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CORD-19:a44cd4fe9a7c450cf0e3e0fe955d43f824bb3ebe JSONTXT 7 Projects

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Id Subject Object Predicate Lexical cue
T1 0-199 Sentence denotes CCL2 transgene expression in the central nervous system directs diffuse infiltration of CD45 high CD11b + monocytes and enhanced Theiler's murine encephalomyelitis virus-induced demyelinating disease
T2 201-209 Sentence denotes Abstract
T3 210-425 Sentence denotes CCL2 is a member of the CC chemokine family that mediates the migration and recruitment of monocytes and T cells and has been identified in the central nervous system (CNS) during several neuroinflammatory diseases.
T4 426-650 Sentence denotes In order to examine the biological effect of constitutive CCL2 expression in the CNS, the authors engineered a mouse that expressed CCL2 in the CNS under control of the human glial fibrillary acidic protein (hGFAP) promoter.
T5 651-785 Sentence denotes The results demonstrated that transgenic expression of CCL2 in the CNS resulted in diffuse CNS monocyte infiltration and accumulation.
T6 786-887 Sentence denotes Transgenic CCL2 expression did not alter normal development, differentiation, or function of T cells.
T7 888-1021 Sentence denotes There was no evidence of overt CNS disease or other pathologic phenotype when mice were left unchallenged with antigen or uninfected.
T8 1022-1193 Sentence denotes However, when CCL2 transgenic mice were given a peripheral challenge of lipopolysaccharide (LPS), an inflammatory infiltrate with organized perivascular lesions developed.
T9 1194-1370 Sentence denotes Infection of the transgenic mice with Theiler's murine encephalomyelitis virus (TMEV) resulted in accelerated onset and increased severity of clinical and histological disease.
T10 1371-1537 Sentence denotes These results suggest that CCL2 expression in the CNS is a major pathogenic factor that drives macrophage accumulation in the development of CNS inflammatory disease.
T11 1538-1581 Sentence denotes Journal of NeuroVirology (2003) 9, 623-636.
T12 1582-1723 Sentence denotes CCL2 mediates enhanced TMEV-induced demyelination 624 JL Bennett et al CCL2 mediates enhanced TMEV-induced demyelination JL Bennett et al 625
T13 1725-2037 Sentence denotes Chemokines are small chemotactic molecules that play an important role in immunology, including regulation of leukocyte migration, T-cell differentiation (Huang et al, 2001; , organ system development and vascularization, and numerous tissue-specific inflammatory responses (Murphy et al, 2000; Gu et al, 2000) .
T14 2038-2213 Sentence denotes These molecules represent a superfamily that can be classified into four groups based on the position of the conserved cysteine residues at the amino terminus of the molecule.
T15 2214-2362 Sentence denotes The chemokine subfamilies include CCL, CXCL, CL, and CX 3 CL, with the X representing a nonconserved amino acid residue (Zlotnik and Yoshie, 2000) .
T16 2363-2513 Sentence denotes Corresponding receptors are appropriately named CCR, CXCR, CR, and CX 3 CR based on the family of chemokines to which they bind (Murphy et al, 2000) .
T17 2514-2656 Sentence denotes The binding pattern of chemokine ligands and their receptors suggests an intricate system for directing cell localization throughout the body.
T18 2657-2859 Sentence denotes Multiple ligands may bind the same receptor or one ligand may bind multiple receptors, providing redundancy and complexity for cellular recruitment in different physiological processes (Rollins, 1997) .
T19 2860-3000 Sentence denotes Although there is considerable overlap in ligand-receptor binding, ligands are generally restricted to binding receptors of the same family.
T20 3001-3169 Sentence denotes Chemokine receptors signal through seven-transmembrane spanning, G-proteincoupled receptors expressed on the surface of numerous cell populations (Murphy et al, 2000) .
T21 3170-3355 Sentence denotes Regulation and expression of corresponding receptors is critical for cells to respond to chemokine gradients produced at sites of tissue inflammation and remodeling (Baggiolini, 1998) .
T22 3356-3454 Sentence denotes Murine CCL2 (monocyte chemoattractant protein [MCP]-1) is a member of the CC family of chemokines.
T23 3455-3614 Sentence denotes This ligand binds CCR2, which is expressed by monocytes, T cells, B cells, natural killer (NK) cells, fibroblasts, and endothelial cells (Boring et al, 1996) .
T24 3615-3840 Sentence denotes CCL2 was first described in mice as a platelet-derived growth factor (PDGF)-inducible gene in fibroblasts, named JE (Cochran et al, 1983) , and later identified as a recruitment factor for monocytes (Matsushima et al, 1989) .
T25 3841-4044 Sentence denotes CCL2 was also shown to have a role in recruitment of T cells (Carr et al, 1994) , polarization of a Th2-type T-cell response , regulation of interleukin (IL)-4 expression , and down-regulation of IL-12 .
T26 4045-4133 Sentence denotes In the absence of CCL2 expression, Th2 responses are impaired in vivo (Gu et al, 2000) .
T27 4134-4308 Sentence denotes Intracerebral infection of susceptible inbred strains of mice with Theiler's murine encephalomyelitis virus (TMEV) leads to a chronic, immune-mediated demyelinating disease .
T28 4309-4578 Sentence denotes Because of the suspected viral etiology, chronic pathology, and primary demyelination accompanied by mononuclear cell infiltration seen in multiple sclerosis (MS), TMEV-induced demyelinating disease (TMEV-IDD) is considered to be an excellent experimental model of MS .
T29 4579-4856 Sentence denotes Virus is rapidly cleared from the peripheral circulation; however, TMEV persists within the central nervous system (CNS) of susceptible mice for their lifetime (Clatch et al, 1990) , serving as a reservoir for chronic activation of virus-specific T cells (Lipton et al, 1995) .
T30 4857-5055 Sentence denotes Although TMEV has been shown to preferentially reside in CNS macrophages, the role of TMEV infection in the chronic activation of macrophages to secrete soluble proinflammatory cytokines is unknown.
T31 5056-5304 Sentence denotes As a result of CNS TMEV infection, increasing virus-specific (DTH-delayed type hypersensitivity) has been shown to correlate with development of increasing disease severity, supporting a role for Th1-mediated CNS inflammation (Clatch et al, 1986) .
T32 5305-5521 Sentence denotes Virus replication occurs in the spinal cord (Yamada et al, 1990; Ozden et al, 1991) and focal inflammation, consisting primarily of T cells and macrophages, is limited to the spinal cord white matter (Lipton, 1975) .
