The ileum, excised from both normal and 8-week-infected [representative of the acute phase of schistosomiasis (3)] WT (n = 6) and Mcpt-1−/− mice (n = 6), was washed in Krebs solution. Three 10-mm segments were removed at the distal end of each ileum. One segment was formalin-fixed followed by paraffin embedding and 5-μm-thick paraffin sections were stained with haematoxylin and eosin (HE). The second segment was processed for cryosectioning. Briefly, the segment was fixed for 2 h at room temperature in 4% paraformaldehyde (PFA) in 0·1 M phosphate buffer (pH 7·0). Subsequently, it was rinsed in 0·01 M phosphate-buffered saline (PBS; pH 7·4), transferred to

PBS FGFR inhibitor containing 20% sucrose and stored overnight at 4°C. Next, it was embedded in OCT-embedding medium (Pelko, Torrance, CA, USA), cryostat-sectioned at 12 μm and thaw-mounted on poly-l-lysine-coated slides. Sections were allowed to air-dry and selleck screening library were immediately used for mMCP-1 and mMCP-2 immunostaining. The mMCP-2 staining was applied to identify and count MMC in Mcpt-1−/−. The

third segment was embedded in OCT-medium, frozen in liquid nitrogen-cooled isopentane and stored at −80°C. Subsequently, 60-μm-thick tangential sections were made by cryostat sectioning, and allowed to air-dry and fixed for 10 min in ice-cold acetone followed by rehydration in 0·01 M PBS and finally used for immunostaining of the TJ proteins claudin-3, occludin and ZO-1. All incubations were performed

at room temperature. The primary and secondary antibodies (Table 1) were diluted in PBS Suplatast tosilate containing 10% normal goat serum, 0·01% bovine serum albumin, 0·05% thimerosal and 0·01% sodium azide (PBS*). The sections were pre-treated for 30 min with PBS* containing 1% Triton X-100. Next, they were incubated for 90 min with a primary antibody. Subsequently, after rinsing in PBS, they were incubated with an appropriate secondary antibody for 30 min. For negative controls, primary antisera were omitted in the protocol. The specificity of the primary antibodies was tested by performing immunoblotting and pre-absorption tests. The effect of S. mansoni infection on intestinal barrier integrity of the ileum was assessed by measuring the electrical resistance and transepithelial flux of Na-fluorescein (NaFl; Sigma, Zwijndrecht, the Netherlands) in Ussing chambers. The electrical resistance is mainly determined by the TJs in the epithelium. Alterations in the resistance are thought to reflect opening (in case of reduced resistance) or closing (increased resistance) of TJs of the epithelial paracellular pathway, rather than an alteration in the transcellular pathway. Alterations in the transepithelial flux of NaFl indicate changes in the permeability of the epithelial barrier for small molecules (24). Each of the four groups (non-infected WT and Mcpt-1−/− mice; 8-week-infected WT and Mcpt-1−/− mice) consisted of seven animals.

1) 4, 5. This association and resultant activation of the inflammasome leads to the activation of caspase-1 from its inactive zymogen pro-caspase-1. Active caspase-1 cleaves the pro-forms of the cytokines IL-1β and IL-18 to their active and secreted forms. Caspase-1 may Ulixertinib possess additional functions including regulation of glycolysis pathways 6 and unconventional protein secretion 7; however, in vivo studies demonstrating a role for NLRP3 in these processes are lacking to date. In addition to NLRP3, two other NLR family members have been demonstrated to form inflammasomes and activate caspase-1. The NLRP1 inflammasome is a key mediator

of cell death due to anthrax lethal toxin 8 and the NLRC4 inflammasome is activated by numerous Gram-negative bacteria possessing either a type III or type IV secretion system 9–11. NLRC4 may also interact with another cytosolic NLR, Naip5 to activate caspase-1 in response to cytosolic flagellin 12. Recent studies have

also demonstrated that the cytosolic nucleic acid recognition receptors AIM2 and RIG-I can interact with ASC to form caspase-1 activating inflammasomes 13–17. The NLRP3 inflammasome can be activated in response to a wide array of stimuli (Fig. 1). These activators lack structural or functional similarity making it unlikely that their activation is through Adriamycin supplier direct interaction with NLRP3. Rather, a common endogenous molecule upon which these pathways converge is likely the actual ligand for NLRP3. Numerous microbes including various bacteria, viruses, fungi and protozoan parasites can activate the NLRP3 inflammasome (reviewed in 18). In addition to microbial activators, endogenous danger signals such as ATP, monosodium urate and amyloid-β have been demonstrated to activate the NLRP3 inflammasome. It is interesting to speculate that NLRP3, or its evolutionary ancestor, originally served a primary role in host

