“
“The objective of this study is to evaluate urinary high mobility

group box 1 (HMGB1) levels as markers for active nephritis in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in comparison with urinary CD4+ effector memory T cells and urinary monocyte chemoattractant protein-1 (MCP-1). Twenty-four AAV patients with active nephritis and 12 healthy controls (HC) were evaluated. In nine patients, samples were also obtained during remission. Urinary levels of HMGB1 were measured by Western blot. CD4+ T cells and CD4+ effector memory T cells find more (CD4+CD45RO+CCR7-) were determined in urine and whole blood by flow cytometry. Measurement of urinary levels of MCP-1 and serum HMGB1 levels were performed by enzyme-linked immunosorbent assay (ELISA). AAV patients with active nephritis had higher median intensity of HMGB1 in urine than HC [10·3 (7·05–18·50) versus 5·8 (4·48–7·01); P = 0·004]. Both urinary HMGB1 and MCP-1 levels decreased significantly from active nephritis to remission. The urinary MCP-1/creatinine ratio correlated with Birmingham Vasculitis Activity Score (BVAS) (P = 0·042). No correlation was found between the HMGB1/creatinine ratio and 24-h proteinuria, estimated glomerular filtration rate (eGFR), MCP-1/creatinine ratio, BVAS and serum HMGB1. A positive correlation was found between urinary HMGB1/creatinine ratio and CD4+

T cells/creatinine ratio (P = 0·028) and effector memory T cells/creatinine ratio (P = 0·039) in urine. Urinary HMGB1 levels are increased in AAV patients with active nephritis when selleck chemical compared with HC and patients in remission, and urinary HMGB1 levels are associated

with CD4+ T cells and CD4+ effector memory T cells in urine. Measurement of urinary HMGB1 may be of additional value in identifying active glomerulonephritis in AAV patients. “
“IFN-γ-activated keratinocytes are key contributors to the pathogenetic mechanisms leading to type-1 immune-mediated skin disorders. In these epidermal cells, SOCS1 negatively regulates the molecular cascades Janus kinase (JAK) triggered by IFN-γ by disabling JAK2 phosphorylation through its kinase inhibitory region (KIR). Aimed at potentiating the SOCS1 inhibitory function on JAK2/STAT1 axis in keratinocytes, we recently developed a set of peptides mimicking the SOCS1 KIR domain, which are capable of efficiently binding JAK2 in vitro. Here, the effects of one such SOCS1 KIR mimetic named PS-5 on IFN-γ-activated human keratinocytes were evaluated. We found that IFN-γ-activated keratinocytes treated with PS-5 exhibited impaired JAK2, IFN-γRα, and STAT1 phosphorylation. We also observed reduced levels of the IRF-1 transcription factor, and a strong reduction in ICAM-1, HLA-DR, CXCL10, and CCL2 inflammatory gene expression. ICAM-1 reduced expression resulted in an impaired adhesiveness of T lymphocytes to autologous keratinocytes.

4). This can be due to Daporinad solubility dmso a reduced apoptotic activity in Lcn2−/− mice as reported [6, 17] or an overwhelming growth of bacteria in Lcn2−/− mice leading to increased PMNs mobilization over time despite mechanistically problems. The current paradigm of leukocyte migration suggests that following selectin-induced rolling neutrophils are activated by chemokines, resulting in a conformational change of β2 integrins to their active form [39]. This results in neutrophil adhesion to the epithelium and allows the transendothelial migration of these cells. Leukocytes

are then guided to the sites of inflammation by chemotactic factors. The results presented herein suggest that Lcn2 is one of these important chemoattractants

