentence denotes 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats.
T149 16415-16584 Sentence denotes Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D).
T150 16585-16751 Sentence denotes Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control.
T151 16752-16844 Sentence denotes Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F).
T152 16846-16905 Sentence denotes Degraded CGN Induced-TNF-α Production by Monocytes In Vitro

events-check-again

Id Subject Object Predicate Lexical cue
T654 109-118 Positive_regulation denotes increased
T655 119-125 Protein denotes ICAM-1
T656 126-136 Gene_expression denotes expression
T657 344-354 Positive_regulation denotes stimulated
T658 344-354 Positive_regulation denotes stimulated
T659 355-360 Protein denotes TNF-α
T660 361-371 Gene_expression denotes expression
T661 376-385 Localization denotes secretion
T2022 1904-1908 Protein denotes IL-8
T2023 1909-1919 Gene_expression denotes production
T2024 1974-1977 Positive_regulation denotes via
T2025 3264-3273 Positive_regulation denotes increased
T2026 3274-3280 Protein denotes ICAM-1
T2027 3281-3291 Gene_expression denotes expression
T2028 3304-3310 Protein denotes ICAM-1
T2029 3347-3357 Positive_regulation denotes stimulated
T2030 3347-3357 Positive_regulation denotes stimulated
T2031 3358-3363 Protein denotes TNF-α
T2032 3364-3374 Gene_expression denotes expression
T2033 3379-3388 Localization denotes secretion
T3652 7509-7524 Protein denotes myeloperoxidase
T3653 7526-7529 Protein denotes MPO
T3654 7585-7588 Protein denotes MPO
T3655 7597-7606 Gene_expression denotes expressed
T4725 10098-10101 Protein denotes TNF
T4726 10295-10300 Protein denotes TNF-α
T4727 10301-10302 Protein denotes β
T4728 10399-10402 Protein denotes TNF
T5210 11176-11183 Protein denotes β-actin
T5211 11311-11316 Protein denotes TNF-α
T5212 11447-11453 Protein denotes ICAM-1
T5452 11843-11853 Protein denotes luciferase
T5453 12034-12044 Protein denotes Luciferase
T6440 12730-12735 Protein denotes I-κBα
T7124 15365-15380 Protein denotes myeloperoxidase
T8141 13-16866 Positive_regulation denotes inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced
T8142 16867-16872 Protein denotes TNF-α
T8143 16873-16883 Gene_expression denotes Production
R530 T655 T656 themeOf ICAM-1,expression
R531 T656 T654 themeOf expression,increased
R532 T659 T660 themeOf TNF-α,expression
R533 T659 T661 themeOf TNF-α,secretion
R534 T660 T658 themeOf expression,stimulated
R535 T661 T657 themeOf secretion,stimulated
R1759 T2022 T2023 themeOf IL-8,production
R1760 T2023 T2024 themeOf production,via
R1761 T2026 T2027 themeOf ICAM-1,expression
R1762 T2027 T2025 themeOf expression,increased
R1763 T2031 T2032 themeOf TNF-α,expression
R1764 T2031 T2033 themeOf TNF-α,secretion
R1765 T2032 T2029 themeOf expression,stimulated
R1766 T2033 T2030 themeOf secretion,stimulated
R3269 T3653 T3652 equivalentTo MPO,myeloperoxidase
R3270 T3654 T3655 themeOf MPO,expressed
R7185 T8142 T8143 themeOf TNF-α,Production
R7186 T8143 T8141 themeOf Production,"inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced"

bionlp-st-ge-2016-reference-tees

Id Subject Object Predicate Lexical cue
T671 126-136 Gene_expression denotes expression
T672 109-118 Positive_regulation denotes increased
T673 224-228 Protein denotes dCGN
T674 308-328 Protein denotes anti-ICAM-1 antibody
T675 339-343 Protein denotes dCGN
T676 355-360 Protein denotes TNF-α
T677 361-371 Gene_expression denotes expression
T678 344-354 Positive_regulation denotes stimulated
T679 448-453 Protein denotes NF-κB
T680 454-464 Positive_regulation denotes activation
T681 521-524 Protein denotes CGN
T682 503-511 Protein_catabolism denotes degraded
T2034 1978-1995 Protein denotes Nuclear Factor-κB
T2035 1997-2002 Protein denotes NF-κB
T2036 2004-2014 Positive_regulation denotes activation
T2037 2004-2014 Positive_regulation denotes activation