defense against pathogens. But rather than sensing specific conserved PAMP as the TLR do, it is capable of detecting a wide swath of divergent pathogens Inositol monophosphatase 1 by detecting one of the major consequences of infection, namely, cellular damage. Sequencing of the sea urchin Strongylocentrotus purpuratus genome revealed 222 TLR and 203 NLR, demonstrating the importance of these innate immune receptors in lower species such as the echinoderms 19. As species evolved and vertebrates developed adaptive immune systems some of these early innate NLR involved in pathogen surveillance have likely been co-opted to serve other functions such as responding to metabolic stress, ischemia and trauma. Recent studies suggest that the NLRP3 inflammasome may play a significant role in metabolic disorders and sterile inflammatory responses including type II diabetes mellitus, gout, Alzheimer’s disease and ischemia 6, 20–23.

Renal biopsy can be used to determine whether the patients are associated with idiopathic or secondary renal glomerular disease and identify the pathological type of glomerulopathy. However, the anatomical structure of HSK and complex relations to adjoining great vessels and organs increase the difficulty and risk of renipuncture,[5] which is the primary reason for why there are fewer HSK cases who receive renal biopsy. We believe that renal glomerular disease of HSK is one of the possible

factors leading to proteinuria, haematuria and renal dysfunction. Therefore, that the pathological type of glomerulopathy is determined by renal biopsy will benefit treatment and prognosis, but it LDK378 order is essential to evaluate the value and GW-572016 in vivo risks of renal biopsy and to select an appropriate puncture site via imaging. The right renal lower pole is generally the best site for normal kidneys, but the bilateral lower renal poles of HSK are close to the abdominal aorta, and thus, the upper poles may be relatively more secure

than the lower poles. There have been some case reports about the occurrence of glomerulopathy in HSK in the literature. It is believed by the authors of these reports that the co-occurrence of HSK and glomerulopathy may be a coincidence or HSK can predispose glomerular diseases because it facilitates immune complex deposition and amyloid formation.[6-13] But

because few patients with HSKs receive a renal biopsy, there is a lack of evidence elucidating the causal relationship of glomerulopathy and HSK.[10] We appeal for further study to identify the relationship between horseshoe kdieny and glomerulopathy. We conclude that glomerulopathy as immunoglobulin A nephropathy is a possible explanation for the association of HSK with heavy proteinuria. Renal biopsy may be valuable for HSK patients with heavy proteinuria to identify the type of glomerulopathy Alanine-glyoxylate transaminase and facilitate further treatment. Moreover, renal biopsy performed by experienced doctors at the renal upper pole using a standard needle biopsy gun under renal ultrasonic guidance may be viable. However, it is necessary to sufficiently evaluate the value, risk and appropriate puncture site before renipuncture. After percutaneous renipuncture, it is also crucial to pay close attention to potential postoperative complications, especially massive haemorrhage. This work was supported by a grant (2011CB944004) from the National Basic Research Program of China, a grant (2012AA02A512) from 863 program and a grant (2011BAI10B00) from the Twelfth Five-Year National Key Technology R&D Program of China. All the authors declare no competing interests. “
“Polyomavirus BK nephropathy (BKVN) is an important infectious complication in kidney transplantation.

One example of a detrimental fungal Th2-cell response in the lung is that generated by allergic bronchopulmonary aspergillosis, which can result from inhalation of the fungal spores of Aspergillus spp. [133]. Indeed, the severity of asthma is

associated with the presence of Alternaria, Aspergillus, Cladosporium, and Penicillium species in the lung, exposure to which may occur indoors, outdoors, or both [118]. In order to improve upon current treatments for invasive fungal infections, it is imperative to understand the nature of fungal pathogenesis not only in the context of the diversity of fungal strains present in the lung [134] but also the complex interplay between lung-colonizing AZD1208 clinical trial microbial communities and invading pathogens. As mentioned before, one notable component of the lung mycobiota of a healthy Tanespimycin ic50 individual is Pneumocystis spp. [135]. New molecular surveys are revealing that Pneumocystis is carried at low levels, even in healthy individuals. This fungus can be spread from individual to individual through airborne transmission, but it can also cause pneumonia following overgrowth in HIV-immunocompromised hosts [136]. Pneumocystis has also been implicated as a cofactor of chronic obstructive pulmonary disease [137]. Thus, Pneumocystis appears to exist as a very low level commensal