T33 5522-5654 Sentence denotes From 2 to 5 months following infection, extensive demyelination occurs, leading to clinical paralysis (Dal Canto and Lipton, 1975) .
T34 5655-5767 Sentence denotes One of the major characteristics of TMEV-IDD includes CNS mononuclear cell infiltration over the disease course.
T35 5768-5932 Sentence denotes Immune responses originate in peripheral lymphoid tissue and once activated, the specific lymphocytes leave the organized lymphoid tissue and accumulate in the CNS.
T36 5933-6268 Sentence denotes In order to further understand the biological role of CCL2 expression in a CNS inflammatory disease such as TMEV-IDD, we generated a transgenic mouse line where astrocytes constitutively expressed CCL2 at low levels (Huang et al, 2002) under control of the human glial fibrillary acidic protein (hGFAP) promoter (Brenner et al, 1994) .
T37 6269-6435 Sentence denotes Astrocytes have been reported as a principle source of CCL2 production in vivo during several CNS inflammatory responses (Glabinski et al, 1997; Berman et al, 1996) .
T38 6436-6657 Sentence denotes Therefore, transgenic CCL2 expression controlled by the hGFAP promoter provides the appropriate spatial chemokine distribution for investigation of its role in several neuroinflammatory disease models, including TMEV-IDD.
T39 6658-6784 Sentence denotes In order to study the regulation of CNS demyelinating diseases by specific chemokines, we constructed a CCL2 transgenic mouse.
T40 6785-6932 Sentence denotes The hGFAP-CCL2 transgene construct ( Figure 1A ) was used to produce six transgenic founders, of which four transmitted the transgene to offspring.
T41 6933-7013 Sentence denotes In the present report, we used the JE32 (MCP-1 low ) mouse line for our studies.
T42 7014-7117 Sentence denotes Expression of the hGFAP promoter is known to target CCL2 production to astrocytes (Huang et al, 2002) .
T43 7118-7262 Sentence denotes The presence of hGFAP-CCL2 was confirmed by Southern blotting and polymerase chain reaction (PCR) analysis of tail DNA samples (data not shown).
T44 7263-7426 Sentence denotes To demonstrate tissue-specific expression levels of CCL2 mRNA, RNase protection assay (RPA) was performed on CNS tissue recovered from mice at 6 to 9 weeks of age.
T45 7427-7618 Sentence denotes As shown in Figure 1B , there was a dramatic increase in CCL2 levels in the forebrain, hindbrain, and spinal cord of JE32 transgenic mice compared to minimal expression in wild-type controls.
T46 7619-7668 Sentence denotes All other tissues were negative (data not shown).
T47 7669-7762 Sentence denotes Astrocytespecific expression of CCL2 was confirmed by in situ hybridization (data not shown).
T48 7763-7875 Sentence denotes Additionally, there was an increase in CXCL10 (Crg-2) mRNA expression in the JE32 transgenic mice ( Figure 1B ).
T49 7876-8043 Sentence denotes Because RPA analysis indicated high levels of CCL2 mRNA expression in the CNS, we wanted to know if the level of chemokine protein was also increased in the periphery.
T50 8044-8261 Sentence denotes To assess this possibility, serum was recovered from control mice at 32 weeks of age and from JE32 mice at both 9 and 36 weeks of age and analyzed by enzyme-linked immunosorbent assay (ELISA) for the presence of CCL2.
T51 8262-8362 Sentence denotes In wild-type mice at 32 weeks of age, very little CCL2 could be detected in the serum ( Figure 1C ).
T52 8363-8471 Sentence denotes However, there was a significant increase in serum CCL2 levels in JE32 mice at 9 weeks of age ( Figure 1C ).
T53 8472-8632 Sentence denotes JE32 mice that were 36 weeks of age showed a more pronounced level of CCL2 in the serum when compared to either wild-type control or 9-week-old transgenic mice.
T54 8633-8812 Sentence denotes These data indicate that the CCL2 transgene was expressed in the CNS, specifically by astrocytes, and resulted in a translated protein product that could be detected in the serum.
T55 8813-9112 Sentence denotes Given the role of chemokines in regulating lymphocyte migration and accumulation throughout the body (Baggiolini, 1998) , it was important to determine if overexpression of CCL2 in the CNS affected the number or distribution of lymphocytes in other organs as a result of a developmental abnormality.
T56 9113-9259 Sentence denotes Therefore, we examined spleen, peripheral lymph node, thymus, and peripheral blood of JE32 mice for relative numbers of lymphocytes and monocytes.
T57 9260-9509 Sentence denotes There were increases noted in the size of individual spleen and lymph nodes obtained from JE32 mice, but the average weight of organs and average number of mononuclear leukocytes per milligram of tissue was not remarkably different (data not shown).
T58 9510-9636 Sentence denotes This indicated that there were no apparent developmental defects in the JE32 transgenic mice when compared to normal controls.
T59 9637-9814 Sentence denotes We next analyzed the effect of CCL2 transgene expression in the CNS on mononuclear cell infiltration in JE32 mice that had not been immunized with antigen or infected with TMEV.
T60 9815-10031 Sentence denotes The CNS of wild-type control (SJL×SWR) and JE32 mice was analyzed by flow cytometry for the presence of microglia (CD45 low CD11b + ), monocytes/macrophages (CD45 high CD11b + ), and lymphocytes (CD45 high CD11b − ).
T61 10032-10383 Sentence denotes Figure 2 shows that the CNS of JE32 mice that have not been immunized or The JE32 CCL2transgenic mouse line was made using the construct shown, consisting of a human GFAP (glial fibrilary acidic protein) promoter directing expression of murine CCL2. (B) CNS chemokine expression was determined for both control and JE32 CCL2-transgenic mice using RPA.
T62 10384-10584 Sentence denotes The data indicate densitometric evaluation of the RPA analysis. (C) Serum levels were determined for normal control mice (32 weeks of age) and normal JE32 (9 and 36 weeks of age) using specific ELISA.
T63 10585-10669 Sentence denotes The data represent serum CCL2 concentration + SD of three individual mice per group.
T64 10670-10839 Sentence denotes Cells isolated from the spinal cord of normal control or CCL2transgenic animals were separated by Percoll density-gradient centrifugation and analyzed by flow cytometry.
T65 10840-11034 Sentence denotes Antibodies to CD4, CD8, CD45, and CD11b were used to identify infiltrating T cells (CD4 + and CD8 + ), monocytes/macrophages (CD45 high CD11b + ), and resident CNS microglia (CD45 low CD11b + ).
T66 11035-11159 Sentence denotes Data are expressed as percent of CD45 + -gated cell population and are representative of at least three similar experiments.