T2038 2027-2032 Protein denotes NF-κB
T2039 2064-2073 Regulation denotes regulates
T2040 3274-3280 Protein denotes ICAM-1
T2041 3304-3310 Protein denotes ICAM-1
T2042 3358-3363 Protein denotes TNF-α
T2043 3281-3291 Gene_expression denotes expression
T2044 3364-3374 Gene_expression denotes expression
T2045 3264-3273 Positive_regulation denotes increased
T2046 3347-3357 Positive_regulation denotes stimulated
T2047 3473-3478 Protein denotes NF-κB
T2048 3479-3489 Positive_regulation denotes activation
T2049 3674-3677 Protein denotes CGN
T2050 3656-3664 Protein_catabolism denotes degraded
T2808 5636-5641 Protein denotes Sigma
T2809 5643-5653 Protein denotes St Quentin
T3656 7509-7524 Protein denotes myeloperoxidase
T3657 7526-7529 Protein denotes MPO
T3658 7495-7504 Positive_regulation denotes collected
T3659 7495-7504 Positive_regulation denotes collected
T3660 7585-7588 Protein denotes MPO
T3661 7597-7606 Gene_expression denotes expressed
T3662 7710-7713 Protein denotes MPO
T3663 7742-7753 Protein_catabolism denotes degradation
T4230 8956-8965 Protein denotes EPICS XL2
T4558 9294-9309 Protein denotes Surface Antigen
T4559 9310-9320 Gene_expression denotes Expression
T4560 9599-9611 Protein denotes Fc receptors
T4561 9588-9595 Binding denotes binding
T4562 9779-9782 Protein denotes IgG
T4563 9899-9908 Protein denotes EPICS XL2
T4729 10098-10110 Protein denotes TNF Activity
T4730 10295-10302 Protein denotes TNF-α/β
T4731 10399-10402 Protein denotes TNF
T4732 10422-10431 Gene_expression denotes expressed
T5213 10610-10637 Protein denotes M-MLV reverse transcriptase
T5214 10892-10915 Protein denotes Goldstar DNA polymerase
T5215 10950-10973 Protein denotes Goldstar DNA polymerase
T5216 11176-11183 Protein denotes β-actin
T5217 11311-11316 Protein denotes TNF-α
T5218 11447-11453 Protein denotes ICAM-1
T5454 11767-11772 Protein denotes NF-κB
T5455 11801-11818 Protein denotes MHC class I locus
T5456 11843-11858 Protein denotes luciferase gene
T5457 12034-12044 Protein denotes Luciferase
T6515 14570-14577 Entity denotes Nuclear
T6441 12582-12585 Protein denotes Rad
T6442 12730-12743 Protein denotes I-κBα protein
T6443 12837-12859 Protein denotes horseradish peroxidase
T6444 12943-12946 Protein denotes IκB
T6445 13104-13113 Protein denotes α-Tubulin
T6446 13159-13172 Protein denotes nuclear NF-κB
T6447 13787-13818 Protein denotes Rabbit polyclonal antibody anti
T6448 13819-13824 Protein denotes NF-κB
T6449 13825-13836 Protein denotes p50 subunit
T6450 13851-13873 Protein denotes anti-NF-κB p65 subunit
T6451 14047-14090 Protein denotes HRP-conjugated F(ab')2 goat anti-rabbit IgG
T6452 14499-14502 Protein denotes ERK
T6512 14535-14540 Protein denotes NF-κB
T6513 14541-14551 Positive_regulation denotes Activation
T6514 14592-14597 Protein denotes NF−κΒ
T6516 14578-14588 Positive_regulation denotes activation
T7125 15365-15380 Protein denotes myeloperoxidase
T8185 9-16872 Protein denotes CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced-TNF-α
T8186 0-8 Protein_catabolism denotes Degraded
T8187 16873-16883 Gene_expression denotes Production
T15793 16549-16564 Protein denotes Myeloperoxidase
T15794 16566-16569 Protein denotes MPO
T670 119-125 Protein denotes ICAM-1
R543 T671 T672 themeOf expression,increased
R544 T675 T678 causeOf dCGN,stimulated
R545 T676 T677 themeOf TNF-α,expression
R546 T677 T678 themeOf expression,stimulated
R547 T679 T680 themeOf NF-κB,activation
R548 T681 T682 themeOf CGN,degraded
R555 T670 T671 themeOf ICAM-1,expression
R1767 T2034 T2036 themeOf Nuclear Factor-κB,activation
R1768 T2035 T2037 themeOf NF-κB,activation
R1769 T2038 T2039 themeOf NF-κB,regulates
R1770 T2040 T2043 themeOf ICAM-1,expression
R1771 T2042 T2044 themeOf TNF-α,expression
R1772 T2043 T2045 themeOf expression,increased
R1773 T2044 T2046 themeOf expression,stimulated
R1774 T2047 T2048 themeOf NF-κB,activation
R1775 T2049 T2050 themeOf CGN,degraded