in the lung microbiota when the host is healthy and becomes pathogenic when the host becomes immunocompromised. Cystic fibrosis (CF) provides an important example of 17-DMAG (Alvespimycin) HCl the need to enhance our knowledge of the composition of the microbial community in order to improve management of patients susceptible to pulmonary infections. Using pyrosequencing, Delhaes et al. [138] extensively explored the diversity and dynamics of fungal and prokaryotic populations in the lower airways of CF patients. The authors identified 30 genera, including 24 micromycetes, such as Pneumocystis jirovecii or Malassezia sp., and six basidiomycetous fungi [138]. Among the organisms identified, filamentous fungi belonging to the genera

Aspergillus and Penicillium had previously been suggested as pathogens in CF patients [139]. Candida albicans and C. parapsilosis were also recently described as colonizer organisms of CF patients [140, 141]. A significant proportion of other identified species were fungi also detected in patients with asthma (Didymella exitialis, Penicillium camemberti), allergic responses (A. penicilloides and Eurotium halophilicum) [142, 143], or infectious diseases (Kluyveromyces lactis, Malassezia sp., Cryptococci non-neoformans, Chalara sp.) [144]. Fungal colonization (especially repeated or chronic colonization) may thus have a substantial impact on the development of CF and other pulmonary diseases, but more studies are required to determine the real risk relative to the fungal component of the lung microbiota, especially because the coexistence of the bacterial component must be taken into account.

The cardiac troponins

and B-type natriuretic peptide are among the best studied of these biochemical markers of cardiovascular disease. However, controversy remains regarding the interpretation of such results and the subsequent clinical application of these biomarkers, particularly when abnormal in patients with end-stage kidney disease. This review addresses some of the important issues to consider with the interpretation of abnormal cardiac troponin and B-type natriuretic peptide results in patients undergoing dialysis. Many pathological processes contribute to the excess cardiovascular BAY 73-4506 price morbidity and mortality of patients with end-stage kidney disease (ESKD). This cardiac pathophysiology is associated with specific changes in the levels of ‘cardiac biomarkers’.1 The key questions regarding cardiac biomarkers are: (i) Do the changes in cardiac biomarker serum levels directly reflect cardiac disease or does altered metabolism in renal failure influence the levels? (ii) Can cardiac biomarkers be used in the clinical setting

to detect cardiac pathology and allow early intervention with improved clinical outcome? The promise of clinical application of cardiac biomarkers in ESKD cannot be realized without a detailed understanding of cardiac biomarkers and the significance of changes with evolving cardiac https://www.selleckchem.com/products/VX-809.html disease. Two important cardiac pathophysiological processes in patients with ESKD are myocardial ischaemia and abnormal left ventricular structure and function. Myocardial ischaemia is associated Calpain with an elevated cardiac troponin level in serum and available assays measure either cardiac troponin I (cTnI) or cardiac troponin T (cTnT). Abnormal left ventricular structure and function is associated with increased concentration of B-type natriuretic peptide (referred to generally as ‘BNP’) and available assays measure the active hormone,

referred to as BNP-32, and the inactive N-terminal component, referred to as NT-BNP-76, which is often also referred to as ‘NT-proBNP’. These markers have previously been demonstrated to have significant associations with cardiac abnormalities and adverse outcomes in ESKD.2–5 The cardiac troponins and BNP have parallel features (Table 1), which include: (i) assays are available that measure two forms of each marker; (ii) both cardiac troponin and BNP are frequently abnormal in asymptomatic patients with ESKD; and (iii) the relative importance of cardiac pathology and reduced renal clearance in contributing to abnormal levels of these cardiac biomarkers remains controversial. This leads to clinical dilemmas regarding the appropriate management of asymptomatic, as well as symptomatic, patients with abnormal levels of these markers.

