PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T188	0-155	Sentence	denotes	β-(1→3)-D-glucan modulates DNA binding of nuclear factors κB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1β/IL-1 receptor antagonist ratio
T1	0-155	Sentence	denotes	β-(1→3)-D-glucan modulates DNA binding of nuclear factors κB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1β/IL-1 receptor antagonist ratio
T2	157-165	Sentence	denotes	Abstract
T189	166-176	Sentence	denotes	Background
T3	166-176	Sentence	denotes	Background
T190	177-287	Sentence	denotes	β-1→3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses.
T4	177-287	Sentence	denotes	β-1→3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses.
T191	288-610	Sentence	denotes	Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1).
T5	288-610	Sentence	denotes	Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1).
T192	612-619	Sentence	denotes	Results
T6	612-619	Sentence	denotes	Results
T193	620-845	Sentence	denotes	Despite an activation of nuclear factor (NF)κB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1β, IL-6, tumor necrosis factor α or interferon γ induced by glucan phosphate.
T7	620-845	Sentence	denotes	Despite an activation of nuclear factor (NF)κB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1β, IL-6, tumor necrosis factor α or interferon γ induced by glucan phosphate.
T194	846-933	Sentence	denotes	Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h:
T8	846-933	Sentence	denotes	Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h:
T195	934-1018	Sentence	denotes	5000 pg/ml), likely due to binding of NFκB to a consensus site in the IL-8 promoter.
T9	934-1018	Sentence	denotes	5000 pg/ml), likely due to binding of NFκB to a consensus site in the IL-8 promoter.
T196	1019-1087	Sentence	denotes	An increase in IL-1receptor antagonist(RA) production (peak at 24 h:
T10	1019-1087	Sentence	denotes	An increase in IL-1receptor antagonist(RA) production (peak at 24 h:
T197	1088-1174	Sentence	denotes	12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels.
T11	1088-1174	Sentence	denotes	12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels.
T198	1175-1337	Sentence	denotes	Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments.
T12	1175-1337	Sentence	denotes	Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments.
T199	1338-1574	Sentence	denotes	When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFκB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA.
T13	1338-1574	Sentence	denotes	When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFκB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA.
T200	1575-1684	Sentence	denotes	Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1β and IL-6.
T14	1575-1684	Sentence	denotes	Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1β and IL-6.
T201	1685-1805	Sentence	denotes	As a consequence, glucan phosphate shifted the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype.
T15	1685-1805	Sentence	denotes	As a consequence, glucan phosphate shifted the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype.
T202	1806-1901	Sentence	denotes	Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2.
T16	1806-1901	Sentence	denotes	Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2.
T203	1903-1913	Sentence	denotes	Conclusion
T17	1903-1913	Sentence	denotes	Conclusion
T204	1914-2127	Sentence	denotes	Thus, β-1→3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction.
T18	1914-2127	Sentence	denotes	Thus, β-1→3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction.
T205	2128-2255	Sentence	denotes	Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a β-1→3-D-glucan.
T19	2128-2255	Sentence	denotes	Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a β-1→3-D-glucan.
T20	2257-2267	Sentence	denotes	Background
T1072	2268-2446	Sentence	denotes	β-1→3-D-glucans occur as a principal component of microbial cell walls or can be secreted from both, non-pathogenic and pathogenic fungi such as S. cerevisae and C. albicans [1].
T21	2268-2446	Sentence	denotes	β-1→3-D-glucans occur as a principal component of microbial cell walls or can be secreted from both, non-pathogenic and pathogenic fungi such as S. cerevisae and C. albicans [1].
T1073	2447-2564	Sentence	denotes	These β-1→3-D-linked glucose polymers are characterized as a fungal pathogen-associated molecular pattern (PAMP) [2].
T22	2447-2564	Sentence	denotes	These β-1→3-D-linked glucose polymers are characterized as a fungal pathogen-associated molecular pattern (PAMP) [2].
T1074	2565-2707	Sentence	denotes	The primary cellular recognition of β-1→3-D-glucans is mediated by several β-1→3-D-glucan receptors on phagocytes [3,4] and other cells [5,6].
T23	2565-2707	Sentence	denotes	The primary cellular recognition of β-1→3-D-glucans is mediated by several β-1→3-D-glucan receptors on phagocytes [3,4] and other cells [5,6].
T1075	2708-2962	Sentence	denotes	Human as well as murine Dectin-1 has been demonstrated to be the major pattern recognition receptor (PRR) for intact yeast and β-1→3-D-glucan-containing particles (i.e. zymosan) on monocytes/macrophages as well as neutrophils and on primary cells [7-11].
T24	2708-2962	Sentence	denotes	Human as well as murine Dectin-1 has been demonstrated to be the major pattern recognition receptor (PRR) for intact yeast and β-1→3-D-glucan-containing particles (i.e. zymosan) on monocytes/macrophages as well as neutrophils and on primary cells [7-11].
T1076	2963-3111	Sentence	denotes	In the murine system, binding of zymosan to Dectin-1 resulted in production of TNFα through Toll-like receptor 2 and the adaptor protein MyD88 [12].
T25	2963-3111	Sentence	denotes	In the murine system, binding of zymosan to Dectin-1 resulted in production of TNFα through Toll-like receptor 2 and the adaptor protein MyD88 [12].
T1077	3112-3238	Sentence	denotes	Another water-soluble β-1→3-D-glucan (PGG-glucan) has been described to activate NFκB and NFIL-6 in murine cell lines [13,14].
T26	3112-3238	Sentence	denotes	Another water-soluble β-1→3-D-glucan (PGG-glucan) has been described to activate NFκB and NFIL-6 in murine cell lines [13,14].
T1078	3239-3484	Sentence	denotes	Similarly, it has been shown that β-1→3-D-glucans activate NFκB in a human monocyte-like cell line [15] and in human polymorphonuclear neutrophils (PMN), in the latter case without secretion of pro-inflammatory cytokines (IL-1, IL-6, TNFα) [16].
T27	3239-3484	Sentence	denotes	Similarly, it has been shown that β-1→3-D-glucans activate NFκB in a human monocyte-like cell line [15] and in human polymorphonuclear neutrophils (PMN), in the latter case without secretion of pro-inflammatory cytokines (IL-1, IL-6, TNFα) [16].
T1079	3485-3662	Sentence	denotes	One study proposed that the production of the anti-inflammatory IL-1RA, but not IL-1 by human monocytes may be a potentially protective mechanism induced by β-1→3-D-glucan [17].
T28	3485-3662	Sentence	denotes	One study proposed that the production of the anti-inflammatory IL-1RA, but not IL-1 by human monocytes may be a potentially protective mechanism induced by β-1→3-D-glucan [17].
T1080	3663-3845	Sentence	denotes	Three other investigations have reported that human leukocytes and human vascular endothelial cells produce IL-8 in response to zymosan [18] or a water-soluble β-1→3-D-glucan [6,19].
T29	3663-3845	Sentence	denotes	Three other investigations have reported that human leukocytes and human vascular endothelial cells produce IL-8 in response to zymosan [18] or a water-soluble β-1→3-D-glucan [6,19].
T1081	3846-3957	Sentence	denotes	In addition, β-1→3-D-glucans seem to be able to modify the response to pro-inflammatory stimuli or even sepsis.
T30	3846-3957	Sentence	denotes	In addition, β-1→3-D-glucans seem to be able to modify the response to pro-inflammatory stimuli or even sepsis.
T1082	3958-4185	Sentence	denotes	In a murine polymicrobial sepsis model, β-1→3-D-glucan [20] treatment resulted in decreased septic morbidity and mortality mediated via inhibition of NFκB and stimulation of the phosphoinositide-3-kinase (PI3K) pathway [21,22].
T31	3958-4185	Sentence	denotes	In a murine polymicrobial sepsis model, β-1→3-D-glucan [20] treatment resulted in decreased septic morbidity and mortality mediated via inhibition of NFκB and stimulation of the phosphoinositide-3-kinase (PI3K) pathway [21,22].
T1083	4186-4341	Sentence	denotes	These and other animal studies [23,24] as well as a clinical trial [25] support a protective role of β-1→3-D-glucan in certain pro-inflammatory conditions.
T32	4186-4341	Sentence	denotes	These and other animal studies [23,24] as well as a clinical trial [25] support a protective role of β-1→3-D-glucan in certain pro-inflammatory conditions.
T1084	4342-4461	Sentence	denotes	The mechanisms underlying these beneficial effects of β-1→3-D-glucan are only partially resolved, especially in humans.
T33	4342-4461	Sentence	denotes	The mechanisms underlying these beneficial effects of β-1→3-D-glucan are only partially resolved, especially in humans.
T1085	4462-4692	Sentence	denotes	Thus, the aim of this study was to elucidate molecular and cellular mechanisms of β-1→3-D-glucans on human leukocytes in pro-inflammatory conditions with special emphasis on the cytokine profile and its transcriptional regulation.
T34	4462-4692	Sentence	denotes	Thus, the aim of this study was to elucidate molecular and cellular mechanisms of β-1→3-D-glucans on human leukocytes in pro-inflammatory conditions with special emphasis on the cytokine profile and its transcriptional regulation.
T1086	4693-4962	Sentence	denotes	For this purpose, peripheral blood mononuclear cells (PBMC) were exposed to a well-defined β-1→3-D-glucan, i.e. glucan phosphate (GP) [20,26], alone or simultaneously with LPS from gram-negative bacteria or the superantigen TSST-1 from gram-positive bacteria over 48 h.
T35	4693-4962	Sentence	denotes	For this purpose, peripheral blood mononuclear cells (PBMC) were exposed to a well-defined β-1→3-D-glucan, i.e. glucan phosphate (GP) [20,26], alone or simultaneously with LPS from gram-negative bacteria or the superantigen TSST-1 from gram-positive bacteria over 48 h.
T1087	4963-5149	Sentence	denotes	Because of the potential effect of β-1→3-D-glucan on cytokine production [12,16-19], TNFα, IL-1β, IL-6, IL-8 and IL-1RA were measured as well as IFNγ, IL-2, IL-4, IL-10, IL-12 and TGFβ1.
T36	4963-5149	Sentence	denotes	Because of the potential effect of β-1→3-D-glucan on cytokine production [12,16-19], TNFα, IL-1β, IL-6, IL-8 and IL-1RA were measured as well as IFNγ, IL-2, IL-4, IL-10, IL-12 and TGFβ1.
T1088	5150-5404	Sentence	denotes	Correspondingly, four NFκB sites from the TNFα promoter (κ consensus, κ1, κ2, κ3) [27], a κ consensus site from the IL-8 promoter [28], an NFAT site from the IFNγ promoter (ATP2) [29] and a consensus NFIL-6 site from the IL-6 promoter [13] were examined.
T37	5150-5404	Sentence	denotes	Correspondingly, four NFκB sites from the TNFα promoter (κ consensus, κ1, κ2, κ3) [27], a κ consensus site from the IL-8 promoter [28], an NFAT site from the IFNγ promoter (ATP2) [29] and a consensus NFIL-6 site from the IL-6 promoter [13] were examined.
T1089	5405-5524	Sentence	denotes	Because of the anti-inflammatory role of IL-1RA, we focused on binding of transcription factors to the IL-1RA promoter.
T38	5405-5524	Sentence	denotes	Because of the anti-inflammatory role of IL-1RA, we focused on binding of transcription factors to the IL-1RA promoter.
T1090	5525-5726	Sentence	denotes	An inhibitory element and three positive-acting LPS-response elements (LRE-1, LRE-2 and LRE-3) in the IL-1RA promoter, including NFκB, PU.1 and NFIL-6 sites, have been characterized previously [30-33].
T39	5525-5726	Sentence	denotes	An inhibitory element and three positive-acting LPS-response elements (LRE-1, LRE-2 and LRE-3) in the IL-1RA promoter, including NFκB, PU.1 and NFIL-6 sites, have been characterized previously [30-33].
T1091	5727-5838	Sentence	denotes	Using computational analysis for homology search [34], we looked for new binding motifs in the IL-1RA promoter.
T40	5727-5838	Sentence	denotes	Using computational analysis for homology search [34], we looked for new binding motifs in the IL-1RA promoter.
T41	5840-5847	Sentence	denotes	Results
T3157	5849-6032	Sentence	denotes	Binding activities of NFκB, NFIL-6 and NFAT from human PBMC to the TNFα, IL-6 and IFNγ promoters following an in vitro stimulation (1 h) with LPS, TSST-1, GP, GP + LPS and GP + TSST-1
T42	5849-6032	Sentence	denotes	Binding activities of NFκB, NFIL-6 and NFAT from human PBMC to the TNFα, IL-6 and IFNγ promoters following an in vitro stimulation (1 h) with LPS, TSST-1, GP, GP + LPS and GP + TSST-1
T3158	6033-6296	Sentence	denotes	GP induced band shifts, indicating binding of NFκB as well as of NFIL-6 to the corresponding DNA oligonucleotides (κ consensus, κ1, κ2, κ3 sites from the TNFα promoter; κ consensus from the IL-8 promoter; NFIL-6consensus from the IL-6 promoter; Table 1; Fig. 1A).
T43	6033-6296	Sentence	denotes	GP induced band shifts, indicating binding of NFκB as well as of NFIL-6 to the corresponding DNA oligonucleotides (κ consensus, κ1, κ2, κ3 sites from the TNFα promoter; κ consensus from the IL-8 promoter; NFIL-6consensus from the IL-6 promoter; Table 1; Fig. 1A).
T3159	6297-6414	Sentence	denotes	The extent of the band shifts induced by GP was not statistically different when compared to LPS or TSST-1 (Fig. 1B).
T44	6297-6414	Sentence	denotes	The extent of the band shifts induced by GP was not statistically different when compared to LPS or TSST-1 (Fig. 1B).
T3160	6415-6588	Sentence	denotes	A supershift, conducted for an NFκB consensus site from the IL-8 promoter, demonstrated a GP-induced predominant binding of NFκB p65 and to a lesser degree of p50 (Fig. 1A).
T45	6415-6588	Sentence	denotes	A supershift, conducted for an NFκB consensus site from the IL-8 promoter, demonstrated a GP-induced predominant binding of NFκB p65 and to a lesser degree of p50 (Fig. 1A).
T3161	6589-6757	Sentence	denotes	Accordingly, an immunoblot of nuclear extracts from GP-treated PBMC showed a strong binding of NFκB p65 and a weaker reaction of p50, whereas p52 was negative (Fig. 2).
T46	6589-6757	Sentence	denotes	Accordingly, an immunoblot of nuclear extracts from GP-treated PBMC showed a strong binding of NFκB p65 and a weaker reaction of p50, whereas p52 was negative (Fig. 2).
T3162	6758-7147	Sentence	denotes	Simultaneous co-treatment of PBMC with GP did not change the LPS-induced NFκB binding to oligos from the TNFα promoter significantly (Fig. 1B), but substantially decreased the TSST-1-induced NFκB binding when compared to TSST-1 or GP (100 μg) and thus differed completely from the theoretical value of GP/TSST-1 calc. (n = 4; p < 0.05 vs. GP/TSST-1 calc. and p = 0.07 vs. TSST-1; Fig. 1B).
T47	6758-7147	Sentence	denotes	Simultaneous co-treatment of PBMC with GP did not change the LPS-induced NFκB binding to oligos from the TNFα promoter significantly (Fig. 1B), but substantially decreased the TSST-1-induced NFκB binding when compared to TSST-1 or GP (100 μg) and thus differed completely from the theoretical value of GP/TSST-1 calc. (n = 4; p < 0.05 vs. GP/TSST-1 calc. and p = 0.07 vs. TSST-1; Fig. 1B).
T3163	7148-7285	Sentence	denotes	GP was also able to induce band shifts indicative of binding of particular variants of NFAT to an oligo from the IFNγ promoter (Fig. 1A).
T48	7148-7285	Sentence	denotes	GP was also able to induce band shifts indicative of binding of particular variants of NFAT to an oligo from the IFNγ promoter (Fig. 1A).
T3164	7286-7408	Sentence	denotes	An immunoblot for two NFAT subunits demonstrated a GP-induced binding of NFATc2 and to a lesser degree of NFATc1 (Fig. 2).
T49	7286-7408	Sentence	denotes	An immunoblot for two NFAT subunits demonstrated a GP-induced binding of NFATc2 and to a lesser degree of NFATc1 (Fig. 2).
T50	7409-7465	Sentence	denotes	Table 1 Sense strand of oligonucleotides used in EMSAs.
T26163	7418-7465	Sentence	denotes	Sense strand of oligonucleotides used in EMSAs.
T51	7466-7596	Sentence	denotes	Oligonucleotidesa Base sequences (5'- XXX -3') binding site/cytokine gene promoter (position of the cytokine gene from the TSS)b
T52	7597-7683	Sentence	denotes	NFκBcS GAT CCT CAG AGG GGA CTT TCCc GAT G NFκB consensus sequence/TNFα promoter [27]
T53	7684-7750	Sentence	denotes	NFκB1S GAT CCT GGG ACA GCC CAG NFκB1 sequence/TNFα promoter [27]
T54	7751-7815	Sentence	denotes	NFκB2S GAT CCG GGG TAT CCT G NFκB2 sequence/TNFα promoter [27]
T55	7816-7881	Sentence	denotes	NFκB3S GAT CCT GGG TTT CTC CG NFκB3 sequence/TNFα promoter [27]
T56	7882-7961	Sentence	denotes	IL-8κBcS ATC GTG GAA TTT CCT CTG A NFκB consensus sequence/IL-8 promoter [28]
T57	7962-8037	Sentence	denotes	NFATP2S GAT CTA AAA TTT CCA GTC CTT GA NFATP2 sequence/IFNγ promoter [29]
T58	8038-8119	Sentence	denotes	NFIL-6S TGC AGA TTG CGC AAT CTG CA NFIL-6 consensus sequence/IL-6 promoter [13]
T59	8120-8213	Sentence	denotes	NFκBcS GCG AGG AGG GTA TTT CCG CTT between -80 and -100 NFκB consensus/IL-1RA promoter [30]
T60	8214-8306	Sentence	denotes	NFκB3S ACA ACA GCA AGG GTT TCT CTT TTT GGA AAT between -100 and -130 NFκB3/IL-1RA promoter
T61	8307-8397	Sentence	denotes	NFκBcS AGT AGG GAG TTT GGT between -266 and -280 NFκB consensus sequence/IL-1RA promoter
T62	8398-8483	Sentence	denotes	NFκB2/3S ACT CTG GGT ACC TGT between -288 and -302 NFκB2/3 sequence/IL-1RA promoter
T63	8484-8591	Sentence	denotes	NFATP2/3S GGC GCA CAA AAC CTA AAA TAT TTA CTA TCT between -471 and -500 NFATP2/3 sequence/IL-1RA promoter
T64	8592-8700	Sentence	denotes	NFIL-6S TTA CAA CAC TCC ATT GCG ACA CTT AGT GGG between -140 and -170 NFIL-6 sequence/IL-1RA promoter [31]
T65	8701-8869	Sentence	denotes	Oct-1 DNAS AAT TGC ATT GCC TGC AGG TCG ACT CTA GAG GAT CCA TGC AAA TGG ATC CCC GGG TAC CGA GCT C exclusion of unspecific bindings, unrelated DNA, (Amersham Pharmacia)
T66	8870-8873	Sentence	denotes	(a:
T67	8874-8948	Sentence	denotes	S = sense; b: TSS = transcription start site;c: ___ = main binding motif).
T68	8949-9009	Sentence	denotes	Figure 1 A, GP led to DNA binding of NFAT, NFκB and NFIL-6.
T21461	8959-9009	Sentence	denotes	A, GP led to DNA binding of NFAT, NFκB and NFIL-6.
T69	9010-9093	Sentence	denotes	Human PBMC were incubated with medium control or GP (1 and 100 μg) for 1 h at 37°C.
T21462	9010-9271	Sentence	denotes	Human PBMC were incubated with medium control or GP (1 and 100 μg) for 1 h at 37°C. Nuclear extracts were incubated with a 32P-labeled NFAT, NFκB, NFIL-6 or NFκB-IL-8 oligonucleotide probe corresponding to the IFNγ, TNFα, IL-6 and IL-8 gene promoters (Table 1).
T70	9094-9271	Sentence	denotes	Nuclear extracts were incubated with a 32P-labeled NFAT, NFκB, NFIL-6 or NFκB-IL-8 oligonucleotide probe corresponding to the IFNγ, TNFα, IL-6 and IL-8 gene promoters (Table 1).
T21463	9272-9426	Sentence	denotes	Arrows with the black head indicate migrational location of the NFAT-DNA, NFκB-DNA, NFIL-6-DNA or NFκB-IL-8-DNA complex compared to free probe (no shift).
T71	9272-9426	Sentence	denotes	Arrows with the black head indicate migrational location of the NFAT-DNA, NFκB-DNA, NFIL-6-DNA or NFκB-IL-8-DNA complex compared to free probe (no shift).
T21464	9427-9524	Sentence	denotes	Arrow with the open head indicates a supershift of NFκB-IL-8-DNA-anti-p65 and -p50, respectively.
T72	9427-9524	Sentence	denotes	Arrow with the open head indicates a supershift of NFκB-IL-8-DNA-anti-p65 and -p50, respectively.
T21465	9525-9592	Sentence	denotes	An autoradiogram from a representative experiment is shown (n = 7).
T73	9525-9592	Sentence	denotes	An autoradiogram from a representative experiment is shown (n = 7).
T21466	9593-9686	Sentence	denotes	B, Decreased DNA binding of NFκB following GP + TSST-1, compared to TSST-1, GP, or TSST-1/GP.
T74	9593-9686	Sentence	denotes	B, Decreased DNA binding of NFκB following GP + TSST-1, compared to TSST-1, GP, or TSST-1/GP.
T75	9687-9801	Sentence	denotes	Human PBMC were incubated with medium control, LPS, TSST-1, GP (100 μg), GP + TSST-1 and GP + LPS for 1 h at 37°C.
T21467	9687-9861	Sentence	denotes	Human PBMC were incubated with medium control, LPS, TSST-1, GP (100 μg), GP + TSST-1 and GP + LPS for 1 h at 37°C. An autoradiogram from a representative experiment is shown.
T76	9802-9861	Sentence	denotes	An autoradiogram from a representative experiment is shown.
T21468	9862-10074	Sentence	denotes	Schematic representation of NFκB-DNA binding activity following GP (1 and 100 μg) or TSST-1 treatment (250 ng) as well as simultaneous administration of both compounds and the theoretical value (GP/TSST-1 calc.).
T77	9862-10074	Sentence	denotes	Schematic representation of NFκB-DNA binding activity following GP (1 and 100 μg) or TSST-1 treatment (250 ng) as well as simultaneous administration of both compounds and the theoretical value (GP/TSST-1 calc.).
T21469	10075-10204	Sentence	denotes	Medium control levels were set equal to 1± SEM and significances (* = p < 0.05) are shown with respect to medium control (n = 4).
T78	10075-10204	Sentence	denotes	Medium control levels were set equal to 1± SEM and significances (* = p < 0.05) are shown with respect to medium control (n = 4).
T79	10205-10282	Sentence	denotes	Figure 2 NFκB p65/p50 and NFATc2/c1 were involved in GP-induced DNA binding.
T22368	10215-10282	Sentence	denotes	NFκB p65/p50 and NFATc2/c1 were involved in GP-induced DNA binding.
T22369	10283-10419	Sentence	denotes	In order to determine whether the DNA binding proteins activated by GP were related to NFκB and NFAT, an immuno(dot) blot was performed.
T80	10283-10419	Sentence	denotes	In order to determine whether the DNA binding proteins activated by GP were related to NFκB and NFAT, an immuno(dot) blot was performed.
T22370	10420-10780	Sentence	denotes	A representative dot blot showing staining of negative and positive controls (recombinant proteins NFκB p52, c-rel; nuclear extracts of PMA-treated Jurkat cells positive for NFκB p50, p65 and for NFATc1, c2) in the upper panel and staining of nuclear extracts of GP-treated PBMC (positive for NFκB p65, p50 and NFAT c2, c1) in the lower panel is shown (n = 3).
T81	10420-10780	Sentence	denotes	A representative dot blot showing staining of negative and positive controls (recombinant proteins NFκB p52, c-rel; nuclear extracts of PMA-treated Jurkat cells positive for NFκB p50, p65 and for NFATc1, c2) in the upper panel and staining of nuclear extracts of GP-treated PBMC (positive for NFκB p65, p50 and NFAT c2, c1) in the lower panel is shown (n = 3).
T4719	10782-10897	Sentence	denotes	Cytokine profile of human PBMC following a 48 h in vitro stimulation with LPS, TSST-1, GP, GP + LPS and GP + TSST-1
T82	10782-10897	Sentence	denotes	Cytokine profile of human PBMC following a 48 h in vitro stimulation with LPS, TSST-1, GP, GP + LPS and GP + TSST-1
T4720	10898-11137	Sentence	denotes	Because of the GP-induced band shifts to seven sites from the TNFα, IL-8, IL-6 and IFNγ promoters, it could be assumed that GP treatment of human PBMC would lead to production of TNFα, IL-8, IL-6, IFNγ and other pro-inflammatory mediators.
T83	10898-11137	Sentence	denotes	Because of the GP-induced band shifts to seven sites from the TNFα, IL-8, IL-6 and IFNγ promoters, it could be assumed that GP treatment of human PBMC would lead to production of TNFα, IL-8, IL-6, IFNγ and other pro-inflammatory mediators.
T4721	11138-11256	Sentence	denotes	Previous studies reported that β-1→3-D-glucans induced only a limited cytokine secretion of human blood cells [15-18].
T84	11138-11256	Sentence	denotes	Previous studies reported that β-1→3-D-glucans induced only a limited cytokine secretion of human blood cells [15-18].
T4722	11257-11679	Sentence	denotes	Corroborating and extending these singular findings in terms of examining pro- (eight) as well as anti- (three) inflammatory cytokines over time (48 h) and six additional transcription factor binding sites, our comprehensive analysis revealed the following cytokine profile of human leukocytes in response to a highly purified water soluble β-1→3-D-glucan and in comparison to two pro-inflammatory mediators (LPS, TSST-1):
T85	11257-11679	Sentence	denotes	Corroborating and extending these singular findings in terms of examining pro- (eight) as well as anti- (three) inflammatory cytokines over time (48 h) and six additional transcription factor binding sites, our comprehensive analysis revealed the following cytokine profile of human leukocytes in response to a highly purified water soluble β-1→3-D-glucan and in comparison to two pro-inflammatory mediators (LPS, TSST-1):
T4723	11681-11690	Sentence	denotes	(i) IL-1β
T86	11681-11690	Sentence	denotes	(i) IL-1β
T4724	11691-11778	Sentence	denotes	There was no IL-1β production detectable following PBMC treatment with 1 nor 100 μg GP.
T87	11691-11778	Sentence	denotes	There was no IL-1β production detectable following PBMC treatment with 1 nor 100 μg GP.
T4725	11779-11879	Sentence	denotes	An insignificant up-regulation of IL-1β production for GP + LPS was observed in comparison with LPS.
T88	11779-11879	Sentence	denotes	An insignificant up-regulation of IL-1β production for GP + LPS was observed in comparison with LPS.
T4726	11880-12075	Sentence	denotes	For GP + TSST-1 we found a significant reduction in IL-1β from 18 h – 24 h, for the latter by about 40% when compared to TSST-1 or the theoretical value of GP/TSST-1 calc. (n = 6; both p = 0.01).
T89	11880-12075	Sentence	denotes	For GP + TSST-1 we found a significant reduction in IL-1β from 18 h – 24 h, for the latter by about 40% when compared to TSST-1 or the theoretical value of GP/TSST-1 calc. (n = 6; both p = 0.01).
T4727	12076-12175	Sentence	denotes	On the whole, GP mediated a reduction of the TSST-1-induced amount of IL-1β by about 50% (Fig. 3A).
T90	12076-12175	Sentence	denotes	On the whole, GP mediated a reduction of the TSST-1-induced amount of IL-1β by about 50% (Fig. 3A).
T91	12176-12274	Sentence	denotes	Figure 3 A, IL-1β was down-regulated (12 h – 24 h) following GP + TSST-1 when compared to TSST-1.
T22957	12186-12274	Sentence	denotes	A, IL-1β was down-regulated (12 h – 24 h) following GP + TSST-1 when compared to TSST-1.
T22958	12275-12430	Sentence	denotes	Time course (48 h) of IL-1β production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS.
T92	12275-12430	Sentence	denotes	Time course (48 h) of IL-1β production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS.
T22959	12431-12558	Sentence	denotes	At 18 h and 24 h there was a marked reduction in IL-1β production following GP + TSST-1 when compared to TSST-1 (* = p < 0.05).
T93	12431-12558	Sentence	denotes	At 18 h and 24 h there was a marked reduction in IL-1β production following GP + TSST-1 when compared to TSST-1 (* = p < 0.05).
T22960	12559-12651	Sentence	denotes	Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are also shown.
T94	12559-12651	Sentence	denotes	Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are also shown.
T22961	12652-12744	Sentence	denotes	IL-1β levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3).
T95	12652-12744	Sentence	denotes	IL-1β levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3).
T22962	12745-12831	Sentence	denotes	B, IL-1RA was exaggerated following GP + TSST-1 (12 h – 48 h) when compared to TSST-1.
T96	12745-12831	Sentence	denotes	B, IL-1RA was exaggerated following GP + TSST-1 (12 h – 48 h) when compared to TSST-1.
T22963	12832-12988	Sentence	denotes	Time course (48 h) of IL-1RA production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS.
T97	12832-12988	Sentence	denotes	Time course (48 h) of IL-1RA production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS.
T22964	12989-13205	Sentence	denotes	Between 18 h and 48 h a simultaneous treatment of PBMC with GP + TSST-1 led to a synergistic effect, i.e. a higher IL-1RA production when compared to an addition of the single values for TSST-1 and GP (* = p < 0.05).
T98	12989-13205	Sentence	denotes	Between 18 h and 48 h a simultaneous treatment of PBMC with GP + TSST-1 led to a synergistic effect, i.e. a higher IL-1RA production when compared to an addition of the single values for TSST-1 and GP (* = p < 0.05).
T22965	13206-13296	Sentence	denotes	Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are included.
T99	13206-13296	Sentence	denotes	Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are included.
T22966	13297-13390	Sentence	denotes	IL-1RA levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3).
T100	13297-13390	Sentence	denotes	IL-1RA levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3).
T4728	13392-13401	Sentence	denotes	(ii) IL-6
T101	13392-13401	Sentence	denotes	(ii) IL-6
T4729	13402-13522	Sentence	denotes	GP induced only a small amount of IL-6 and there was no significant alteration of the LPS-induced IL-6 production by GP.
T102	13402-13522	Sentence	denotes	GP induced only a small amount of IL-6 and there was no significant alteration of the LPS-induced IL-6 production by GP.
T4730	13523-13723	Sentence	denotes	On the contrary, we observed a decreased IL-6 production for GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc., especially at 24 h by about 40% (n = 5; both p = 0.02), (data not shown in detail).
T103	13523-13723	Sentence	denotes	On the contrary, we observed a decreased IL-6 production for GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc., especially at 24 h by about 40% (n = 5; both p = 0.02), (data not shown in detail).
T4731	13725-13735	Sentence	denotes	(iii) IL-8
T104	13725-13735	Sentence	denotes	(iii) IL-8
T4732	13736-13865	Sentence	denotes	We found that GP induced a substantial IL-8 production, in comparison with medium control, especially at 24 h (n = 13; p < 0.01).
T105	13736-13865	Sentence	denotes	We found that GP induced a substantial IL-8 production, in comparison with medium control, especially at 24 h (n = 13; p < 0.01).
T4733	13866-13988	Sentence	denotes	A combination of GP and LPS resulted in a non-significant increase of IL-8 production when compared to LPS or GP/LPS calc.