R3271 T3656 T3658 themeOf myeloperoxidase,collected
R3272 T3657 T3659 themeOf MPO,collected
R3273 T3660 T3661 themeOf MPO,expressed
R3274 T3662 T3663 themeOf MPO,degradation
R4090 T4558 T4559 themeOf Surface Antigen,Expression
R4091 T4560 T4561 themeOf Fc receptors,binding
R4212 T4731 T4732 themeOf TNF,expressed
R5797 T6512 T6513 themeOf NF-κB,Activation
R5798 T6514 T6516 themeOf NF−κΒ,activation
R5799 T6515 T6516 Site Nuclear,activation
R7214 T8185 T8186 themeOf "CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced-TNF-α",Degraded
R7215 T8185 T8187 themeOf "CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced-TNF-α",Production

bionlp-st-ge-2016-reference

Id Subject Object Predicate Lexical cue
T63 109-118 Positive_regulation denotes increased
T64 119-125 Protein denotes ICAM-1
T65 126-136 Gene_expression denotes expression
T66 344-354 Positive_regulation denotes stimulated
T67 344-354 Positive_regulation denotes stimulated
T68 355-360 Protein denotes TNF-α
T69 361-371 Gene_expression denotes expression
T70 376-385 Localization denotes secretion
T741 1904-1908 Protein denotes IL-8
T742 1909-1919 Gene_expression denotes production
T743 1974-1977 Positive_regulation denotes via
T744 3264-3273 Positive_regulation denotes increased
T745 3274-3280 Protein denotes ICAM-1
T746 3281-3291 Gene_expression denotes expression
T747 3304-3310 Protein denotes ICAM-1
T748 3347-3357 Positive_regulation denotes stimulated
T749 3347-3357 Positive_regulation denotes stimulated
T750 3358-3363 Protein denotes TNF-α
T751 3364-3374 Gene_expression denotes expression
T752 3379-3388 Localization denotes secretion
T3114 7526-7529 Protein denotes MPO
T3115 7585-7588 Protein denotes MPO
T3116 7597-7606 Gene_expression denotes expressed
T4581 10301-10302 Protein denotes β
T4582 10399-10402 Protein denotes TNF
T5474 12730-12735 Protein denotes I-κBα
T7226 13-16866 Positive_regulation denotes inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced
T7227 16867-16872 Protein denotes TNF-α
T7228 16873-16883 Gene_expression denotes Production
T4579 10098-10101 Protein denotes TNF
T4580 10295-10300 Protein denotes TNF-α
T6611 15365-15380 Protein denotes myeloperoxidase
T5233 11843-11853 Protein denotes luciferase
T5234 12034-12044 Protein denotes Luciferase
T4742 11176-11183 Protein denotes β-actin
T4743 11311-11316 Protein denotes TNF-α
T4744 11447-11453 Protein denotes ICAM-1
T3113 7509-7524 Protein denotes myeloperoxidase
R571 T741 T742 themeOf IL-8,production
R572 T742 T743 themeOf production,via
R573 T745 T746 themeOf ICAM-1,expression
R574 T746 T744 themeOf expression,increased
R575 T750 T751 themeOf TNF-α,expression
R576 T750 T752 themeOf TNF-α,secretion
R577 T751 T748 themeOf expression,stimulated
R578 T752 T749 themeOf secretion,stimulated
R22 T64 T65 themeOf ICAM-1,expression
R23 T65 T63 themeOf expression,increased
R24 T68 T69 themeOf TNF-α,expression
R25 T68 T70 themeOf TNF-α,secretion
R26 T69 T67 themeOf expression,stimulated
R27 T70 T66 themeOf secretion,stimulated
R2770 T3114 T3113 equivalentTo MPO,myeloperoxidase
R2771 T3115 T3116 themeOf MPO,expressed
R6412 T7227 T7228 themeOf TNF-α,Production
R6413 T7228 T7226 themeOf Production,"inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced"

bionlp-st-ge-2016-uniprot

Id Subject Object Predicate Lexical cue
T322 119-125 P05362 denotes ICAM-1
T323 313-319 P05362 denotes ICAM-1
T324 355-358 P01375 denotes TNF
T325 355-360 P01375 denotes TNF-α
T1339 1904-1908 P10145 denotes IL-8
T1340 3274-3280 P05362 denotes ICAM-1
T1341 3304-3310 P05362 denotes ICAM-1
T1342 3358-3361 P01375 denotes TNF
T1343 3358-3363 P01375 denotes TNF-α
T2944 6389-6392 Q15025 denotes Van
T3815 8524-8530 P05362 denotes ICAM-1
T4381 10044-10052 Q04864 denotes relative
T4644 10098-10101 P01375 denotes TNF
T4645 10295-10298 P01375 denotes TNF
T4646 10295-10300 P01375 denotes TNF-α
T4647 10399-10402 P01375 denotes TNF