In this study, we depleted NK cells when studied TCRVβ clonal deletion. This treatment might be redundant as the NK cells should not affect the TCR Vβ clonal constitution and NKT cells mainly recognize the CD1 molecule and constitute less than 0.5% of the overall T-cell population [[53]]. Besides T MK-1775 cell line cells, NK cells pose another obstacle in establishing mixed chimerism [[54, 55]]. Total body irradiation in combination with anti-NK cell depletion can induce BM allograft

survival [[20-23, 31]]. CD4+FOXP3+ Treg cells could inhibit NK-cell function and transfer of donor CD4+FOXP3+ Treg cells promoted mixed chimerism [[56, 57]]. In a recent study, we have found that in sublethally irradiated mice, donor-derived DN Treg cells can suppress NK cell-mediated allogeneic BM graft rejection [[24]]. In current study, the early phase rejection of donor BM cells Gemcitabine by NK cells can be mostly abrogated by DN Treg-cell transfer (Fig. 4C and D). Consistent with our previous finding [[24]], perforin plays a crucial role for DN Treg cells (Fig. 4D and E). Hence, DN Treg cell-mediated NK-cell suppression can be achieved in BM transplantation an irradiation-free condition. In summary, with the unique capability to

overcome NK-cell and T-cell responses, DN Treg cells facilitate the mixed chimerism and achieve donor-specific tolerance in a nonmyeloablative regimen while minimizing the cytotoxicity side effects. This study sheds light to a possible pathway out of the classical rejection-suppression dilemma and may have potential in the future of transplantation therapy. C57BL/6

(H-2b), BALB/c (H-2d), FasLnull gld, Fas null MRL/lpr, and perforin null mice were purchased from Jackson Laboratories DOK2 (Bar Harbor, ME) and Charles River Laboratories (Wilmington, MA). The animals were maintained in the animal facility at the University of Western Ontario. All animal procedures in this study have been approved by the Animal Use Subcommittee, the Council on Animal Care, The University of Western Ontario (Approval ID #2007-096-10). DN Treg cells were characterized with fluorescent-conjugated monoclonal antibodies that specifically recognize CD3, CD4, CD8, NK1.1, and TCR γδ (eBioscience, San Diego, CA). To detect receptor-derived TCR Vβ clonal deletion, the mouse Vβ TCR screening panel kit (BD Pharmingen, San Diego, CA) was used. Data were acquired and analyzed on the FC500 flow cytometer (Beckman Coulter, Missassauga, Canada). Anti-CD4 (GK1.5) and anti-CD8 (YTS169.4) were used to deplete CD4+ or CD8+ T cells (BioXcell, West Lebanon, NH). NK-cell depletion antibody, anti-Asialo GM1(8.S.007), was purchased from Cedarlane (Burlington, ON, Canada). To confirm the depletion efficiency of T cells or NK cells, cells from spleen or lymph nodes were analyzed by anti-CD4 (H129.19), anti-CD8 (53-6.7), and anti-NK1.1 (PK136).

Intact, antigenic proteins are thus prevented from reaching the LP [16,17]. Tight junctions between the apical pole of enterocytes are another factor that contributes to shielding LP against the intestinal lumen content [18]. These junctions are formed of transmembrane proteins – claudins, occludins and junction-associated molecules, connected to the cytoskeleton by another protein structure, zonula occludens. The tight junctional complexes allow only small molecules, less than 500 Daltons in molecular mass, to cross Smoothened antagonist between cells [19]. These

types of small molecules are usually not immunogenic. Tight junctions differ in permeability along the intestine, being more permeable in the large bowel than in the jejunum. They are also sensitive to the immune medium in the intestinal mucosa, manifesting an increased leakiness after MLN0128 cell line prolonged exposure

of epithelial cells to high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-13 or low levels of IL-10 [20]. An increased transcytosis of intact proteins was found in animal models of allergic diseases, which supports the importance of the intestinal epithelium as a mechanical barrier [21]. In these animals, epithelial permeability of allergens seems to be mediated by CD23/FcεRII and is antigen-specific, given the involvement of immunoglobulin (Ig)E [22]. CD23 is a molecule normally present on the surface of enterocytes, click here both in humans and in rodents [23]. A high rate of CD23-mediated IgE transfer from the basal to the

apical pole of the enterocyte was found in allergic individuals, followed by intraluminal allergen binding and return of the antigen–antibody complex in LP, with the possibility of mast cell activation [24]. The epithelial barrier protects the internal medium not only from food antigens, but also from bacteria. The distal small bowel, caecum and colon have higher bacterial colonization levels than the proximal regions, reaching 1012 colony-forming units per gram of intestinal content in the colon. Sixty per cent of the faecal matter mass in humans is due to bacteria. The small intestine contains lower numbers of commensal bacteria as a result of stomach acid, pancreatic enzymes and motility patterns [25]. Instead, the small intestine contains higher levels of nutrients, available for absorption. The distribution of the immune structures is correlated inversely with the density of luminal bacteria. The small intestine has higher numbers of intraepithelial T cells than the colon; it also harbours lymphoid structures such as Peyer’s patches, which are absent in the large intestine. Paneth cells, which produce anti-microbial peptides, are almost confined to the small intestine, being only marginally encountered in the caecum and appendix.