by stimulating PMN migration and adherence. In addition, recent data indicate that different composition of leukocyte subset result in alterations of circulating lipocalin levels [40, 41], which is in a line with a role of Lcn2 as a regulator for the proliferation of hematopoetic cells [42]. In summary, the production of Lcn2 by PMNs and epithelial cells appears to be an important and immediate effector pathway of innate immune function by attracting PMNs and likewise also monocytes to the sides of infection or tissue damage. C57BL/6 WT male mice and C57BL/6 Lcn2 KO (6–8 weeks) male mice were kept on standard rodent diet (C2010 Altromin, Munich, Germany). The animals had free access to food and water and were kept according institutional and governmental guidelines in the KU-60019 supplier quarters of Medical University of Innsbruck with a 12 h dark–light cycle and an average temperature of 20 ± 1°C. The animal experiments were approved by the Austrian Federal Ministry of Science and Research (BMWF-66.011/0011-II/10b/2010). PMNs were obtained

by peripheral blood of healthy volunteers by Ficoll density gradient centrifugation, followed by dextran sedimentation and hypotonic lysis of contaminating erythrocytes. Cell preparation yielded >95% neutrophils (by morphology in GIEMSA stains) with a viability of >99% (estimated by trypan blue exclusion). Heparin-anticoagulated blood Atezolizumab of three to four mice was pooled and used for PMNs isolation with Histopaque-1083 and Histopaque-1119 (Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s protocol with small modifications. In brief, 1.5 mL of Histopaque-1119 was added to a 1.5 mL conical centrifuge tube, 1.5 mL Histopaque-1083 was layered onto Histopaque-1119 and 3 mL of pooled blood was carefully layered onto the upper gradient of the tube. The tube was centrifuged at 700 × g for 30 min at 24°C. Two distinct opaque layers can be observed after centrifugation, of which the second one represents PMNs.

Hence, immunoregulation may revolve around highly specific host–microbial molecular interactions, presumably reflecting a long and intimate co-evolution of the symbiotic relationship. The vitamin A metabolite, retinoic acid (RA), plays a major role in the GI tract, via its capacity to enhance the TGF-β-mediated generation of forkhead box P3 (FoxP3+) Tregs from naive T cells by gut DCs [42]. Reciprocally, RA can inhibit the generation of Th17 cells [43], suggesting that it may play an important role in maintaining the balance between effector and regulatory populations in the GI tract. Several populations of mucosal APC can induce Tregs via RA,

although only the CD103 subset is equipped with the enzymatic machinery to generate RA. Retinoic acid can also imprint gut homing this website molecules on various populations of lymphocytes. Defined microenvironments may have evolved self-contained strategies in which local mediators (such as RA) can imprint homing properties while also favouring the induction or function of Tregs. It is therefore tempting to speculate Angiogenesis inhibitor that a link between homing and regulatory function induction may represent a more general mechanism.

Such a strategy could allow the constant generation and migration of Tregs to defined compartments. These Tregs would be expected to have the prerequisite antigen specificities (e.g. persistent microorganisms, flora antigens), status of activation and survival requirement that Exoribonuclease allow them to regulate a defined microenvironment. Although the capacity of gut-associated lymphoid tissue (GALT) DCs or macrophages to imprint gut-homing receptors and induce FoxP3+ Tregs is associated with their capacity to release RA, it remains unclear if these cells are the main producers of this metabolite in the gut. Synthesis of RA from stored or dietary retinol depends on

the direct expression of the appropriate enzymes by GALT DCs. Certainly, DCs from Peyer’s patches and mesenteric lymph nodes (MLNs) express Aldh1a1 and Aldh1a2, respectively, and CD103+ DCs from the lamina propria express a large array of this family of enzymes; moreover, Peyer’s patch and MLN DCs can convert retinol directly to RA in culture. However, other cells, including IELs, can express enzymes associated with vitamin A metabolism, suggesting that DCs may also acquire retinoic acid from other sources and store it. A recent study demonstrated that monocyte-derived DCs pretreated with RA can acquire several attributes characteristic of mucosal DCs, such as secretion of TGF-β and IL-6, and the capacity to augment mucosal homing receptor expression and IgA responses in lymphocytes [44]. In this particular study, these gut-derived features acquired by DCs were associated with the capacity of DCs to become carriers and not producers of RA.