T4947 11176-11183 P60709 denotes β-actin
T4948 11311-11314 P01375 denotes TNF
T4949 11311-11316 P01375 denotes TNF-α
T4950 11447-11453 P05362 denotes ICAM-1
T5333 11843-11853 P08659 denotes luciferase
T5334 12034-12044 P08659 denotes Luciferase
T5928 12336-12338 P0A7Z4 denotes pH
T5929 13825-13828 P19838 denotes p50
T5930 13825-13836 P19838 denotes p50 subunit
T5931 13862-13865 Q04206 denotes p65
T5932 13862-13865 P21579 denotes p65
T7627 16867-16870 P01375 denotes TNF
T7628 16867-16872 P01375 denotes TNF-α

test2

Id Subject Object Predicate Lexical cue Speculation
T43 109-118 Positive_regulation denotes increased
T44 119-125 Protein denotes ICAM-1
T45 126-136 Gene_expression denotes expression
T46 344-354 Positive_regulation denotes stimulated
T47 355-360 Protein denotes TNF-α
T48 361-371 Gene_expression denotes expression
T49 376-385 Localization denotes secretion
T719 1904-1908 Protein denotes IL-8
T720 1909-1919 Gene_expression denotes production
T721 3264-3273 Positive_regulation denotes increased
T722 3274-3280 Protein denotes ICAM-1
T723 3281-3291 Gene_expression denotes expression
T724 3293-3303 Positive_regulation denotes stimulated
T725 3304-3310 Protein denotes ICAM-1
T726 3311-3320 Regulation denotes dependent
T727 3347-3357 Positive_regulation denotes stimulated
T728 3358-3363 Protein denotes TNF-α
T729 3364-3374 Gene_expression denotes expression
T730 3379-3388 Localization denotes secretion
T3096 7509-7524 Protein denotes myeloperoxidase
T3097 7526-7529 Protein denotes MPO
T3098 7585-7588 Protein denotes MPO
T3099 7597-7606 Gene_expression denotes expressed
T4231 9588-9595 Binding denotes binding
T4574 10098-10101 Protein denotes TNF
T4575 10295-10300 Protein denotes TNF-α
T4576 10301-10302 Protein denotes β
T4577 10399-10402 Protein denotes TNF
T4739 11176-11183 Protein denotes β-actin
T4740 11311-11316 Protein denotes TNF-α
T4741 11447-11453 Protein denotes ICAM-1
T5231 11843-11853 Protein denotes luciferase
T5232 12034-12044 Protein denotes Luciferase
T5472 12730-12735 Protein denotes I-κBα
T6602 15365-15380 Protein denotes myeloperoxidase
T7185 13-16866 Positive_regulation denotes inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced
T7186 16867-16872 Protein denotes TNF-α
T7187 16873-16883 Gene_expression denotes Production true
R17 T44 T45 themeOf ICAM-1,expression
R18 T45 T43 themeOf expression,increased
R19 T47 T48 themeOf TNF-α,expression
R20 T48 T46 themeOf expression,stimulated
R21 T49 T46 themeOf secretion,stimulated
R564 T719 T720 themeOf IL-8,production
R565 T722 T723 themeOf ICAM-1,expression
R566 T723 T721 themeOf expression,increased
R567 T726 T724 themeOf dependent,stimulated
R568 T728 T729 themeOf TNF-α,expression
R569 T729 T727 themeOf expression,stimulated
R570 T730 T727 themeOf secretion,stimulated
R2768 T3097 T3096 equivalentTo MPO,myeloperoxidase
R2769 T3098 T3099 themeOf MPO,expressed
R6388 T7186 T7187 themeOf TNF-α,Production
R6389 T7187 T7185 themeOf Production,"inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype. Introduction Carrageenan (CGN) is a high molecular weight sulphated polysaccharide (>200 kDa) derived from red algae (Rhodophyceae). Three main forms of CGN have been identified: kappa, iota, and lambda. They differ from each other in sulphation degree and solubility [1], [2]. Native CGN is thought to be harmless and is widely used as a food additive to improve texture. It is also used in cosmetics and pharmaceuticals. However, acid treatment at high temperature (80°C) triggers CGN hydrolysis to lower molecular weight (<50 kDa) compounds known as poligeenan or degraded CGN (dCGN). These dCGNs induce inflammation and have been widely used as models of colitis in several species, including rats [3], rabbits [4] and guinea pigs [5]. The role of dCGN as a tumor-promoting factor remains controversial [4], [6]–[8]. Although the native form is thought