T67 11160-11312 Sentence denotes infected contains microglia, monocytes/macrophages, and a small population of lymphocytes compared to normal control mice, which only contain microglia.
T68 11313-11420 Sentence denotes The low-level CNS lymphocyte infiltrate in the JE32 mice consisted primarily of CD4 + T cells ( Figure 2 ).
T69 11421-11609 Sentence denotes This small population of CD4 + T cells localized to the CNS of naïve CCL2-transgenic mice have the phenotype CD62L high , CD25 − , and CD44 + as determined by flow cytometry ( Figure 3A ).
T70 11610-11695 Sentence denotes These cells are also negative for the T-cell activation marker CD69 (data not shown).
T71 11696-11837 Sentence denotes We were also interested in determining the activation state of the CD45 high CD11b + cells observed in the CNS of naïve CCL2-transgenic mice.
T72 11838-11922 Sentence denotes An absence of disease symptoms suggested that these cells were likely not activated.
T73 11923-12255 Sentence denotes Cells isolated from the CNS were further separated by flow cytometric sorting into CD45 low CD11b + and CD45 high CD11b + populations. cDNA prepared from the purified cell populations was analyzed by PCR for expression of inducible nitric oxide synthase (iNOS), a welldocumented marker of activated macrophages (Lyons et al, 1992) .
T74 12256-12403 Sentence denotes Controls for iNOS expression included purified macrophages treated with 75 μg lipopolysaccharide (LPS) or phosphate-buffered saline (PBS) for 24 h.
T75 12404-12635 Sentence denotes The results in Figure 3B show that both resident microglia (CD45 low CD11b + ) and infiltrating monocyte/macrophage (CD45 high CD11b + ) populations in CCL2-transgenic mice are not activated, similar to transgene negative controls.
T76 12636-12733 Sentence denotes This result is consistent with the absence of clinical disease in untreated CCL2-transgenic mice.
T77 12734-12988 Sentence denotes These data demonstrate that transgenic expression of CCL2 in the murine CNS mediates accumulation of mononuclear cells, predominantly monocytes/macrophages, in the absence of inflammatory stimuli, and that these cells have a naïve, unactivated phenotype.
T78 12989-13106 Sentence denotes CCL2 has been previously shown to induce in vitro Th2 polarization and in vivo Th2 differentiation (Gu et al, 2000) .
T79 13107-13214 Sentence denotes Therefore, we wanted to assess T-cell proliferative and cytokine responsiveness from JE32 and control mice.
T80 13215-13389 Sentence denotes Purified T cells were isolated from spleen and lymph node of control and JE32 mice and stimulated by immobilized anti-CD3 and anti-CD28 as described in Materials and methods.
T81 13390-13523 Sentence denotes T cells from either the spleen or peripheral lymph nodes of JE32 mice proliferated equally as well as control cells (data not shown).
T82 13524-13723 Sentence denotes In addition, these cells are also capable of producing both interferon (IFN)-γ and IL-2 and do not produce detectable levels of IL-4 in response to T-cell receptor (TCR) stimulation (data not shown).
T83 13724-13957 Sentence denotes This suggests that transgenic expression of CCL2 in the CNS does not change the fundamental ability of T cells to proliferate or diminish their ability to respond to TCR polyclonal stimulation by production of Th1-specific cytokines.
T84 13958-14109 Sentence denotes To this point, our data suggested that transgenic expression of CCL2 in the CNS resulted in an accumulation of CD45 high CD11b + monocytes/macrophages.
T85 14110-14276 Sentence denotes To assess the ability of recruited monocytes in the CNS to initiate inflammation, JE32 and control animals were challenged by intraperitoneal (IP) LPS administration.
T86 14277-14416 Sentence denotes Three days later, spinal cord tissue was examined by standard hematoxylin and eosin (H&E) histology to determine the level of inflammation.
T87 14417-14564 Sentence denotes Mononuclear cell accumulation and inflammatory lesions were not detected in PBS-treated ( Figure 4A ) or LPS-treated ( Figure 4B ) control animals.
T88 14565-14688 Sentence denotes In PBStreated JE32 animals, there was a diffuse mononuclear cell infiltrate and few small, organized lesions ( Figure 4C ).
T89 14689-14848 Sentence denotes However, following LPS administration, distinct perivascular lesions were observed in the JE32 spinal cord tissue, primarily in the white matter ( Figure 4D ).
T90 14849-14942 Sentence denotes Tracts of mononuclear cell invasion from the meninges into the white matter was also visible.
T91 14943-15168 Sentence denotes Collectively, these results demonstrate that CNS transgenic expression of CCL2 results in an accumulation of monocytes/macrophages that have the capacity to induce CNS inflammation when challenged with an activating stimulus.
T92 15169-15235 Sentence denotes Activation state of CNS-infiltrating cells in CCL2transgenic mice.
T93 15236-15375 Sentence denotes Cells isolated from pooled spinal cords of naïve SJL×SWR and CCL2-transgenic mice were purified by Percoll density-gradient centrifugation.
T94 15376-15471 Sentence denotes Magnetic antibody coated beads (anti-CD4) were used to further purify CD4 + T cells followed by
T95 15472-15781 Sentence denotes To test the hypothesis that the presence of CCL2 in the CNS could regulate an immune-mediated, macrophage-dependent demyelinating disease, we infected either control, JE32, or JE95 (described previously; Huang et al [2002] ) transgenic mice with TMEV and assessed the animals for resulting clinical paralysis.
T96 15782-15955 Sentence denotes JE32 (MCP-1 low ) transgenic mice express low levels of CCL2 in the CNS whereas JE95 (MCP-1 high ) mice express high levels as measured by RNA analysis (Huang et al, 2002) .
T97 15956-16164 Sentence denotes The data shown in Figure 5 demonstrate that transgenic overexpression of CCL2 in the CNS results in earlier onset of paralytic disease and more severe paralysis during the first 35 days of the disease course.
T98 16165-16271 Sentence denotes Eventually, the severity of disease in either JE32 or JE95 mice becomes equal to that of the control mice.
T99 16272-16467 Sentence denotes These data suggest that CCL2-mediated accumulation of monocytes/macrophages in the CNS prior to viral infection can result in a more severe disease phenotype once the mice are infected with TMEV.
T100 16468-16633 Sentence denotes The effect of CCL2 overexpression on CNS histologic disease following TMEV infection was tested by examining spinal cord tissue from control and CCL2transgenic mice.
T101 16634-16777 Sentence denotes TMEV-infected (SJL×SWR) control mice develop scattered perivascular mononuclear cell lesions, primarily in the white matter ( Figure 6C and D).
