The factors that trigger EC apoptosis in PAH remain unclear. Autoimmune factors may be among them [12, 30]. Recently, we reported that the majority of PAH patients have circulating AECA specifically targeting cell surface antigens of ECs [13]. To study the specificity of AECA towards ECs in our study we determined the reactivity

of our patients’ sera towards human fibroblasts by means of a cyto-ELISA with unfixed normal human dermal fibroblast (NHDF). The sera of the AECA-positive PAH patients did not show any reactivity towards NHDF compared to the sera of the healthy controls (data not shown). We also demonstrated that IgG from AECA-positive patients with SLE nephritis induce EC apoptosis in vitro by a mechanism as yet unknown [18]. In avian SSc, AECA have been shown to induce CH5424802 EC apoptosis, which is considered a primary pathogenic event in SSc [31]. However, conflicting data have been published concerning the mechanisms by which AECA exert EC apoptosis in human SSc [17, 32]. AECA in SSc have selleckchem been shown to directly induce

apoptosis [17]. Alternatively, EC apoptosis may be induced by antibody-dependent cell-mediated cytotoxicity (ADCC) [32]. Irrespective of the mechanism, AECA have been shown to exert pro-apoptotic activity on ECs. Hence we hypothesized that AECA could be the trigger leading to the development of PAH by inducing EC apoptosis which subsequently activates a cascade culminating in EC proliferation. In the present study we demonstrate, surprisingly, that in contrast to IgG from AECA-positive

SLE patients the IgG from AECA-positive PAH patients do not induce apoptosis of EC. We confirmed this finding by employing three different methods, of which the RT–CES™ technology is a new method, to measure cell viability by high-throughput screening [28]. The lack of apoptosis-inducing activity of purified IgG from AECA-positive PAH patients suggests that other circulating factors may trigger EC apoptosis. Kahaleh et al. suggested serum-mediated selleck chemicals endothelial injury and demonstrated the presence of granular enzymes (granzymes) in sera of SSc patients [33]. Granzymes gain access to the cells following cellular membrane damage by perforin [34]. We tested sera from PAH patients on their ability to induce EC apoptosis in vitro to assess whether serum factors other than IgG could induce EC apoptosis. However, none of the tested sera from AECA-positive PAH expressed EC apoptosis-inducing activity (data not shown). ADCC is another proposed mechanism of EC apoptosis in SSc [32]. This mechanism of EC apoptosis requires antibodies and appropriate effector cells. Sgonc et al. found activated natural killer (NK) cells to be absolutely necessary for the AECA-dependent apoptosis induction in EC cultures [32]. In the present study we did not address this mechanism of EC apoptosis in PAH.

This contributes to disease pathology, in part via positive feedback loops between T and myeloid cells [49, 50]. The percentage of CD4+ cells expressing the

activation marker CD69 was elevated compared with that in WT in lyn–/–, but not lyn–/–IL-21–/– mice (Fig. 6C and Supporting Information Fig. 4). However, the frequency of IFN-γ, IL-4, and IL-17-producing cells among CD4+ T cells was similar in aged lyn–/– and lyn–/–IL-21–/– mice (Fig. 8D, Supporting Information Fig. 4). In the myeloid compartment, we observed an elevated frequency of CD11b+ cells in both lyn–/– and lyn–/–IL-21–/– spleens (Fig. 7). This increase was primarily in the CD11b+Gr1+CD11c− subset (Fig. 7). Because of variability in the total number of splenocytes in aged lyn–/– and lyn–/–IL-21–/– mice (Supporting Information Fig. 5), it was difficult to detect significant changes in the total number of T and myeloid cell subpopulations. buy Acalabrutinib However, since the relative frequency of myeloid cells is increased significantly in both lyn–/– and lyn–/–IL-21–/– mice, other cell types will have greater exposure to them and the factors they produce than in WT mice. Finally, we asked whether IL-21 mediates kidney damage in lyn–/– mice. Despite the lack of anti-DNA IgG, aged lyn–/–IL-21–/– mice experienced severe GN (Fig. 8A and B). They also demonstrated an increased frequency of CD11b+ (both CD11c−/lo and CD11c+ subsets) and CD8+ cells in the