T106	13866-13988	Sentence	denotes	A combination of GP and LPS resulted in a non-significant increase of IL-8 production when compared to LPS or GP/LPS calc.
T4734	13989-14136	Sentence	denotes	There was no significant alteration in IL-8 production following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail).
T107	13989-14136	Sentence	denotes	There was no significant alteration in IL-8 production following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail).
T4735	14138-14149	Sentence	denotes	(iv) IL-1RA
T108	14138-14149	Sentence	denotes	(iv) IL-1RA
T4736	14150-14300	Sentence	denotes	Besides IL-8, IL-1RA was the only mediator which was produced in significant quantities following GP treatment, especially at 24 h (n = 14, p < 0.01).
T109	14150-14300	Sentence	denotes	Besides IL-8, IL-1RA was the only mediator which was produced in significant quantities following GP treatment, especially at 24 h (n = 14, p < 0.01).
T4737	14301-14418	Sentence	denotes	When compared to LPS stimulation or GP/LPS calc., GP + LPS did not alter the kinetic course of the IL-1RA production.
T110	14301-14418	Sentence	denotes	When compared to LPS stimulation or GP/LPS calc., GP + LPS did not alter the kinetic course of the IL-1RA production.
T4738	14419-14653	Sentence	denotes	However, following GP + TSST-1 we found a synergistic increase in IL-1RA production from 18 h to 48 h, when compared to TSST-1 (for instance at 24 h: n = 6; p = 0.01) or to the theoretical value of GP/TSST-1 calc. (from 18 h to 36 h).
T111	14419-14653	Sentence	denotes	However, following GP + TSST-1 we found a synergistic increase in IL-1RA production from 18 h to 48 h, when compared to TSST-1 (for instance at 24 h: n = 6; p = 0.01) or to the theoretical value of GP/TSST-1 calc. (from 18 h to 36 h).
T4739	14654-14764	Sentence	denotes	Over the time course of 48 h, GP elevated the TSST-1-induced amount of IL-1RA by approximately 200% (Fig. 3B).
T112	14654-14764	Sentence	denotes	Over the time course of 48 h, GP elevated the TSST-1-induced amount of IL-1RA by approximately 200% (Fig. 3B).
T4740	14765-14833	Sentence	denotes	Positive correlation between GP-induced IL-8 and IL-1RA productions.
T113	14765-14833	Sentence	denotes	Positive correlation between GP-induced IL-8 and IL-1RA productions.
T4741	14834-14931	Sentence	denotes	Following stimulation with GP (24 h), we observed a positive correlation between IL-8 and IL-1RA.
T114	14834-14931	Sentence	denotes	Following stimulation with GP (24 h), we observed a positive correlation between IL-8 and IL-1RA.
T4742	14932-15115	Sentence	denotes	Moreover, we found this correlation to be dose-dependent, since 100 μg of GP induced larger amounts of IL-8 and IL-1RA than 1 μg GP (n = 5, p ≤ 0.002, r = 0.9 and 0.98, respectively).
T115	14932-15115	Sentence	denotes	Moreover, we found this correlation to be dose-dependent, since 100 μg of GP induced larger amounts of IL-8 and IL-1RA than 1 μg GP (n = 5, p ≤ 0.002, r = 0.9 and 0.98, respectively).
T4743	15117-15125	Sentence	denotes	(v) TNFα
T116	15117-15125	Sentence	denotes	(v) TNFα
T4744	15126-15262	Sentence	denotes	With respect to TNFα production, 100 μg of GP yielded minor, statistically not distinguishable amounts, when compared to medium control.
T117	15126-15262	Sentence	denotes	With respect to TNFα production, 100 μg of GP yielded minor, statistically not distinguishable amounts, when compared to medium control.
T4745	15263-15359	Sentence	denotes	GP + LPS did not change the TNFα production when compared to LPS supplementation or GP/LPS calc.
T118	15263-15359	Sentence	denotes	GP + LPS did not change the TNFα production when compared to LPS supplementation or GP/LPS calc.
T4746	15360-15583	Sentence	denotes	When combined with TSST-1 or GP/TSST-1 calc., GP seemed to exert a synergistic effect on TNFα secretion after 36 h (n = 3; p < 0.05), but overall an enhancement of only 10% was observed (p > 0.05; data not shown in detail).
T119	15360-15583	Sentence	denotes	When combined with TSST-1 or GP/TSST-1 calc., GP seemed to exert a synergistic effect on TNFα secretion after 36 h (n = 3; p < 0.05), but overall an enhancement of only 10% was observed (p > 0.05; data not shown in detail).
T4747	15585-15594	Sentence	denotes	(vi) IFNγ
T120	15585-15594	Sentence	denotes	(vi) IFNγ
T4748	15595-15672	Sentence	denotes	No IFNγ production was detectable following treatment of PBMC with 100 μg GP.
T121	15595-15672	Sentence	denotes	No IFNγ production was detectable following treatment of PBMC with 100 μg GP.
T4749	15673-15914	Sentence	denotes	There was a minor increase in IFNγ production following GP + LPS at 36 h, when compared to LPS or GP/LPS calc., and a slight down-regulation following GP + TSST-1 at 24 h and 36 h, when compared to TSST-1 or GP/TSST-1 calc. (data not shown).
T122	15673-15914	Sentence	denotes	There was a minor increase in IFNγ production following GP + LPS at 36 h, when compared to LPS or GP/LPS calc., and a slight down-regulation following GP + TSST-1 at 24 h and 36 h, when compared to TSST-1 or GP/TSST-1 calc. (data not shown).
T4750	15916-15953	Sentence	denotes	(vii) IL-2, IL-12, IL-4, IL-10, TGFβ1
T123	15916-15953	Sentence	denotes	(vii) IL-2, IL-12, IL-4, IL-10, TGFβ1
T4751	15954-16030	Sentence	denotes	The production of IL-2, IL-12, IL-4, IL-10 and TGFβ 1 was not induced by GP.
T124	15954-16030	Sentence	denotes	The production of IL-2, IL-12, IL-4, IL-10 and TGFβ 1 was not induced by GP.
T4752	16031-16200	Sentence	denotes	There were also no significant differences between medium control, LPS, TSST-1, GP, GP + LPS, GP/LPS calc. and GP + TSST-1 or GP/TSST-1 calc. over 48 h (data not shown).
T125	16031-16200	Sentence	denotes	There were also no significant differences between medium control, LPS, TSST-1, GP, GP + LPS, GP/LPS calc. and GP + TSST-1 or GP/TSST-1 calc. over 48 h (data not shown).
T8008	16202-16328	Sentence	denotes	Role of NFκB, NFIL-6 and NFAT binding in LPS-, TSST-1-, GP-, GP + LPS- and GP + TSST-1-induced IL-1RA expression by human PBMC
T126	16202-16328	Sentence	denotes	Role of NFκB, NFIL-6 and NFAT binding in LPS-, TSST-1-, GP-, GP + LPS- and GP + TSST-1-induced IL-1RA expression by human PBMC
T8009	16329-16519	Sentence	denotes	Because GP induced band shifts to NFκB, NFIL-6 and NFAT sites, but no TNFα, IL-6 or IFNγ, we hypothesized that the activated transcription factors might bind to sites in the IL-1RA promoter.
T127	16329-16519	Sentence	denotes	Because GP induced band shifts to NFκB, NFIL-6 and NFAT sites, but no TNFα, IL-6 or IFNγ, we hypothesized that the activated transcription factors might bind to sites in the IL-1RA promoter.
T8010	16520-16710	Sentence	denotes	We investigated previoulsy described and newly discovered transcription factor binding sites of the IL-1RA promoter (Table 1) using the program OMIGA (v.1.1.3, Oxford Molecular, Oxford, UK).
T128	16520-16710	Sentence	denotes	We investigated previoulsy described and newly discovered transcription factor binding sites of the IL-1RA promoter (Table 1) using the program OMIGA (v.1.1.3, Oxford Molecular, Oxford, UK).
T8011	16711-16910	Sentence	denotes	The LPS responsive κB consensus site of LRE-1 (between -84 and -93) [30] displayed band shifts, but no significant alterations of its binding activity, irrespective of the treatment (data not shown).
T129	16711-16910	Sentence	denotes	The LPS responsive κB consensus site of LRE-1 (between -84 and -93) [30] displayed band shifts, but no significant alterations of its binding activity, irrespective of the treatment (data not shown).
T8012	16911-17327	Sentence	denotes	A new κB3 site (-100 and -130) within the LRE-1 [30] showed increased binding induced by TSST-1 (+30% vs. control), but GP co-treatment up-regulated the LPS (+65% vs. control)- and the TSST-1 (+110% vs. control)-induced binding in a superadditive synergistic fashion, when compared to medium control as well as to LPS, TSST-1 or GP or the theoretical values of GP/LPS calc. or GP/TSST-1 calc. (all p < 0.05; Fig. 4).
T130	16911-17327	Sentence	denotes	A new κB3 site (-100 and -130) within the LRE-1 [30] showed increased binding induced by TSST-1 (+30% vs. control), but GP co-treatment up-regulated the LPS (+65% vs. control)- and the TSST-1 (+110% vs. control)-induced binding in a superadditive synergistic fashion, when compared to medium control as well as to LPS, TSST-1 or GP or the theoretical values of GP/LPS calc. or GP/TSST-1 calc. (all p < 0.05; Fig. 4).
T8013	17328-17457	Sentence	denotes	Binding to a new κB consensus site (between -266 and -280) was significantly elevated for GP + TSST-1 (p < 0.05; data not shown).
T131	17328-17457	Sentence	denotes	Binding to a new κB consensus site (between -266 and -280) was significantly elevated for GP + TSST-1 (p < 0.05; data not shown).
T8014	17458-17639	Sentence	denotes	The LPS-, TSST-1- and GP + LPS-induced binding to the new κB2/3 site (-288 and -302) was down-regulated by about 20–35% in comparison with medium control (p < 0.05; data not shown).
T132	17458-17639	Sentence	denotes	The LPS-, TSST-1- and GP + LPS-induced binding to the new κB2/3 site (-288 and -302) was down-regulated by about 20–35% in comparison with medium control (p < 0.05; data not shown).
T8015	17640-17817	Sentence	denotes	The NFIL-6 site (-147 and -170) close to LRE-2 [31,32] showed an up-regulation of binding for all compounds and combinations vs. medium control, ranging from 25–110% (p < 0.05).
T133	17640-17817	Sentence	denotes	The NFIL-6 site (-147 and -170) close to LRE-2 [31,32] showed an up-regulation of binding for all compounds and combinations vs. medium control, ranging from 25–110% (p < 0.05).
T8016	17818-17967	Sentence	denotes	Further, GP + TSST-1 induced an increased binding to the NFIL-6 site in comparison with TSST-1 (1.3fold) or GP (1.6fold; p < 0.05), (data not shown).
T134	17818-17967	Sentence	denotes	Further, GP + TSST-1 induced an increased binding to the NFIL-6 site in comparison with TSST-1 (1.3fold) or GP (1.6fold; p < 0.05), (data not shown).
T8017	17968-18208	Sentence	denotes	A new binding site for NFATP2/3 was found more distal of the transcription start site, between -471 and -490, displaying again a substantial increase of binding for all compounds and combinations, when compared to medium control (p < 0.05).
T135	17968-18208	Sentence	denotes	A new binding site for NFATP2/3 was found more distal of the transcription start site, between -471 and -490, displaying again a substantial increase of binding for all compounds and combinations, when compared to medium control (p < 0.05).
T8018	18209-18506	Sentence	denotes	In addition, binding to the NFATP2/3 site following both, GP + LPS (1.4fold of GP) and GP + TSST-1 (1.5fold of GP, 1.25fold of TSST-1) was elevated (p < 0.05; Fig. 5), but only in an additive synergistic manner, i.e. the experimental results were not different from the theoretical values (calc.).
T136	18209-18506	Sentence	denotes	In addition, binding to the NFATP2/3 site following both, GP + LPS (1.4fold of GP) and GP + TSST-1 (1.5fold of GP, 1.25fold of TSST-1) was elevated (p < 0.05; Fig. 5), but only in an additive synergistic manner, i.e. the experimental results were not different from the theoretical values (calc.).
T137	18507-18605	Sentence	denotes	Figure 4 GP modulated LPS- and TSST-1-induced binding to a new NFκB3 site in the IL-1RA promoter.
T23876	18517-18605	Sentence	denotes	GP modulated LPS- and TSST-1-induced binding to a new NFκB3 site in the IL-1RA promoter.
T23877	18606-18767	Sentence	denotes	Binding was enhanced for GP + LPS and GP + TSST-1, when compared to LPS, TSST-1, GP, GP/LPS calc. or GP/TSST-1 calc., respectively (* = p < 0.05; ** = p < 0.01).
T138	18606-18767	Sentence	denotes	Binding was enhanced for GP + LPS and GP + TSST-1, when compared to LPS, TSST-1, GP, GP/LPS calc. or GP/TSST-1 calc., respectively (* = p < 0.05; ** = p < 0.01).
T23878	18768-18824	Sentence	denotes	Medium control levels were set equal to 1 ± SEM (n = 6).
T139	18768-18824	Sentence	denotes	Medium control levels were set equal to 1 ± SEM (n = 6).
T23879	18825-18872	Sentence	denotes	DNA binding was assessed by EMSA (see methods).
T140	18825-18872	Sentence	denotes	DNA binding was assessed by EMSA (see methods).
T141	18873-18969	Sentence	denotes	Figure 5 GP modulated TSST-1-induced DNA binding to a new NFATP2/3 site in the IL-1RA promoter.
T24196	18883-18969	Sentence	denotes	GP modulated TSST-1-induced DNA binding to a new NFATP2/3 site in the IL-1RA promoter.
T24197	18970-19107	Sentence	denotes	Binding was up-regulated for GP + LPS and GP + TSST-1, when compared to GP and, in the latter case, to both GP and TSST-1 (* = p < 0.05).
T142	18970-19107	Sentence	denotes	Binding was up-regulated for GP + LPS and GP + TSST-1, when compared to GP and, in the latter case, to both GP and TSST-1 (* = p < 0.05).
T143	19108-19213	Sentence	denotes	Human PBMC were incubated with medium control, LPS, TSST-1, GP, GP + TSST-1 and GP + LPS for 1 h at 37°C.
T24198	19108-19316	Sentence	denotes	Human PBMC were incubated with medium control, LPS, TSST-1, GP, GP + TSST-1 and GP + LPS for 1 h at 37°C. Schematic representation of the elevated NFAT-DNA binding activity to the site in the IL-1RA promoter.
T144	19214-19316	Sentence	denotes	Schematic representation of the elevated NFAT-DNA binding activity to the site in the IL-1RA promoter.
T24199	19317-19402	Sentence	denotes	Bars depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are added.
T145	19317-19402	Sentence	denotes	Bars depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are added.
T24200	19403-19459	Sentence	denotes	Medium control levels were set equal to 1 ± SEM (n = 6).
T146	19403-19459	Sentence	denotes	Medium control levels were set equal to 1 ± SEM (n = 6).
T24201	19460-19656	Sentence	denotes	An autoradiogram of a representative experiment is shown in the lower panel (lane 1: water; lane 2: medium control; lane 3: GP; lane 4: LPS; lane 5: TSST-1, lane 6: GP + LPS; lane 7: GP + TSST-1).
T147	19460-19656	Sentence	denotes	An autoradiogram of a representative experiment is shown in the lower panel (lane 1: water; lane 2: medium control; lane 3: GP; lane 4: LPS; lane 5: TSST-1, lane 6: GP + LPS; lane 7: GP + TSST-1).
T9554	19658-19747	Sentence	denotes	GP modulated the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype
T148	19658-19747	Sentence	denotes	GP modulated the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype
T9555	19748-19866	Sentence	denotes	In a simplified approach, IL-1β and IL-1RA might represent two sides of a coin, i.e. pro- or anti-inflammatory action.
T149	19748-19866	Sentence	denotes	In a simplified approach, IL-1β and IL-1RA might represent two sides of a coin, i.e. pro- or anti-inflammatory action.
T9556	19867-19958	Sentence	denotes	Therefore, we applied the IL-1β/IL-1RA ratio as an indicator of the degree of inflammation.
T150	19867-19958	Sentence	denotes	Therefore, we applied the IL-1β/IL-1RA ratio as an indicator of the degree of inflammation.
T9557	19959-20077	Sentence	denotes	Both, LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA at early time points, i.e. up to 24 h.
T151	19959-20077	Sentence	denotes	Both, LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA at early time points, i.e. up to 24 h.
T9558	20078-20255	Sentence	denotes	Comparing LPS vs. GP + LPS, we did not find substantial alterations over a time course of 48 h in the IL-1β/IL-1RA ratio, only a – non-significant – 2.5fold higher ratio at 6 h.
T152	20078-20255	Sentence	denotes	Comparing LPS vs. GP + LPS, we did not find substantial alterations over a time course of 48 h in the IL-1β/IL-1RA ratio, only a – non-significant – 2.5fold higher ratio at 6 h.
T9559	20256-20563	Sentence	denotes	With respect to TSST-1 or the theoretical value of GP/TSST-1 calc. vs. GP + TSST-1, we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h of incubation, i.e. a higher production of IL-1RA than of IL-1β (n = 3; both p < 0.05; Fig. 6).
T153	20256-20563	Sentence	denotes	With respect to TSST-1 or the theoretical value of GP/TSST-1 calc. vs. GP + TSST-1, we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h of incubation, i.e. a higher production of IL-1RA than of IL-1β (n = 3; both p < 0.05; Fig. 6).
T9560	20564-20800	Sentence	denotes	A biological relevance of this result was suggested by a decrease of the IL-1 dependent release of IL-2 from murine EL-4 cells following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail, n = 4; p < 0.07).
T154	20564-20800	Sentence	denotes	A biological relevance of this result was suggested by a decrease of the IL-1 dependent release of IL-2 from murine EL-4 cells following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail, n = 4; p < 0.07).
T155	20801-20886	Sentence	denotes	Figure 6 Decreased IL-1β/IL-1RA ratio following GP + TSST-1 when compared to TSST-1.
T24819	20811-20886	Sentence	denotes	Decreased IL-1β/IL-1RA ratio following GP + TSST-1 when compared to TSST-1.
T24820	20887-21039	Sentence	denotes	Time course (48 h) of the IL-1β/IL-1RA ratio following incubation of human PBMC with LPS, TSST-1, GP + LPS, GP/LPS calc., GP + TSST-1 or GP/TSST-1 calc.
T156	20887-21039	Sentence	denotes	Time course (48 h) of the IL-1β/IL-1RA ratio following incubation of human PBMC with LPS, TSST-1, GP + LPS, GP/LPS calc., GP + TSST-1 or GP/TSST-1 calc.
T24821	21040-21125	Sentence	denotes	LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA up to 24 h.
T157	21040-21125	Sentence	denotes	LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA up to 24 h.
T24822	21126-21249	Sentence	denotes	When comparing LPS or GP/LPS calc. vs GP + LPS we did not find significant alterations in the IL-1β/IL-1RA ratio over 48 h.
T158	21126-21249	Sentence	denotes	When comparing LPS or GP/LPS calc. vs GP + LPS we did not find significant alterations in the IL-1β/IL-1RA ratio over 48 h.
T24823	21250-21500	Sentence	denotes	With respect to TSST-1 or GP/TSST-1 calc. vs GP + TSST-1 we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h, i.e. a higher production of IL-1RA than of IL-1β (* = p < 0.05).
T159	21250-21500	Sentence	denotes	With respect to TSST-1 or GP/TSST-1 calc. vs GP + TSST-1 we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h, i.e. a higher production of IL-1RA than of IL-1β (* = p < 0.05).
T24824	21501-21538	Sentence	denotes	Data are shown as mean ± SEM (n = 3).
T160	21501-21538	Sentence	denotes	Data are shown as mean ± SEM (n = 3).
T10436	21540-21604	Sentence	denotes	Inhibition of transcription factors led to reduced IL-1RA levels
T161	21540-21604	Sentence	denotes	Inhibition of transcription factors led to reduced IL-1RA levels
T10437	21605-21767	Sentence	denotes	Pharmacological inhibitors were used to demonstrate that the transcription factor sites within the IL-1RA promoter are relevant for the induction of IL-1RA by GP.
T162	21605-21767	Sentence	denotes	Pharmacological inhibitors were used to demonstrate that the transcription factor sites within the IL-1RA promoter are relevant for the induction of IL-1RA by GP.
T10438	21768-22054	Sentence	denotes	As expected, an inhibition of NFκB via CAPE [35] and CyA, NFAT via CyA and NFIL-6 via CHX [36] could not be overruled by GP, leading to a down-regulated binding activity of the transcription factors to the IL-1RA sites, and a reduction of IL-1RA mRNA and IL-1RA protein levels (Fig. 7).
T163	21768-22054	Sentence	denotes	As expected, an inhibition of NFκB via CAPE [35] and CyA, NFAT via CyA and NFIL-6 via CHX [36] could not be overruled by GP, leading to a down-regulated binding activity of the transcription factors to the IL-1RA sites, and a reduction of IL-1RA mRNA and IL-1RA protein levels (Fig. 7).
T10439	22055-22217	Sentence	denotes	The extent of reduction in NFATP2/3 binding is exemplarily shown in an autoradiogram (Fig. 7A, left side) and graphically summarized (n = 4, Fig. 7A, right side).
T164	22055-22217	Sentence	denotes	The extent of reduction in NFATP2/3 binding is exemplarily shown in an autoradiogram (Fig. 7A, left side) and graphically summarized (n = 4, Fig. 7A, right side).
T10440	22218-22298	Sentence	denotes	The results for the NFκB and NFIL-6 binding sites were similar (data not shown).
T165	22218-22298	Sentence	denotes	The results for the NFκB and NFIL-6 binding sites were similar (data not shown).
T10441	22299-22410	Sentence	denotes	Because the highest IL-1RA amount was found at 24 h, mRNA was examined following 1 h inhibition and 18 h of GP.
T166	22299-22410	Sentence	denotes	Because the highest IL-1RA amount was found at 24 h, mRNA was examined following 1 h inhibition and 18 h of GP.
T10442	22411-22544	Sentence	denotes	A representative gel demonstrating a decrease in IL-1RA mRNA after inhibition with CAPE, CyA and CHX is displayed in Fig. 7B (n = 4).
T167	22411-22544	Sentence	denotes	A representative gel demonstrating a decrease in IL-1RA mRNA after inhibition with CAPE, CyA and CHX is displayed in Fig. 7B (n = 4).
T10443	22545-22640	Sentence	denotes	In addition, corresponding IL-1RA protein levels after 24 h of GP are shown in Fig. 7C (n = 3).
T168	22545-22640	Sentence	denotes	In addition, corresponding IL-1RA protein levels after 24 h of GP are shown in Fig. 7C (n = 3).
T10444	22641-22805	Sentence	denotes	Altogether, NFκB inhibition by CAPE was more pronounced at the binding activity and the transcription, whereas CyA and CHX mainly led to a decreased IL-1RA release.
T169	22641-22805	Sentence	denotes	Altogether, NFκB inhibition by CAPE was more pronounced at the binding activity and the transcription, whereas CyA and CHX mainly led to a decreased IL-1RA release.
T170	22806-22898	Sentence	denotes	Figure 7 Inhibition of GP-induced NFκB, NFAT and NFIL-6 DNA binding to the IL-1RA promoter.
T25500	22816-22898	Sentence	denotes	Inhibition of GP-induced NFκB, NFAT and NFIL-6 DNA binding to the IL-1RA promoter.
T25501	22899-23274	Sentence	denotes	A, A representative autoradiogram (lane 1: control, lane 2: GP, lane 3: GP + unlabeled mutated NFATP2/3 oligo, lane 4: GP + CAPE, lane 5: GP + CyA, lane 6: GP + CHX, lane 7: GP + unlabeled NFATP2/3 oligo, lane 8: water) as well as a graphical summary for the NFATP2/3 site, depicting a decrease in binding following inhibition when compared to GP is displayed (* = p < 0.05).
T171	22899-23274	Sentence	denotes	A, A representative autoradiogram (lane 1: control, lane 2: GP, lane 3: GP + unlabeled mutated NFATP2/3 oligo, lane 4: GP + CAPE, lane 5: GP + CyA, lane 6: GP + CHX, lane 7: GP + unlabeled NFATP2/3 oligo, lane 8: water) as well as a graphical summary for the NFATP2/3 site, depicting a decrease in binding following inhibition when compared to GP is displayed (* = p < 0.05).
T25502	23275-23312	Sentence	denotes	Data are shown as mean ± SEM (n = 4).
T172	23275-23312	Sentence	denotes	Data are shown as mean ± SEM (n = 4).
T25503	23313-23428	Sentence	denotes	B, Inhibition of NFκB, NFAT and NFIL-6 resulted in decreased IL-1RA mRNA following CHX, CAPE or CyA compared to GP.
T173	23313-23428	Sentence	denotes	B, Inhibition of NFκB, NFAT and NFIL-6 resulted in decreased IL-1RA mRNA following CHX, CAPE or CyA compared to GP.
T25504	23429-23495	Sentence	denotes	GAPDH is used as housekeeping control (representative gel, n = 4).
T174	23429-23495	Sentence	denotes	GAPDH is used as housekeeping control (representative gel, n = 4).
T25505	23496-23688	Sentence	denotes	C, Inhibition of NFκB, NFAT and NFIL-6 significantly reduced the production of IL-1RA by human PBMC following treatment with CHX, CAPE or CyA when compared to GP (* = p < 0.05; ** = p < 0.01).
T175	23496-23688	Sentence	denotes	C, Inhibition of NFκB, NFAT and NFIL-6 significantly reduced the production of IL-1RA by human PBMC following treatment with CHX, CAPE or CyA when compared to GP (* = p < 0.05; ** = p < 0.01).
T25506	23689-23726	Sentence	denotes	Data are shown as mean ± SEM (n = 3).
T176	23689-23726	Sentence	denotes	Data are shown as mean ± SEM (n = 3).
T177	23728-23738	Sentence	denotes	Discussion
T11851	23739-23923	Sentence	denotes	Animal models of sepsis and myocardial injury suggest that a β-1→3-D-glucan like GP mediates its protective mechanisms, in part, via a rapid shift from NFκB- to PI3K-signaling [22,37].
T178	23739-23923	Sentence	denotes	Animal models of sepsis and myocardial injury suggest that a β-1→3-D-glucan like GP mediates its protective mechanisms, in part, via a rapid shift from NFκB- to PI3K-signaling [22,37].
T11852	23924-24145	Sentence	denotes	Our study provides novel evidence that downstream of recognition and signaling pro-inflammatory transcription factor binding and cytokine expression of human leukocytes is switched to an anti-inflammatory phenotype by GP.
T179	23924-24145	Sentence	denotes	Our study provides novel evidence that downstream of recognition and signaling pro-inflammatory transcription factor binding and cytokine expression of human leukocytes is switched to an anti-inflammatory phenotype by GP.
T11853	24146-24333	Sentence	denotes	We confirmed and extended previous results [13-16] indicating that, in the absence of other stimuli, β-1→3-D-glucans induced binding of NFκB-, NFIL-6- and NFAT-mers to cytokine promoters.
T180	24146-24333	Sentence	denotes	We confirmed and extended previous results [13-16] indicating that, in the absence of other stimuli, β-1→3-D-glucans induced binding of NFκB-, NFIL-6- and NFAT-mers to cytokine promoters.
T11854	24334-24536	Sentence	denotes	Because of the multiple band shifts observed for NFAT binding (Fig. 1A), one could speculate that there is activation of several different NFAT isoforms, probably derived from alternative splicing [38].
T181	24334-24536	Sentence	denotes	Because of the multiple band shifts observed for NFAT binding (Fig. 1A), one could speculate that there is activation of several different NFAT isoforms, probably derived from alternative splicing [38].
T11855	24537-24685	Sentence	denotes	Interestingly, the GP-induced transcription factor binding transformed only into a very limited cytokine response, namely IL-8 and IL-1RA (Fig. 3B).
T182	24537-24685	Sentence	denotes	Interestingly, the GP-induced transcription factor binding transformed only into a very limited cytokine response, namely IL-8 and IL-1RA (Fig. 3B).
T11856	24686-24820	Sentence	denotes	Hence, our data are in aggreement with the few reports describing a β-1→3-D-glucan-mediated IL-8 [6,16,19] and IL-1RA production [17].
T183	24686-24820	Sentence	denotes	Hence, our data are in aggreement with the few reports describing a β-1→3-D-glucan-mediated IL-8 [6,16,19] and IL-1RA production [17].
T184	24821-25020	Sentence	denotes	In addition, our EMSA/supershift and immunoblotting results demonstrated a GP-mediated predominant binding of NFκB p65 and to a lesser extent of p50 to a κB consensus site of the IL-8 promoter (Figs.
T11857	24821-25028	Sentence	denotes	In addition, our EMSA/supershift and immunoblotting results demonstrated a GP-mediated predominant binding of NFκB p65 and to a lesser extent of p50 to a κB consensus site of the IL-8 promoter (Figs. 1A, 2).
T185	25021-25028	Sentence	denotes	1A, 2).
T11858	25029-25268	Sentence	denotes	Results by Schulte and colleagues [28] pointed to an induction of IL-8 transcription depending on activation via an NFκB p65/65 homodimer, rather than via p65/50 heterodimers, which might be the case for the GP-mediated IL-8 transcription.
T186	25029-25268	Sentence	denotes	Results by Schulte and colleagues [28] pointed to an induction of IL-8 transcription depending on activation via an NFκB p65/65 homodimer, rather than via p65/50 heterodimers, which might be the case for the GP-mediated IL-8 transcription.
T11859	25269-25446	Sentence	denotes	A GP-mediated IL-8 transcription based upon a cooperation between transactivated NFκB p65 and NFIL-6 [13,39] or NFATc2 dimer binding to the IL-8κB site [38] seems also possible.
T187	25269-25446	Sentence	denotes	A GP-mediated IL-8 transcription based upon a cooperation between transactivated NFκB p65 and NFIL-6 [13,39] or NFATc2 dimer binding to the IL-8κB site [38] seems also possible.
T11860	25447-25756	Sentence	denotes	The IL-8κB consensus site exhibits a preferentially p65 binding half site and thus differs from the κB half site described for TNFα and IL-1β [27,40], supporting the idea of regulating NFκB binding through combinatorial associations of the subunits and the specific sequence of the decameric κB motif [41,42].
T188	25447-25756	Sentence	denotes	The IL-8κB consensus site exhibits a preferentially p65 binding half site and thus differs from the κB half site described for TNFα and IL-1β [27,40], supporting the idea of regulating NFκB binding through combinatorial associations of the subunits and the specific sequence of the decameric κB motif [41,42].
T11861	25757-25908	Sentence	denotes	Unlike LPS or TSST-1, we found that GP did not induce IL-1β, but it strongly induced IL-1RA, suggesting an immediate anti-inflammatory potential of GP.
T189	25757-25908	Sentence	denotes	Unlike LPS or TSST-1, we found that GP did not induce IL-1β, but it strongly induced IL-1RA, suggesting an immediate anti-inflammatory potential of GP.
T11862	25909-26156	Sentence	denotes	Analysing the IL-1RA promoter [34], we discovered four new binding sites (Fig. 8): an NFκB3 site (between -100 and -130), an NFκB consensus site (-266 and -280), another NFκB2/3 site (-288 and -302) and a more distal NFATP2/3 site (-471 and -490).
T190	25909-26156	Sentence	denotes	Analysing the IL-1RA promoter [34], we discovered four new binding sites (Fig. 8): an NFκB3 site (between -100 and -130), an NFκB consensus site (-266 and -280), another NFκB2/3 site (-288 and -302) and a more distal NFATP2/3 site (-471 and -490).
T11863	26157-26437	Sentence	denotes	Our data indicated that GP leads to production of IL-1RA primarily via induction of NFATP2/3 and NFIL-6 DNA binding, which might be due to differences in the binding motif or the composition of the activated transcription factors (NFAT) between the IFNγ, IL-6 and IL-1RA promoter.