6b). The inhibition of PI3K and JAKs reduced, but did not abolish, the enhanced MCP-1 secretion, which was induced after monocytes were treated with PAR2-cAP together with IFN-γ (Fig. 5a). This reduced level of secreted MCP-1 was similar to the level reached after monocytes were stimulated with PAR2-cAP alone (Fig. 5a,b). These data indicate that PAR2-cAP effects on MCP-1 secretion by human monocytes are mediated not only via a signalling Selleckchem Ibrutinib pathway involving PI3K activation, but also via another

pathway (Fig. 6b). Surprisingly, the PKCδ inhibitor rottlerin enhanced the effect of PAR2-cAP and IFN-γ on MCP-1 release by monocytes (Fig. 5a). Rottlerin also synergized with PAR2-cAP in its action on MCP-1 secretion (Fig. 5b). Moreover, rottlerin, when applied alone, enhanced MCP-1 secretion by human monocytes (Fig. 5c). Treatment with the p38

inhibitor SB203580 did not influence the increased MCP-1 secretion caused by either PAR2-cAP stimulation or combined application of PAR2-cAP and IFN-γ (Fig. 5a,b). The levels of secreted MCP-1 after IFN-γ stimulation were below the threshold in the neutrophil samples and could therefore not be determined (Fig. 3a,b). The treatment of human monocytes with IFN-γ yielded no significant changes in MCP-1 levels (Fig. 3c). Hence, the effects of the inhibitors of signalling molecules at MCP-1 release were not studied after IFN-γ stimulation of human monocytes and neutrophils. Altogether, the results of our experiments allowed us to suggest a possible scheme of signalling events involved in the enhancement of MCP-1 secretion triggered after combined stimulation of human neutrophils and monocytes Y-27632 ic50 with PAR2-cAP and IFN-γ (Fig. 6a,b). In summary, our study demonstrates that PAR2 agonist acting alone can enhance a bactericidal response of human neutrophils Cyclic nucleotide phosphodiesterase and monocytes in vitro. However, PAR2 agonist is unable to synergize with IFN-γ in the enhancement of the bactericidal response. On the other hand, PAR2 agonist and IFN-γ do synergize to increase MCP-1 secretion by human neutrophils and monocytes during the late phase (after 24 hr)

of the inflammatory response. This synergistic action of PAR2 agonist and IFN-γ on MCP-1 release apparently involves the activation of PI3 kinase and JAKs in neutrophils and monocytes. The work was supported by grants from the IZKF Münster (Stei3/034/09), German Research Foundation (SFB 293-A14, STE 1014/2-2), CERIES (Paris), Weston Haven foundation San Francisco USA (to M.S.), SFB 293 (S.L.), IMF grant SH 120709 (University of Münster, Germany) (to V.M.S.), IMF grant FE 110905 (University of Münster, Germany) (to M.F.) as well as Canadian Institutes of Health Research (Operating and Proteinases and Inflammation Network grants to M.D.H.), Transregional Collaborative Research Centre 34 (C12) (to D.H. and J.R.) and IMF grant HO 220912 (University of Münster, Germany) (to D.H.). The position of V.M.

There is no proven vaccination technique that can prevent and/or cure endogenous ag–caused disorders [28, 31, 61–65]. However, CH5424802 concentration some recently instituted vaccination techniques provide a glimmer of hope in providing future possibilities for the prevention and treatment of chronic ailments [66–71]. E.g. one of the vaccination techniques – being able to induce oral tolerance – proved itself to be effective in animal experiments, especially in preventing and delaying the occurrence of autoimmune diseases; but its effectiveness in treating humans with autoimmune conditions so far has not resulted

in significant clinical improvements [67]. For this reason, endogenous ag–initiated disorders are treated with cytotoxic and immunosuppressive agents. These treatment modalities provide no specific cures and often have undesirable side effects.