kidneys (Fig. 8C RXDX-106 and Supporting Information Fig. 6). Each of these populations has been shown to be elevated in the nephritic kidneys of other lupus models [51, 52]. IgG deposits were observed in four of four lyn–/–IL-21–/– kidneys examined (Fig. 8B and Supporting Information Fig. 6), likely due to residual autoreactive IgG against non-DNA Ags (Fig. 5). Tubular interstitial nephritis was minimal, although mildly elevated (Supporting Information Fig. 6). These results are consistent with a predominant role for immune complex-mediated

kidney damage. IL-21 is associated with lupus in both humans and mice [18, 29-36]. While IL-21 mRNA is not significantly elevated in Lyn-deficient mice, several manipulations that reduce autoantibodies also dampen IL-21 expression. This suggested a role for IL-21 in the autoimmune phenotype of lyn–/– mice. Indeed, we show that IL-21 is required for IgG against Epothilone B (EPO906, Patupilone) DNA and some other, but not all, self-Ags in lyn–/– mice. However, IL-21 is dispensable for kidney damage in these animals. IL-21 could promote autoreactive B-cell class switching in two ways; by directly acting on B cells [18, 19, 21, 25-28], and/or by maintaining ICOS+CXCR5− and ICOS+CXCR5+ CD4+ T cells. These subsets are efficient B-cell helpers in extrafollicular and GC responses, respectively [29, 30]. Autoreactive B cells are likely activated in an extrafollicular response in lyn–/– mice. These animals fail to form GCs, either spontaneously or in response to immunization [4, 47, 48].

All subjects provided informed consent under the auspices of the appropriate research and ethics committees. CD4+ and CD8+ T cell counts were measured using a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA). A single-platform lyse-no-wash procedure was performed using Trucount tubes 17-AAG molecular weight and TriTEST anti-CD4-FITC/CD8-PE/CD3-PerCP reagents (BD). TruCOUNT Control

Beads (low, median and high beads; BD) were used to control the quality and accuracy of the CD4+ T cell true count test. HIV RNA in plasma was measured by RT-PCR using the COBAS Amplicor HIV Monitor 1.5 (Roche Molecular Systems, Branchbury, NJ, USA). The detection limit of the assay was from 400-copies/mL to 750 000 copies/mL.

The HIV RNA copy number was calculated based on the manufacturer’s reference standards. Peripheral venous blood samples were collected in EDTA-containing tubes. The blood samples were immediately stained and analyzed using a LSRII flow cytometer. find more A mixture of four antibodies, consisting of anti-CD3, anti-CD8, anti-NKG2A, and anti-NKG2D or anti-CD3, anti-CD8, anti-KIR3DL1, and anti-NKG2D, was used for staining. Phycoerythrin-Cy7-conjugated anti-CD3, peridinin-chlorophyll protein-conjugated anti-CD8 and allophycocyanin-conjugated anti-NKG2D were from BD Bioscience, while phycoerythrin-conjugated anti-NKG2A and phycoerythrin-conjugated anti-KIR3DL1 were from R&D Systems (Minneapolis, MN, USA). The appropriate antibody isotypes were used for multicolor compensation and as negative controls for gating. Rainbow Beads were used for daily quality control of the flow cytometer. Events were collected in the different lymphocyte gates and analyzed. CD8+ T cells were defined as CD3+CD8+ cells, while CD4+ T cells could only be analyzed indirectly by gating of the CD3+CD8− population (22). The gating strategies used to identify NKRs on T cell populations are depicted in Figure 1. CD3+CD8+ or CD3+CD8− cells were analyzed for surface expression of NKG2D, NKG2A, and KIR3DL1. Analyses were

performed using SPTLC1 GraphPad Prism software. The nonparametric Kruskal–Wallis test was used followed by the Dunn post-test to compare four groups. Correlations between variables were evaluated using the Spearman’s rank correlation test. P < 0.05 was considered significant. In the present study, CD4+ T cell counts were used to categorize individuals into four different groups, after which the absolute number of CD8+ T cells of each of the groups was determined. CD8+ T cell counts were higher in the HIV group than in the HIV-negative normal control group (P < 0.05), while in the AIDS group, CD8+ T cell counts were similar to that of the normal controls. Meanwhile, there were no significant differences in CD8+ T cell counts among the normal control group, the AIDS group and the HAART group (Fig. 2a).