His group had shown earlier that the CD3 subunits of the αβ TCR undergo a conformational change only upon multivalent antigen-binding to the TCR, and that this change is required for CD3 phosphorylation [13]. Based on these findings they now used a combination of pMHC tetramer-TCR binding data and mathematical modelling, which suggested that the necessity of multivalent binding contributes to the distinction

of low from high affinity pMHC ligands for the αβ TCR. Asking whether CD3 subunits of the γδ TCR undergo this conformational change, Elaine Dopfer (Freiburg, Germany) demonstrated that stimulation with some anti-CD3 antibodies, but not others, leads to this structural change in human γδ TCRs. However, and in contrast to all αβ TCR-pMHC interactions, the binding of the MHC-like T22 molecule to murine γδ G8 TCR does not result in the CD3 conformational change. Thus, the G8 TCR may be activated by a different mechanism than BAY 80-6946 purchase the αβ TCRs. Whether this holds true for other γδ TCRs is currently unclear. To investigate the impact of this CD3 structural change in vivo, Balbino Alarcón (Madrid, Spain) generated a mutant CD3ε knock-in mouse

strain, in which CD3 cannot undergo this change. αβ T cells in these mice display a complete block at the DN3 stage, suggesting that the pre-TCR also needs the conformational change for active signalling. Likewise, some γδ T-cell subsets (such as Vγ2+) are completely absent, whereas others (such as Vγ1.1+) are present in normal numbers, suggesting distinct requirements for the TCR conformational change among γδ T-cell subsets. Riitta Lahesmaa (Turku, MK-8669 Finland) presented a holistic systems biology approach using state-of-the-art transcriptomics to identify the genes that are up- or downregulated

during human T-cell differentiation. Purified primary cord blood (naïve) CD4+ T cells that were differentiated in vitro into Th1, Th2 or Th17 lineages were used to examine the PIM kinases that are upregulated during Th1 differentiation and that lead to the activation of the Th1 promoting pathways IFN-γ/T-bet and IL-12/STAT4. Building on Casein kinase 1 the well-established anti-CMV function of human γδ T cells, two independent groups — Michael Mach (Erlangen, Germany) and Myriam Capone (Bordeaux, France) — developed mouse models to study new aspects of the γδ T-cell response to mouse cytomegalovirus (MCMV). They both demonstrated, using distinct experimental set-ups, that γδ T cells are a key component of the (largely redundant) anti-viral T-cell effector compartment. Moreover, γδ T cells are uniquely capable of killing MCMV-infected cells ex vivo, and their adoptive transfer in vivo significantly reduces viral titers in all organs examined, ultimately saving the recipient animals from the lethal course of infection. Gang Qin and Wenwei Tu (Hong Kong) established chimeric humanised mouse models to investigate the γδ T-cell response to human and avian influenza infections.

We analyzed the effect of IQGAP1 knockdown on actin and MT of confluent EC. The results indicate that IQGAP1 knockdown in EC monolayers decreases MT captured at the interendothelial junctions and decreases lymphocyte diapedesis. Further, drug-induced MT depolymerization decreases paracellular lymphocyte diapedesis. These results indicate that endothelial IQGAP1 tethers MT to interendothelial junctions and participates in junction remodeling during lymphocyte TEM. IQGAP1 has been shown to colocalize with AJ cadherin complex and regulate cadherin-mediated cell–cell

adhesion 24, 26, 27. In EC, we observed IQGAP1 enrichment at the interendothelial junctions (Fig. 1B). To study the role of EC IQGAP1 in lymphocyte TEM, endothelial IQGAP1 expression was inhibited by RNAi. IQGAP1 siRNA transfection of HUVEC consistently reduced IQGAP1 protein expression more than 80% (Fig. 1A–C). However, confluent Wnt inhibitor IQGAP1-knockdown EC monolayers developed normal AJ, reflected by β-catenin (Fig. 1E) and VE-cadherin (Fig. 2D) localization at the junctions, similar to the control monolayers (Figs. 1D and 2C). Further, analysis of cell surface expression of VE-cadherin and PECAM-1 by flow cytometry identified no change in IQGAP1-knockdown versus control cells (data not shown). Functionally,