T191	26157-26437	Sentence	denotes	Our data indicated that GP leads to production of IL-1RA primarily via induction of NFATP2/3 and NFIL-6 DNA binding, which might be due to differences in the binding motif or the composition of the activated transcription factors (NFAT) between the IFNγ, IL-6 and IL-1RA promoter.
T11864	26438-26724	Sentence	denotes	The differential decrease of NFκB, NFAT and NFIL-6 binding to sites in the IL-1RA promoter as well as of IL-1RA mRNA and protein induced by selective inhibitors prior to GP treatment might suggest that these steps are linked to each other and necessary for induction of IL-1RA (Fig. 7).
T192	26438-26724	Sentence	denotes	The differential decrease of NFκB, NFAT and NFIL-6 binding to sites in the IL-1RA promoter as well as of IL-1RA mRNA and protein induced by selective inhibitors prior to GP treatment might suggest that these steps are linked to each other and necessary for induction of IL-1RA (Fig. 7).
T11865	26725-26834	Sentence	denotes	Regarding cellular sources, both, monocytes as well as neutrophils have been reported to produce IL-1RA [32].
T193	26725-26834	Sentence	denotes	Regarding cellular sources, both, monocytes as well as neutrophils have been reported to produce IL-1RA [32].
T11866	26835-26988	Sentence	denotes	Flow cytometric experiments seemed to confirm that monocytes and neutrophils were able to produce IL-8 just as IL-1RA in response to GP (data not shown).
T194	26835-26988	Sentence	denotes	Flow cytometric experiments seemed to confirm that monocytes and neutrophils were able to produce IL-8 just as IL-1RA in response to GP (data not shown).
T11867	26989-27175	Sentence	denotes	This GP-induced cytokine profile was substantially more restricted than that of LPS or TSST-1, which is likely due to differences in recognition and signaling between LPS, TSST-1 and GP.
T195	26989-27175	Sentence	denotes	This GP-induced cytokine profile was substantially more restricted than that of LPS or TSST-1, which is likely due to differences in recognition and signaling between LPS, TSST-1 and GP.
T11868	27176-27357	Sentence	denotes	Recognition of LPS is mainly mediated through Toll-like receptor 4 and subsequent signaling via the NFκB pathway, leading to expression of pro-inflammatory mediators like TNFα [43].
T196	27176-27357	Sentence	denotes	Recognition of LPS is mainly mediated through Toll-like receptor 4 and subsequent signaling via the NFκB pathway, leading to expression of pro-inflammatory mediators like TNFα [43].
T11869	27358-27595	Sentence	denotes	A bacterial superantigen like TSST-1 acts through binding to MHC-II molecules and subsequently the T cell receptor, leading to release of mainly IFNγ and TNFα, the latter via both, PI3K and p38 mitogen-activated kinase signaling [44,45].
T197	27358-27595	Sentence	denotes	A bacterial superantigen like TSST-1 acts through binding to MHC-II molecules and subsequently the T cell receptor, leading to release of mainly IFNγ and TNFα, the latter via both, PI3K and p38 mitogen-activated kinase signaling [44,45].
T11870	27596-27824	Sentence	denotes	Altogether, the induction of the neutrophil-attracting IL-8 and the anti-inflammatory IL-1RA by fungal carbohydrates (GP) may well fit to a benign PAMP response, mounting defensive mechanisms against a possible microbial attack.
T198	27596-27824	Sentence	denotes	Altogether, the induction of the neutrophil-attracting IL-8 and the anti-inflammatory IL-1RA by fungal carbohydrates (GP) may well fit to a benign PAMP response, mounting defensive mechanisms against a possible microbial attack.
T199	27825-27886	Sentence	denotes	Figure 8 Cartoon of the sense strand of the IL-1RA promoter.
T26184	27835-27886	Sentence	denotes	Cartoon of the sense strand of the IL-1RA promoter.
T26185	27887-28065	Sentence	denotes	Relative locations of previously (LRE-1) [30-33] and newly described (boxed), positively (black) and negatively (gray) regulatory transcription factor binding sites are depicted.
T200	27887-28065	Sentence	denotes	Relative locations of previously (LRE-1) [30-33] and newly described (boxed), positively (black) and negatively (gray) regulatory transcription factor binding sites are depicted.
T11871	28066-28341	Sentence	denotes	Potentially even more relevant than the specific cytokine panel induced by GP, we found that, in the presence of other stimuli (LPS or TSST-1), GP ameliorated their pro-inflammatory immune reactions, similar to the effects in murine models of sepsis and inflammation [21-24].
T201	28066-28341	Sentence	denotes	Potentially even more relevant than the specific cytokine panel induced by GP, we found that, in the presence of other stimuli (LPS or TSST-1), GP ameliorated their pro-inflammatory immune reactions, similar to the effects in murine models of sepsis and inflammation [21-24].
T11872	28342-28547	Sentence	denotes	Mainly, GP altered the TSST-1-induced IL-1β/IL-1RA ratio from a pro- to an anti-inflammatory profile via down-regulation of IL-1β and IL-6, at the same time there was a synergistic up-regulation of IL-1RA.
T202	28342-28547	Sentence	denotes	Mainly, GP altered the TSST-1-induced IL-1β/IL-1RA ratio from a pro- to an anti-inflammatory profile via down-regulation of IL-1β and IL-6, at the same time there was a synergistic up-regulation of IL-1RA.
T11873	28548-28788	Sentence	denotes	In part, this change may be caused by GP-mediated modulations of the TSST-1 induced binding of NFκB, NFIL-6 and NFAT to known and up to now unknown sites within the IL-8 and IL-1RA promoter instead of the TNFα, IL-1β, IL-6 or IFNγ promoter.
T203	28548-28788	Sentence	denotes	In part, this change may be caused by GP-mediated modulations of the TSST-1 induced binding of NFκB, NFIL-6 and NFAT to known and up to now unknown sites within the IL-8 and IL-1RA promoter instead of the TNFα, IL-1β, IL-6 or IFNγ promoter.
T11874	28789-29026	Sentence	denotes	Accordingly, we found that the TSST-1-induced binding of NFκB to sites from the TNFα promoter negatively correlated with the GP-mediated enhancement of the TSST-1-induced IL-1RA production (r = -0.88; p < 0.01; data not shown in detail).
T204	28789-29026	Sentence	denotes	Accordingly, we found that the TSST-1-induced binding of NFκB to sites from the TNFα promoter negatively correlated with the GP-mediated enhancement of the TSST-1-induced IL-1RA production (r = -0.88; p < 0.01; data not shown in detail).
T11875	29027-29232	Sentence	denotes	Of the four examined NFκB sites from the IL-1RA promoter, mostly the TSST-1- and LPS-induced binding to the NFκB3 and the TSST-1-induced binding to the new NFκB consensus site seemed to be increased by GP.
T205	29027-29232	Sentence	denotes	Of the four examined NFκB sites from the IL-1RA promoter, mostly the TSST-1- and LPS-induced binding to the NFκB3 and the TSST-1-induced binding to the new NFκB consensus site seemed to be increased by GP.
T11876	29233-29448	Sentence	denotes	So, while we observed a GP-mediated down-regulation of the LPS- and the TSST-1-induced NFκB binding to sites of the TNFα promoter, there was an up-regulation to NFκB3 and NFκB consensus sites of the IL-1RA promoter.
T206	29233-29448	Sentence	denotes	So, while we observed a GP-mediated down-regulation of the LPS- and the TSST-1-induced NFκB binding to sites of the TNFα promoter, there was an up-regulation to NFκB3 and NFκB consensus sites of the IL-1RA promoter.
T11877	29449-29830	Sentence	denotes	These seemingly contradictory data could be explained by differences in either NFκB subunits or conserved nucleotides (#1, 2, 3, 10) within the decameric NFκB binding motif between the TNFα and the IL-1RA promoter (for NFκB3 the IL-1RA motif contains a T on position 10 instead of the conserved C in the TNFα motif, see Table 1), probably leading to differences in binding [41,42].
T207	29449-29830	Sentence	denotes	These seemingly contradictory data could be explained by differences in either NFκB subunits or conserved nucleotides (#1, 2, 3, 10) within the decameric NFκB binding motif between the TNFα and the IL-1RA promoter (for NFκB3 the IL-1RA motif contains a T on position 10 instead of the conserved C in the TNFα motif, see Table 1), probably leading to differences in binding [41,42].
T11878	29831-29996	Sentence	denotes	Despite the location of the new NFκB consensus site (-266 and -280) in the inhibitory element (-250 and -294) of the IL-1RA promoter [30], we observed no inhibition.
T208	29831-29996	Sentence	denotes	Despite the location of the new NFκB consensus site (-266 and -280) in the inhibitory element (-250 and -294) of the IL-1RA promoter [30], we observed no inhibition.
T11879	29997-30215	Sentence	denotes	On the other side, we found an inhibitory NFκB2/3 site (-288 and -302) towards the end of the inhibitory element, demonstrating down-regulations of the LPS- and TSST-1-induced binding, which could not be altered by GP.
T209	29997-30215	Sentence	denotes	On the other side, we found an inhibitory NFκB2/3 site (-288 and -302) towards the end of the inhibitory element, demonstrating down-regulations of the LPS- and TSST-1-induced binding, which could not be altered by GP.
T11880	30216-30334	Sentence	denotes	In our opinion, this site may therefore represent at least a part of the previoulsy described inhibitory element [30].
T210	30216-30334	Sentence	denotes	In our opinion, this site may therefore represent at least a part of the previoulsy described inhibitory element [30].
T11881	30335-30570	Sentence	denotes	The GP-modulated increase in TSST-1-induced binding to the new NFκB3 and NFκB consensus site, the NFIL-6 site [32] as well as to the novel NFATP2/3 site may explain the synergistic up-regulation of the TSST-1-induced IL-1RA production.
T211	30335-30570	Sentence	denotes	The GP-modulated increase in TSST-1-induced binding to the new NFκB3 and NFκB consensus site, the NFIL-6 site [32] as well as to the novel NFATP2/3 site may explain the synergistic up-regulation of the TSST-1-induced IL-1RA production.
T11882	30571-30718	Sentence	denotes	We think that this GP-modulated activation of transcription was reflected by the decrease of the IL-1β/IL-1RA ratio following GP + TSST-1 (Fig. 6).
T212	30571-30718	Sentence	denotes	We think that this GP-modulated activation of transcription was reflected by the decrease of the IL-1β/IL-1RA ratio following GP + TSST-1 (Fig. 6).
T11883	30719-30864	Sentence	denotes	In this context, it has been postulated that in vitro a 100fold excess of IL-1RA over IL-1β might control the biological effects of IL-1 [46,47].
T213	30719-30864	Sentence	denotes	In this context, it has been postulated that in vitro a 100fold excess of IL-1RA over IL-1β might control the biological effects of IL-1 [46,47].
T11884	30865-31040	Sentence	denotes	Since, in fact, the IL-1β/IL-1RA ratio following GP + TSST-1 is partially less than 0.01, it is not unreasonable to assume that IL-1β bioactivity is inactivated in our system.
T214	30865-31040	Sentence	denotes	Since, in fact, the IL-1β/IL-1RA ratio following GP + TSST-1 is partially less than 0.01, it is not unreasonable to assume that IL-1β bioactivity is inactivated in our system.
T11885	31041-31159	Sentence	denotes	Indeed, GP reduced the TSST-1-induced, IL-1-dependent IL-2 production of murine EL-4 cells (data not shown in detail).
T215	31041-31159	Sentence	denotes	Indeed, GP reduced the TSST-1-induced, IL-1-dependent IL-2 production of murine EL-4 cells (data not shown in detail).
T11886	31160-31361	Sentence	denotes	The weaker modulating effects of GP on the LPS-induced immune response observed in this study, may be attributed to delicately balanced differences in signaling pathways between LPS and TSST-1 [43-45].
T216	31160-31361	Sentence	denotes	The weaker modulating effects of GP on the LPS-induced immune response observed in this study, may be attributed to delicately balanced differences in signaling pathways between LPS and TSST-1 [43-45].
T11887	31362-31484	Sentence	denotes	TSST-1 has been shown to use the PI3K pathway for signaling [44] and this effect may be sustained by GP treatment [22,37].
T217	31362-31484	Sentence	denotes	TSST-1 has been shown to use the PI3K pathway for signaling [44] and this effect may be sustained by GP treatment [22,37].
T11888	31485-31624	Sentence	denotes	It has been demonstrated that in septic/LPS-adapted leukocytes the PI3K pathway selectively controls sIL-1RA but not IL-1β production [48].
T218	31485-31624	Sentence	denotes	It has been demonstrated that in septic/LPS-adapted leukocytes the PI3K pathway selectively controls sIL-1RA but not IL-1β production [48].
T11889	31625-31719	Sentence	denotes	Signaling via PI3K has been reported to be involved in the activation of NFAT in T cells [49].
T219	31625-31719	Sentence	denotes	Signaling via PI3K has been reported to be involved in the activation of NFAT in T cells [49].
T11890	31720-31898	Sentence	denotes	Activation of NFκB can also take place via PI3K [50], which may offer an explanation for a difference in signaling between GP (PI3K) and LPS (mitogen-activated kinase signaling).
T220	31720-31898	Sentence	denotes	Activation of NFκB can also take place via PI3K [50], which may offer an explanation for a difference in signaling between GP (PI3K) and LPS (mitogen-activated kinase signaling).
T11891	31899-32193	Sentence	denotes	This idea may be supported by another study showing that despite the use of similar PRR, LPS and peptidoglycan activated the IL-1RA gene through different mechanisms/DNA-binding proteins and acted synergistically in combination, suggestive of signals which are not equivalent in all parts [51].
T221	31899-32193	Sentence	denotes	This idea may be supported by another study showing that despite the use of similar PRR, LPS and peptidoglycan activated the IL-1RA gene through different mechanisms/DNA-binding proteins and acted synergistically in combination, suggestive of signals which are not equivalent in all parts [51].
T222	32195-32205	Sentence	denotes	Conclusion
T17105	32206-32500	Sentence	denotes	In summary, our data demonstrated that in vitro glucan phosphate induced a transcription factor binding and a subsequent cytokine profile different from LPS and also from TSST-1 and, moreover, switched a pro-inflammatory TSST-1-induced response to an IL-1RA-mediated anti-inflammatory reaction.
T223	32206-32500	Sentence	denotes	In summary, our data demonstrated that in vitro glucan phosphate induced a transcription factor binding and a subsequent cytokine profile different from LPS and also from TSST-1 and, moreover, switched a pro-inflammatory TSST-1-induced response to an IL-1RA-mediated anti-inflammatory reaction.
T17106	32501-32743	Sentence	denotes	Our results also generated new insights into a very complex interplay of transcription factor binding to various known and newly identified sites in the IL-1RA promoter (Fig. 8), which can be regulated differentially by a fungal carbohydrate.
T224	32501-32743	Sentence	denotes	Our results also generated new insights into a very complex interplay of transcription factor binding to various known and newly identified sites in the IL-1RA promoter (Fig. 8), which can be regulated differentially by a fungal carbohydrate.
T17107	32744-32967	Sentence	denotes	Together with the in vivo studies [22,37], our findings might support the concept of protective effects mediated by glucan phosphate in pro-inflammatory conditions, especially with a dysregulated IL-1β/IL-1RA ratio [52,53].
T225	32744-32967	Sentence	denotes	Together with the in vivo studies [22,37], our findings might support the concept of protective effects mediated by glucan phosphate in pro-inflammatory conditions, especially with a dysregulated IL-1β/IL-1RA ratio [52,53].
T226	32969-32976	Sentence	denotes	Methods
T17461	32978-32987	Sentence	denotes	Materials
T227	32978-32987	Sentence	denotes	Materials
T17462	32988-33070	Sentence	denotes	Water soluble phosphorylated β-1→3-D-glucan (GP) was prepared as done before [20].
T228	32988-33070	Sentence	denotes	Water soluble phosphorylated β-1→3-D-glucan (GP) was prepared as done before [20].
T17463	33071-33160	Sentence	denotes	The physicochemical characteristics of GP were determined as reported previously [20,26].
T229	33071-33160	Sentence	denotes	The physicochemical characteristics of GP were determined as reported previously [20,26].
T17464	33161-33388	Sentence	denotes	GP was dissolved in aqueous media, filter-sterilized (0.2 μm) and screened for endotoxin contamination with the Endospecy assay (Seigakaku, Tokyo, Japan), which is specific for endotoxin but does not respond to β-1→3-D-glucans.
T230	33161-33388	Sentence	denotes	GP was dissolved in aqueous media, filter-sterilized (0.2 μm) and screened for endotoxin contamination with the Endospecy assay (Seigakaku, Tokyo, Japan), which is specific for endotoxin but does not respond to β-1→3-D-glucans.
T17618	33390-33413	Sentence	denotes	Isolation of human PBMC
T231	33390-33413	Sentence	denotes	Isolation of human PBMC
T232	33414-33585	Sentence	denotes	Buffy coats were obtained from healthy blood donors in compliance with the Helsinki declaration and with the approval of the ethics committee of the University of Luebeck.
T17619	33414-33684	Sentence	denotes	Buffy coats were obtained from healthy blood donors in compliance with the Helsinki declaration and with the approval of the ethics committee of the University of Luebeck. The buffy coats were separated over a Ficoll-Hypaque (Biochrom, Berlin, Germany) gradient [54,55].
T233	33586-33684	Sentence	denotes	The buffy coats were separated over a Ficoll-Hypaque (Biochrom, Berlin, Germany) gradient [54,55].
T17620	33685-34131	Sentence	denotes	Following isolation, PBMC were cultured at a final concentration of 3 × 106 cells/ml (for ELISA measurements) or 5 × 106/ml (for gel shifts) or 1 × 106 cells/ml (for flow cytometry) in RPMI 1640 medium (BioWhittaker, Heidelberg, Germany, LPS-free), containing 10% heat-inactivated fetal calf serum (FCS, low-LPS, Invitrogen, Karlsruhe, Germany), 1% penicillin (10000 U/ml), 1% streptomycin (10000 μg/ml), and 1% 200 mM L-glutamine (BioWhittaker).
T234	33685-34131	Sentence	denotes	Following isolation, PBMC were cultured at a final concentration of 3 × 106 cells/ml (for ELISA measurements) or 5 × 106/ml (for gel shifts) or 1 × 106 cells/ml (for flow cytometry) in RPMI 1640 medium (BioWhittaker, Heidelberg, Germany, LPS-free), containing 10% heat-inactivated fetal calf serum (FCS, low-LPS, Invitrogen, Karlsruhe, Germany), 1% penicillin (10000 U/ml), 1% streptomycin (10000 μg/ml), and 1% 200 mM L-glutamine (BioWhittaker).
T17939	34133-34155	Sentence	denotes	Stimulation conditions
T235	34133-34155	Sentence	denotes	Stimulation conditions
T17940	34156-34325	Sentence	denotes	For induction of transcription factors and cytokines, PBMC were cultured in a volume of 4 ml in sterile pyrogen-free 6-well culture plates (Falcon, Heidelberg, Germany).
T236	34156-34325	Sentence	denotes	For induction of transcription factors and cytokines, PBMC were cultured in a volume of 4 ml in sterile pyrogen-free 6-well culture plates (Falcon, Heidelberg, Germany).
T17941	34326-34604	Sentence	denotes	Cultures were stimulated for 1 h (EMSA) or from 0 h to 48 h (ELISA) with 1 or 100 μg per 106 cells of GP, with 250 ng/106 cells TSST-1 (Toxin Technologies, Sarasota, FL, USA) or with 250 ng/106 cells wild-type LPS from Escherichia coli serotype 0111:B4 (Sigma, Munich, Germany).
T237	34326-34604	Sentence	denotes	Cultures were stimulated for 1 h (EMSA) or from 0 h to 48 h (ELISA) with 1 or 100 μg per 106 cells of GP, with 250 ng/106 cells TSST-1 (Toxin Technologies, Sarasota, FL, USA) or with 250 ng/106 cells wild-type LPS from Escherichia coli serotype 0111:B4 (Sigma, Munich, Germany).
T17942	34605-34745	Sentence	denotes	In costimulatory experiments, PBMC were supplemented simultaneously with a combination of GP and TSST-1 or with a combination of GP and LPS.
T238	34605-34745	Sentence	denotes	In costimulatory experiments, PBMC were supplemented simultaneously with a combination of GP and TSST-1 or with a combination of GP and LPS.
T18267	34747-34793	Sentence	denotes	Oligonucleotides (Oligos) and 5'-32-P-Labeling
T239	34747-34793	Sentence	denotes	Oligonucleotides (Oligos) and 5'-32-P-Labeling
T18268	34794-35228	Sentence	denotes	The complementary double-stranded (ds) oligonucleotides (oligos) from the TNFα, IL-8, IFNγ and IL-6 promoters were synthesised from single stranded (ss) oligos (illustrated in Table 1), (TIB Molbiol, Berlin, Germany) and 32P-labeled with 5'γ-P-ATP (3,000 Ci/mmol, Amersham, Braunschweig, Germany) using the Ready-To-Go-Polynucleotide-Kinase Kit (PNK Kit, Pharmacia LKB, Freiburg, Germany) according to the manufacturer's instructions.
T240	34794-35228	Sentence	denotes	The complementary double-stranded (ds) oligonucleotides (oligos) from the TNFα, IL-8, IFNγ and IL-6 promoters were synthesised from single stranded (ss) oligos (illustrated in Table 1), (TIB Molbiol, Berlin, Germany) and 32P-labeled with 5'γ-P-ATP (3,000 Ci/mmol, Amersham, Braunschweig, Germany) using the Ready-To-Go-Polynucleotide-Kinase Kit (PNK Kit, Pharmacia LKB, Freiburg, Germany) according to the manufacturer's instructions.
T18269	35229-35326	Sentence	denotes	To test the binding specificity of the oligos, mutated oligos were used as an additional control.
T241	35229-35326	Sentence	denotes	To test the binding specificity of the oligos, mutated oligos were used as an additional control.
T18270	35327-35565	Sentence	denotes	Mutations in the P2 site of the IFNγ promoter [29] and in the κ consensus sequence of the TNFα promoter [55] have been reported to interfere with sequences, which seem to be crucial for the binding of NFAT and NFκB proteins, respectively.
T242	35327-35565	Sentence	denotes	Mutations in the P2 site of the IFNγ promoter [29] and in the κ consensus sequence of the TNFα promoter [55] have been reported to interfere with sequences, which seem to be crucial for the binding of NFAT and NFκB proteins, respectively.
T18271	35566-35719	Sentence	denotes	Afterwards, the ds oligos were purified via gel filtration using Probe Quant G-50 Micro Columns (Pharmacia) according to the manufacturer's instructions.
T243	35566-35719	Sentence	denotes	Afterwards, the ds oligos were purified via gel filtration using Probe Quant G-50 Micro Columns (Pharmacia) according to the manufacturer's instructions.
T18788	35721-35801	Sentence	denotes	Preparation of nuclear extracts and electrophoretic mobility shift assays (EMSA)
T244	35721-35801	Sentence	denotes	Preparation of nuclear extracts and electrophoretic mobility shift assays (EMSA)
T18789	35802-35952	Sentence	denotes	Nuclear extracts of purified PBMCs were prepared according to a technique described by Trede et al. [27] and modified for our experimental setup [56].
T245	35802-35952	Sentence	denotes	Nuclear extracts of purified PBMCs were prepared according to a technique described by Trede et al. [27] and modified for our experimental setup [56].
T18790	35953-36033	Sentence	denotes	The intensity of shifted bands was normalized to bands of unstimulated controls.
T246	35953-36033	Sentence	denotes	The intensity of shifted bands was normalized to bands of unstimulated controls.
T18791	36034-36229	Sentence	denotes	Competition experiments with labeled and unlabeled mutated oligos of the cytokine promoter binding sites (see above) were carried out in order to prevent non-specific binding to nuclear proteins.
T247	36034-36229	Sentence	denotes	Competition experiments with labeled and unlabeled mutated oligos of the cytokine promoter binding sites (see above) were carried out in order to prevent non-specific binding to nuclear proteins.
T18792	36230-36324	Sentence	denotes	For such experiments, an excess (5–50×) of unlabeled oligos was added to the nuclear extracts.
T248	36230-36324	Sentence	denotes	For such experiments, an excess (5–50×) of unlabeled oligos was added to the nuclear extracts.
T18793	36325-36444	Sentence	denotes	For specificity of binding, supershift assays for NFκB with a combined p65/50 antibody were performed (data not shown).
T249	36325-36444	Sentence	denotes	For specificity of binding, supershift assays for NFκB with a combined p65/50 antibody were performed (data not shown).
T18794	36445-36595	Sentence	denotes	To exclude non-specific reactions, a 30-fold molar excess of Oct-1 DNA (unrelated DNA, Table 1) was used, which did not compete with specific binding.
T250	36445-36595	Sentence	denotes	To exclude non-specific reactions, a 30-fold molar excess of Oct-1 DNA (unrelated DNA, Table 1) was used, which did not compete with specific binding.
T19195	36597-36614	Sentence	denotes	Immuno (Dot) blot
T251	36597-36614	Sentence	denotes	Immuno (Dot) blot
T19196	36615-37422	Sentence	denotes	To determine whether the DNA binding proteins were related to NFκB and NFAT, dot blots using a SRC96D SNS minifold I dot blotter (Schleicher & Schüll, Dassel, Germany) were performed with the following positive controls and antibodies (all from Santa Cruz Biotechnology, Heidelberg, Germany): positive controls (10 ng/dot): NFκB p52 (80 kD) sc-4095WB, c-Rel (61 kD) sc-4030WB; Jurkat nuclear extract, PMA-stimulated; primary antibodies (1 μg/dot): anti-NFκB p50 (NLS) sc-114, anti-NFκB p65 (A) sc-109, anti-NFκB p52 (447) sc-848, a rabbit polyclonal IgG1 antiserum, anti-c-rel (N466) sc-272, anti-NFATc2 (M-20) sc-1151, anti-NFATc1 (K-18) sc-1149, two goat polyclonal IgG1 antisera; secondary antibodies (0.8 μg/dot): goat anti-rabbit IgG AP-conjugate sc-2007, and donkey anti-goat IgG AP-conjugate sc-2022.
T252	36615-37422	Sentence	denotes	To determine whether the DNA binding proteins were related to NFκB and NFAT, dot blots using a SRC96D SNS minifold I dot blotter (Schleicher & Schüll, Dassel, Germany) were performed with the following positive controls and antibodies (all from Santa Cruz Biotechnology, Heidelberg, Germany): positive controls (10 ng/dot): NFκB p52 (80 kD) sc-4095WB, c-Rel (61 kD) sc-4030WB; Jurkat nuclear extract, PMA-stimulated; primary antibodies (1 μg/dot): anti-NFκB p50 (NLS) sc-114, anti-NFκB p65 (A) sc-109, anti-NFκB p52 (447) sc-848, a rabbit polyclonal IgG1 antiserum, anti-c-rel (N466) sc-272, anti-NFATc2 (M-20) sc-1151, anti-NFATc1 (K-18) sc-1149, two goat polyclonal IgG1 antisera; secondary antibodies (0.8 μg/dot): goat anti-rabbit IgG AP-conjugate sc-2007, and donkey anti-goat IgG AP-conjugate sc-2022.
T19197	37423-37498	Sentence	denotes	The nuclear proteins were blotted onto a nitrocellulose membrane (Bio-Rad).
T253	37423-37498	Sentence	denotes	The nuclear proteins were blotted onto a nitrocellulose membrane (Bio-Rad).
T19198	37499-37761	Sentence	denotes	After blocking with PBS/3% BSA (Fluka, Deisenhofen, Germany), the blot was incubated overnight with the primary antibodies diluted 1:2000 in PBS/1% BSA, washed again and incubated with the alkaline phosphatase-conjugated secondary antibody (1:500 in PBS/1% BSA).
T254	37499-37761	Sentence	denotes	After blocking with PBS/3% BSA (Fluka, Deisenhofen, Germany), the blot was incubated overnight with the primary antibodies diluted 1:2000 in PBS/1% BSA, washed again and incubated with the alkaline phosphatase-conjugated secondary antibody (1:500 in PBS/1% BSA).
T255	37762-37845	Sentence	denotes	The blot was developed using the Vectastain® staining kit (Vector Laboratories Inc.
T19199	37762-37867	Sentence	denotes	The blot was developed using the Vectastain® staining kit (Vector Laboratories Inc. Burlingame, CA, USA).
T256	37846-37867	Sentence	denotes	Burlingame, CA, USA).
T19743	37869-37890	Sentence	denotes	Cytokine measurements
T257	37869-37890	Sentence	denotes	Cytokine measurements
T19744	37891-38048	Sentence	denotes	Supernatants of cell cultures were harvested 3, 6, 9, 12, 18, 24, 30, 36 and 48 hours after stimulation and stored at -80 °C until used and thawed only once.
T258	37891-38048	Sentence	denotes	Supernatants of cell cultures were harvested 3, 6, 9, 12, 18, 24, 30, 36 and 48 hours after stimulation and stored at -80 °C until used and thawed only once.
T19745	38049-38279	Sentence	denotes	IFNγ, TNFα, IL-1 and IL-6 were determined using an ELISA from Bender Systems (Vienna, Austria), IL-1RA and IL-8 by an ELISA from R&D Systems (Wiesbaden-Nordenstadt, Germany), and TGFβ 1 using an ELISA as previously described [57].
T259	38049-38279	Sentence	denotes	IFNγ, TNFα, IL-1 and IL-6 were determined using an ELISA from Bender Systems (Vienna, Austria), IL-1RA and IL-8 by an ELISA from R&D Systems (Wiesbaden-Nordenstadt, Germany), and TGFβ 1 using an ELISA as previously described [57].
T19746	38280-38385	Sentence	denotes	Quantification of IL-2 as an indicator for the bioactivity of IL-1 was done as previously described [46].
T260	38280-38385	Sentence	denotes	Quantification of IL-2 as an indicator for the bioactivity of IL-1 was done as previously described [46].
T19747	38386-38457	Sentence	denotes	Samples were diluted in the same buffer as that used for the standards.
T261	38386-38457	Sentence	denotes	Samples were diluted in the same buffer as that used for the standards.
T19748	38458-38581	Sentence	denotes	All cytokines were quantified using an ELISA plate reader (Anthos Labotec, Salzburg, Austria or Microplate Reader, BioRad).
T262	38458-38581	Sentence	denotes	All cytokines were quantified using an ELISA plate reader (Anthos Labotec, Salzburg, Austria or Microplate Reader, BioRad).
T19749	38582-38647	Sentence	denotes	Cytokine amounts were all within the range of the standard curve.
T263	38582-38647	Sentence	denotes	Cytokine amounts were all within the range of the standard curve.
T19750	38648-38763	Sentence	denotes	Only stimulation of PBMC observed in donors showing no spontaneous cytokine release was included in the statistics.
T264	38648-38763	Sentence	denotes	Only stimulation of PBMC observed in donors showing no spontaneous cytokine release was included in the statistics.
T20261	38765-38800	Sentence	denotes	Inhibition of transcription factors