This murine model is at present the only one reported to recapitulate the IFN signature in peripheral blood (PB) and, similar to its proposed role in human SLE, IFN signaling is required for the production of pathogenic autoantibodies and glomerulonephritis [[25]]. As such, we assessed changes in immune status associated with Irf5 loss in this model. KU-60019 ic50 Autoantibodies directed against nuclear components, such as

DNA/protein or RNA/protein macromolecular complexes, are a diagnostic feature of SLE and contribute to disease pathogenesis [[1]]. Pristane induces the production of lupus autoantibodies ∼4–6 month postperitoneal injection [[27]]. At 10 months postinjection, Savitsky et al. reported a decrease in antinuclear antibodies (ANAs) in the sera of pristane-injected Irf5−/− mice that was, in part, due to a decrease in anti-dsDNA and anti-Sm IgG2a and IgG2b lupus autoantibodies [[24]]. We observed a similar SCH 900776 decrease in sera ANAs 6 months postinjection by HEp-2 immunostaining (Fig. 1A); ten of 12 Irf5−/− mice had no detectable ANA

staining while the remaining two lacked cytoplasmic staining and gave weak positive homogenous nuclear staining (data not shown). To extend upon the repertoire of lupus autoantibodies that may be affected by loss of Irf5, we analyzed additional autoantibodies (anti-Ribosomal Phosphoprotein P0 (anti-RiboP0), anti-U1A, anti-sn RNP BB′ (RNP BB′), and anti-histone)

that are present in pristane-induced SLE [[25, 28]]. This analysis confirmed a marked reduction in IgG autoantibody levels of Irf5−/− mice targeted against a variety of autoantigens (Fig. 1B). Furthermore, we show that IgM autoantibodies are unaffected by loss of Irf5. Pristane-induced lupus is associated with hypergammaglobulin-emia and marked polyclonal B-cell activation [[29]]. In mice, IgG2a/c autoantibodies are considered to be the most Fossariinae pathogenic, while IgG1 displays the poorest pathogenicity [[30]]. Of the total sera IgG produced in response to pristane, IgG2a/c predominates, with relatively smaller differences observed in IgG1 levels between pristane- and PBS-injected mice [[31]]. Examination of total serum IgG subclasses (IgG1, IgG2a/c, IgG2b, and IgG3) in wild-type and Irf5−/- mice revealed significant decreases in both IgG2a/c and IgG2b levels of Irf5-deficient mice; in addition, we observed a striking increase in IgG1 levels of Irf5−/− mice (Fig. 2A). The decrease in total IgG2a/c and IgG2b levels correlated with significant decreases in specific lupus autoantibodies (Supporting Information Fig. 1A). T cells are required for IgG1 and IgG2a/c hypergammaglobulinemia in pristane-injected mice [[32]]. While data in Fig.

Importantly,

we found that proinflammatory cytokines may be dysregulated by a decreased STAT5. STAT5 normally stimulates an inflammatory response during bacterial infection [[36]]. Park et al. [[37]] have shown that Cav1 is a negative regulator of JAK2/STAT5a signaling in the mammary gland. This negative regulation may occur through direct molecular interaction owing to structural homology between Cav1 and SOCS-1 or SOCS3 [[38]]. Our data suggest that the GSK3β−β-catenin−Akt axis may be related click here to a decreased STAT5 profile, making a connection from Cav1 deficiency to the exacerbated inflammatory response. Although the above research begins to hint at some important answers, it is not known why decreased STAT5 functionality leads to an increased proinflammatory cytokine profile. Previous reports have shown that Akt can connect