electrical impedance across an IQGAP1-knockdown versus the control monolayer was unchanged (data not shown). ABT-263 purchase Taken together, these data indicate that IQGAP1 is not required for the surface expression or assembly of endothelial junction components. Next, we sought to characterize the effect of IQGAP1 knockdown on EC cytoskeletal

components since IQGAP1 regulates dynamic filamentous-actin (F-actin) polymerization 23, 35, 36 and MT capture at the cell cortex 21–23. Biochemical analysis of free and polymerized tubulin within EC determined IQGAP1 knockdown decreased the ratio of polymerized tubulin to free tubulin levels in the cytosolic extracts selleck chemicals (Fig. 2A and B). Further, measurements of MT density underlying junctions by immunofluorescent double-staining of VE-cadherin and tubulin indicated that tubulin fluorescence intensity per μm2 area adjacent to the VE-cadherin band among IQGAP1 knockdown EC (Fig. 2D and C) decreased by ∼40% (Fig. 2E). These data indicate that IQGAP1 knockdown induced loss of polymerized MT at the interendothelial junctions. To evaluate the effect of IQGAP1 knockdown on the actin cytoskeleton of confluent EC, the population of F-actin and globular-actin (G-actin) in cells was measured. Quantification of results by densitometry did not show any effect in F-actin content by IQGAP1 knockdown (Fig. 2F). Consistent with the biochemical assay, F-actin distribution did not change between IQGAP1 knockdown cells versus control cells by immunofluorescence microscopy (Fig. 2G and H).

, 2000) and this has an impact on the PK/PD

parameters of biofilm killing. The PK/PD parameter for the beta-lactam killing of biofilms formed by P. aeruginosa expressing low basal levels of beta-lactamase is, as for planktonically grown cells, the time above MIC but higher concentrations of antibiotics and longer periods of action are required to eliminate biofilm compared with planktonically grown cells (Hengzhuang et al., 2011, 2012). Continuous administration of ceftazidime would thus be better for biofilm treatment, which in this way will be exposed for longer to concentrations above the MIC (T > MIC). Compared with intermittent infusion, continuous infusion at normal daily doses is more likely to achieve optimal T > MIC PD goals for intermediate and borderline resistant organisms with find more an MIC of ceftazidime up to 16 mg L−1 (Prescott

et al., 2011). Although the results of studies comparing the efficacy and safety of continuous-infusion and intermittent-infusion antipseudomonal BGB324 solubility dmso beta-lactam therapy are promising, there is insufficient evidence to recommend continuous infusion for routine use. However, continuous-infusion dosing with ceftazidime does appear to be a reasonable option for patients who have not responded to traditional dosing methods or who have multidrug-resistant P. aeruginosa isolates. In the case of biofilms formed by P. aeruginosa expressing high basal levels of beta-lactamase,

a concentration-dependent killing of the biofilm was observed, supporting the idea of impaired penetration of beta-lactam antibiotics in the biofilm due to inactivation of the beta-lactam molecules by hydrolysing enzymes (our unpublished data). A similar effect was observed in biofilms of nfxB mutants of P. aeruginosa which show an increased PI-1840 extracellular level of AmpC beta-lactamase that impaired biofilm killing (Mulet et al., 2011). Treatment with beta-lactamase-stable compounds such as meropenem or combinations with beta-lactamase inhibitors might improve penetration of the drug into the biofilm and ensure a better effect of treatment with beta-lactams. This effect was observed in vitro during treatment of biofilm-grown P. aeruginosa with combination ceftazidime and aztreonam (Hoiby et al., 2010), probably because aztreonam acts as a beta-lactamase inhibitor (Giwercman et al., 1992), and with meropenem (Moskowitz et al., 2004; Hill et al., 2005). Efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY, which play an important role in the resistance to antibiotics of planktonic P. aeruginosa, have been considered to have no impact on biofilm tolerance (De Kievit et al., 2001). However, recent studies are starting to modify this perception, as it has been suggested that MexAB-OprM and MexCD-oprJ are involved in biofilm tolerance to the macrolide azithromycin (Gillis et al.