T188

Sentence

denotes

β-(1→3)-D-glucan modulates DNA binding of nuclear factors κB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1β/IL-1 receptor antagonist ratio

T1

0-155

Sentence

denotes

β-(1→3)-D-glucan modulates DNA binding of nuclear factors κB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1β/IL-1 receptor antagonist ratio

T2

157-165

Sentence

denotes

Abstract

T189

166-176

Sentence

denotes

Background

T3

166-176

Sentence

denotes

Background

T190

177-287

Sentence

denotes

β-1→3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses.

T4

177-287

Sentence

denotes

β-1→3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses.

T191

288-610

Sentence

denotes

Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1).

T5

288-610

Sentence

denotes

Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1).

T192

612-619

Sentence

denotes

Results

T6

612-619

Sentence

denotes

Results

T193

620-845

Sentence

denotes

Despite an activation of nuclear factor (NF)κB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1β, IL-6, tumor necrosis factor α or interferon γ induced by glucan phosphate.

T7

620-845

Sentence

denotes

Despite an activation of nuclear factor (NF)κB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1β, IL-6, tumor necrosis factor α or interferon γ induced by glucan phosphate.

T194

846-933

Sentence

denotes

Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h:

T8

846-933

Sentence

denotes

Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h:

T195

934-1018

Sentence

denotes

5000 pg/ml), likely due to binding of NFκB to a consensus site in the IL-8 promoter.

T9

934-1018

Sentence

denotes

5000 pg/ml), likely due to binding of NFκB to a consensus site in the IL-8 promoter.

T196

1019-1087

Sentence

denotes

An increase in IL-1receptor antagonist(RA) production (peak at 24 h:

T10

1019-1087

Sentence

denotes

An increase in IL-1receptor antagonist(RA) production (peak at 24 h:

T197

1088-1174

Sentence

denotes

12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels.

T11

1088-1174

Sentence

denotes

12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels.

T198

1175-1337

Sentence

denotes

Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments.

T12

1175-1337

Sentence

denotes

Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments.

T199

1338-1574

Sentence

denotes

When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFκB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA.

T13

1338-1574

Sentence

denotes

When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFκB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA.

T200

1575-1684

Sentence

denotes

Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1β and IL-6.

T14

1575-1684

Sentence

denotes

Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1β and IL-6.

T201

1685-1805

Sentence

denotes

As a consequence, glucan phosphate shifted the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype.

T15

1685-1805

Sentence

denotes

As a consequence, glucan phosphate shifted the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype.

T202

1806-1901

Sentence

denotes

Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2.

T16

1806-1901

Sentence

denotes

Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2.

T203

1903-1913

Sentence

denotes

Conclusion

T17

1903-1913

Sentence

denotes

Conclusion

T204

1914-2127

Sentence

denotes

Thus, β-1→3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction.

T18

1914-2127

Sentence

denotes

Thus, β-1→3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction.

T205

2128-2255

Sentence

denotes

Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a β-1→3-D-glucan.

T19

2128-2255

Sentence

denotes

Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a β-1→3-D-glucan.

T20

2257-2267

Sentence

denotes

Background

T1072

2268-2446

Sentence

denotes

β-1→3-D-glucans occur as a principal component of microbial cell walls or can be secreted from both, non-pathogenic and pathogenic fungi such as S. cerevisae and C. albicans [1].

T21

2268-2446

Sentence

denotes

β-1→3-D-glucans occur as a principal component of microbial cell walls or can be secreted from both, non-pathogenic and pathogenic fungi such as S. cerevisae and C. albicans [1].

T1073

2447-2564

Sentence

denotes

These β-1→3-D-linked glucose polymers are characterized as a fungal pathogen-associated molecular pattern (PAMP) [2].

T22

2447-2564

Sentence

denotes

These β-1→3-D-linked glucose polymers are characterized as a fungal pathogen-associated molecular pattern (PAMP) [2].