to be harmless for human consumption, small amounts of dCGN are probably produced by acid hydrolysis during gastric digestion [9], [10] or interaction with intestinal bacteria [11], [12]. Whereas the effects of native and dCGN on intestinal inflammation have been extensively analyzed in animal models, only few studies have been conducted using human cell lines. Recent studies have shown a link between exposure to native form CGN and IL-8 production by the human intestinal epithelial cell line, NCM460, via Nuclear Factor-κB (NF-κB) activation [13], [14]. NF-κB is a transcription factor that regulates the expression of genes associated with inflammation [15], [16]. Macrophage infiltration and accumulation is a common characteristic of intestinal diseases [17]. Macrophages represent 10% of total lamina propria cells, secrete a wide range of biologically active compounds and express cell-adhesion molecules. The immune cell response to an inflammatory stimulus seems to be amplified or directly generated by cells exposed to sulphated polysaccharides such as carrageenans. Indeed, inflammation induced by dCGN was associated with recruitment of macrophages to inflammation sites [18], [19]. Also, inflammation induced by Dextran Sulphate Sodium (DSS), another sulphated compound, was directly associated with macrophages recruitment [20], since DSS still provoked inflammation after T-lymphocyte and NK cell depletion [20]. Although inflammation can be induced by dCGN, there are no data on human monocyte responses to dCGN exposure. Therefore, to investigate the effects of dCGN on human monocytes, normal Peripheral Blood Monocytes (PBM) and tumoral monocyte/macrophage THP-1 cells were exposed to 10 kDa and 40 kDa dCGN. We found that dCGN inhibited THP-1 cell proliferation in vitro, increased ICAM-1 expression, stimulated ICAM-1-dependent monocyte aggregation, and stimulated TNF-α expression and secretion. These responses were more pronounced after 40 kDa dCGN exposure and were linked to NF-κB activation. In addition, the 40 kDa dCGN, but not the 10 kDa dCGN induced in vivo colitis as shown by the inflammatory response in the rat colon. These results suggest that the degraded forms of CGN have an important effect on monocytes resulting in an inflammatory phenotype. Materials and Methods Preparation of Degraded Carrageenan Two preparations of degraded carrageenan with low, (∼10 kDa; C10), and medium, (∼40 kDa; C40) molecular weight were prepared from native iota-carrageenan extracted from Euchema spinosum (generously provided by Sanofi Biosystems Industry, Boulogne-Billancourt, France). Native carrageenan was dissolved in distilled water (5% w/v) under vigorous stirring and heated to 60°C. Then, the carrageenan solution was submitted to two different treatments to obtain both low and medium molecular weight fractions. Briefly, for the low molecular weight fraction, carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 15 min at 80°C. After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 56°C. Animals, Chemicals and Diet Male Wistar rats (150 g average weight) were housed under standard conditions and fed ad libitum with standard rodent laboratory chow. Degraded iota-carrageenans were administered in the drinking water (5% w/v) for 55 days to 2 groups of six animals each. The first group received the low molecular weight carrageenan (10 kDa dCGN) and the second received the medium molecular weight carrageenan (40 kDa dCGN). An additional group of four rats were maintained on regular tap water (control group). To increase palatability 0.2% sucrose was added to the drinking water of all groups (Van der Waaji et al., [23]). Fresh carrageenan solutions were prepared daily. Evaluation of Colitis Body weight, liquid and food consumption, diarrhea and rectal bleeding (detected by eye inspection) were recorded throughout the feeding