The presence and the expression of the transgene were identified in founder Selleckchem MDV3100 CalpTG mice by PCR and RT-PCR analysis, respectively 12. All CalpTG mice used in these studies were backcrossed into the C57BL/6 background more than nine generations. Full thickness tail skin grafts (∼1 cm2) from donor mice were transplanted onto the dorsal thorax of recipient mice and secured with a bandage for 7 d. Graft survival was assessed by daily visual inspection, and rejection was defined as the 90% loss of viable tissue grafts. Where

indicated, WT recipients of skin graft received a daily i.p. injection of the specific calpain inhibitor PD150606 (Calbiochem) at the dose of 3 mg/kg BW or the vehicle alone (DMSO 0.3%). At the time of skin transplantation,

RAG-1−/− mice were reconstituted intravenously with 107 lymphocytes purified from the spleen of either WT or CalpTG mice and resuspended in 200 μL phosphate-buffered saline. Paraffin-embedded sections of the human kidney tissue (3 μm thick) were fixed and incubated with 5% normal goat serum to block non-specific binding. After blockade of endogenous peroxidase, the sections were immunostained with polyclonal antobodies for μ-calpain (H-65, Santa Cruz) or CD3 (Dako) at 1/100 dilution, which were revealed by goat anti-rabbit IgG at 1/2000 dilution, and counterstained with hematoxylin. Four-micrometer-thick cryostat sections of skin graft tissue were fixed with acetone for 4 min. buy Abiraterone After blockade of endogenous peroxidase, they were stained

with hematoxylin and immunostained with primary antibodies for CD3 (Serotec), CD4 (BD Pharmingen), CD8 (Serotec), NK (BD Pharmingen), and F4/80 (Serotec). The number of allograft-infiltrating CD3+, CD4+, and CD8+ T cells in WT and CalpTG mice was counted in four high-power fields (HPFs) per skin allograft section. Four-micrometer-thick cryostat sections of human kidney tissue were fixed with acetone for 4 min. They were immunostained with primary antibodies for CD3 (Dako) at 1/200 dilution and μ-calpain (Santa Cruz) at 1/100 dilution, which were revealed by anti-rabbit antibody (Alexafluor, Invitrogen) at 1/1000 dilution and anti-goat antibody (Alexafluor, Demeclocycline Invitrogen) at 1/1000 dilution, respectively. Confocal microscopy was performed using a Leica TCS laser scanning confocal microscope (Lasertechnik, GmbH, Wetzlar, Germany). Spleen CD3+ T cells (5×105) from WT and CalpTG mice were incubated in the upper chamber of Transwell 5 μm pore size filters (Costar) and 20 ng/mL recombinant mouse MCP-1 (R&D) or 100 ng/mL recombinant mouse SDF-1 (R&D) were added in lower chamber. After 4 h, cells were fixed in frozen methanol and cells that migrated from the upper to the lower chamber were counted at 200×magnification after violet crystal staining. Results are presented as the average number of cells migrated per HPF.

4%, 8 h after UV treatment (Fig. 1B). Therefore, we chose to use cells immediately after UV treatment as apoptotic DC for further experiments. Similarly, apoptosis was induced in splenocytes via UV radiation and 1 h after UV treatment, approximately 40% of splenocytes were annexin V+PI–, indicative of apoptotic