T1074

2565-2707

Sentence

denotes

The primary cellular recognition of β-1→3-D-glucans is mediated by several β-1→3-D-glucan receptors on phagocytes [3,4] and other cells [5,6].

T23

2565-2707

Sentence

denotes

The primary cellular recognition of β-1→3-D-glucans is mediated by several β-1→3-D-glucan receptors on phagocytes [3,4] and other cells [5,6].

T1075

2708-2962

Sentence

denotes

Human as well as murine Dectin-1 has been demonstrated to be the major pattern recognition receptor (PRR) for intact yeast and β-1→3-D-glucan-containing particles (i.e. zymosan) on monocytes/macrophages as well as neutrophils and on primary cells [7-11].

T24

2708-2962

Sentence

denotes

Human as well as murine Dectin-1 has been demonstrated to be the major pattern recognition receptor (PRR) for intact yeast and β-1→3-D-glucan-containing particles (i.e. zymosan) on monocytes/macrophages as well as neutrophils and on primary cells [7-11].

T1076

2963-3111

Sentence

denotes

In the murine system, binding of zymosan to Dectin-1 resulted in production of TNFα through Toll-like receptor 2 and the adaptor protein MyD88 [12].

T25

2963-3111

Sentence

denotes

In the murine system, binding of zymosan to Dectin-1 resulted in production of TNFα through Toll-like receptor 2 and the adaptor protein MyD88 [12].

T1077

3112-3238

Sentence

denotes

Another water-soluble β-1→3-D-glucan (PGG-glucan) has been described to activate NFκB and NFIL-6 in murine cell lines [13,14].

T26

3112-3238

Sentence

denotes

Another water-soluble β-1→3-D-glucan (PGG-glucan) has been described to activate NFκB and NFIL-6 in murine cell lines [13,14].

T1078

3239-3484

Sentence

denotes

Similarly, it has been shown that β-1→3-D-glucans activate NFκB in a human monocyte-like cell line [15] and in human polymorphonuclear neutrophils (PMN), in the latter case without secretion of pro-inflammatory cytokines (IL-1, IL-6, TNFα) [16].

T27

3239-3484

Sentence

denotes

Similarly, it has been shown that β-1→3-D-glucans activate NFκB in a human monocyte-like cell line [15] and in human polymorphonuclear neutrophils (PMN), in the latter case without secretion of pro-inflammatory cytokines (IL-1, IL-6, TNFα) [16].

T1079

3485-3662

Sentence

denotes

One study proposed that the production of the anti-inflammatory IL-1RA, but not IL-1 by human monocytes may be a potentially protective mechanism induced by β-1→3-D-glucan [17].

T28

3485-3662

Sentence

denotes

One study proposed that the production of the anti-inflammatory IL-1RA, but not IL-1 by human monocytes may be a potentially protective mechanism induced by β-1→3-D-glucan [17].

T1080

3663-3845

Sentence

denotes

Three other investigations have reported that human leukocytes and human vascular endothelial cells produce IL-8 in response to zymosan [18] or a water-soluble β-1→3-D-glucan [6,19].

T29

3663-3845

Sentence

denotes

Three other investigations have reported that human leukocytes and human vascular endothelial cells produce IL-8 in response to zymosan [18] or a water-soluble β-1→3-D-glucan [6,19].

T1081

3846-3957

Sentence

denotes

In addition, β-1→3-D-glucans seem to be able to modify the response to pro-inflammatory stimuli or even sepsis.

T30

3846-3957

Sentence

denotes

In addition, β-1→3-D-glucans seem to be able to modify the response to pro-inflammatory stimuli or even sepsis.

T1082

3958-4185

Sentence

denotes

In a murine polymicrobial sepsis model, β-1→3-D-glucan [20] treatment resulted in decreased septic morbidity and mortality mediated via inhibition of NFκB and stimulation of the phosphoinositide-3-kinase (PI3K) pathway [21,22].

T31

3958-4185

Sentence

denotes

In a murine polymicrobial sepsis model, β-1→3-D-glucan [20] treatment resulted in decreased septic morbidity and mortality mediated via inhibition of NFκB and stimulation of the phosphoinositide-3-kinase (PI3K) pathway [21,22].

T1083

4186-4341

Sentence

denotes

These and other animal studies [23,24] as well as a clinical trial [25] support a protective role of β-1→3-D-glucan in certain pro-inflammatory conditions.

T32

4186-4341

Sentence

denotes

These and other animal studies [23,24] as well as a clinical trial [25] support a protective role of β-1→3-D-glucan in certain pro-inflammatory conditions.

T1084

4342-4461

Sentence

denotes

The mechanisms underlying these beneficial effects of β-1→3-D-glucan are only partially resolved, especially in humans.

T33

4342-4461

Sentence

denotes

The mechanisms underlying these beneficial effects of β-1→3-D-glucan are only partially resolved, especially in humans.

T1085

4462-4692

Sentence

denotes

Thus, the aim of this study was to elucidate molecular and cellular mechanisms of β-1→3-D-glucans on human leukocytes in pro-inflammatory conditions with special emphasis on the cytokine profile and its transcriptional regulation.

T34

4462-4692

Sentence

denotes

Thus, the aim of this study was to elucidate molecular and cellular mechanisms of β-1→3-D-glucans on human leukocytes in pro-inflammatory conditions with special emphasis on the cytokine profile and its transcriptional regulation.

T1086

4693-4962

Sentence

denotes

For this purpose, peripheral blood mononuclear cells (PBMC) were exposed to a well-defined β-1→3-D-glucan, i.e. glucan phosphate (GP) [20,26], alone or simultaneously with LPS from gram-negative bacteria or the superantigen TSST-1 from gram-positive bacteria over 48 h.

T35

4693-4962

Sentence

denotes

For this purpose, peripheral blood mononuclear cells (PBMC) were exposed to a well-defined β-1→3-D-glucan, i.e. glucan phosphate (GP) [20,26], alone or simultaneously with LPS from gram-negative bacteria or the superantigen TSST-1 from gram-positive bacteria over 48 h.

T1087

4963-5149

Sentence

denotes

Because of the potential effect of β-1→3-D-glucan on cytokine production [12,16-19], TNFα, IL-1β, IL-6, IL-8 and IL-1RA were measured as well as IFNγ, IL-2, IL-4, IL-10, IL-12 and TGFβ1.

T36

4963-5149

Sentence

denotes

Because of the potential effect of β-1→3-D-glucan on cytokine production [12,16-19], TNFα, IL-1β, IL-6, IL-8 and IL-1RA were measured as well as IFNγ, IL-2, IL-4, IL-10, IL-12 and TGFβ1.

T1088

5150-5404

Sentence

denotes

Correspondingly, four NFκB sites from the TNFα promoter (κ consensus, κ1, κ2, κ3) [27], a κ consensus site from the IL-8 promoter [28], an NFAT site from the IFNγ promoter (ATP2) [29] and a consensus NFIL-6 site from the IL-6 promoter [13] were examined.

T37

5150-5404

Sentence

denotes

Correspondingly, four NFκB sites from the TNFα promoter (κ consensus, κ1, κ2, κ3) [27], a κ consensus site from the IL-8 promoter [28], an NFAT site from the IFNγ promoter (ATP2) [29] and a consensus NFIL-6 site from the IL-6 promoter [13] were examined.

T1089

5405-5524

Sentence

denotes

Because of the anti-inflammatory role of IL-1RA, we focused on binding of transcription factors to the IL-1RA promoter.

T38

5405-5524

Sentence

denotes

Because of the anti-inflammatory role of IL-1RA, we focused on binding of transcription factors to the IL-1RA promoter.

T1090

5525-5726

Sentence

denotes

An inhibitory element and three positive-acting LPS-response elements (LRE-1, LRE-2 and LRE-3) in the IL-1RA promoter, including NFκB, PU.1 and NFIL-6 sites, have been characterized previously [30-33].

T39

5525-5726

Sentence

denotes

An inhibitory element and three positive-acting LPS-response elements (LRE-1, LRE-2 and LRE-3) in the IL-1RA promoter, including NFκB, PU.1 and NFIL-6 sites, have been characterized previously [30-33].

T1091

5727-5838

Sentence

denotes

Using computational analysis for homology search [34], we looked for new binding motifs in the IL-1RA promoter.

T40

5727-5838

Sentence

denotes

Using computational analysis for homology search [34], we looked for new binding motifs in the IL-1RA promoter.

T41

5840-5847

Sentence

denotes

Results

T3157

5849-6032

Sentence

denotes

Binding activities of NFκB, NFIL-6 and NFAT from human PBMC to the TNFα, IL-6 and IFNγ promoters following an in vitro stimulation (1 h) with LPS, TSST-1, GP, GP + LPS and GP + TSST-1

T42

5849-6032

Sentence

denotes

Binding activities of NFκB, NFIL-6 and NFAT from human PBMC to the TNFα, IL-6 and IFNγ promoters following an in vitro stimulation (1 h) with LPS, TSST-1, GP, GP + LPS and GP + TSST-1

T3158

6033-6296

Sentence

denotes

GP induced band shifts, indicating binding of NFκB as well as of NFIL-6 to the corresponding DNA oligonucleotides (κ consensus, κ1, κ2, κ3 sites from the TNFα promoter; κ consensus from the IL-8 promoter; NFIL-6consensus from the IL-6 promoter; Table 1; Fig. 1A).

T43

6033-6296

Sentence

denotes

GP induced band shifts, indicating binding of NFκB as well as of NFIL-6 to the corresponding DNA oligonucleotides (κ consensus, κ1, κ2, κ3 sites from the TNFα promoter; κ consensus from the IL-8 promoter; NFIL-6consensus from the IL-6 promoter; Table 1; Fig. 1A).

T3159

6297-6414

Sentence

denotes

The extent of the band shifts induced by GP was not statistically different when compared to LPS or TSST-1 (Fig. 1B).

T44

6297-6414

Sentence

denotes

The extent of the band shifts induced by GP was not statistically different when compared to LPS or TSST-1 (Fig. 1B).

T3160

6415-6588

Sentence

denotes

A supershift, conducted for an NFκB consensus site from the IL-8 promoter, demonstrated a GP-induced predominant binding of NFκB p65 and to a lesser degree of p50 (Fig. 1A).

T45

6415-6588

Sentence

denotes

A supershift, conducted for an NFκB consensus site from the IL-8 promoter, demonstrated a GP-induced predominant binding of NFκB p65 and to a lesser degree of p50 (Fig. 1A).

T3161

6589-6757

Sentence

denotes

Accordingly, an immunoblot of nuclear extracts from GP-treated PBMC showed a strong binding of NFκB p65 and a weaker reaction of p50, whereas p52 was negative (Fig. 2).

T46

6589-6757

Sentence

denotes

Accordingly, an immunoblot of nuclear extracts from GP-treated PBMC showed a strong binding of NFκB p65 and a weaker reaction of p50, whereas p52 was negative (Fig. 2).

T3162

6758-7147

Sentence

denotes

Simultaneous co-treatment of PBMC with GP did not change the LPS-induced NFκB binding to oligos from the TNFα promoter significantly (Fig. 1B), but substantially decreased the TSST-1-induced NFκB binding when compared to TSST-1 or GP (100 μg) and thus differed completely from the theoretical value of GP/TSST-1 calc. (n = 4; p < 0.05 vs. GP/TSST-1 calc. and p = 0.07 vs. TSST-1; Fig. 1B).

T47

6758-7147

Sentence

denotes

Simultaneous co-treatment of PBMC with GP did not change the LPS-induced NFκB binding to oligos from the TNFα promoter significantly (Fig. 1B), but substantially decreased the TSST-1-induced NFκB binding when compared to TSST-1 or GP (100 μg) and thus differed completely from the theoretical value of GP/TSST-1 calc. (n = 4; p < 0.05 vs. GP/TSST-1 calc. and p = 0.07 vs. TSST-1; Fig. 1B).

T3163

7148-7285

Sentence

denotes

GP was also able to induce band shifts indicative of binding of particular variants of NFAT to an oligo from the IFNγ promoter (Fig. 1A).

T48

7148-7285

Sentence

denotes

GP was also able to induce band shifts indicative of binding of particular variants of NFAT to an oligo from the IFNγ promoter (Fig. 1A).

T3164

7286-7408

Sentence

denotes

An immunoblot for two NFAT subunits demonstrated a GP-induced binding of NFATc2 and to a lesser degree of NFATc1 (Fig. 2).

T49

7286-7408

Sentence

denotes

An immunoblot for two NFAT subunits demonstrated a GP-induced binding of NFATc2 and to a lesser degree of NFATc1 (Fig. 2).

T50

7409-7465

Sentence

denotes

Table 1 Sense strand of oligonucleotides used in EMSAs.

T26163

7418-7465

Sentence

denotes

Sense strand of oligonucleotides used in EMSAs.

T51

7466-7596

Sentence

denotes

Oligonucleotidesa Base sequences (5'- XXX -3') binding site/cytokine gene promoter (position of the cytokine gene from the TSS)b

T52

7597-7683

Sentence

denotes

NFκBcS GAT CCT CAG AGG GGA CTT TCCc GAT G NFκB consensus sequence/TNFα promoter [27]

T53

7684-7750

Sentence

denotes

NFκB1S GAT CCT GGG ACA GCC CAG NFκB1 sequence/TNFα promoter [27]

T54

7751-7815

Sentence

denotes

NFκB2S GAT CCG GGG TAT CCT G NFκB2 sequence/TNFα promoter [27]

T55

7816-7881

Sentence

denotes

NFκB3S GAT CCT GGG TTT CTC CG NFκB3 sequence/TNFα promoter [27]

T56

7882-7961

Sentence

denotes

IL-8κBcS ATC GTG GAA TTT CCT CTG A NFκB consensus sequence/IL-8 promoter [28]

T57

7962-8037

Sentence

denotes

NFATP2S GAT CTA AAA TTT CCA GTC CTT GA NFATP2 sequence/IFNγ promoter [29]

T58

8038-8119

Sentence

denotes

NFIL-6S TGC AGA TTG CGC AAT CTG CA NFIL-6 consensus sequence/IL-6 promoter [13]

T59

8120-8213

Sentence

denotes

NFκBcS GCG AGG AGG GTA TTT CCG CTT between -80 and -100 NFκB consensus/IL-1RA promoter [30]

T60

8214-8306

Sentence

denotes

NFκB3S ACA ACA GCA AGG GTT TCT CTT TTT GGA AAT between -100 and -130 NFκB3/IL-1RA promoter

T61

8307-8397

Sentence

denotes

NFκBcS AGT AGG GAG TTT GGT between -266 and -280 NFκB consensus sequence/IL-1RA promoter

T62

8398-8483

Sentence

denotes

NFκB2/3S ACT CTG GGT ACC TGT between -288 and -302 NFκB2/3 sequence/IL-1RA promoter

T63

8484-8591

Sentence

denotes

NFATP2/3S GGC GCA CAA AAC CTA AAA TAT TTA CTA TCT between -471 and -500 NFATP2/3 sequence/IL-1RA promoter

T64

8592-8700

Sentence

denotes

NFIL-6S TTA CAA CAC TCC ATT GCG ACA CTT AGT GGG between -140 and -170 NFIL-6 sequence/IL-1RA promoter [31]

T65

8701-8869

Sentence

denotes

Oct-1 DNAS AAT TGC ATT GCC TGC AGG TCG ACT CTA GAG GAT CCA TGC AAA TGG ATC CCC GGG TAC CGA GCT C exclusion of unspecific bindings, unrelated DNA, (Amersham Pharmacia)

T66

8870-8873

Sentence

denotes

(a:

T67

8874-8948

Sentence

denotes

S = sense; b: TSS = transcription start site;c: ___ = main binding motif).

T68

8949-9009

Sentence

denotes

Figure 1 A, GP led to DNA binding of NFAT, NFκB and NFIL-6.

T21461

8959-9009

Sentence

denotes

A, GP led to DNA binding of NFAT, NFκB and NFIL-6.

T69

9010-9093

Sentence

denotes

Human PBMC were incubated with medium control or GP (1 and 100 μg) for 1 h at 37°C.

T21462

9010-9271

Sentence

denotes

Human PBMC were incubated with medium control or GP (1 and 100 μg) for 1 h at 37°C. Nuclear extracts were incubated with a 32P-labeled NFAT, NFκB, NFIL-6 or NFκB-IL-8 oligonucleotide probe corresponding to the IFNγ, TNFα, IL-6 and IL-8 gene promoters (Table 1).

T70

9094-9271

Sentence

denotes

Nuclear extracts were incubated with a 32P-labeled NFAT, NFκB, NFIL-6 or NFκB-IL-8 oligonucleotide probe corresponding to the IFNγ, TNFα, IL-6 and IL-8 gene promoters (Table 1).

T21463

9272-9426

Sentence

denotes

Arrows with the black head indicate migrational location of the NFAT-DNA, NFκB-DNA, NFIL-6-DNA or NFκB-IL-8-DNA complex compared to free probe (no shift).

T71

9272-9426

Sentence

denotes

Arrows with the black head indicate migrational location of the NFAT-DNA, NFκB-DNA, NFIL-6-DNA or NFκB-IL-8-DNA complex compared to free probe (no shift).

T21464

9427-9524

Sentence

denotes

Arrow with the open head indicates a supershift of NFκB-IL-8-DNA-anti-p65 and -p50, respectively.

T72

9427-9524

Sentence

denotes

Arrow with the open head indicates a supershift of NFκB-IL-8-DNA-anti-p65 and -p50, respectively.

T21465

9525-9592

Sentence

denotes

An autoradiogram from a representative experiment is shown (n = 7).

T73

9525-9592

Sentence

denotes

An autoradiogram from a representative experiment is shown (n = 7).

T21466

9593-9686

Sentence

denotes

B, Decreased DNA binding of NFκB following GP + TSST-1, compared to TSST-1, GP, or TSST-1/GP.

T74

9593-9686

Sentence

denotes

B, Decreased DNA binding of NFκB following GP + TSST-1, compared to TSST-1, GP, or TSST-1/GP.

T75

9687-9801

Sentence

denotes

Human PBMC were incubated with medium control, LPS, TSST-1, GP (100 μg), GP + TSST-1 and GP + LPS for 1 h at 37°C.

T21467

9687-9861

Sentence

denotes

Human PBMC were incubated with medium control, LPS, TSST-1, GP (100 μg), GP + TSST-1 and GP + LPS for 1 h at 37°C. An autoradiogram from a representative experiment is shown.

T76

9802-9861

Sentence

denotes

An autoradiogram from a representative experiment is shown.

T21468

9862-10074

Sentence

denotes

Schematic representation of NFκB-DNA binding activity following GP (1 and 100 μg) or TSST-1 treatment (250 ng) as well as simultaneous administration of both compounds and the theoretical value (GP/TSST-1 calc.).

T77

9862-10074

Sentence

denotes

Schematic representation of NFκB-DNA binding activity following GP (1 and 100 μg) or TSST-1 treatment (250 ng) as well as simultaneous administration of both compounds and the theoretical value (GP/TSST-1 calc.).

T21469

10075-10204

Sentence

denotes

Medium control levels were set equal to 1± SEM and significances (* = p < 0.05) are shown with respect to medium control (n = 4).

T78

10075-10204

Sentence

denotes

Medium control levels were set equal to 1± SEM and significances (* = p < 0.05) are shown with respect to medium control (n = 4).

T79

10205-10282

Sentence

denotes

Figure 2 NFκB p65/p50 and NFATc2/c1 were involved in GP-induced DNA binding.

T22368

10215-10282

Sentence

denotes

NFκB p65/p50 and NFATc2/c1 were involved in GP-induced DNA binding.

T22369

10283-10419

Sentence

denotes

In order to determine whether the DNA binding proteins activated by GP were related to NFκB and NFAT, an immuno(dot) blot was performed.

T80

10283-10419

Sentence

denotes

In order to determine whether the DNA binding proteins activated by GP were related to NFκB and NFAT, an immuno(dot) blot was performed.

T22370

10420-10780

Sentence

denotes

A representative dot blot showing staining of negative and positive controls (recombinant proteins NFκB p52, c-rel; nuclear extracts of PMA-treated Jurkat cells positive for NFκB p50, p65 and for NFATc1, c2) in the upper panel and staining of nuclear extracts of GP-treated PBMC (positive for NFκB p65, p50 and NFAT c2, c1) in the lower panel is shown (n = 3).

T81

10420-10780

Sentence

denotes

A representative dot blot showing staining of negative and positive controls (recombinant proteins NFκB p52, c-rel; nuclear extracts of PMA-treated Jurkat cells positive for NFκB p50, p65 and for NFATc1, c2) in the upper panel and staining of nuclear extracts of GP-treated PBMC (positive for NFκB p65, p50 and NFAT c2, c1) in the lower panel is shown (n = 3).

T4719

10782-10897

Sentence

denotes

Cytokine profile of human PBMC following a 48 h in vitro stimulation with LPS, TSST-1, GP, GP + LPS and GP + TSST-1

T82

10782-10897

Sentence

denotes

Cytokine profile of human PBMC following a 48 h in vitro stimulation with LPS, TSST-1, GP, GP + LPS and GP + TSST-1

T4720

10898-11137

Sentence

denotes

Because of the GP-induced band shifts to seven sites from the TNFα, IL-8, IL-6 and IFNγ promoters, it could be assumed that GP treatment of human PBMC would lead to production of TNFα, IL-8, IL-6, IFNγ and other pro-inflammatory mediators.

T83

10898-11137

Sentence

denotes

Because of the GP-induced band shifts to seven sites from the TNFα, IL-8, IL-6 and IFNγ promoters, it could be assumed that GP treatment of human PBMC would lead to production of TNFα, IL-8, IL-6, IFNγ and other pro-inflammatory mediators.

T4721

11138-11256

Sentence

denotes

Previous studies reported that β-1→3-D-glucans induced only a limited cytokine secretion of human blood cells [15-18].

T84

11138-11256

Sentence

denotes

Previous studies reported that β-1→3-D-glucans induced only a limited cytokine secretion of human blood cells [15-18].

T4722

11257-11679

Sentence

denotes

Corroborating and extending these singular findings in terms of examining pro- (eight) as well as anti- (three) inflammatory cytokines over time (48 h) and six additional transcription factor binding sites, our comprehensive analysis revealed the following cytokine profile of human leukocytes in response to a highly purified water soluble β-1→3-D-glucan and in comparison to two pro-inflammatory mediators (LPS, TSST-1):

T85

11257-11679

Sentence

denotes

Corroborating and extending these singular findings in terms of examining pro- (eight) as well as anti- (three) inflammatory cytokines over time (48 h) and six additional transcription factor binding sites, our comprehensive analysis revealed the following cytokine profile of human leukocytes in response to a highly purified water soluble β-1→3-D-glucan and in comparison to two pro-inflammatory mediators (LPS, TSST-1):

T4723

11681-11690

Sentence

denotes

(i) IL-1β

T86

11681-11690

Sentence

denotes

(i) IL-1β

T4724

11691-11778

Sentence

denotes

There was no IL-1β production detectable following PBMC treatment with 1 nor 100 μg GP.

T87

11691-11778

Sentence

denotes

There was no IL-1β production detectable following PBMC treatment with 1 nor 100 μg GP.

T4725

11779-11879

Sentence

denotes

An insignificant up-regulation of IL-1β production for GP + LPS was observed in comparison with LPS.

T88

11779-11879

Sentence

denotes

An insignificant up-regulation of IL-1β production for GP + LPS was observed in comparison with LPS.

T4726

11880-12075

Sentence

denotes

For GP + TSST-1 we found a significant reduction in IL-1β from 18 h – 24 h, for the latter by about 40% when compared to TSST-1 or the theoretical value of GP/TSST-1 calc. (n = 6; both p = 0.01).

T89

11880-12075

Sentence

denotes

For GP + TSST-1 we found a significant reduction in IL-1β from 18 h – 24 h, for the latter by about 40% when compared to TSST-1 or the theoretical value of GP/TSST-1 calc. (n = 6; both p = 0.01).

T4727

12076-12175

Sentence

denotes

On the whole, GP mediated a reduction of the TSST-1-induced amount of IL-1β by about 50% (Fig. 3A).

T90

12076-12175

Sentence

denotes

On the whole, GP mediated a reduction of the TSST-1-induced amount of IL-1β by about 50% (Fig. 3A).

T91

12176-12274

Sentence

denotes

Figure 3 A, IL-1β was down-regulated (12 h – 24 h) following GP + TSST-1 when compared to TSST-1.

T22957

12186-12274

Sentence

denotes

A, IL-1β was down-regulated (12 h – 24 h) following GP + TSST-1 when compared to TSST-1.

T22958

12275-12430

Sentence

denotes

Time course (48 h) of IL-1β production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS.

T92

12275-12430

Sentence

denotes

Time course (48 h) of IL-1β production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS.