to STAT5 and regulate neuroprotective activity or cancer development [[39]]. However, little is known as to the specific functions of the GSK3β−β-catenin−Akt axis in bacterial infection. We hypothesized that decreased STAT5 may be regulated by changes in GSK3β or from the loss of Akt/β-catenin activity (at middle or late phases of infection), since our in vitro assays indicated an increase in pSTAT5 at early phases of infection. Following PIP3 and PI3K activation, Akt activation is required to regulate apoptosis against LPS or other oxidants [[40]], which could also be associated with a heightened inflammatory response. Akt is negatively regulated under Cav1 deficiency, while GSK3β is upregulated. INCB024360 supplier As feedback, Akt can inhibit GSK3β, thereby reducing the negative regulation of GSK3β in cellular processes. We assumed that an excessive inflammatory response and inefficient apoptotic clearance of dead cells lead to severe lung injury. Thus, an interaction between Akt and Cav1 may broadly impact the cytokine production and disease process.

Downregulation of Akt and STAT5 was initiated to counteract the loss of Cav1, but failed to eradicate the invading bugs. As a result, IL-6 and related cytokines could not be properly controlled by feedback signaling, next contributing to the severe infection seen in cav1 KO mice. In summary, our studies illustrate a typical phenotype in cav1 KO mice following K. pneumoniae infection, characterized by increased bacterial burdens in the lung, decreased survival, severe lung injury, and increased inflammatory response. Furthermore, the increased impairment of the immune system in these KO mice is at least in part attributed to a regulatory function of the STAT5 pathway, which is, in turn, influenced by a GSK3β−β-catenin−Akt axis. Our studies have also characterized a novel role of Cav1 in infection resistance and explored its involvement with the Akt-STAT5 cross-talk, whose underlying mechanisms warrant further study. More specifically, our data may shed light on the pathogenesis of K. pneumoniae infection and suggest a novel therapeutic target.

As a consequence, LPS-treatment enhanced the migratory activity along a chemokine (CCL21)-gradient in WT, but not in TLR4-deficient BMDCs suggesting that the LPS/TLR4-induced BMS-777607 mouse swelling response facilitates DC migration. Moreover, the role of calcium-activated potassium

channels (KCa3.1) as putative regulators of immune cell volume regulation and migration was analyzed in LPS-challenged BMDCs. We found that the LPS-induced swelling of KCa3.1-deficient DCs was impaired when compared to WT DCs. Accordingly, the LPS-induced increase in [Ca2+]i detected in WT DCs was reduced in KCa3.1-deficient DCs. Finally, directed migration of LPS-challenged KCa3.1-deficient DCs was low compared to WT DCs indicating that activation of KCa3.1 is involved in LPS-induced DC migration. These findings suggest that both TLR4 and KCa3.1 contribute to the migration of LPS-activated DCs as an important feature of the adaptive immune response. Dendritic cells (DCs) are the most potent antigen-presenting cells that play a key role in regulating T-cell-mediated adaptive immune responses [1]. Immature DCs placed in peripheral tissues act as sensors for microbial pathogens, stress, or inflammatory signals. Uptake of antigens or exposure to inflammatory stimuli AZD1208 clinical trial at peripheral sites causes maturation of DCs including the up-regulation of MHC and co-stimulatory

molecules and the conversion to a migratory phenotype [1]. Migration of DCs to the draining lymph nodes and presentation of the antigen to T cells can initiate a protective immune response or promote regulatory T cell responses that help to maintain tolerance against the antigen [2]. Recognition of LPS, a cell wall component of gram-negative bacteria by DCs is mediated mainly by Toll-like receptor