Would we be able to terminate the pathogenic IgG aab response in an autoimmune disease e.g. in SPHN, then the continuance of the disease process would come to a halt and a recovery from the disease would ensue. According to some scientists, once an autoimmune disease is initiated and maintained, e.g. by emerging autoreactive T cells or by pathogenic IgG aabs [72, 73] (produced by long lived plasma cells), the autoimmune disease causing process cannot be halted, only interfered with somewhat by anti-inflammatory medications. However, there are those who believe that ag-specific downregulation Lenvatinib clinical trial of autoimmune diseases is possible, e.g. if the inciting agent is removed (it could be a drug) [24], or if the target ag is presented in a suitable

tuclazepam format (which only works if the ag is presented in a soluble form prior to induction of an experimental autoimmune disease) [36–41]. We share this belief that ag-specific downregulation or upregulation of immune responses in certain autoimmune disorders (i.e. autoimmune disease and cancer) are possible and our experiments have shown these to be true through the utilization of the modified vaccination technique (MVT) [21, 44, 51]. We have shown that by a predetermined ab inducing/maintaining technique:  specific IgM aabs can be produced to eliminate disease contributing aag [44, 51, 52]; and similarly To achieve desired corrective immune responses, the etiologies and pathogenesis of the autoimmune disorders must be understood as well as how to produce the essential components that are able to evoke the appropriate preventative and/or therapeutic outcomes. The immune system unconditionally responds to the right antigenic ‘information’. The challenge was to find how the normally functioning immune system could be affected – by the presentation of the antigenic ‘information’– to respond and correct endogenous ag–caused mishaps.

The first mammalian glycolipid ligand (isoglobotrihexosylceramide, or iGb3) was not discovered until after a decade of research on iNKT cells [25]. Our hypothesis was that the character of hepatic lipids changes in a manner that increases their capacity c-Met inhibitor to stimulate iNKT cells. An alternate, but not mutually exclusive, hypothesis is that the expression level of CD1d increases, thereby enabling enhanced iNKT cell activation. In the current study, we utilized adoptive cell transfer techniques in several

strains of knockout mice to demonstrate that hepatic lipids isolated from wild-type mice 30 min after sensitization are significantly more stimulatory to naïve hepatic iNKT cells than hepatic lipids isolated after sham sensitization. These stimulatory hepatic lipids specifically affect iNKT cells and not B-1 B cells, consistent with our hypothesis. Our data suggest that iNKT cell activation occurs in a CD1d-dependent manner involving lipid presentation by cells other than hepatocytes. These findings begin to clarify the mystery of rapid iNKT cell response and may carry future implications for a multitude of clinical diseases including CS, NAFLD and cancer, with potential for dietary and medical interventions affecting immune stimulation and lipid metabolism. Mice.  Six- to 12-week-old pathogen-free CD1d−/−, CBA/N-xid (H-2k), BALB/c (H-2d) and CBA/J mice were obtained from The Jackson Laboratory (Bar

Epacadostat Harbor, ME, USA). Breeders of pan-B cell-deficient JH−/− C-X-C chemokine receptor type 7 (CXCR-7) mice (CB.17, H-2d) [26] were kindly provided by Mark Shlomchik of Yale University School of Medicine, New Haven, CT. Breeders for Jα18−/− (H-2d) mice were obtained from Masaru Taniguchi (Chiba University, Chiba, Japan). Deficiencies are as follows: CD1d−/− lack CD1d and iNKT cells; Jα18−/− lack iNKT cells; JH−/− lack B cells; CBA/N-xid lack B-1 B cells. Experiments were conducted according to guidelines of the Yale Animal Care and Use Committee. Reagents.  Trinitrophenyl chloride (TNP-Cl) (Nacalai

Tesque, Kyoto, Japan) was recrystallized twice and stored protected from light. α-GalCer (KRN7000) was provided by the Pharmaceutical Research Laboratory of Kirin Brewery Company (Tokyo, Japan) [27]. α-GalCer was diluted to 220 μg/ml in 0.5% polysorbate-20 in sterile pyrogen-free 0.9% NaCl (Abbot Labs, Chicago, IL, USA) and used as an iNKT cell-stimulatory positive control. For flow cytometry analysis, we used fluorescein isothiocyanate (FITC)-anti-CD1d antibody (BD Biosciences Pharmingen, San Diego, CA, USA), anti-TCR-β antibody (BD), anti-CD1d antibody (BD) and PE-α-GalCer-CD1d tetramers (Mitch Kronenberg, La Jolla Institute for Allergy and Immunology, San Diego, CA, USA). Sensitization and elicitation of CS.  Mice were actively contact-sensitized on day 0 with 150 μl of 5% TNP-Cl in absolute ethanol and acetone (4:1) on the shaved chest, abdomen and footpads.