T22959

12431-12558

Sentence

denotes

At 18 h and 24 h there was a marked reduction in IL-1β production following GP + TSST-1 when compared to TSST-1 (* = p < 0.05).

T93

12431-12558

Sentence

denotes

At 18 h and 24 h there was a marked reduction in IL-1β production following GP + TSST-1 when compared to TSST-1 (* = p < 0.05).

T22960

12559-12651

Sentence

denotes

Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are also shown.

T94

12559-12651

Sentence

denotes

Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are also shown.

T22961

12652-12744

Sentence

denotes

IL-1β levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3).

T95

12652-12744

Sentence

denotes

IL-1β levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3).

T22962

12745-12831

Sentence

denotes

B, IL-1RA was exaggerated following GP + TSST-1 (12 h – 48 h) when compared to TSST-1.

T96

12745-12831

Sentence

denotes

B, IL-1RA was exaggerated following GP + TSST-1 (12 h – 48 h) when compared to TSST-1.

T22963

12832-12988

Sentence

denotes

Time course (48 h) of IL-1RA production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS.

T97

12832-12988

Sentence

denotes

Time course (48 h) of IL-1RA production (pg/ml) by human PBMC incubated with medium control, 100 μg GP, 250 ng TSST-1, GP + TSST-1, 250 ng LPS and GP + LPS.

T22964

12989-13205

Sentence

denotes

Between 18 h and 48 h a simultaneous treatment of PBMC with GP + TSST-1 led to a synergistic effect, i.e. a higher IL-1RA production when compared to an addition of the single values for TSST-1 and GP (* = p < 0.05).

T98

12989-13205

Sentence

denotes

Between 18 h and 48 h a simultaneous treatment of PBMC with GP + TSST-1 led to a synergistic effect, i.e. a higher IL-1RA production when compared to an addition of the single values for TSST-1 and GP (* = p < 0.05).

T22965

13206-13296

Sentence

denotes

Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are included.

T99

13206-13296

Sentence

denotes

Graphs depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are included.

T22966

13297-13390

Sentence

denotes

IL-1RA levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3).

T100

13297-13390

Sentence

denotes

IL-1RA levels in the supernatnant were obtained by Elisa and are shown as mean ± SEM (n = 3).

T4728

13392-13401

Sentence

denotes

(ii) IL-6

T101

13392-13401

Sentence

denotes

(ii) IL-6

T4729

13402-13522

Sentence

denotes

GP induced only a small amount of IL-6 and there was no significant alteration of the LPS-induced IL-6 production by GP.

T102

13402-13522

Sentence

denotes

GP induced only a small amount of IL-6 and there was no significant alteration of the LPS-induced IL-6 production by GP.

T4730

13523-13723

Sentence

denotes

On the contrary, we observed a decreased IL-6 production for GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc., especially at 24 h by about 40% (n = 5; both p = 0.02), (data not shown in detail).

T103

13523-13723

Sentence

denotes

On the contrary, we observed a decreased IL-6 production for GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc., especially at 24 h by about 40% (n = 5; both p = 0.02), (data not shown in detail).

T4731

13725-13735

Sentence

denotes

(iii) IL-8

T104

13725-13735

Sentence

denotes

(iii) IL-8

T4732

13736-13865

Sentence

denotes

We found that GP induced a substantial IL-8 production, in comparison with medium control, especially at 24 h (n = 13; p < 0.01).

T105

13736-13865

Sentence

denotes

We found that GP induced a substantial IL-8 production, in comparison with medium control, especially at 24 h (n = 13; p < 0.01).

T4733

13866-13988

Sentence

denotes

A combination of GP and LPS resulted in a non-significant increase of IL-8 production when compared to LPS or GP/LPS calc.

T106

13866-13988

Sentence

denotes

A combination of GP and LPS resulted in a non-significant increase of IL-8 production when compared to LPS or GP/LPS calc.

T4734

13989-14136

Sentence

denotes

There was no significant alteration in IL-8 production following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail).

T107

13989-14136

Sentence

denotes

There was no significant alteration in IL-8 production following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail).

T4735

14138-14149

Sentence

denotes

(iv) IL-1RA

T108

14138-14149

Sentence

denotes

(iv) IL-1RA

T4736

14150-14300

Sentence

denotes

Besides IL-8, IL-1RA was the only mediator which was produced in significant quantities following GP treatment, especially at 24 h (n = 14, p < 0.01).

T109

14150-14300

Sentence

denotes

Besides IL-8, IL-1RA was the only mediator which was produced in significant quantities following GP treatment, especially at 24 h (n = 14, p < 0.01).

T4737

14301-14418

Sentence

denotes

When compared to LPS stimulation or GP/LPS calc., GP + LPS did not alter the kinetic course of the IL-1RA production.

T110

14301-14418

Sentence

denotes

When compared to LPS stimulation or GP/LPS calc., GP + LPS did not alter the kinetic course of the IL-1RA production.

T4738

14419-14653

Sentence

denotes

However, following GP + TSST-1 we found a synergistic increase in IL-1RA production from 18 h to 48 h, when compared to TSST-1 (for instance at 24 h: n = 6; p = 0.01) or to the theoretical value of GP/TSST-1 calc. (from 18 h to 36 h).

T111

14419-14653

Sentence

denotes

However, following GP + TSST-1 we found a synergistic increase in IL-1RA production from 18 h to 48 h, when compared to TSST-1 (for instance at 24 h: n = 6; p = 0.01) or to the theoretical value of GP/TSST-1 calc. (from 18 h to 36 h).

T4739

14654-14764

Sentence

denotes

Over the time course of 48 h, GP elevated the TSST-1-induced amount of IL-1RA by approximately 200% (Fig. 3B).

T112

14654-14764

Sentence

denotes

Over the time course of 48 h, GP elevated the TSST-1-induced amount of IL-1RA by approximately 200% (Fig. 3B).

T4740

14765-14833

Sentence

denotes

Positive correlation between GP-induced IL-8 and IL-1RA productions.

T113

14765-14833

Sentence

denotes

Positive correlation between GP-induced IL-8 and IL-1RA productions.

T4741

14834-14931

Sentence

denotes

Following stimulation with GP (24 h), we observed a positive correlation between IL-8 and IL-1RA.

T114

14834-14931

Sentence

denotes

Following stimulation with GP (24 h), we observed a positive correlation between IL-8 and IL-1RA.

T4742

14932-15115

Sentence

denotes

Moreover, we found this correlation to be dose-dependent, since 100 μg of GP induced larger amounts of IL-8 and IL-1RA than 1 μg GP (n = 5, p ≤ 0.002, r = 0.9 and 0.98, respectively).

T115

14932-15115

Sentence

denotes

Moreover, we found this correlation to be dose-dependent, since 100 μg of GP induced larger amounts of IL-8 and IL-1RA than 1 μg GP (n = 5, p ≤ 0.002, r = 0.9 and 0.98, respectively).

T4743

15117-15125

Sentence

denotes

(v) TNFα

T116

15117-15125

Sentence

denotes

(v) TNFα

T4744

15126-15262

Sentence

denotes

With respect to TNFα production, 100 μg of GP yielded minor, statistically not distinguishable amounts, when compared to medium control.

T117

15126-15262

Sentence

denotes

With respect to TNFα production, 100 μg of GP yielded minor, statistically not distinguishable amounts, when compared to medium control.

T4745

15263-15359

Sentence

denotes

GP + LPS did not change the TNFα production when compared to LPS supplementation or GP/LPS calc.

T118

15263-15359

Sentence

denotes

GP + LPS did not change the TNFα production when compared to LPS supplementation or GP/LPS calc.

T4746

15360-15583

Sentence

denotes

When combined with TSST-1 or GP/TSST-1 calc., GP seemed to exert a synergistic effect on TNFα secretion after 36 h (n = 3; p < 0.05), but overall an enhancement of only 10% was observed (p > 0.05; data not shown in detail).

T119

15360-15583

Sentence

denotes

When combined with TSST-1 or GP/TSST-1 calc., GP seemed to exert a synergistic effect on TNFα secretion after 36 h (n = 3; p < 0.05), but overall an enhancement of only 10% was observed (p > 0.05; data not shown in detail).

T4747

15585-15594

Sentence

denotes

(vi) IFNγ

T120

15585-15594

Sentence

denotes

(vi) IFNγ

T4748

15595-15672

Sentence

denotes

No IFNγ production was detectable following treatment of PBMC with 100 μg GP.

T121

15595-15672

Sentence

denotes

No IFNγ production was detectable following treatment of PBMC with 100 μg GP.

T4749

15673-15914

Sentence

denotes

There was a minor increase in IFNγ production following GP + LPS at 36 h, when compared to LPS or GP/LPS calc., and a slight down-regulation following GP + TSST-1 at 24 h and 36 h, when compared to TSST-1 or GP/TSST-1 calc. (data not shown).

T122

15673-15914

Sentence

denotes

There was a minor increase in IFNγ production following GP + LPS at 36 h, when compared to LPS or GP/LPS calc., and a slight down-regulation following GP + TSST-1 at 24 h and 36 h, when compared to TSST-1 or GP/TSST-1 calc. (data not shown).

T4750

15916-15953

Sentence

denotes

(vii) IL-2, IL-12, IL-4, IL-10, TGFβ1

T123

15916-15953

Sentence

denotes

(vii) IL-2, IL-12, IL-4, IL-10, TGFβ1

T4751

15954-16030

Sentence

denotes

The production of IL-2, IL-12, IL-4, IL-10 and TGFβ 1 was not induced by GP.

T124

15954-16030

Sentence

denotes

The production of IL-2, IL-12, IL-4, IL-10 and TGFβ 1 was not induced by GP.

T4752

16031-16200

Sentence

denotes

There were also no significant differences between medium control, LPS, TSST-1, GP, GP + LPS, GP/LPS calc. and GP + TSST-1 or GP/TSST-1 calc. over 48 h (data not shown).

T125

16031-16200

Sentence

denotes

There were also no significant differences between medium control, LPS, TSST-1, GP, GP + LPS, GP/LPS calc. and GP + TSST-1 or GP/TSST-1 calc. over 48 h (data not shown).

T8008

16202-16328

Sentence

denotes

Role of NFκB, NFIL-6 and NFAT binding in LPS-, TSST-1-, GP-, GP + LPS- and GP + TSST-1-induced IL-1RA expression by human PBMC

T126

16202-16328

Sentence

denotes

Role of NFκB, NFIL-6 and NFAT binding in LPS-, TSST-1-, GP-, GP + LPS- and GP + TSST-1-induced IL-1RA expression by human PBMC

T8009

16329-16519

Sentence

denotes

Because GP induced band shifts to NFκB, NFIL-6 and NFAT sites, but no TNFα, IL-6 or IFNγ, we hypothesized that the activated transcription factors might bind to sites in the IL-1RA promoter.

T127

16329-16519

Sentence

denotes

Because GP induced band shifts to NFκB, NFIL-6 and NFAT sites, but no TNFα, IL-6 or IFNγ, we hypothesized that the activated transcription factors might bind to sites in the IL-1RA promoter.

T8010

16520-16710

Sentence

denotes

We investigated previoulsy described and newly discovered transcription factor binding sites of the IL-1RA promoter (Table 1) using the program OMIGA (v.1.1.3, Oxford Molecular, Oxford, UK).

T128

16520-16710

Sentence

denotes

We investigated previoulsy described and newly discovered transcription factor binding sites of the IL-1RA promoter (Table 1) using the program OMIGA (v.1.1.3, Oxford Molecular, Oxford, UK).

T8011

16711-16910

Sentence

denotes

The LPS responsive κB consensus site of LRE-1 (between -84 and -93) [30] displayed band shifts, but no significant alterations of its binding activity, irrespective of the treatment (data not shown).

T129

16711-16910

Sentence

denotes

The LPS responsive κB consensus site of LRE-1 (between -84 and -93) [30] displayed band shifts, but no significant alterations of its binding activity, irrespective of the treatment (data not shown).

T8012

16911-17327

Sentence

denotes

A new κB3 site (-100 and -130) within the LRE-1 [30] showed increased binding induced by TSST-1 (+30% vs. control), but GP co-treatment up-regulated the LPS (+65% vs. control)- and the TSST-1 (+110% vs. control)-induced binding in a superadditive synergistic fashion, when compared to medium control as well as to LPS, TSST-1 or GP or the theoretical values of GP/LPS calc. or GP/TSST-1 calc. (all p < 0.05; Fig. 4).

T130

16911-17327

Sentence

denotes

A new κB3 site (-100 and -130) within the LRE-1 [30] showed increased binding induced by TSST-1 (+30% vs. control), but GP co-treatment up-regulated the LPS (+65% vs. control)- and the TSST-1 (+110% vs. control)-induced binding in a superadditive synergistic fashion, when compared to medium control as well as to LPS, TSST-1 or GP or the theoretical values of GP/LPS calc. or GP/TSST-1 calc. (all p < 0.05; Fig. 4).

T8013

17328-17457

Sentence

denotes

Binding to a new κB consensus site (between -266 and -280) was significantly elevated for GP + TSST-1 (p < 0.05; data not shown).

T131

17328-17457

Sentence

denotes

Binding to a new κB consensus site (between -266 and -280) was significantly elevated for GP + TSST-1 (p < 0.05; data not shown).

T8014

17458-17639

Sentence

denotes

The LPS-, TSST-1- and GP + LPS-induced binding to the new κB2/3 site (-288 and -302) was down-regulated by about 20–35% in comparison with medium control (p < 0.05; data not shown).

T132

17458-17639

Sentence

denotes

The LPS-, TSST-1- and GP + LPS-induced binding to the new κB2/3 site (-288 and -302) was down-regulated by about 20–35% in comparison with medium control (p < 0.05; data not shown).

T8015

17640-17817

Sentence

denotes

The NFIL-6 site (-147 and -170) close to LRE-2 [31,32] showed an up-regulation of binding for all compounds and combinations vs. medium control, ranging from 25–110% (p < 0.05).

T133

17640-17817

Sentence

denotes

The NFIL-6 site (-147 and -170) close to LRE-2 [31,32] showed an up-regulation of binding for all compounds and combinations vs. medium control, ranging from 25–110% (p < 0.05).

T8016

17818-17967

Sentence

denotes

Further, GP + TSST-1 induced an increased binding to the NFIL-6 site in comparison with TSST-1 (1.3fold) or GP (1.6fold; p < 0.05), (data not shown).

T134

17818-17967

Sentence

denotes

Further, GP + TSST-1 induced an increased binding to the NFIL-6 site in comparison with TSST-1 (1.3fold) or GP (1.6fold; p < 0.05), (data not shown).

T8017

17968-18208

Sentence

denotes

A new binding site for NFATP2/3 was found more distal of the transcription start site, between -471 and -490, displaying again a substantial increase of binding for all compounds and combinations, when compared to medium control (p < 0.05).

T135

17968-18208

Sentence

denotes

A new binding site for NFATP2/3 was found more distal of the transcription start site, between -471 and -490, displaying again a substantial increase of binding for all compounds and combinations, when compared to medium control (p < 0.05).

T8018

18209-18506

Sentence

denotes

In addition, binding to the NFATP2/3 site following both, GP + LPS (1.4fold of GP) and GP + TSST-1 (1.5fold of GP, 1.25fold of TSST-1) was elevated (p < 0.05; Fig. 5), but only in an additive synergistic manner, i.e. the experimental results were not different from the theoretical values (calc.).

T136

18209-18506

Sentence

denotes

In addition, binding to the NFATP2/3 site following both, GP + LPS (1.4fold of GP) and GP + TSST-1 (1.5fold of GP, 1.25fold of TSST-1) was elevated (p < 0.05; Fig. 5), but only in an additive synergistic manner, i.e. the experimental results were not different from the theoretical values (calc.).

T137

18507-18605

Sentence

denotes

Figure 4 GP modulated LPS- and TSST-1-induced binding to a new NFκB3 site in the IL-1RA promoter.

T23876

18517-18605

Sentence

denotes

GP modulated LPS- and TSST-1-induced binding to a new NFκB3 site in the IL-1RA promoter.

T23877

18606-18767

Sentence

denotes

Binding was enhanced for GP + LPS and GP + TSST-1, when compared to LPS, TSST-1, GP, GP/LPS calc. or GP/TSST-1 calc., respectively (* = p < 0.05; ** = p < 0.01).

T138

18606-18767

Sentence

denotes

Binding was enhanced for GP + LPS and GP + TSST-1, when compared to LPS, TSST-1, GP, GP/LPS calc. or GP/TSST-1 calc., respectively (* = p < 0.05; ** = p < 0.01).

T23878

18768-18824

Sentence

denotes

Medium control levels were set equal to 1 ± SEM (n = 6).

T139

18768-18824

Sentence

denotes

Medium control levels were set equal to 1 ± SEM (n = 6).

T23879

18825-18872

Sentence

denotes

DNA binding was assessed by EMSA (see methods).

T140

18825-18872

Sentence

denotes

DNA binding was assessed by EMSA (see methods).

T141

18873-18969

Sentence

denotes

Figure 5 GP modulated TSST-1-induced DNA binding to a new NFATP2/3 site in the IL-1RA promoter.

T24196

18883-18969

Sentence

denotes

GP modulated TSST-1-induced DNA binding to a new NFATP2/3 site in the IL-1RA promoter.

T24197

18970-19107

Sentence

denotes

Binding was up-regulated for GP + LPS and GP + TSST-1, when compared to GP and, in the latter case, to both GP and TSST-1 (* = p < 0.05).

T142

18970-19107

Sentence

denotes

Binding was up-regulated for GP + LPS and GP + TSST-1, when compared to GP and, in the latter case, to both GP and TSST-1 (* = p < 0.05).

T143

19108-19213

Sentence

denotes

Human PBMC were incubated with medium control, LPS, TSST-1, GP, GP + TSST-1 and GP + LPS for 1 h at 37°C.

T24198

19108-19316

Sentence

denotes

Human PBMC were incubated with medium control, LPS, TSST-1, GP, GP + TSST-1 and GP + LPS for 1 h at 37°C. Schematic representation of the elevated NFAT-DNA binding activity to the site in the IL-1RA promoter.

T144

19214-19316

Sentence

denotes

Schematic representation of the elevated NFAT-DNA binding activity to the site in the IL-1RA promoter.

T24199

19317-19402

Sentence

denotes

Bars depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are added.

T145

19317-19402

Sentence

denotes

Bars depicting the theoretical values for GP/LPS calc. and GP/TSST-1 calc. are added.

T24200

19403-19459

Sentence

denotes

Medium control levels were set equal to 1 ± SEM (n = 6).

T146

19403-19459

Sentence

denotes

Medium control levels were set equal to 1 ± SEM (n = 6).

T24201

19460-19656

Sentence

denotes

An autoradiogram of a representative experiment is shown in the lower panel (lane 1: water; lane 2: medium control; lane 3: GP; lane 4: LPS; lane 5: TSST-1, lane 6: GP + LPS; lane 7: GP + TSST-1).

T147

19460-19656

Sentence

denotes

An autoradiogram of a representative experiment is shown in the lower panel (lane 1: water; lane 2: medium control; lane 3: GP; lane 4: LPS; lane 5: TSST-1, lane 6: GP + LPS; lane 7: GP + TSST-1).

T9554

19658-19747

Sentence

denotes

GP modulated the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype

T148

19658-19747

Sentence

denotes

GP modulated the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype

T9555

19748-19866

Sentence

denotes

In a simplified approach, IL-1β and IL-1RA might represent two sides of a coin, i.e. pro- or anti-inflammatory action.

T149

19748-19866

Sentence

denotes

In a simplified approach, IL-1β and IL-1RA might represent two sides of a coin, i.e. pro- or anti-inflammatory action.

T9556

19867-19958

Sentence

denotes

Therefore, we applied the IL-1β/IL-1RA ratio as an indicator of the degree of inflammation.

T150

19867-19958

Sentence

denotes

Therefore, we applied the IL-1β/IL-1RA ratio as an indicator of the degree of inflammation.

T9557

19959-20077

Sentence

denotes

Both, LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA at early time points, i.e. up to 24 h.

T151

19959-20077

Sentence

denotes

Both, LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA at early time points, i.e. up to 24 h.

T9558

20078-20255

Sentence

denotes

Comparing LPS vs. GP + LPS, we did not find substantial alterations over a time course of 48 h in the IL-1β/IL-1RA ratio, only a – non-significant – 2.5fold higher ratio at 6 h.

T152

20078-20255

Sentence

denotes

Comparing LPS vs. GP + LPS, we did not find substantial alterations over a time course of 48 h in the IL-1β/IL-1RA ratio, only a – non-significant – 2.5fold higher ratio at 6 h.

T9559

20256-20563

Sentence

denotes

With respect to TSST-1 or the theoretical value of GP/TSST-1 calc. vs. GP + TSST-1, we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h of incubation, i.e. a higher production of IL-1RA than of IL-1β (n = 3; both p < 0.05; Fig. 6).

T153

20256-20563

Sentence

denotes

With respect to TSST-1 or the theoretical value of GP/TSST-1 calc. vs. GP + TSST-1, we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h of incubation, i.e. a higher production of IL-1RA than of IL-1β (n = 3; both p < 0.05; Fig. 6).

T9560

20564-20800

Sentence

denotes

A biological relevance of this result was suggested by a decrease of the IL-1 dependent release of IL-2 from murine EL-4 cells following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail, n = 4; p < 0.07).

T154

20564-20800

Sentence

denotes

A biological relevance of this result was suggested by a decrease of the IL-1 dependent release of IL-2 from murine EL-4 cells following GP + TSST-1 when compared to TSST-1 or GP/TSST-1 calc. (data not shown in detail, n = 4; p < 0.07).

T155

20801-20886

Sentence

denotes

Figure 6 Decreased IL-1β/IL-1RA ratio following GP + TSST-1 when compared to TSST-1.

T24819

20811-20886

Sentence

denotes

Decreased IL-1β/IL-1RA ratio following GP + TSST-1 when compared to TSST-1.

T24820

20887-21039

Sentence

denotes

Time course (48 h) of the IL-1β/IL-1RA ratio following incubation of human PBMC with LPS, TSST-1, GP + LPS, GP/LPS calc., GP + TSST-1 or GP/TSST-1 calc.

T156

20887-21039

Sentence

denotes

Time course (48 h) of the IL-1β/IL-1RA ratio following incubation of human PBMC with LPS, TSST-1, GP + LPS, GP/LPS calc., GP + TSST-1 or GP/TSST-1 calc.

T24821

21040-21125

Sentence

denotes

LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA up to 24 h.

T157

21040-21125

Sentence

denotes

LPS and GP + LPS treatments resulted in generating more IL-1β than IL-1RA up to 24 h.

T24822

21126-21249

Sentence

denotes

When comparing LPS or GP/LPS calc. vs GP + LPS we did not find significant alterations in the IL-1β/IL-1RA ratio over 48 h.

T158

21126-21249

Sentence

denotes

When comparing LPS or GP/LPS calc. vs GP + LPS we did not find significant alterations in the IL-1β/IL-1RA ratio over 48 h.

T24823

21250-21500

Sentence

denotes

With respect to TSST-1 or GP/TSST-1 calc. vs GP + TSST-1 we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h, i.e. a higher production of IL-1RA than of IL-1β (* = p < 0.05).

T159

21250-21500

Sentence

denotes

With respect to TSST-1 or GP/TSST-1 calc. vs GP + TSST-1 we observed a pronounced anti-inflammatory action of GP, as demonstrated by an about 10–100 fold reduced ratio at 18 h and 24 h, i.e. a higher production of IL-1RA than of IL-1β (* = p < 0.05).

T24824

21501-21538

Sentence

denotes

Data are shown as mean ± SEM (n = 3).

T160

21501-21538

Sentence

denotes

Data are shown as mean ± SEM (n = 3).

T10436

21540-21604

Sentence

denotes

Inhibition of transcription factors led to reduced IL-1RA levels

T161

21540-21604

Sentence

denotes

Inhibition of transcription factors led to reduced IL-1RA levels

T10437

21605-21767

Sentence

denotes

Pharmacological inhibitors were used to demonstrate that the transcription factor sites within the IL-1RA promoter are relevant for the induction of IL-1RA by GP.

T162

21605-21767

Sentence

denotes

Pharmacological inhibitors were used to demonstrate that the transcription factor sites within the IL-1RA promoter are relevant for the induction of IL-1RA by GP.

T10438

21768-22054

Sentence

denotes

As expected, an inhibition of NFκB via CAPE [35] and CyA, NFAT via CyA and NFIL-6 via CHX [36] could not be overruled by GP, leading to a down-regulated binding activity of the transcription factors to the IL-1RA sites, and a reduction of IL-1RA mRNA and IL-1RA protein levels (Fig. 7).