period. After 55 days, animals were sacrificed by cervical dislocation. The length of the colon was measured as described by Okayashu et al. [24]. Then, each colon was ligated in sections of 2 cm and 1 to 2 ml of 10% formalin was infused into the intestinal lumen. The moderately distended segment was sectioned and fixed in 10% formalin. The following day, the intestinal content was removed by vortexing. The fixed segment was kept in 10% formalin at 4°C until the paraffin embedding procedure. To evaluate the degree of inflammation, this segment of colon was opened longitudinally and macroscopic and histological scores of inflammation were recorded as previously described [25], [26]. The toluidine blue staining was used for identification of sulphated polysaccharides in the intestinal mucosa. On the day of sacrifice, a fresh sample of each colon (50 mg) was collected for myeloperoxidase (MPO) assay according to Krawisz et al., [27]. The level of MPO, mainly expressed by neutrophils, indicates the rate of recruitment of neutrophils to the intestinal mucosa. One unit of MPO activity corresponds to the degradation of 1 µmol of peroxide per minute at 25°C. Cell Culture All tissue culture reagents were from Invitrogen (Cergy Pontoise, France). THP-1 human monocytic cells were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM L -glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C in a 5% CO2 incubator. Human peripheral blood mononuclear cells were obtained from heparinized blood by Ficoll-Hypaque density gradient. Monocytes were then isolated by adherence to culture flasks as described [28]. For cell aggregation, monocytes were cultured in the presence or absence of C10 or C40 for 72 h. Cell colonies were monitored under an inverted phase contrast microscope coupled through a video camera to a computer. In some wells, neutralizing monoclonal antibody to ICAM-1 (2.5 µg/ml) (Tebu, Le Perray en Yvelines, France) was added. Cell Cycle Analysis THP-1 cells in exponential growth phase were exposed to complete medium in the presence or absence of carrageenans for 24 h before being stained with propidium iodide using the DNA-Prep Coulter kit according to the manufacturer's instruction (Beckman-Coulter, Villepinte, France). Cell DNA content was then analyzed by flow cytometry using an EPICS XL2 (Beckman-Coulter). Raw data for the distribution of DNA content of 30,000 cells retrieved from the cytometer were expressed as the percentage of G0/G1 through G2/M populations. Multicycle AV software (Phoenix Flow Systems, San Diego, CA) was used to generate DNA content frequency histograms and facilitate data analysis. Cell Surface Antigen Expression Analysis Peripheral Blood Monocytes or THP-1 cells were exposed to complete medium in the presence or absence of carrageenan for 36 h. After two washes in PBS without Ca2+ and Mg2+, cells were incubated in PBS containing 0.1% gelatin and 8% AB human serum to prevent binding to Fc receptors. Then, 5×105 cells were incubated with primary antibodies at 4°C for 30 min. Two other washes in PBS preceded incubation with FITC-conjugated goat antibody anti-mouse IgG diluted 1/1000 at 4°C for 30 min (Tebu). After two additional washes, analysis of stained cells was performed on an EPICS XL2 (Beckman-Coulter). The cell population was gated according to its forward and wide-angle light scattering. Data were expressed as mean relative fluorescence intensity (MFI) of 3000 cells. TNF Activity Bioassay Monocytes or THP-1 cells were cultured with or without different concentrations of CGNs or LPS (Salmonella typhosa, Sigma) for 24 h or the indicated time. Biologically active TNF-α/β in tissue culture supernatant was measured using the WEHI 164 clone 13-cell killing assay [29]. TNF concentrations are expressed as pg/ml. RT-PCR Analysis Total RNA from monocytes was isolated using TRIzol Reagent™ (Invitrogen). cDNA