T102 16778-17029 Sentence denotes Spinal cord tissue from TMEV-infected CCL2transgenic mouse strains (JE32 and JE95) demonstrates more severe mononuclear cell infiltration and development of numerous perivascular lesions as well as meningial accumulation (JE32 shown, Figure 6A and B).
T103 17030-17186 Sentence denotes CNS tissue from control and JE32 mice was also analyzed for degree of demyelination by epon-embedded, toluidine blue-stained thin sections (data not shown).
T104 17187-17415 Sentence denotes The results of the demyelination studies were consistent with the H&E histology results, showing slightly higher, though not significantly different, demyelination scores at 2 to 3 weeks post TMEV infection compared to controls.
T105 17416-17804 Sentence denotes These data support the clinical results ( Figure 4) where disease onset is more rapid in the CCL2-transgenic mice compared to control mice. incubation with anti-CD45 and anti-CD11b antibodies and flow cytometric sorting in to CD45 low CD11b + and CD45 high CD11b + pools. (A) Purified T cells were incubated with antibodies to CD62L, CD25, CD45, and TcRα/β and analyzed by flow cytometry.
T106 17805-17889 Sentence denotes Histogram plots show CD62L and CD25 peaks (shaded) with isotype controls (unshaded).
T107 17890-17976 Sentence denotes Cells are gated out of forward scatter versus side scatter, CD45 high , and TcRα/β + .
T108 17977-18195 Sentence denotes CD62L high CD25 − staining correlates with a naïve T cell phenotype. (B) cDNA isolated from resident microglia (CD45 low CD11b + ) and monocytes/macrophages (CD45 high CD11b + ) was analyzed by PCR for iNOS expression.
T109 18196-18286 Sentence denotes Controls for iNOS up-regulation are purified macrophages treated with PBS or LPS for 24 h.
T110 18287-18412 Sentence denotes The results indicate that microglia and monocytes in both SJL×SWR and CCL2-transgenic spinal cord tissue do not express iNOS.
T111 18413-18469 Sentence denotes The data are representative of at least two experiments.
T112 18470-18723 Sentence denotes To determine the constituents of the inflammatory infiltrate in both control and JE32 mice, we performed flow cytometric analysis of cells recovered from spinal cords of mice 4 weeks post infection, at a time when there was evidence of clinical disease.
T113 18724-18898 Sentence denotes Figure 7 shows that the TMEV-infected JE32 mice showed a greater percentage of both lymphocytes (CD45 high CD11b − ) and monocytes (CD45 high CD11b + ) than the control mice.
T114 18899-19002 Sentence denotes In both control and JE32 mice, the lymphocyte infiltrate consisted of a mixture of CD4 and CD8 T cells.
T115 19003-19196 Sentence denotes The CCL2 transgenic mice appeared to have a lower percentage of microglia (CD45 low CD11b + ); however, this was simply due to the increased fraction of lymphocytes and monocytes in the sample.
T116 19197-19312 Sentence denotes These data support the idea of CCL2mediated enhancement of clinical and histological disease at the cellular level.
T117 19313-19432 Sentence denotes Two distinct possibilities exist to explain the enhanced clinical and histological disease in the CCL2 transgenic mice.
T118 19433-19575 Sentence denotes One possibility is enhanced tissue damage mediated by an increased mononuclear cell infiltrate consisting of T cells and effector macrophages.
T119 19576-19710 Sentence denotes The second is augmented TMEV replication and viral presence in the CNS, possibly inducing direct cytolytic events in oligodendrocytes.
T120 19711-19940 Sentence denotes To determine between these two possibilities, we infected both control (SJL×SWR) and JE32 mice with TMEV, allowed disease to develop, and harvested spinal cord tissue for analysis of the presence of virus by plaque forming assay.
T121 19941-20057 Sentence denotes The results in Figure 8 demonstrate no significant difference in the viral titers among control and transgenic mice.
T122 20058-20167 Sentence denotes These data suggests that CNS CCL2 transgenic expression does not enhance or diminish the replication of TMEV.
T123 20168-20348 Sentence denotes Rather, the data support the idea that CNS CCL2 transgenic expression enhances mononuclear cell infiltration, which leads to enhanced clinical and histologic demyelinating disease.
T124 20349-20505 Sentence denotes Our data support the idea that accelerated disease after TMEV infection is due to CCL2-mediated monocyte/macrophage recruitment and not altered TMEV levels.
T125 20506-20743 Sentence denotes However, it could be possible that the presence of any virus in the CNS of CCL2-transgenic mice would be a sufficient stimulus to activate the accumulated macrophage pool, initiating inflammation and development of demyelinating disease.
T126 20744-21392 Sentence denotes To test this possibility, SJL×SWR control and CCL2transgenic mice were infected intracranially with mCMV (Smith strain) or TMEV and monitored for disease development. mCMV is a double-stranded DNA β-herpesvirus that normally replicates in the liver and spleen of susceptible strains of mice dur-ing the replicative phase (approximately 14 days) before entering a latent phase. mCMV can infect astrocytes, neurons, endothelial cells, radial glia, ependymal cells, microglia, and cells from the meninges and choroid in the mouse CNS (Tsutsui et al, 1989; Shinmura et al, 1997; van Den Pol et al, 1999) as well as macrophages (Brautigam et al, 1979) .
T127 21393-21602 Sentence denotes The results shown in Figure 9 demonstrate that TMEV-infected CCL2-transgenic mice developed an earlier-onset disease compared to mCMV-infected CCL2-transgenic mice and and TMEV-infected wild-type control mice.
T128 21603-21918 Sentence denotes Following onset of disease in TMEV-infected CCL2transgenic mice and the appearance of clinical symptoms in TMEV-infected SJL×SWR mice, all groups were analyzed by histology ( Figure 9B ) and flow cytometry ( Figure 9C ) to determine whether infection with mCMV resulted in altered pathology and cellular infiltrate.
T129 21919-22122 Sentence denotes Spinal cords of mCMV-infected SJL×SWR mice showed no focal lesions ( Figure 9B , a), whereas TMEV-infected SJL×SWR showed scattered small perivascular lesions and meningial accumulation ( Figure 9B, b) .
T130 22123-22427 Sentence denotes Spinal cords of mCMVinfected CCL2-transgenic mice had a diffuse infiltrate with few scattered focal lesions and meningial accumulation ( Figure 9B , c) but did not demonstrate the level of pathology associated with ongoing demyelinating disease, as in TMEV-infected CCL2-transgenic mice ( Figure 9B, d) .