(TLR) 4 [3, 4]. Binding of LPS to TLR4 causes maturation and migration of DCs [5]. However, the underlying mechanisms of LPS-induced DC migration are not well understood. In DCs stimulated with LPS dissolution of cell adhesion structures in a TLR4-dependent manner has been described [6] suggesting that TLR4 signaling and actin-driven cytoskeletal rearrangement are involved Liothyronine Sodium in LPS-induced DC migration. Additionally, it has been demonstrated that ion channels contribute to the conversion of DCs towards a migratory phenotype [7]. Accordingly, DCs respond to LPS with a fast increase in free cytosolic calcium ions originating from both intracellular and extracellular calcium stores [7]. Moreover, activation of voltage-gated potassium channels (Kv1.3 and Kv1.5) and sustained increase in [Ca2+]i via store-operated calcium channels (ICRAC) have been shown to play an important role for LPS-induced DC maturation and migration [7]. In addition to voltage-gated K+ channels several members of Ca2+-activated K+ channels like BK (KCa1.1), SK3 (KCa2.3), and in particular SK4 (KCa3.1, IK1, KCNN4) are involved in cell migration [8].

An organism’s environment is ultimately as unique as its genetic code. The current wave of interest in the gut microbiota and host–microbe interactons in health and disease has been accelerated in large part by technological advances, including molecular methods, such as metagenomics

and compositional sequencing. These have facilitated the study of mixed microbial communities, particularly the non-cultivable sector, and have revealed greater microbial diversity in the gut, in health and disease, than contemplated previously [2]. Of the other key drivers of research interest in host–microbe interactions in the gut, the discovery of Helicobacter pylori as a cause of peptic ICG-001 supplier ulceration and gastric cancer provided the most salutary lessons. First, it showed that successive generations of epidemiologists missed https://www.selleckchem.com/products/PD-0325901.html the involvement of a transmissible agent in such a common disease. Perhaps this reflects the limitations of traditional

epidemiological approaches, described by one critic as ‘risk-factor epidemiology’ without rapprochement with concepts of disease mechanisms [3]. How many other chronic disorders are due to infections waiting to be discovered? The second lesson was that generations of biologists also missed the essential participation of an infectious component to the pathogenesis of disease. Arguably, this was due to a lack of convergent thinking or scientists capable of latitudinal thinking across the artificial boundaries of disparate research disciplines. Thirdly, it showed that

a single microbial agent can underlie seemingly complex and heterogeneous chronic diseases, and that regardless of variations in host genetic susceptibility, a lasting solution can be secured if an essential environmental trigger is eliminated. Finally, and most importantly, the story of H. pylori and peptic disease showed that some diseases can never be solved by research focused exclusively upon the host response, without due consideration of the interface between the human and microbial components of what is, in fact, a composite super-organism. The major milestone in inflammatory bowel disease research within the past decade has been the discovery that genetic risk factors for Crohn’s Ibrutinib disease include mutant genes which normally code for proteins that are either sensors of the microbial environment [e.g. as nucleotide-binding oligomerization domain/caspase-recruitment domain (NOD2/CARD15)] or are regulators of host responses to the microbiota [e.g. interleukin (IL)-23R, autophagy][4,5]. However, regardless of genetic susceptibility, the relative contribution of lifestyle or environmental factors is shown by the abrupt increase in frequency of Crohn’s disease and ulcerative colitis in modern societies and by the concordance rate for these conditions in monozygotic twins (less than 50% in Crohn’s disease and less than 10% for ulcerative colitis) [6,7].

Although the baseline characteristics of the participants were similar, both groups showed a significant reduction in pain level and hyperaemia on the tongue mucosa (P = 0.000) after 4-week application. However, despite the reduction in hyperaemia

in the probiotic group, these improvements did not display statistically significant differences. The detection rate of Candida spp. was 100% before treatment and 8.21% in the experimental group and 34.6% in the control group after treatment. The detection rate of Candida spp. decreased (P = 0.000) in both groups and was significantly lower in the probiotic group than the control group (P = 0.038). Other analysed micro-organisms, including the decreased detection rate for Lactobacillus spp. (P = 0.049) and the increased detection rate for Staphylococcus LY294002 in vivo epidermidis (P = 0.019), did not display consistent change trends in the probiotics group. Compared with conventional antifungal