splenocytes (Fig. 1C). In order to assess the uptake of apoptotic DC by viable DC, apoptotic DC were labeled with CFSE and incubated with immature viable DC. Eight hours later, FACS analysis was performed to assess uptake of CFSE-labeled apoptotic DC by live DC (PI–CD11c+) (Fig. 2A). Results indicate that approximately 50% of viable DC had taken up apoptotic DC (Fig. 2). In order to confirm that there were no contaminating CFSE+ PI– apoptotic DC, a parallel experiment was performed where apoptotic DC were labeled progestogen antagonist with CFSE, cultured for 8 h, and subsequently stained with PI; approximately 98% of the DC were PI+ (data not shown), indicating that gating for PI– cells would gate out any CFSE+ apoptotic DC. Furthermore, in order to distinguish binding of apoptotic DC to live DC from uptake of apoptotic DC by live DC, the co-culture experiments were carried https://www.selleckchem.com/HDAC.html out in the presence of cytochalasin D,

a known inhibitor of phagocytosis (Fig. 2). In the presence of cytochalasin D, only 12% of the cells were CFSE+, which is probably indicative of apoptotic DC that bound to live DC. Collectively, the results indicate that immature viable DC have the ability to phagocytose apoptotic DC. In ADP ribosylation factor order to assess the effects of apoptotic or necrotic DC on viable DC, viable immature DC were incubated with mature apoptotic, immature apoptotic and necrotic DC. In order to generate mature apoptotic DC, bone-marrow-derived DC were treated with LPS for 24 h to induce maturation followed by exposure to UV radiation. Viable immature DC were

characterized as CD11c+ DC with low levels of CD86, CD80 and MHC II expression. LPS treatment of viable immature DC resulted in the upregulation of CD86, CD80 and MHC II (Fig. 3A). Furthermore, viable immature DC do not produce any IL-12; however, in response to LPS, approximately 30% of DC were IL-12+, as expected (Fig. 3B). However, treatment with immature or mature apoptotic DC did not result in the upregulation of CD86, CD80 or MHC II; nor was there any induction of IL-12 production. Similar results were also observed upon treatment of immature viable DC with necrotic DC. Taken together, these findings indicate that immature/mature apoptotic or necrotic DC do not induce maturation of viable immature DC. We next assessed the effects of uptake of necrotic/apoptotic DC by viable immature DC on subsequent treatment with LPS (Fig. 4). In the absence of inflammatory stimuli, viable immature DC express very low levels of CD86, with approximately only 20% cells being CD86+. This proportion increases to 50–60% upon treatment with LPS with a concomitant increase in the intensity of CD86 expression (Fig. 4B).

Importantly, no significant side effects have been reported so far, thus corroborating the apparent safety of sTRAIL treatment in humans. In addition, a number of agonistic antibodies (HGS-ETR1, HGS-ETR2, HGS-TR2J, LBY135, CS-1008, AMG 655) that selectively target TRAIL-R1 or TRAIL-R2 have been developed. All of these antibodies have potent tumouricidal activity in vitro and in vivo and appear to have a low toxicity profile in early-phase clinical studies Poziotinib cost [33,36–39]. An obvious difference between these TRAIL receptor-selective mAbs and TRAIL is the fact that TRAIL interacts with both of its agonistic receptors. This might provide TRAIL either with a wider

therapeutic spectrum or a narrower and more unpredictable therapeutic window, especially in light of its additional interaction with decoy TRAIL receptors. It is interesting to note that several groups have pursued the design of sTRAIL variants Ceritinib manufacturer that show selectivity for TRAIL-R1 or TRAIL-R2

[40–43]. Although the precise fine specificity of some of these variants remains a matter of debate, the use of TRAIL receptor-selective variants for the treatment of a specific tumour type may prove valuable. For instance, CLL appears to be preferentially sensitive to TRAIL-R1 apoptotic signalling, whereas certain solid tumours appear to preferentially signal via TRAIL-R2. Rational integration of TRAIL receptor-selective sTRAIL variants may in those cases help to optimize efficacy. Importantly, as will be described in more detail below, normal cells can be sensitized to sTRAIL by certain other anti-cancer drugs. These side effects are likely due to a sensitizing effect by the co-administered drug on normal cells for the ubiquitous priming of TRAIL-R1 by sTRAIL trimers, as sTRAIL trimers are fully capable of TRAIL-R1 activation. In contrast, TRAIL-R2 is not/minimally activated by homotrimeric sTRAIL. Therefore, it seems a reasonable assumption that TRAIL-R1 signalling Fossariinae is the main