T131 22428-22649 Sentence denotes Finally, flow cytometric analysis of CNS-infiltrating cells shows that mCMV infection results in far fewer CD4 + T cells and monocytes/ macrophages in SJL×SWR control mice compared to TMEV-infected controls ( Figure 9C ).
T132 22650-22725 Sentence denotes This correlates with the absence of demyelinating disease in these animals.
T133 22726-22983 Sentence denotes Surprisingly, mCMV-infected CCL2transgenic mice showed no clinical disease despite the presence of both CD4 + and CD8 + T cells and a similar ratio of CD11b + CD45 high cells in the spinal cord tissue compared to TMEV-infected transgenic mice ( Figure 9C ).
T134 22984-23174 Sentence denotes These data demonstrate that the effect of CCL2 on demyelinating disease initiated by TMEV is not only due to the enhanced accumulation of macrophages, but is also specific to TMEV infection.
T135 23175-23378 Sentence denotes In the present report, we studied the role of CCL2 in the pathogenesis of CNS inflammatory demyelinating disease by creating a transgenic mouse where the astrocytes overexpress our chemokine of interest.
T136 23379-23509 Sentence denotes The results demonstrated that CNS transgenic expression of CCL2 induces a monocyte/macrophage accumulation in the CNS (Figure 2 ).
T137 23510-23727 Sentence denotes This cellular accumulation was benign in that the accumulating cells were not activated and no overt histological or clinical inflammatory disease was induced in the absence of an inciting stimulus (Figures 3 and 4) .
T138 23728-23861 Sentence denotes However, when the transgenic mice were challenged with LPS, there was evidence of organized CNS mononuclear cell lesions (Figure 4) .
T139 23862-24024 Sentence denotes Moreover, when the transgenic mice were infected with TMEV, clinical disease onset ( Figure 5 ) was more rapid and histologic disease was more severe (Figure 6 ).
T140 24025-24244 Sentence denotes CCL2 has been previously overexpressed in the CNS using the myelin basic protein promoter (Fuentes et al, 1995) where the cellular distribution of the gene product was limited to oligodendrocytes rather than astrocytes.
T141 24245-24416 Sentence denotes Administration of LPS to these mice initiated inflammation, presumably by inducing macrophage accumulation the CNS, similar to what we have observed in the present report.
T142 24417-24593 Sentence denotes Our results support the idea that CCL2-mediated monocyte/macrophage accumulation in the CNS is a pathogenic factor in the development of CNS inflammatory demyelinating disease.
T143 24594-24694 Sentence denotes There are two major possibilities to explain the effect of CCL2 on TMEV-induced disease development.
T144 24695-24833 Sentence denotes The first is that overexpression of CCL2 by astrocytes results in an influx and accumulation of normal monocytes/macrophages into the CNS.
T145 24834-24971 Sentence denotes These cells are not activated and there is no evidence of inflammatory lesions or spontaneous inflammatory disease or histologic disease.
T146 24972-25144 Sentence denotes These monocytes/macrophages have the capacity to be activated as a peripheral challenge of the JE32 transgenic mice with LPS-induced organized CNS mononuclear cell lesions.
T147 25145-25288 Sentence denotes These lesions are presumably due to the activation of monocytes/macrophages previously recruited to the CNS as a result of CCL2 overexpression.
T148 25289-25498 Sentence denotes During the normal TMEV disease process, CCL2 is expressed in the CNS of infected mice (Hoffman et al, 1999) and anti-CCL2 treatment results in decreased monocyte accumulation in the CNS (manuscript submitted).
T149 25499-25643 Sentence denotes These findings support the idea that enhanced disease as a result of transgenic overexpression of CCL2 is due to enhanced monocyte accumulation.
T150 25644-25859 Sentence denotes However, from the histology data, it cannot be ruled out that either LPS challenge or TMEV infection induced the activation of microglia to form inflammatory foci resembling an accumulation of monocytes/macrophages.
T151 25860-25965 Sentence denotes The second possibility is that overexpression of CCL2 in the CNS results in enhanced T-cell accumulation.
T152 25966-26157 Sentence denotes Because CCL2 can function as a chemoattractant for T cells (Carr et al, 1994) and T cells Figure 7 Flow cytometric analysis of CNS infiltrate in TMEVinfected control and CCL2-transgenic mice.
T153 26158-26335 Sentence denotes Cells isolated from the spinal cord of TMEV-infected control or JE32 transgenic animals were separated by Percoll density-gradient centrifugation and analyzed by flow cytometry.
T154 26336-26529 Sentence denotes Antibodies to CD4, CD8, CD45, and CD11b were used to identify infiltrating T cells (CD4 + and CD8 + ), monocytes/macrophages (CD45 high CD11b + ) and resident CNS microglia (CD45 low CD11b + ).
T155 26530-26718 Sentence denotes Data are expressed as percent of CD45 +gated cell population (T cells) or CD11b + CD45 + (microglia and monocytes/macrophages) and are representative of at least three similar experiments.
T156 26719-26935 Sentence denotes are required for development of TMEV-IDD (Clatch et al, 1986) , overexpression of CCL2 in both the JE32 and JE95 mice could promote the accumulation of effector Th1 cells that drive the demyelinating disease process.
T157 26936-27000 Sentence denotes However, our present data ( Figure 2 does not support this idea.
T158 27001-27186 Sentence denotes In unchallenged, uninfected JE32 transgenic mice, there is little T-cell (CD4 + or CD8 + ) accumulation but there is a significant monocyte/macrophage (CD45 high CD11b + ) accumulation.
T159 27187-27291 Sentence denotes This low level of T-cell accumulation could be the result of upregulation of CXCL10 (Crg-2, Figure 1B ).
T160 27292-27426 Sentence denotes CD44 expression is usually associated with memory T-cells, but there is no indication of previous activation by other surface markers.
T161 27427-27569 Sentence denotes CD44 upregulation on unactivated cells with no CD25 or CD69 expression and unchanged CD62L have been previously reported (Viret et al, 2000) .
T162 27570-27821 Sentence denotes Further, CD44 expression is thought to play a role in leukocyte migration into tissue, as up-regulation of the molecule has been associated with binding to its ligand, hyaluronan, and rolling or adhesion before extravasation (DeGrendele et al, 1997) .
T163 27822-28042 Sentence denotes CD44 has been correlated with CNS infiltration by CD4 + T cells (Brocke et al, 1999) , suggesting that up-regulation may be required for entry and retention of the small number of T cells observed in CCL-transgenic mice.
T164 28043-28112 Sentence denotes These cells do not appear to be activated except for CD44 expression.
T165 28113-28205 Sentence denotes Significant accumulation of T cells in the CNS occurs only after TMEV infection (Figure 7) .