T163

21768-22054

Sentence

denotes

As expected, an inhibition of NFκB via CAPE [35] and CyA, NFAT via CyA and NFIL-6 via CHX [36] could not be overruled by GP, leading to a down-regulated binding activity of the transcription factors to the IL-1RA sites, and a reduction of IL-1RA mRNA and IL-1RA protein levels (Fig. 7).

T10439

22055-22217

Sentence

denotes

The extent of reduction in NFATP2/3 binding is exemplarily shown in an autoradiogram (Fig. 7A, left side) and graphically summarized (n = 4, Fig. 7A, right side).

T164

22055-22217

Sentence

denotes

The extent of reduction in NFATP2/3 binding is exemplarily shown in an autoradiogram (Fig. 7A, left side) and graphically summarized (n = 4, Fig. 7A, right side).

T10440

22218-22298

Sentence

denotes

The results for the NFκB and NFIL-6 binding sites were similar (data not shown).

T165

22218-22298

Sentence

denotes

The results for the NFκB and NFIL-6 binding sites were similar (data not shown).

T10441

22299-22410

Sentence

denotes

Because the highest IL-1RA amount was found at 24 h, mRNA was examined following 1 h inhibition and 18 h of GP.

T166

22299-22410

Sentence

denotes

Because the highest IL-1RA amount was found at 24 h, mRNA was examined following 1 h inhibition and 18 h of GP.

T10442

22411-22544

Sentence

denotes

A representative gel demonstrating a decrease in IL-1RA mRNA after inhibition with CAPE, CyA and CHX is displayed in Fig. 7B (n = 4).

T167

22411-22544

Sentence

denotes

A representative gel demonstrating a decrease in IL-1RA mRNA after inhibition with CAPE, CyA and CHX is displayed in Fig. 7B (n = 4).

T10443

22545-22640

Sentence

denotes

In addition, corresponding IL-1RA protein levels after 24 h of GP are shown in Fig. 7C (n = 3).

T168

22545-22640

Sentence

denotes

In addition, corresponding IL-1RA protein levels after 24 h of GP are shown in Fig. 7C (n = 3).

T10444

22641-22805

Sentence

denotes

Altogether, NFκB inhibition by CAPE was more pronounced at the binding activity and the transcription, whereas CyA and CHX mainly led to a decreased IL-1RA release.

T169

22641-22805

Sentence

denotes

Altogether, NFκB inhibition by CAPE was more pronounced at the binding activity and the transcription, whereas CyA and CHX mainly led to a decreased IL-1RA release.

T170

22806-22898

Sentence

denotes

Figure 7 Inhibition of GP-induced NFκB, NFAT and NFIL-6 DNA binding to the IL-1RA promoter.

T25500

22816-22898

Sentence

denotes

Inhibition of GP-induced NFκB, NFAT and NFIL-6 DNA binding to the IL-1RA promoter.

T25501

22899-23274

Sentence

denotes

A, A representative autoradiogram (lane 1: control, lane 2: GP, lane 3: GP + unlabeled mutated NFATP2/3 oligo, lane 4: GP + CAPE, lane 5: GP + CyA, lane 6: GP + CHX, lane 7: GP + unlabeled NFATP2/3 oligo, lane 8: water) as well as a graphical summary for the NFATP2/3 site, depicting a decrease in binding following inhibition when compared to GP is displayed (* = p < 0.05).

T171

22899-23274

Sentence

denotes

A, A representative autoradiogram (lane 1: control, lane 2: GP, lane 3: GP + unlabeled mutated NFATP2/3 oligo, lane 4: GP + CAPE, lane 5: GP + CyA, lane 6: GP + CHX, lane 7: GP + unlabeled NFATP2/3 oligo, lane 8: water) as well as a graphical summary for the NFATP2/3 site, depicting a decrease in binding following inhibition when compared to GP is displayed (* = p < 0.05).

T25502

23275-23312

Sentence

denotes

Data are shown as mean ± SEM (n = 4).

T172

23275-23312

Sentence

denotes

Data are shown as mean ± SEM (n = 4).

T25503

23313-23428

Sentence

denotes

B, Inhibition of NFκB, NFAT and NFIL-6 resulted in decreased IL-1RA mRNA following CHX, CAPE or CyA compared to GP.

T173

23313-23428

Sentence

denotes

B, Inhibition of NFκB, NFAT and NFIL-6 resulted in decreased IL-1RA mRNA following CHX, CAPE or CyA compared to GP.

T25504

23429-23495

Sentence

denotes

GAPDH is used as housekeeping control (representative gel, n = 4).

T174

23429-23495

Sentence

denotes

GAPDH is used as housekeeping control (representative gel, n = 4).

T25505

23496-23688

Sentence

denotes

C, Inhibition of NFκB, NFAT and NFIL-6 significantly reduced the production of IL-1RA by human PBMC following treatment with CHX, CAPE or CyA when compared to GP (* = p < 0.05; ** = p < 0.01).

T175

23496-23688

Sentence

denotes

C, Inhibition of NFκB, NFAT and NFIL-6 significantly reduced the production of IL-1RA by human PBMC following treatment with CHX, CAPE or CyA when compared to GP (* = p < 0.05; ** = p < 0.01).

T25506

23689-23726

Sentence

denotes

Data are shown as mean ± SEM (n = 3).

T176

23689-23726

Sentence

denotes

Data are shown as mean ± SEM (n = 3).

T177

23728-23738

Sentence

denotes

Discussion

T11851

23739-23923

Sentence

denotes

Animal models of sepsis and myocardial injury suggest that a β-1→3-D-glucan like GP mediates its protective mechanisms, in part, via a rapid shift from NFκB- to PI3K-signaling [22,37].

T178

23739-23923

Sentence

denotes

Animal models of sepsis and myocardial injury suggest that a β-1→3-D-glucan like GP mediates its protective mechanisms, in part, via a rapid shift from NFκB- to PI3K-signaling [22,37].

T11852

23924-24145

Sentence

denotes

Our study provides novel evidence that downstream of recognition and signaling pro-inflammatory transcription factor binding and cytokine expression of human leukocytes is switched to an anti-inflammatory phenotype by GP.

T179

23924-24145

Sentence

denotes

Our study provides novel evidence that downstream of recognition and signaling pro-inflammatory transcription factor binding and cytokine expression of human leukocytes is switched to an anti-inflammatory phenotype by GP.

T11853

24146-24333

Sentence

denotes

We confirmed and extended previous results [13-16] indicating that, in the absence of other stimuli, β-1→3-D-glucans induced binding of NFκB-, NFIL-6- and NFAT-mers to cytokine promoters.

T180

24146-24333

Sentence

denotes

We confirmed and extended previous results [13-16] indicating that, in the absence of other stimuli, β-1→3-D-glucans induced binding of NFκB-, NFIL-6- and NFAT-mers to cytokine promoters.

T11854

24334-24536

Sentence

denotes

Because of the multiple band shifts observed for NFAT binding (Fig. 1A), one could speculate that there is activation of several different NFAT isoforms, probably derived from alternative splicing [38].

T181

24334-24536

Sentence

denotes

Because of the multiple band shifts observed for NFAT binding (Fig. 1A), one could speculate that there is activation of several different NFAT isoforms, probably derived from alternative splicing [38].

T11855

24537-24685

Sentence

denotes

Interestingly, the GP-induced transcription factor binding transformed only into a very limited cytokine response, namely IL-8 and IL-1RA (Fig. 3B).

T182

24537-24685

Sentence

denotes

Interestingly, the GP-induced transcription factor binding transformed only into a very limited cytokine response, namely IL-8 and IL-1RA (Fig. 3B).

T11856

24686-24820

Sentence

denotes

Hence, our data are in aggreement with the few reports describing a β-1→3-D-glucan-mediated IL-8 [6,16,19] and IL-1RA production [17].

T183

24686-24820

Sentence

denotes

Hence, our data are in aggreement with the few reports describing a β-1→3-D-glucan-mediated IL-8 [6,16,19] and IL-1RA production [17].

T184

24821-25020

Sentence

denotes

In addition, our EMSA/supershift and immunoblotting results demonstrated a GP-mediated predominant binding of NFκB p65 and to a lesser extent of p50 to a κB consensus site of the IL-8 promoter (Figs.

T11857

24821-25028

Sentence

denotes

In addition, our EMSA/supershift and immunoblotting results demonstrated a GP-mediated predominant binding of NFκB p65 and to a lesser extent of p50 to a κB consensus site of the IL-8 promoter (Figs. 1A, 2).

T185

25021-25028

Sentence

denotes

1A, 2).

T11858

25029-25268

Sentence

denotes

Results by Schulte and colleagues [28] pointed to an induction of IL-8 transcription depending on activation via an NFκB p65/65 homodimer, rather than via p65/50 heterodimers, which might be the case for the GP-mediated IL-8 transcription.

T186

25029-25268

Sentence

denotes

Results by Schulte and colleagues [28] pointed to an induction of IL-8 transcription depending on activation via an NFκB p65/65 homodimer, rather than via p65/50 heterodimers, which might be the case for the GP-mediated IL-8 transcription.

T11859

25269-25446

Sentence

denotes

A GP-mediated IL-8 transcription based upon a cooperation between transactivated NFκB p65 and NFIL-6 [13,39] or NFATc2 dimer binding to the IL-8κB site [38] seems also possible.

T187

25269-25446

Sentence

denotes

A GP-mediated IL-8 transcription based upon a cooperation between transactivated NFκB p65 and NFIL-6 [13,39] or NFATc2 dimer binding to the IL-8κB site [38] seems also possible.

T11860

25447-25756

Sentence

denotes

The IL-8κB consensus site exhibits a preferentially p65 binding half site and thus differs from the κB half site described for TNFα and IL-1β [27,40], supporting the idea of regulating NFκB binding through combinatorial associations of the subunits and the specific sequence of the decameric κB motif [41,42].

T188

25447-25756

Sentence

denotes

The IL-8κB consensus site exhibits a preferentially p65 binding half site and thus differs from the κB half site described for TNFα and IL-1β [27,40], supporting the idea of regulating NFκB binding through combinatorial associations of the subunits and the specific sequence of the decameric κB motif [41,42].

T11861

25757-25908

Sentence

denotes

Unlike LPS or TSST-1, we found that GP did not induce IL-1β, but it strongly induced IL-1RA, suggesting an immediate anti-inflammatory potential of GP.

T189

25757-25908

Sentence

denotes

Unlike LPS or TSST-1, we found that GP did not induce IL-1β, but it strongly induced IL-1RA, suggesting an immediate anti-inflammatory potential of GP.

T11862

25909-26156

Sentence

denotes

Analysing the IL-1RA promoter [34], we discovered four new binding sites (Fig. 8): an NFκB3 site (between -100 and -130), an NFκB consensus site (-266 and -280), another NFκB2/3 site (-288 and -302) and a more distal NFATP2/3 site (-471 and -490).

T190

25909-26156

Sentence

denotes

Analysing the IL-1RA promoter [34], we discovered four new binding sites (Fig. 8): an NFκB3 site (between -100 and -130), an NFκB consensus site (-266 and -280), another NFκB2/3 site (-288 and -302) and a more distal NFATP2/3 site (-471 and -490).

T11863

26157-26437

Sentence

denotes

Our data indicated that GP leads to production of IL-1RA primarily via induction of NFATP2/3 and NFIL-6 DNA binding, which might be due to differences in the binding motif or the composition of the activated transcription factors (NFAT) between the IFNγ, IL-6 and IL-1RA promoter.

T191

26157-26437

Sentence

denotes

Our data indicated that GP leads to production of IL-1RA primarily via induction of NFATP2/3 and NFIL-6 DNA binding, which might be due to differences in the binding motif or the composition of the activated transcription factors (NFAT) between the IFNγ, IL-6 and IL-1RA promoter.

T11864

26438-26724

Sentence

denotes

The differential decrease of NFκB, NFAT and NFIL-6 binding to sites in the IL-1RA promoter as well as of IL-1RA mRNA and protein induced by selective inhibitors prior to GP treatment might suggest that these steps are linked to each other and necessary for induction of IL-1RA (Fig. 7).

T192

26438-26724

Sentence

denotes

The differential decrease of NFκB, NFAT and NFIL-6 binding to sites in the IL-1RA promoter as well as of IL-1RA mRNA and protein induced by selective inhibitors prior to GP treatment might suggest that these steps are linked to each other and necessary for induction of IL-1RA (Fig. 7).

T11865

26725-26834

Sentence

denotes

Regarding cellular sources, both, monocytes as well as neutrophils have been reported to produce IL-1RA [32].

T193

26725-26834

Sentence

denotes

Regarding cellular sources, both, monocytes as well as neutrophils have been reported to produce IL-1RA [32].

T11866

26835-26988

Sentence

denotes

Flow cytometric experiments seemed to confirm that monocytes and neutrophils were able to produce IL-8 just as IL-1RA in response to GP (data not shown).

T194

26835-26988

Sentence

denotes

Flow cytometric experiments seemed to confirm that monocytes and neutrophils were able to produce IL-8 just as IL-1RA in response to GP (data not shown).

T11867

26989-27175

Sentence

denotes

This GP-induced cytokine profile was substantially more restricted than that of LPS or TSST-1, which is likely due to differences in recognition and signaling between LPS, TSST-1 and GP.

T195

26989-27175

Sentence

denotes

This GP-induced cytokine profile was substantially more restricted than that of LPS or TSST-1, which is likely due to differences in recognition and signaling between LPS, TSST-1 and GP.

T11868

27176-27357

Sentence

denotes

Recognition of LPS is mainly mediated through Toll-like receptor 4 and subsequent signaling via the NFκB pathway, leading to expression of pro-inflammatory mediators like TNFα [43].

T196

27176-27357

Sentence

denotes

Recognition of LPS is mainly mediated through Toll-like receptor 4 and subsequent signaling via the NFκB pathway, leading to expression of pro-inflammatory mediators like TNFα [43].

T11869

27358-27595

Sentence

denotes

A bacterial superantigen like TSST-1 acts through binding to MHC-II molecules and subsequently the T cell receptor, leading to release of mainly IFNγ and TNFα, the latter via both, PI3K and p38 mitogen-activated kinase signaling [44,45].

T197

27358-27595

Sentence

denotes

A bacterial superantigen like TSST-1 acts through binding to MHC-II molecules and subsequently the T cell receptor, leading to release of mainly IFNγ and TNFα, the latter via both, PI3K and p38 mitogen-activated kinase signaling [44,45].

T11870

27596-27824

Sentence

denotes

Altogether, the induction of the neutrophil-attracting IL-8 and the anti-inflammatory IL-1RA by fungal carbohydrates (GP) may well fit to a benign PAMP response, mounting defensive mechanisms against a possible microbial attack.

T198

27596-27824

Sentence

denotes

Altogether, the induction of the neutrophil-attracting IL-8 and the anti-inflammatory IL-1RA by fungal carbohydrates (GP) may well fit to a benign PAMP response, mounting defensive mechanisms against a possible microbial attack.

T199

27825-27886

Sentence

denotes

Figure 8 Cartoon of the sense strand of the IL-1RA promoter.

T26184

27835-27886

Sentence

denotes

Cartoon of the sense strand of the IL-1RA promoter.

T26185

27887-28065

Sentence

denotes

Relative locations of previously (LRE-1) [30-33] and newly described (boxed), positively (black) and negatively (gray) regulatory transcription factor binding sites are depicted.

T200

27887-28065

Sentence

denotes

Relative locations of previously (LRE-1) [30-33] and newly described (boxed), positively (black) and negatively (gray) regulatory transcription factor binding sites are depicted.

T11871

28066-28341

Sentence

denotes

Potentially even more relevant than the specific cytokine panel induced by GP, we found that, in the presence of other stimuli (LPS or TSST-1), GP ameliorated their pro-inflammatory immune reactions, similar to the effects in murine models of sepsis and inflammation [21-24].

T201

28066-28341

Sentence

denotes

Potentially even more relevant than the specific cytokine panel induced by GP, we found that, in the presence of other stimuli (LPS or TSST-1), GP ameliorated their pro-inflammatory immune reactions, similar to the effects in murine models of sepsis and inflammation [21-24].

T11872

28342-28547

Sentence

denotes

Mainly, GP altered the TSST-1-induced IL-1β/IL-1RA ratio from a pro- to an anti-inflammatory profile via down-regulation of IL-1β and IL-6, at the same time there was a synergistic up-regulation of IL-1RA.

T202

28342-28547

Sentence

denotes

Mainly, GP altered the TSST-1-induced IL-1β/IL-1RA ratio from a pro- to an anti-inflammatory profile via down-regulation of IL-1β and IL-6, at the same time there was a synergistic up-regulation of IL-1RA.

T11873

28548-28788

Sentence

denotes

In part, this change may be caused by GP-mediated modulations of the TSST-1 induced binding of NFκB, NFIL-6 and NFAT to known and up to now unknown sites within the IL-8 and IL-1RA promoter instead of the TNFα, IL-1β, IL-6 or IFNγ promoter.

T203

28548-28788

Sentence

denotes

In part, this change may be caused by GP-mediated modulations of the TSST-1 induced binding of NFκB, NFIL-6 and NFAT to known and up to now unknown sites within the IL-8 and IL-1RA promoter instead of the TNFα, IL-1β, IL-6 or IFNγ promoter.

T11874

28789-29026

Sentence

denotes

Accordingly, we found that the TSST-1-induced binding of NFκB to sites from the TNFα promoter negatively correlated with the GP-mediated enhancement of the TSST-1-induced IL-1RA production (r = -0.88; p < 0.01; data not shown in detail).

T204

28789-29026

Sentence

denotes

Accordingly, we found that the TSST-1-induced binding of NFκB to sites from the TNFα promoter negatively correlated with the GP-mediated enhancement of the TSST-1-induced IL-1RA production (r = -0.88; p < 0.01; data not shown in detail).

T11875

29027-29232

Sentence

denotes

Of the four examined NFκB sites from the IL-1RA promoter, mostly the TSST-1- and LPS-induced binding to the NFκB3 and the TSST-1-induced binding to the new NFκB consensus site seemed to be increased by GP.

T205

29027-29232

Sentence

denotes

Of the four examined NFκB sites from the IL-1RA promoter, mostly the TSST-1- and LPS-induced binding to the NFκB3 and the TSST-1-induced binding to the new NFκB consensus site seemed to be increased by GP.

T11876

29233-29448

Sentence

denotes

So, while we observed a GP-mediated down-regulation of the LPS- and the TSST-1-induced NFκB binding to sites of the TNFα promoter, there was an up-regulation to NFκB3 and NFκB consensus sites of the IL-1RA promoter.

T206

29233-29448

Sentence

denotes

So, while we observed a GP-mediated down-regulation of the LPS- and the TSST-1-induced NFκB binding to sites of the TNFα promoter, there was an up-regulation to NFκB3 and NFκB consensus sites of the IL-1RA promoter.

T11877

29449-29830

Sentence

denotes

These seemingly contradictory data could be explained by differences in either NFκB subunits or conserved nucleotides (#1, 2, 3, 10) within the decameric NFκB binding motif between the TNFα and the IL-1RA promoter (for NFκB3 the IL-1RA motif contains a T on position 10 instead of the conserved C in the TNFα motif, see Table 1), probably leading to differences in binding [41,42].

T207

29449-29830

Sentence

denotes

These seemingly contradictory data could be explained by differences in either NFκB subunits or conserved nucleotides (#1, 2, 3, 10) within the decameric NFκB binding motif between the TNFα and the IL-1RA promoter (for NFκB3 the IL-1RA motif contains a T on position 10 instead of the conserved C in the TNFα motif, see Table 1), probably leading to differences in binding [41,42].

T11878

29831-29996

Sentence

denotes

Despite the location of the new NFκB consensus site (-266 and -280) in the inhibitory element (-250 and -294) of the IL-1RA promoter [30], we observed no inhibition.

T208

29831-29996

Sentence

denotes

Despite the location of the new NFκB consensus site (-266 and -280) in the inhibitory element (-250 and -294) of the IL-1RA promoter [30], we observed no inhibition.

T11879

29997-30215

Sentence

denotes

On the other side, we found an inhibitory NFκB2/3 site (-288 and -302) towards the end of the inhibitory element, demonstrating down-regulations of the LPS- and TSST-1-induced binding, which could not be altered by GP.

T209

29997-30215

Sentence

denotes

On the other side, we found an inhibitory NFκB2/3 site (-288 and -302) towards the end of the inhibitory element, demonstrating down-regulations of the LPS- and TSST-1-induced binding, which could not be altered by GP.

T11880

30216-30334

Sentence

denotes

In our opinion, this site may therefore represent at least a part of the previoulsy described inhibitory element [30].

T210

30216-30334

Sentence

denotes

In our opinion, this site may therefore represent at least a part of the previoulsy described inhibitory element [30].

T11881

30335-30570

Sentence

denotes

The GP-modulated increase in TSST-1-induced binding to the new NFκB3 and NFκB consensus site, the NFIL-6 site [32] as well as to the novel NFATP2/3 site may explain the synergistic up-regulation of the TSST-1-induced IL-1RA production.

T211

30335-30570

Sentence

denotes

The GP-modulated increase in TSST-1-induced binding to the new NFκB3 and NFκB consensus site, the NFIL-6 site [32] as well as to the novel NFATP2/3 site may explain the synergistic up-regulation of the TSST-1-induced IL-1RA production.

T11882

30571-30718

Sentence

denotes

We think that this GP-modulated activation of transcription was reflected by the decrease of the IL-1β/IL-1RA ratio following GP + TSST-1 (Fig. 6).

T212

30571-30718

Sentence

denotes

We think that this GP-modulated activation of transcription was reflected by the decrease of the IL-1β/IL-1RA ratio following GP + TSST-1 (Fig. 6).

T11883

30719-30864

Sentence

denotes

In this context, it has been postulated that in vitro a 100fold excess of IL-1RA over IL-1β might control the biological effects of IL-1 [46,47].

T213

30719-30864

Sentence

denotes

In this context, it has been postulated that in vitro a 100fold excess of IL-1RA over IL-1β might control the biological effects of IL-1 [46,47].

T11884

30865-31040

Sentence

denotes

Since, in fact, the IL-1β/IL-1RA ratio following GP + TSST-1 is partially less than 0.01, it is not unreasonable to assume that IL-1β bioactivity is inactivated in our system.

T214

30865-31040

Sentence

denotes

Since, in fact, the IL-1β/IL-1RA ratio following GP + TSST-1 is partially less than 0.01, it is not unreasonable to assume that IL-1β bioactivity is inactivated in our system.

T11885

31041-31159

Sentence

denotes

Indeed, GP reduced the TSST-1-induced, IL-1-dependent IL-2 production of murine EL-4 cells (data not shown in detail).

T215

31041-31159

Sentence

denotes

Indeed, GP reduced the TSST-1-induced, IL-1-dependent IL-2 production of murine EL-4 cells (data not shown in detail).

T11886

31160-31361

Sentence

denotes

The weaker modulating effects of GP on the LPS-induced immune response observed in this study, may be attributed to delicately balanced differences in signaling pathways between LPS and TSST-1 [43-45].

T216

31160-31361

Sentence

denotes

The weaker modulating effects of GP on the LPS-induced immune response observed in this study, may be attributed to delicately balanced differences in signaling pathways between LPS and TSST-1 [43-45].

T11887

31362-31484

Sentence

denotes

TSST-1 has been shown to use the PI3K pathway for signaling [44] and this effect may be sustained by GP treatment [22,37].

T217

31362-31484

Sentence

denotes

TSST-1 has been shown to use the PI3K pathway for signaling [44] and this effect may be sustained by GP treatment [22,37].

T11888

31485-31624

Sentence

denotes

It has been demonstrated that in septic/LPS-adapted leukocytes the PI3K pathway selectively controls sIL-1RA but not IL-1β production [48].

T218

31485-31624

Sentence

denotes

It has been demonstrated that in septic/LPS-adapted leukocytes the PI3K pathway selectively controls sIL-1RA but not IL-1β production [48].

T11889

31625-31719

Sentence

denotes

Signaling via PI3K has been reported to be involved in the activation of NFAT in T cells [49].

T219

31625-31719

Sentence

denotes

Signaling via PI3K has been reported to be involved in the activation of NFAT in T cells [49].

T11890

31720-31898

Sentence

denotes

Activation of NFκB can also take place via PI3K [50], which may offer an explanation for a difference in signaling between GP (PI3K) and LPS (mitogen-activated kinase signaling).

T220

31720-31898

Sentence

denotes

Activation of NFκB can also take place via PI3K [50], which may offer an explanation for a difference in signaling between GP (PI3K) and LPS (mitogen-activated kinase signaling).

T11891

31899-32193

Sentence

denotes

This idea may be supported by another study showing that despite the use of similar PRR, LPS and peptidoglycan activated the IL-1RA gene through different mechanisms/DNA-binding proteins and acted synergistically in combination, suggestive of signals which are not equivalent in all parts [51].