culprit behind potential side effects of sTRAIL trimers. Thus, the rational design and use of TRAIL-R2-selective sTRAIL variants may help to optimize therapeutic efficacy, while minimizing the occurrence of toxic side effects. The available preliminary data indicate that activation of apoptotic TRAIL receptor signalling using sTRAIL or agonistic TRAIL-R antibodies may indeed prove beneficial to cancer patients and certainly warrant further evaluation of this reagent in clinical trials. However, intrinsic and/or acquired resistance to TRAIL receptor signalling is likely to pose a significant hurdle to clinical efficacy. Indeed, almost half of tumour cell lines analysed have intrinsic resistance to TRAIL receptor signalling, which also holds true for GBM cell lines.

To determine whether the cross-reactive WNV S9 epitope was recognized in vivo, we assessed cytotoxicity during acute JEV SA14-14-2 infection. Splenocytes pulsed with decreasing doses of JEV NS4b S9 (JEV S9) were lysed to a similar extent in each of the JEV-immunized mice (Fig. 1B and C). In contrast, the mean percent specific lysis of WNV

S9-pulsed target cells was consistently lower than that seen for the JEV S9 variant for all dose ranges of peptide. Target cells pulsed with a H2-Db-restricted Selleck ABT737 influenza NP epitope (Fig. 1B) and unpulsed splenocytes were not lysed in JEV-immunized or naïve mice (data not shown). These in vivo findings support ex vivo cytotoxicity studies demonstrating the higher cytotoxic activity of the JEV

S9 variant compared with the WNV S9 variant in JEV-immunized mice (data not shown). Functional avidity, defined as T-cell responsiveness to a given epitope and its variants, may be influenced by the infecting virus, resulting in an altered outcome upon secondary heterologous virus infections 17–19. Dose-response experiments revealed that at higher peptide concentrations (1–0.1 μg/mL), the JEV S9 and WNV S9 peptide variants stimulated similar frequencies of IFN-γ+ CD8+ T cells in JEV-immunized mice. At lower peptide concentrations (0.01 μg/mL), the JEV S9 variant stimulated a greater proportion of IFN-γ+ CD8+ T cells than did the WNV S9 variant, suggesting a higher functional avidity for the homologous JEV variant (Fig. 2A). The pattern for TNF-α production was similar to that seen for IFN-γ selleck screening library (data not shown). In WNV-infected mice, at higher peptide concentrations, the homologous WNV S9 variant induced higher frequencies of IFN-γ+ CD8+ T cells compared with the JEV S9 variant but frequencies declined rapidly at lower peptide concentrations (Fig. 2A). In contrast, the frequency of IFN-γ+ CD8+ T cells induced by the heterologous JEV S9 variant was maintained at lower peptide concentrations

(mean±SEM % IFNγ+ CD8+ SPTLC1 T cells at 0.01 μg/mL: JEV S9=1.63±0.31% versus WNV S9=0.45±0.26%). Again, the pattern for TNF-α was similar to that seen for IFN-γ (data not shown). We next examined the frequency of CD8+ T cells that secrete both IFN-γ and TNF-α in the context of the specific stimulating variant as well as infecting virus (JEV versus WNV), in order to determine the contribution of each factor to CD8+ T-cell cytokine profiles. In both JEV SA14-14-2- and WNV-infected mice, we found that stimulation by either the JEV S9 or WNV S9 variant induced both IFN-γ+ and IFN-γ+TNF-α+ CD8+ T cells while single positive TNF-α+ CD8+ T cells were not detected in either JEV SA14-14-2- or WNV-infected mice (Fig. 2B and C). In JEV SA14-14-2-immunized mice, stimulation with the JEV S9 or WNV S9 peptides induced higher frequencies of IFN-γ+ CD8+ T cells than IFN-γ+TNF-α+ CD8+ T cells.