T166 28206-28470 Sentence denotes Further support that the murine CCL2 and CCR2 axis primarily controls CNS monocyte/macrophage and has little regulation over T-cell accumulation has been demonstrated in murine experimental autoimmune encephalomyelitis (EAE) (Fife et al, 2000; Huang et al, 2001) .
T167 28471-28689 Sentence denotes It was also possible that transgenic overexpression of CCL2 in the CNS promoted enhanced viral replication by inducing accumulation of the macrophage, which is the reservoir for viral persistence (Clatch et al, 1990) .
T168 28690-28946 Sentence denotes The presence of more host cells could potentially result in an increased production of infective virus that could lead to direct infection and cytolysis of oligodrendrocytes (Aubert and Brahic, 1995) , culminating in enhanced clinical disease ( Figure 5 ).
T169 28947-29090 Sentence denotes We do not believe that this was the case as there is no apparent difference in CNS viral titer between control and transgenic mice (Figure 8) .
T170 29091-29242 Sentence denotes Therefore, the data argue in favor of the hypothesis that expression of CCL2 promotes CNS accumulation of monocytes/macrophages that are not activated.
T171 29243-29391 Sentence denotes Upon infection with TMEV, transgenic mice develop disease more rapidly than controls due to the presence of the end-stage effector cells in the CNS.
T172 29392-29559 Sentence denotes Eventually, the control mice reach the same level of clinical disease as the transgenic mice as a result of normal accumulation of monocytes/macrophages in this model.
T173 29560-29802 Sentence denotes Further, the mCMV studies demonstrate that the accelerated disease observed in CCL2-transgenic mice is specific to TMEV infection and not due to a nonspecific activation of localized monocytes/macrophages by any virus introduced into the CNS.
T174 29803-29939 Sentence denotes This is consistent with our conclusion that the role of CCL2 in TMEV-induced demyelinating disease is directing macrophage accumulation.
T175 29940-30317 Sentence denotes Figure 9 mCMV infection of SJL×SWR and CCL2-transgenic mice. (A) Control SJL×SWR and CCL2-transgenic mice were infected intracranially with mCMV or TMEV and monitored for development of demyelinating disease symptoms. (B) After 28 days, after disease onset in the CCL2-transgenic TMEV-infected mice, spinal cord tissue was recovered by dissection and analyzed by H&E histology.
T176 30318-30505 Sentence denotes Spinal cords of SJL×SWR mice infected with mCMV showed no focal lesions (a), whereas SJL×SWR infected with TMEV showed scattered small perivascular lesions and meningial accumulation (b).
T177 30506-30651 Sentence denotes Spinal cords of CCL2-transgenic mice infected with mCMV had diffuse infiltrate with a few scattered focal lesions and meningial accumulation (c).
T178 30652-31077 Sentence denotes TMEV-infected CCL2-transgenic mice (d) showed multifocal mononuclear cell infiltration and extensive multilayer meningial accumulation associated with ongoing demyelinating disease. (C) Cells were also isolated from spinal cord tissue at day 28 after infection and analyzed for T-cell infiltrate (CD4 + and CD8 + ) and microglia or monocyte/macrophage accumulation (CD45 low CD11b + and CD45 high CD11b + ) by flow cytometry.
T179 31078-31239 Sentence denotes Data are derived from the forward scatter versus side scatter and CD45 + gates for all cells, as well as the CD11b + gate for migroglia for monocyte/macrophages.
T180 31240-31337 Sentence denotes The chemokine regulation of demyelinating disease has been studied in other virus-induced models.
T181 31338-31434 Sentence denotes In a separate study, TMEV-IDD was induced using different strains of virus: DA, GDVII, and H101.
T182 31435-31555 Sentence denotes These are neurovirulent strains of TMEV that cause different neuropathology and disease when inoculated into SJL/J mice.
T183 31556-31724 Sentence denotes DA virus causes a chronic demyelinating disease, GDVII virus causes an acute fatal encephalomyelitis, and H101 virus causes an acute pachymeningitis with hydrocephalus.
T184 31725-31879 Sentence denotes CCL2, CCL3, CCL4, CCL5, CXCL10, and MIP-2 mRNA expression was detected in both the brain and spinal cord during all three infections (Theil et al, 2000) .
T185 31880-32068 Sentence denotes Murray et al (2000) examined spinal cords during the acute and chronic phases of TMEV infection in mice sus-ceptible (B10.M, H-2f) and resistant (B10, H-2b) to virus-induced demyelination.
T186 32069-32247 Sentence denotes In this model, TMEV infection resulted in robust expression of mRNAs for CCL2, CCL5, and, CXCL10, but not CXCL1, in brains and spinal cords of both strains of mice within 5 days.
T187 32248-32399 Sentence denotes During the chronic, demyelinating phase of infection, there was a resurgence in CCL2, CCL5, and CXCL10 mRNAs in spinal cords of susceptible B10.M mice.
T188 32400-32616 Sentence denotes In both of these studies, the direct function of the chemokines identified as being expressed during the disease process was not directly determined, including whether CCL2 regulated monocyte/macrophage accumulation.
T189 32617-32730 Sentence denotes Lane et al (1998) demonstrated the presence of chemokines in the CNS of mice infected with mouse hepatitis virus.
T190 32731-32867 Sentence denotes By analyzing chemokine mRNA, they found that CCL4, CCL5, and CXCL10 expression was evident at the time of chronic demyelinating disease.
T191 32868-33068 Sentence denotes They went on to demonstrate that CCL5 was required for the development of demyelinating disease , whereas CXCL9 and CXCL10 expression was required for control of viral replication (Liu et al, , 2001 .
T192 33069-33256 Sentence denotes Finally, this same group of investigators demonstrated that CCL2 regulation of monocyte/macrophage accumulation was essential for development of demyelinating disease (Chen et al, 2001) .
T193 33257-33506 Sentence denotes These examples point to the essential functions of chemokines during the pathogenesis of virus-induced demyelinating disease and support our contention that CCL2 regulates the accumulation of monocytes/macrophages during the development of TMEV-IDD.
T194 33507-33690 Sentence denotes We would speculate that CCL2 is a desirable target for intervention in the demyelinating disease process as it regulates the accumulation of a key mononuclear cell subtype in the CNS.
T195 33691-33844 Sentence denotes Development of antagonists to CCR2, the only known receptor for CCL2, could lead to regimens that can interdict during ongoing CNS demyelinating disease.