was generated on 1 µg of total RNA in a reaction volume of 20 µl, using M-MLV reverse transcriptase (Invitrogen). PCR was done in the linear range of amplification (determined for each primer pair-cDNA combination). Standard PCR reactions were performed with 1 µl of the cDNA solution, 50 µM of each primer solution, 10 mM of each dNTP, 25 mM MgCl2, 10X Goldstar DNA polymerase reaction buffer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). First PCR cycle consisted of 1 min at 92°C, 1 min at 58°C and 1 min at 72°C; then each PCR cycle consisted of 40 sec at 92°C, 40 sec at 58°C and 50 sec at 72°C. cDNA for β-actin was amplified for 28 cycles using the oligos: sense 5′-GGCATCGTGATGGACTCCG-3′ and antisense 5′GCTGGAAGGTGGACAGCGA-3′. cDNA for TNF-α was amplified for 35 cycles using the oligos: sense 5′-AAGCCTGTAGCCCATGTTGT-3′ and antisense 5′-CAGATAGATGGGCTCATACC-3′. cDNA for ICAM-1 was amplified for 35 cycles using the oligos sense 5′-GTAGCAGCCGCAGTCATAATGG-3′ and antisense 5′-A TGCTGTTGTATCTGACTGAGG-3′. NF-kB Transcription Reporter Gene Assay The plasmid 3XMHC-luc (a generous gift from Drs. J. Westwick and D.A. Brenner, University of North Carolina, Chapel Hill) contains three copies of NF-κB-responsive element from the MHC class I locus, placed upstream of the luciferase gene. Human monocytic THP-1 cells were transiently transfected as previously described [30], and then cultured for 4 h alone or with increasing concentration of either C10 or C40. Luciferase activity was determined using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Western Blot Analysis THP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control. For nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]. Analysis of NF-κB Activation by Flow Cytometry Nuclear activation of NF−κΒ by flow cytometry was performed as described [31]. Statistical Analysis The results were expressed as the mean value ± S.E.M. of individual experiments. The statistical significance of the differences between mean values was assessed by the Student's t-test and analysis of variance (ANOVA). Results Degraded CGN Induce Colonic Inflammation All rats developed diarrhea during degraded carrageenan administration and gross evidence of blood was frequently detected in the stools. Colon length dramatically decreased in all treated rats with a more pronounced effect being observed in the 40 kDa dCGN treated group (Fig. 1A). Furthermore, prolonged exposure to 40 kDa dCGN resulted in high macroscopic and histological scores of inflammation (Fig. 1B, C). Only weak myeloperoxidase activity was detected in both control and dCGN-treated groups (Fig. 1D), indicating that granulocytes did not play a major role in the inflammation at that stage. Histological examination revealed various degrees of mucosal inflammation. Rats treated with 10 kDa dCGN showed edema, epithelium atrophy and slight lymphocyte infiltration (data not shown). These symptoms were totally absent in the colon of control rats (Fig. 1E). More severe mucosal injuries including ulceration, hyperplastic epithelium, crypt distortion and a strong macrophage infiltration, were observed in the 40 kDa dCGN-treated rats (Fig. 1F). No sulphated polysaccharides were detected by toluidine blue staining of colon mucosa from rats treated with either the 10 or 40 kDa dCGN (not shown). Although we cannot exclude that dCGN mat not have retained in the section during the histology procedure, this indicates that these polymers may not have been phagocytosed. 10.1371/journal.pone.0008666.g001 Figure 1 Degraded CGN induced colon inflammation in rats. Histograms showing the effect of degraded CGN on: colon length (A); macroscopic (B) and histological (C) inflammation score of colon; Myeloperoxidase (MPO) activity (D). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (E), and from 40 kDa dCGN-treated rats (F). Degraded CGN Induced"