therapies for oral candidiasis, AZD4547 in vivo the inclusion of locally administered probiotics helped improve certain clinical conditions and reduced the prevalence of Candida spp., although the impact of probiotics on oral bacterial species remains to be further studied. “
“Faculty of Medicine, University of Ottawa, Roger Guindon Hall, ON, Canada Yeast are among the most frequent pathogens in humans. The dominant yeast causing human infections belong to the genus Candida and Candida albicans is the most frequently isolated species. However, several non-C. albicans species are becoming increasingly common in patients worldwide. The relationships between yeast in humans and the natural

environments remain poorly understood. Furthermore, it is often difficult to identify or exclude the origins of disease-causing yeast from specific environmental reservoirs. In this study, we compared the yeast isolates from tree hollows TCL and from clinics in Hamilton, Ontario, Canada. Our surveys and analyses showed significant differences in yeast species composition, in their temporal dynamics, and in yeast genotypes between isolates from tree hollows and hospitals. Our results are inconsistent with the hypothesis that yeast from trees constitute a significant source of pathogenic yeast in humans in this region. Similarly, the yeast in humans and clinics do not appear to contribute to yeast in tree hollows. “
“Tinea capitis in postpubertal patients is unusual and may be misdiagnosed as dissecting cellulitis. We report a case of a healthy 19-year-old Hispanic male presenting with a 2-month history of a large, painful subcutaneous boggy plaque on the scalp with patchy alopecia, erythematous papules, cysts and pustules. Although initially diagnosed as dissecting cellulitis, potassium hydroxide evaluation (KOH preparation) of the hair from the affected region was positive.

2, 21.8 ± 3.3, 22.0 ± 3.3 (Prend = 0.079); eGFR (mL/min per 1.73 m2) 90 ± 15, 92 ± 16, and 90 ± 15 (Ptrend = 0.082); serum uric acid (mg/dL), 5.0 ± 1.4, 5.1 ± 1.4, and 5.3 ± 1.4. During median 2.9 yr selleck chemical (1.5–4.2) of the observational period, 301 (8.4%),

272 (8.9%), and 144 (10.7%) participants developed proteinuria and their incidence rate per 1000 person-year were 28.3 (95% confidence interval 25.2–31.7), 31.2 (27.6–35.2), and 37.4 (31.6–44.1), respectively (Ptrend = 0.007) (Figure). A multivariate-adjusted Poisson model identified ≥2 drinks/day as a significant predictor of proteinuria (vs. 0 drink/day; 1 drink/day, incidence rate ratio 1.03 (95% confidence interval 0.88–1.22), P = 0.698; 2 drinks/day, 1.28 (1.05–1.56), P < 0.015). Conclusion: Excessive soft drink consumption (≥2 drinks/day) predicts the incidence of proteinuria. KUMA AKIHIRO, TAMURA MASAHITO, HARUKI NOBUHIKO, MIYAMOTO TETSU, SERINO RYOTA, TAKEUCHI MASAAKI, OTSUJI YUTAKA The Second Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health Introduction: It is particularly difficult to treat heart failure (HF) accompanied by chronic kidney disease (CKD). A recent study reported that CKD patients were also more likely to have

sleep apnea syndrome (SAS). Adaptive servo ventilation (ASV) has been indicated that it is effective Selleck Cabozantinib for treating and managing HF with SAS. So here we investigated whether ASV has benefits for cardiac and renal functions. Methods: This single-center retrospective study was conducted enough with non-replacement CKD patients. We selected a subgroup with drug-refractory