T196 33845-34047 Sentence denotes Indeed, antagonists to CCR1 can treat ongoing EAE (Liang et al, 2000) by presumably interfering with the ability of CCL3 and/or CCL5 from inducing accumulation of T cells in the CNS (Fife et al, 2001) .
T197 34048-34210 Sentence denotes This example points to the possibility of interfering with the functions of CCL2 in a similar manner using receptor antagonists to interfere with ongoing disease.
T198 34211-34301 Sentence denotes Mice CCL2-transgenic mice (JE32) were made using standard techniques (Huang et al, 2002) .
T199 34302-34459 Sentence denotes JE32 transgenic mice expressed low levels of CCL2 (MCP-1 low ) whereas JE95 transgenic mice expressed high levels of CCL2 (MCP-1 high ) (Huang et al, 2002) .
T200 34460-34702 Sentence denotes The modified murine CCL2 gene, with replacement of the mRNA cap site, was obtained from Dr. Barrett Rollins (Dana Farber Cancer Institute, Boston, MA) and was placed under the control of the hGFAP promoter to produce the hGFAP-CCL2 transgene.
T201 34703-34866 Sentence denotes Initial microinjections of (SJL×SWR)F1 mice resulted in six transgene positive founders identified by Southern blotting of tail DNA for elements of the hGFAP gene.
T202 34867-34929 Sentence denotes Four founders transmitted the hGFAP-CCL2 transgene to progeny.
T203 34930-34991 Sentence denotes Normal, nontransgenic (SJL×SWR)F2 mice were used as controls.
T204 34992-35070 Sentence denotes SJL×SWR controls were established by crossing female SJL/J with male SWR mice.
T205 35071-35208 Sentence denotes The offspring of this cross were then maintained as a repeatedly intercrossed control SJL×SWR line in parallel with the transgenic lines.
T206 35209-35295 Sentence denotes The biological properties of JE95 mice were previously described (Huang et al, 2002) .
T207 35296-35504 Sentence denotes Animal care and use at Northwestern University and the Cleveland Clinic were approved by the respective institutional animal care and use committees and performed according to Public Health Sevice guidelines.
T208 35505-35549 Sentence denotes Virus was prepared as described previously .
T209 35550-35660 Sentence denotes Briefly, confluent monolayers of BHK-21 cells (ATCC) were infected with the BeAn 8386 strain of TMEV for 72 h.
T210 35661-35760 Sentence denotes Virus was precipitated with NaCl and polyethylene glycol (PEG) and then pelleted by centrifugation.
T211 35761-35871 Sentence denotes Virus was further purified by ultracentrifugation on discontinuous 20% to 70% sucrose and Cs 2 SO 4 gradients.
T212 35872-35971 Sentence denotes Finally, virus was pelleted, resuspended in PBS, and measured for optical absorbance at 260/280 nm.
T213 35972-36036 Sentence denotes Plaque assays of the supernatant were performed on BHK-21 cells.
T214 36037-36220 Sentence denotes Mice were anesthetized with sodium pentobarbital and injected in the right cerebral hemisphere with 3 × 10 6 plaque-forming units (PFU) of TMEV (BeAn strain) in 30 μl of sterile DMEM.
T215 36221-36364 Sentence denotes Mice were examined two to three times per week for the first 3 weeks until all infected animals were exhibiting neurological signs of TMEV-IDD.
T216 36365-36426 Sentence denotes After signs of clinical disease, mice were examined biweekly.
T217 36427-36586 Sentence denotes Clinical symptoms were scored as 1 = waddling gait; 2 = severe waddling gait and righting reflex impairment; 3 = hind limb paralysis with/without incontinence.
T218 36587-36674 Sentence denotes Clinical data have been expressed as the mean clinical score at a particular timepoint.
T219 36675-36793 Sentence denotes Chemokine analysis CNS chemokine mRNA expression was analyzed by RPA as previously described (Glabinski et al, 1998) .
T220 36794-36924 Sentence denotes Serum CCL2 concentrations were determined from JE32 and control mice of different ages by specific ELISA as previously described .
T221 36925-37058 Sentence denotes Histologic evaluation of CNS inflammation was performed using standard H&E methodology as previously described (Karpus et al, 1995) .
T222 37059-37233 Sentence denotes Transgenic and control animals, aged 6 to 8 weeks, were given 50 μg of LPS (Escherichia coli serotype 0111:B4; Sigma, St. Louis, MO) in 100 μl of sterile PBS by IP injection.
T223 37234-37397 Sentence denotes Animals were monitored for 72 h and sacrificed by total body perfusion with PBS through the left ventricle for examination of CNS tissue by standard H&E histology.
T224 37398-37469 Sentence denotes Control treatment consisted of IP administration of 100 μl sterile PBS.
T225 37470-37588 Sentence denotes Spinal cord tissue for histology was recovered by microdissection of the dorsal vertebrae to maintain intact meninges.
T226 37589-37871 Sentence denotes Organ harvest and cell isolation Spleen, peripheral lymph nodes (inguinal, brachial, axillary), thymus, peripheral blood, and spinal cord CCL2 mediates enhanced TMEV-induced demyelination 634 JL Bennett et al tissue were harvested from two to four mice per group, aged 6 to 8 weeks.
T227 37872-37921 Sentence denotes Sample weights were determined before processing.
T228 37922-38109 Sentence denotes Splenocytes were isolated by homogenization through 100-mesh stainless steel screens and red blood cells lysed by incubation with 2 ml/organ of Tris-NH 4 Cl (pH 7.2) at 37 • C for 10 min.
T229 38110-38199 Sentence denotes Lymph nodes and thymi were similarly isolated by homogenization through 100-mesh screens.
T230 38200-38585 Sentence denotes All samples were washed with Hanks buffered salt solution (HBSS) (BioWhittaker, Walkersville, MD) and resuspended in 3 ml Dulbecco's modified Eagle medium containing 5% fetal calf serum (FCS), 1 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 μM nonessential amino acids, and 5 × 10 −5 M 2-ME (complete DMEM-5; all components from Gibco BRL, Grand Island, NY) for culture.
T231 38586-38706 Sentence denotes Peripheral blood was harvested by cardiac puncture into syringes containing 100 μl heparin sulfate (100 U/ml) per mouse.
T232 38707-38881 Sentence denotes Samples were pooled by experimental group and the total volume was determined before overlay of sample on to 3 ml Ficoll (Nycomed Pharma, Oslo, Norway) in polyethylene tubes.
T233 38882-38949 Sentence denotes Gradients were produced by spinning at 22 • C for 15 min, 1200 rpm.
T234 38950-39012 Sentence denotes Buffy coat interface was recovered and washed with 10 ml HBSS.