T221

31899-32193

Sentence

denotes

This idea may be supported by another study showing that despite the use of similar PRR, LPS and peptidoglycan activated the IL-1RA gene through different mechanisms/DNA-binding proteins and acted synergistically in combination, suggestive of signals which are not equivalent in all parts [51].

T222

32195-32205

Sentence

denotes

Conclusion

T17105

32206-32500

Sentence

denotes

In summary, our data demonstrated that in vitro glucan phosphate induced a transcription factor binding and a subsequent cytokine profile different from LPS and also from TSST-1 and, moreover, switched a pro-inflammatory TSST-1-induced response to an IL-1RA-mediated anti-inflammatory reaction.

T223

32206-32500

Sentence

denotes

In summary, our data demonstrated that in vitro glucan phosphate induced a transcription factor binding and a subsequent cytokine profile different from LPS and also from TSST-1 and, moreover, switched a pro-inflammatory TSST-1-induced response to an IL-1RA-mediated anti-inflammatory reaction.

T17106

32501-32743

Sentence

denotes

Our results also generated new insights into a very complex interplay of transcription factor binding to various known and newly identified sites in the IL-1RA promoter (Fig. 8), which can be regulated differentially by a fungal carbohydrate.

T224

32501-32743

Sentence

denotes

Our results also generated new insights into a very complex interplay of transcription factor binding to various known and newly identified sites in the IL-1RA promoter (Fig. 8), which can be regulated differentially by a fungal carbohydrate.

T17107

32744-32967

Sentence

denotes

Together with the in vivo studies [22,37], our findings might support the concept of protective effects mediated by glucan phosphate in pro-inflammatory conditions, especially with a dysregulated IL-1β/IL-1RA ratio [52,53].

T225

32744-32967

Sentence

denotes

Together with the in vivo studies [22,37], our findings might support the concept of protective effects mediated by glucan phosphate in pro-inflammatory conditions, especially with a dysregulated IL-1β/IL-1RA ratio [52,53].

T226

32969-32976

Sentence

denotes

Methods

T17461

32978-32987

Sentence

denotes

Materials

T227

32978-32987

Sentence

denotes

Materials

T17462

32988-33070

Sentence

denotes

Water soluble phosphorylated β-1→3-D-glucan (GP) was prepared as done before [20].

T228

32988-33070

Sentence

denotes

Water soluble phosphorylated β-1→3-D-glucan (GP) was prepared as done before [20].

T17463

33071-33160

Sentence

denotes

The physicochemical characteristics of GP were determined as reported previously [20,26].

T229

33071-33160

Sentence

denotes

The physicochemical characteristics of GP were determined as reported previously [20,26].

T17464

33161-33388

Sentence

denotes

GP was dissolved in aqueous media, filter-sterilized (0.2 μm) and screened for endotoxin contamination with the Endospecy assay (Seigakaku, Tokyo, Japan), which is specific for endotoxin but does not respond to β-1→3-D-glucans.

T230

33161-33388

Sentence

denotes

GP was dissolved in aqueous media, filter-sterilized (0.2 μm) and screened for endotoxin contamination with the Endospecy assay (Seigakaku, Tokyo, Japan), which is specific for endotoxin but does not respond to β-1→3-D-glucans.

T17618

33390-33413

Sentence

denotes

Isolation of human PBMC

T231

33390-33413

Sentence

denotes

Isolation of human PBMC

T232

33414-33585

Sentence

denotes

Buffy coats were obtained from healthy blood donors in compliance with the Helsinki declaration and with the approval of the ethics committee of the University of Luebeck.

T17619

33414-33684

Sentence

denotes

Buffy coats were obtained from healthy blood donors in compliance with the Helsinki declaration and with the approval of the ethics committee of the University of Luebeck. The buffy coats were separated over a Ficoll-Hypaque (Biochrom, Berlin, Germany) gradient [54,55].

T233

33586-33684

Sentence

denotes

The buffy coats were separated over a Ficoll-Hypaque (Biochrom, Berlin, Germany) gradient [54,55].

T17620

33685-34131

Sentence

denotes

Following isolation, PBMC were cultured at a final concentration of 3 × 106 cells/ml (for ELISA measurements) or 5 × 106/ml (for gel shifts) or 1 × 106 cells/ml (for flow cytometry) in RPMI 1640 medium (BioWhittaker, Heidelberg, Germany, LPS-free), containing 10% heat-inactivated fetal calf serum (FCS, low-LPS, Invitrogen, Karlsruhe, Germany), 1% penicillin (10000 U/ml), 1% streptomycin (10000 μg/ml), and 1% 200 mM L-glutamine (BioWhittaker).

T234

33685-34131

Sentence

denotes

Following isolation, PBMC were cultured at a final concentration of 3 × 106 cells/ml (for ELISA measurements) or 5 × 106/ml (for gel shifts) or 1 × 106 cells/ml (for flow cytometry) in RPMI 1640 medium (BioWhittaker, Heidelberg, Germany, LPS-free), containing 10% heat-inactivated fetal calf serum (FCS, low-LPS, Invitrogen, Karlsruhe, Germany), 1% penicillin (10000 U/ml), 1% streptomycin (10000 μg/ml), and 1% 200 mM L-glutamine (BioWhittaker).

T17939

34133-34155

Sentence

denotes

Stimulation conditions

T235

34133-34155

Sentence

denotes

Stimulation conditions

T17940

34156-34325

Sentence

denotes

For induction of transcription factors and cytokines, PBMC were cultured in a volume of 4 ml in sterile pyrogen-free 6-well culture plates (Falcon, Heidelberg, Germany).

T236

34156-34325

Sentence

denotes

For induction of transcription factors and cytokines, PBMC were cultured in a volume of 4 ml in sterile pyrogen-free 6-well culture plates (Falcon, Heidelberg, Germany).

T17941

34326-34604

Sentence

denotes

Cultures were stimulated for 1 h (EMSA) or from 0 h to 48 h (ELISA) with 1 or 100 μg per 106 cells of GP, with 250 ng/106 cells TSST-1 (Toxin Technologies, Sarasota, FL, USA) or with 250 ng/106 cells wild-type LPS from Escherichia coli serotype 0111:B4 (Sigma, Munich, Germany).

T237

34326-34604

Sentence

denotes

Cultures were stimulated for 1 h (EMSA) or from 0 h to 48 h (ELISA) with 1 or 100 μg per 106 cells of GP, with 250 ng/106 cells TSST-1 (Toxin Technologies, Sarasota, FL, USA) or with 250 ng/106 cells wild-type LPS from Escherichia coli serotype 0111:B4 (Sigma, Munich, Germany).

T17942

34605-34745

Sentence

denotes

In costimulatory experiments, PBMC were supplemented simultaneously with a combination of GP and TSST-1 or with a combination of GP and LPS.

T238

34605-34745

Sentence

denotes

In costimulatory experiments, PBMC were supplemented simultaneously with a combination of GP and TSST-1 or with a combination of GP and LPS.

T18267

34747-34793

Sentence

denotes

Oligonucleotides (Oligos) and 5'-32-P-Labeling

T239

34747-34793

Sentence

denotes

Oligonucleotides (Oligos) and 5'-32-P-Labeling

T18268

34794-35228

Sentence

denotes

The complementary double-stranded (ds) oligonucleotides (oligos) from the TNFα, IL-8, IFNγ and IL-6 promoters were synthesised from single stranded (ss) oligos (illustrated in Table 1), (TIB Molbiol, Berlin, Germany) and 32P-labeled with 5'γ-P-ATP (3,000 Ci/mmol, Amersham, Braunschweig, Germany) using the Ready-To-Go-Polynucleotide-Kinase Kit (PNK Kit, Pharmacia LKB, Freiburg, Germany) according to the manufacturer's instructions.

T240

34794-35228

Sentence

denotes

The complementary double-stranded (ds) oligonucleotides (oligos) from the TNFα, IL-8, IFNγ and IL-6 promoters were synthesised from single stranded (ss) oligos (illustrated in Table 1), (TIB Molbiol, Berlin, Germany) and 32P-labeled with 5'γ-P-ATP (3,000 Ci/mmol, Amersham, Braunschweig, Germany) using the Ready-To-Go-Polynucleotide-Kinase Kit (PNK Kit, Pharmacia LKB, Freiburg, Germany) according to the manufacturer's instructions.

T18269

35229-35326

Sentence

denotes

To test the binding specificity of the oligos, mutated oligos were used as an additional control.

T241

35229-35326

Sentence

denotes

To test the binding specificity of the oligos, mutated oligos were used as an additional control.

T18270

35327-35565

Sentence

denotes

Mutations in the P2 site of the IFNγ promoter [29] and in the κ consensus sequence of the TNFα promoter [55] have been reported to interfere with sequences, which seem to be crucial for the binding of NFAT and NFκB proteins, respectively.

T242

35327-35565

Sentence

denotes

Mutations in the P2 site of the IFNγ promoter [29] and in the κ consensus sequence of the TNFα promoter [55] have been reported to interfere with sequences, which seem to be crucial for the binding of NFAT and NFκB proteins, respectively.

T18271

35566-35719

Sentence

denotes

Afterwards, the ds oligos were purified via gel filtration using Probe Quant G-50 Micro Columns (Pharmacia) according to the manufacturer's instructions.

T243

35566-35719

Sentence

denotes

Afterwards, the ds oligos were purified via gel filtration using Probe Quant G-50 Micro Columns (Pharmacia) according to the manufacturer's instructions.

T18788

35721-35801

Sentence

denotes

Preparation of nuclear extracts and electrophoretic mobility shift assays (EMSA)

T244

35721-35801

Sentence

denotes

Preparation of nuclear extracts and electrophoretic mobility shift assays (EMSA)

T18789

35802-35952

Sentence

denotes

Nuclear extracts of purified PBMCs were prepared according to a technique described by Trede et al. [27] and modified for our experimental setup [56].

T245

35802-35952

Sentence

denotes

Nuclear extracts of purified PBMCs were prepared according to a technique described by Trede et al. [27] and modified for our experimental setup [56].

T18790

35953-36033

Sentence

denotes

The intensity of shifted bands was normalized to bands of unstimulated controls.

T246

35953-36033

Sentence

denotes

The intensity of shifted bands was normalized to bands of unstimulated controls.

T18791

36034-36229

Sentence

denotes

Competition experiments with labeled and unlabeled mutated oligos of the cytokine promoter binding sites (see above) were carried out in order to prevent non-specific binding to nuclear proteins.

T247

36034-36229

Sentence

denotes

Competition experiments with labeled and unlabeled mutated oligos of the cytokine promoter binding sites (see above) were carried out in order to prevent non-specific binding to nuclear proteins.

T18792

36230-36324

Sentence

denotes

For such experiments, an excess (5–50×) of unlabeled oligos was added to the nuclear extracts.

T248

36230-36324

Sentence

denotes

For such experiments, an excess (5–50×) of unlabeled oligos was added to the nuclear extracts.

T18793

36325-36444

Sentence

denotes

For specificity of binding, supershift assays for NFκB with a combined p65/50 antibody were performed (data not shown).

T249

36325-36444

Sentence

denotes

For specificity of binding, supershift assays for NFκB with a combined p65/50 antibody were performed (data not shown).

T18794

36445-36595

Sentence

denotes

To exclude non-specific reactions, a 30-fold molar excess of Oct-1 DNA (unrelated DNA, Table 1) was used, which did not compete with specific binding.

T250

36445-36595

Sentence

denotes

To exclude non-specific reactions, a 30-fold molar excess of Oct-1 DNA (unrelated DNA, Table 1) was used, which did not compete with specific binding.

T19195

36597-36614

Sentence

denotes

Immuno (Dot) blot

T251

36597-36614

Sentence

denotes

Immuno (Dot) blot

T19196

36615-37422

Sentence

denotes

To determine whether the DNA binding proteins were related to NFκB and NFAT, dot blots using a SRC96D SNS minifold I dot blotter (Schleicher & Schüll, Dassel, Germany) were performed with the following positive controls and antibodies (all from Santa Cruz Biotechnology, Heidelberg, Germany): positive controls (10 ng/dot): NFκB p52 (80 kD) sc-4095WB, c-Rel (61 kD) sc-4030WB; Jurkat nuclear extract, PMA-stimulated; primary antibodies (1 μg/dot): anti-NFκB p50 (NLS) sc-114, anti-NFκB p65 (A) sc-109, anti-NFκB p52 (447) sc-848, a rabbit polyclonal IgG1 antiserum, anti-c-rel (N466) sc-272, anti-NFATc2 (M-20) sc-1151, anti-NFATc1 (K-18) sc-1149, two goat polyclonal IgG1 antisera; secondary antibodies (0.8 μg/dot): goat anti-rabbit IgG AP-conjugate sc-2007, and donkey anti-goat IgG AP-conjugate sc-2022.

T252

36615-37422

Sentence

denotes

To determine whether the DNA binding proteins were related to NFκB and NFAT, dot blots using a SRC96D SNS minifold I dot blotter (Schleicher & Schüll, Dassel, Germany) were performed with the following positive controls and antibodies (all from Santa Cruz Biotechnology, Heidelberg, Germany): positive controls (10 ng/dot): NFκB p52 (80 kD) sc-4095WB, c-Rel (61 kD) sc-4030WB; Jurkat nuclear extract, PMA-stimulated; primary antibodies (1 μg/dot): anti-NFκB p50 (NLS) sc-114, anti-NFκB p65 (A) sc-109, anti-NFκB p52 (447) sc-848, a rabbit polyclonal IgG1 antiserum, anti-c-rel (N466) sc-272, anti-NFATc2 (M-20) sc-1151, anti-NFATc1 (K-18) sc-1149, two goat polyclonal IgG1 antisera; secondary antibodies (0.8 μg/dot): goat anti-rabbit IgG AP-conjugate sc-2007, and donkey anti-goat IgG AP-conjugate sc-2022.

T19197

37423-37498

Sentence

denotes

The nuclear proteins were blotted onto a nitrocellulose membrane (Bio-Rad).

T253

37423-37498

Sentence

denotes

The nuclear proteins were blotted onto a nitrocellulose membrane (Bio-Rad).

T19198

37499-37761

Sentence

denotes

After blocking with PBS/3% BSA (Fluka, Deisenhofen, Germany), the blot was incubated overnight with the primary antibodies diluted 1:2000 in PBS/1% BSA, washed again and incubated with the alkaline phosphatase-conjugated secondary antibody (1:500 in PBS/1% BSA).

T254

37499-37761

Sentence

denotes

After blocking with PBS/3% BSA (Fluka, Deisenhofen, Germany), the blot was incubated overnight with the primary antibodies diluted 1:2000 in PBS/1% BSA, washed again and incubated with the alkaline phosphatase-conjugated secondary antibody (1:500 in PBS/1% BSA).

T255

37762-37845

Sentence

denotes

The blot was developed using the Vectastain® staining kit (Vector Laboratories Inc.

T19199

37762-37867

Sentence

denotes

The blot was developed using the Vectastain® staining kit (Vector Laboratories Inc. Burlingame, CA, USA).

T256

37846-37867

Sentence

denotes

Burlingame, CA, USA).

T19743

37869-37890

Sentence

denotes

Cytokine measurements

T257

37869-37890

Sentence

denotes

Cytokine measurements

T19744

37891-38048

Sentence

denotes

Supernatants of cell cultures were harvested 3, 6, 9, 12, 18, 24, 30, 36 and 48 hours after stimulation and stored at -80 °C until used and thawed only once.

T258

37891-38048

Sentence

denotes

Supernatants of cell cultures were harvested 3, 6, 9, 12, 18, 24, 30, 36 and 48 hours after stimulation and stored at -80 °C until used and thawed only once.

T19745

38049-38279

Sentence

denotes

IFNγ, TNFα, IL-1 and IL-6 were determined using an ELISA from Bender Systems (Vienna, Austria), IL-1RA and IL-8 by an ELISA from R&D Systems (Wiesbaden-Nordenstadt, Germany), and TGFβ 1 using an ELISA as previously described [57].

T259

38049-38279

Sentence

denotes

IFNγ, TNFα, IL-1 and IL-6 were determined using an ELISA from Bender Systems (Vienna, Austria), IL-1RA and IL-8 by an ELISA from R&D Systems (Wiesbaden-Nordenstadt, Germany), and TGFβ 1 using an ELISA as previously described [57].

T19746

38280-38385

Sentence

denotes

Quantification of IL-2 as an indicator for the bioactivity of IL-1 was done as previously described [46].

T260

38280-38385

Sentence

denotes

Quantification of IL-2 as an indicator for the bioactivity of IL-1 was done as previously described [46].

T19747

38386-38457

Sentence

denotes

Samples were diluted in the same buffer as that used for the standards.

T261

38386-38457

Sentence

denotes

Samples were diluted in the same buffer as that used for the standards.

T19748

38458-38581

Sentence

denotes

All cytokines were quantified using an ELISA plate reader (Anthos Labotec, Salzburg, Austria or Microplate Reader, BioRad).

T262

38458-38581

Sentence

denotes

All cytokines were quantified using an ELISA plate reader (Anthos Labotec, Salzburg, Austria or Microplate Reader, BioRad).

T19749

38582-38647

Sentence

denotes

Cytokine amounts were all within the range of the standard curve.

T263

38582-38647

Sentence

denotes

Cytokine amounts were all within the range of the standard curve.

T19750

38648-38763

Sentence

denotes

Only stimulation of PBMC observed in donors showing no spontaneous cytokine release was included in the statistics.

T264

38648-38763

Sentence

denotes

Only stimulation of PBMC observed in donors showing no spontaneous cytokine release was included in the statistics.

T20261

38765-38800

Sentence

denotes

Inhibition of transcription factors

T265

38765-38800

Sentence

denotes

Inhibition of transcription factors

T20262

38801-39001

Sentence

denotes

To inhibit translocation and binding of transcription factors to the IL-1RA promoter, PBMC were incubated for 1 h with each of the following substances (concentrations were based upon 50% inhibition):

T266

38801-39001

Sentence

denotes

To inhibit translocation and binding of transcription factors to the IL-1RA promoter, PBMC were incubated for 1 h with each of the following substances (concentrations were based upon 50% inhibition):

T20263

39002-39252

Sentence

denotes

50 μg/ml of CAPE (caffeic-3,4-dihydroxycinnamic-acid-phenyl-ester), (Biomol, Hamburg, Germany); 400 ng/ml cyclosporin A (Calbiochem-Merck Biosciences, Bad Soden, Germany); 10 μg/ml cycloheximide (Santa Cruz Biotechnology) prior to incubation with GP.

T267

39002-39252

Sentence

denotes

50 μg/ml of CAPE (caffeic-3,4-dihydroxycinnamic-acid-phenyl-ester), (Biomol, Hamburg, Germany); 400 ng/ml cyclosporin A (Calbiochem-Merck Biosciences, Bad Soden, Germany); 10 μg/ml cycloheximide (Santa Cruz Biotechnology) prior to incubation with GP.

T20264

39253-39505

Sentence

denotes

Band shifts were determined after 1 h incubation with GP as described before, including mutated oligos for NFIL-6 (TTA CAA CAG TGG ATT GCG ACA CTT AGT GGG) and NFATP2/3 (GGC GCA GAA AAG GTA AAA TAT TTA CTA TCT) binding sites within the IL-1RA promoter.

T268

39253-39505

Sentence

denotes

Band shifts were determined after 1 h incubation with GP as described before, including mutated oligos for NFIL-6 (TTA CAA CAG TGG ATT GCG ACA CTT AGT GGG) and NFATP2/3 (GGC GCA GAA AAG GTA AAA TAT TTA CTA TCT) binding sites within the IL-1RA promoter.

T20265

39506-39931

Sentence

denotes

PBMC RNA was isolated with an RNeasy kit (Qiagen, Germany) and messenger RNA was transcribed into cDNA with Reactin Ready First Strand kit (Biomol) and analysed for IL-1RA and GAPDH transcripts by PCR with a HotStart "sweet" PCR mastermix (Biomol) (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec, 25 cycles for IL-1RA and 94°C for 30 sec, 50°C for 30 sec and 72°C for 45 sec, 25 cycles for GAPDH) following 18 h incubation with GP.

T269

39506-39931

Sentence

denotes

PBMC RNA was isolated with an RNeasy kit (Qiagen, Germany) and messenger RNA was transcribed into cDNA with Reactin Ready First Strand kit (Biomol) and analysed for IL-1RA and GAPDH transcripts by PCR with a HotStart "sweet" PCR mastermix (Biomol) (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec, 25 cycles for IL-1RA and 94°C for 30 sec, 50°C for 30 sec and 72°C for 45 sec, 25 cycles for GAPDH) following 18 h incubation with GP.

T20266

39932-40018

Sentence

denotes

IL-1RA protein was measured in the supernatant by ELISA after 24 h incubation with GP.

T270

39932-40018

Sentence

denotes

IL-1RA protein was measured in the supernatant by ELISA after 24 h incubation with GP.

T20981

40020-40040

Sentence

denotes

Statistical analysis

T271

40020-40040

Sentence

denotes

Statistical analysis

T20982

40041-40190

Sentence

denotes

The Kolmogorow-Smirnov test was used to evaluate, whether cytokine amounts and binding activities to transcription factors were normally distributed.

T272

40041-40190

Sentence

denotes

The Kolmogorow-Smirnov test was used to evaluate, whether cytokine amounts and binding activities to transcription factors were normally distributed.

T20983

40191-40351

Sentence

denotes

Correlation coefficients and corresponding significances were analyzed by the Pearson test (normal distribution) or the Spearman test (non-normal distribution).

T273

40191-40351

Sentence

denotes

Correlation coefficients and corresponding significances were analyzed by the Pearson test (normal distribution) or the Spearman test (non-normal distribution).

T20984

40352-40549

Sentence

denotes

To compare experimental data to a theoretical value (for example GP + TSST-1 vs GP/TSST-1 calc.) we calculated the sum of the individual effects and added these values into Fig. 1B, 3, 4, 5, and 6.

T274

40352-40549

Sentence

denotes

To compare experimental data to a theoretical value (for example GP + TSST-1 vs GP/TSST-1 calc.) we calculated the sum of the individual effects and added these values into Fig. 1B, 3, 4, 5, and 6.

T20985

40550-40917

Sentence

denotes

Significances of differences between experimental data (for example GP vs LPS) as well as between experimental data and theoretical values (for example GP + TSST-1 vs GP/TSST-1 calc.) were analysed using the Student's t-test (normal distribution) or the Wilcoxon Signed-Rank test (non-normal distribution), (SPSS for Windows; SPSS Science Software, Erkrath, Germany).

T275

40550-40917

Sentence

denotes

Significances of differences between experimental data (for example GP vs LPS) as well as between experimental data and theoretical values (for example GP + TSST-1 vs GP/TSST-1 calc.) were analysed using the Student's t-test (normal distribution) or the Wilcoxon Signed-Rank test (non-normal distribution), (SPSS for Windows; SPSS Science Software, Erkrath, Germany).

T276

40919-40932

Sentence

denotes

Abbreviations

T277

40933-41388

Sentence

denotes

LPS, lipopolysaccharide; TSST-1, toxic shock syndrome toxin 1; GP, glucan phosphate; PBMC, peripheral blood mononuclear cells; PMN, polymorphonuclear neutrophils; LRE, LPS-responsive element; NF, nuclear factor; IL, interleukin; BSA, bovine serum albumin; PBS, phosphate buffered saline; PMA, phorbol myristate acetate; ATP, adenosine triphosphate; SEM, standard error of the mean; n.s., non-significant (p > 0.05); CyA, cyclosporin A; CHX, cycloheximide.

T278

41390-41412

Sentence

denotes

Authors' contributions

T279

41413-41561

Sentence

denotes

JL participated in the study design and coordination, developed the band shifts assays, performed the statistics and helped to draft the manuscript.

T280

41562-41613

Sentence

denotes

UL carried out band shift assays and the dot blots.

T281

41614-41705

Sentence

denotes

JH, CF and JMB carried out band shift assays and participated in the ELISA and PCR studies.

T282

41706-41769

Sentence

denotes

HH designed the oligos for the analysis of the IL-1RA promoter.

T283

41770-41809

Sentence

denotes

LR participated in the PCR experiments.

T284

41810-41874

Sentence

denotes

PK performed the ELISA tests and the flow cytometry experiments.

T285

41875-41924

Sentence

denotes

NW carried out the IL-1RA inhibition experiments.

T286

41925-41986

Sentence

denotes

DLW conceived of the study and prepared the glucan phosphate.

T287

41987-42091

Sentence

denotes

AM conceived of the study, participated in the study design and coordination and drafted the manuscript.

T288

42092-42143

Sentence

denotes

All authors read and approved the final manuscript.

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