HF and SAS. ASV group patients received ASV (>4 hr/day) for 6 months and control group patients were received medication against for HF, but not ASV therapy. Changes in cardiac and renal function were assessed using the Wilcoxon signed-rank test and correlations by Spearman’s rank correlation. Results: ASV group (n = 23) comprised 16 males (mean age, 66.8 ± 12.2 years) and control group (n = 14) comprised 11 males (mean age, 69.1 ± 14.6 years). Estimated glomerular filtration rate (eGFR; median) of ASV group was 41.8 ml/min per 1.73 m2 before ASV therapy (0 M), 51.5 ml/min per 1.73 m2 1 month after ASV therapy (1 M) (p = 0.0071 vs 0 M), and improved eGFR was maintained for 6 months (6 M; 48.4 ml/min per 1.73 m2) (p = 0.5545 vs 1 M). However eGFR of control group was 49.40 (0 M), 49.45 (1 M) (p = 0.4703 vs 0 M), and tended to decrease for 6 months (6 M; 42.45) (p = 0.0596 vs 0 M). Left ventricular ejection fraction (LVEF; median) of ASV group was 29.1% (0 M), 38.2% (1 M) (p = 0.0019 vs 0 M), and no further change at 6 M (38.5%; p = 0.2166 vs 1 M). LVEF of control group was 40.0% (0 M), 37.6% (1 M) (p = 0.8993 vs 0 M), and no change at 6 M (34.5%; p = 0.7741 vs 1 M). In ASV group, no correlation was observed between baseline eGFR and the improvement in LVEF (0–6 M) (ρ = 0.1610, p = 0.4856).

5). RT–PCR analysis showed significantly elevated MHC-II expression levels in the spinal cords at 16 dpi Alectinib purchase EAE mice compared to CFA mice (P < 0·05). In the spinal cords of EAE mice, MHC-II expression peaked at 16 dpi compared to levels observed at 7 dpi (P < 0·01) and 28 dpi (P < 0·05) (Fig. 4a,b). In order to strengthen the observations in RT–PCR, real-time PCR was employed to determine MHC-II mRNA levels in the spinal cord. The data shown were normalized to GAPDH expression, and the expression levels in the CFA group were set to 1. As shown in Fig. 4c, MHC-II mRNA level

was promoted significantly in the spinal cords at 16 dpi EAE mice compared to either 7 dpi (P < 0·001) or 28 dpi (P < 0·01). MHC-II expression levels were correlated positively with disease progression and IFN-γ production levels in the spinal cord. Double-labelled immunofluorescence staining was employed to localize MHC-II expression on astrocytes. Spinal cords harvested from EAE mice presented with undetectable MHC-II expression levels on astrocytes at 7 dpi, peaked at 16 dpi and then expression was learn more down-regulated at 28 dpi (Fig. 5). MHC-II expression could not be detected on astrocytes

harvested from mice in the CFA group (data not shown). For proliferation assay, astrocytes were treated with different concentrations of IFN-γ ranged from 0 to 200 U/ml for 24 h. They were then co-cultured with lymph node lymphocytes at a lymphocyte : astrocyte ratio of 10:1. Proliferation of lymphocyte was promoted when co-cultured with IFN-γ-treated

astrocytes (P < 0·001). These data indicate that IFN-γ-treated astrocytes could promote the proliferation of MOG35–55-specific lymphocytes (Fig. 6a). Bay 11-7085 For cytokine production assay, astrocytes were treated with 100 U/ml IFN-γ for 24 h. They were then co-cultured with lymph node lymphocytes at a lymphocyte : astrocyte ratio of 10:1. Supernatants were harvested 72 h later and cytokine levels were determined by ELISA. IFN-γ levels in the supernatants of EAE lymphocytes and IFN-γ-treated astrocytes in the co-culture group were elevated significantly (P < 0·001). Levels of IL-4, IL-17 and TGF-β were also elevated compared to levels observed in supernatants from EAE lymphocytes cultured alone. There were no significant differences in cytokine production levels by cells harvested from mice in the CFA group. Levels of the cytokines described above were low in the supernatants of astrocytes cultured (without lymphocytes) in the presence of IFN-γ (Fig. 6b). Astrocytes were treated in the presence or absence of 100 U/ml IFN-γ for 24 h and then co-cultured with lymphocytes at a lymphocyte : astrocyte ratio of 10:1 for 72 h. Total astrocyte RNA was extracted and MHC-II mRNA levels were detected by real-time RT–PCR.