T235 39013-39060 Sentence denotes Samples were resuspended in DMEM-5 for culture.
T236 39061-39235 Sentence denotes Spinal cords were isolated by flushing the vertebral column with 1 × PBS through a blunted 18-gauge needle and homogenized by passage through 100mesh stainless steel screens.
T237 39236-39345 Sentence denotes Single-cell suspensions were washed with HBSS and pelleted by centrifugation at 1200 rpm for 10 min at 4 • C.
T238 39346-39452 Sentence denotes Cells were then resuspended in 5 ml 30% Percoll solution (Amersham Pharmacia Biotech AB, Uppsala, Sweden).
T239 39453-39567 Sentence denotes Percoll solution, 70%, was added by underlay and leukocytes isolated by 10-min spin at 22 • C, 1200 rpm (328 × g).
T240 39568-39639 Sentence denotes Leukocytes were recovered from the interface and washed with 1 ml HBSS.
T241 39640-39807 Sentence denotes Cells were pelleted by quick spin (up to 7000 rpm), resuspended in 1 ml flow buffer (PBS containing 0.05% bovine serum albumin [BSA], 0.01% sodium azide), and counted.
T242 39808-39890 Sentence denotes Samples were stored at 4 • C until antibody staining and flow cytometric analysis.
T243 39891-40040 Sentence denotes Purified T cells were recovered from spleen and lymph nodes as described above and isolated using an AutoMACS machine (Miltenyi Biotech, Auburn, CA).
T244 40041-40276 Sentence denotes Briefly, cell samples were washed with MACS buffer (1 × PBS, 0.5% BSA, 2 mM EDTA, pH 6.5), then resuspended in 2 ml buffer containing 50 μg biotinylated anti-CD4 antibody (L3T4) (Pharmingen, San Diego, CA) and stored on ice for 10 min.
T245 40277-40369 Sentence denotes Following a wash with buffer, AutoMACS streptavidin microbeads were added for 10 min on ice.
T246 40370-40634 Sentence denotes After a final wash, CD4 + T cells were sorted by positive selection in 2-ml samples and analyzed for purity by staining with CD3-FITC (fluorescein isothiocyanate), CD19-PE (phycoerythrin), CD8-PerCP (peridinin chlorophyll protein), and CD11b-APC (allophycocyanin).
T247 40635-40676 Sentence denotes Purity was determined to be at least 97%.
T248 40677-40854 Sentence denotes Cellular composition of lymphoid organs was determined by staining with lymphocyte specific antibodies and analysis on a FACS-Calibur cytometer (Becton Dickinson, San Jose, CA).
T249 40855-41199 Sentence denotes One million cells were incubated with anti-Fc blocking antibody (anti-mouse FcRεII/III, 24G2) for 15 min at 4 • C and incubated with titered dilutions of the following antibodies: CD4-FITC (RMA4-5), CD8-PE (53-6.7), CD45-PerCP (Ly-5), and CD11b-APC (M1/70), CD19-PE (1D3), and CD3-APC (145-2C11) (all antibodies from Pharmingen, San Diego, CA).
T250 41200-41294 Sentence denotes Data were acquired and analyzed using CellQuest Pro software (Becton Dickinson, San Jose, CA).
T251 41295-41382 Sentence denotes Data was gated as a function of CD45 expression and side light scatter characteristics.
T252 41383-41519 Sentence denotes Relative cell composition of organs was estimated by multiplying the percent positive by the total cell count for each individual organ.
T253 41520-41742 Sentence denotes To examine the activation status of T cells and monocytes, CD4 + T cells were depleted from CNSisolated mononuclear cell pools using CD4 Dynabeads according to the manufacturer's instructions (Dynal Biotech, Oslo, Norway).
T254 41743-41962 Sentence denotes CD4 + T cells were washed with flow buffer, filtered, and examine by flow cytometry for expression of CD44-FITC (Ly-24), CD25-PE (3C7), CD62L-FITC (Ly-22), CD69-PE (H1.2F3), CD45 PerCP (Ly-5), and TCR-α/β-APC (H57-597).
T255 41963-42187 Sentence denotes The remaining cell pool was stained with CD11b-APC (M1/70) and CD45-PerCP (Ly-5), sorted in to CD45 high CD11b + and CD45 low CD11b + populations, and reverse transcriptase (RT)-PCR performed for analysis of iNOS expression.
T256 42188-42317 Sentence denotes Proliferative capacity of CD4 + T cells isolated from SJL×SWR and JE32 animals was assayed by conventional mitogenic stimulation.
T257 42318-42398 Sentence denotes Purified T cells were resuspended at a concentration of 10 6 cells/ml in DMEM-5.
T258 42399-42626 Sentence denotes 96-and 24-well plates were coated with 2 μg/well anti-CD3 (145-2C11) and anti-CD28 (37.51) antibodies (Pharmingen), or PBS (pH 7.2) as a control, for 90 min at 37 • C and then washed 3 times with PBS before addition of samples.
T259 42627-42723 Sentence denotes 5 × 10 6 cells/ml were added to the plates in triplicate wells and incubated at 37 • C for 24 h.
T260 42724-42879 Sentence denotes Cells were pulsed with 1 μCi 3 H-TdR (ICN Biochemicals, Irvine, CA), incubated an additional 12 h, and harvested onto glass filters (Packard, Meriden, CT).
T261 42880-43029 Sentence denotes Microscint (Packard, Meriden, CT) fluid was added to the filter and the 3 H-TdR incorporation determined on a Top Count liquid scintillation counter.
T262 43030-43187 Sentence denotes Counts per minute (CPM) was calculated by subtracting average CPM values of unstimulated wells from average CPM values of anti-CD3-and CD28-stimulated wells.
T263 43188-43286 Sentence denotes Cytokine production T cells were analyzed for cytokine response to mitogenic stimulation by ELISA.
T264 43287-43414 Sentence denotes Purified T cells were added at a concentration of 1 × 10 6 cells/ml to anti-CD3-and anti-CD28-coated plates as described above.
T265 43415-43494 Sentence denotes Supernatants were recovered at 24 and 48 h and stored at −20 • C until assayed.
T266 43495-43659 Sentence denotes Cytokine analysis was performed by standard techniques using interferon (IFN)-γ and IL-2 ELISA kits according to manufacturer instructions (Endogen, Cambridge, MA).
T267 43660-43752 Sentence denotes Plates were developed by addition of TMB substrate (Dako, Carpinteria, CA) and 0.18 M H2SO4.
T268 43753-43793 Sentence denotes All samples were analyzed in triplicate.
T269 43794-43806 Sentence denotes Results were