At 4 weeks post-immunization, mice were sacrificed, and their spleens were removed. Splenocytes were restimulated with ESAT-6 protein in vitro, and the number of IFN-γ-secreting cells and the concentration of TNF-α in the supernatant were measured using ELISPOT and ELISA, respectively. No significant differences in the number of IFN-γ-secreting cells or the concentration of TNF-α were observed

between the two groups (Fig. 3B,C). Thus, the addition of CFP-10 to the calreticulin–ESAT-6 fusion did not provide an enhancement of the PI3K inhibitor ESAT-6-specific immune response. We next investigated the ability of the vaccine-induced immune response to reduce the mycobacterial burden after low-dose aerosol infection in the mouse model. Mice were DAPT vaccinated with AdCRT–ESAT-6–CFP10 via the intranasal route and BCG via the subcutaneous route, only once as described in Materials and methods. At 4 weeks post-immunization, mice were infected with M. tuberculosis. Four weeks after challenge, the M. tuberculosis burden of infected animals was determined to evaluate the

protective efficacy in both lung and spleen. The trends were similar in both organs (Fig. 4A,B). BCG caused a reduction in CFU in both the lungs and spleen of infected animals. However, there was no significant difference between mice vaccinated with the adenovirus constructs and the saline-treated group for both organs. The high incidence of TB has Lepirudin stimulated interest in understanding the immune response to infection, resulting in the accelerated identification of novel immunodominant mycobacterial proteins as possible vaccine candidates. Culture filtrates and RD sequences have attracted particular interest as a source of antigens. ESAT-6, TB 10.4, CFP10, MTB12, MTB39 and Ag85 A and B have all been shown to elicit protective immune responses in various animal models of TB [12, 16, 27, 28]. Even though many strategies for vaccination increase the overall immune response, this may not be the ideal solution. When multiple antigens are presented to the immune system, they will compete for

presentation, and the antigens dominating the response will not necessarily be those most relevant for protection. Thus, a targeted approach may be ideal. It has been repeatedly demonstrated that calreticulin can enhance immune responses when linked to antigens in DNA and viral vaccines [23–26]. This suggests that the use of calreticulin may be broadly applicable as a strategy to enhance vaccine efficacy. In addition, several reports have suggested the efficacious use of vaccines against TB in mice using adenoviral vectors expressing different M. tuberculosis antigens [10]. We herein demonstrate the effects of a replication-deficient adenoviral vector that contains the M. tuberculosis ESAT-6 antigen fused to calreticulin and show that there is an increased immune response to this antigen as demonstrated by increased cytokine expression.

The impact of TCR repertoire diversity on Treg-cell function is controversial. Regarding the prevention of autoimmune disease, previous studies on the effective suppression of EAE through Treg cells with

limited TCR repertoires came to divergent conclusions 47, 48. A recent study by Adeegbe et al. found that limited TCR diversity of transferred Treg cells was a risk factor for autoimmune disease in IL-2Rbeta−/− mice 49. Intriguingly, non-obese diabetic mice were recently shown to select a low diversity Treg-cell TCR repertoire 50. Understanding the parameters that govern Treg-cell homeostasis will be critical for the design of future Treg-cell-based intervention strategies. Sufficient availability of organ-specific antigen must be considered in translational attempts to manipulate organ-specific autoimmunity Casein Kinase inhibitor with engineered Treg cells of known self-peptide specificity. Otherwise, exogenous therapeutic Treg cells may be lost quickly after transfer. Previous studies suggested that organ-specific self-antigen preferentially drives the survival and/or expansion of organ-specific Treg-cell clones 11, 13, 21, 22. Our results also support the view that the antigen specificity of Treg cells changes by anatomical location, although

MLN8237 TCR sequences of recovered Treg cells from pLNs and mLNs were largely overlapping. This may be the result of two possible scenarios. Either Treg cells recirculate less than naïve T cells or differences are due to selective local survival. Importantly, our study infers that Treg-cell diversity is connected to diversity and availability of specific self- and foreign-antigen and thus the amount of DCs presenting it on MHC class II. In accord, it was recently shown that DC ablation Calpain reduced Treg-cell frequencies 51, 52, whereas an increase of DC numbers by FLT3L treatment led to expansion of peripheral naturally occurring Treg cells 52,

53. However, in the latter report, it was concluded that Treg-cell proliferation was mainly IL-2 dependent. In our study, we also recognized IL-2 as a master regulator that controls the absolute size of the Treg-cell pool. We propose that an optimal and maximally broad organ-specific Treg-cell TCR repertoire is continuously shaped by inter- and intraclonal competition for diverse antigen. Within a peripheral Treg-cell niche, sufficient population diversity seems to be crucial for proper Treg-cell function. Hence, in future studies, HT-sequencing analysis of Treg-cell diversity may be suitable to predict the relative risk of T-cell-mediated diseases. C57BL/6-Foxp3eGFP (here: WT) 54, C57BL/6-Foxp3.LuciDTR-4 36, and C57BL/6-Tg(TcraTcrb)425Cbn/J (here: OT-II/TCR-Tg) 55 mice have been described. The Thy1.

These disorders indicate that in human neutrophils, NEMO and IRAK4 are required for normal LPS-induced priming of superoxide production. Despite being able to respond normally to phorbol ester stimulation, NEMO-deficient neutrophils failed to produce normal levels of superoxide in response to chemotactic peptide (fMLF) alone and more strikingly fMLF after pretreatment with LPS [82]. Phosphorylation of p47phox Selleck ACP-196 was normal in NEMO-deficient cells, suggesting

that additional regulatory signals, such as p67phox translocation, play a role in regulating NADPH oxidase activity. IRAK4 has also been shown to bind and directly phosphorylate p47phox in neutrophils upon LPS stimulation [83]. Consistent with this finding, p47phox phosphorylation was not detected in response to LPS alone in IRAK4-deficient PMN, but it

was detected in response to fMLF and PMA. More importantly, the clinical syndromes indicate that defective NADPH oxidase activation in NEMO or IRAK4 deficiency play a role during the innate immune response to infection in vivo. Although the defect in NADPH oxidase activation in NEMO deficiency is less dramatic than IRAK4 deficiency in vitro, the consequences may be more severe in the background of altered acquired immunity in EDA-ID caused by NEMO deficiency [82]. G6PD, the key regulatory enzyme in the hexose monophosphate shunt, catalyses the oxidation of glucose-6-phosphate (G6P) to 6-phosphogluconolactone and the production of reducing equivalents in the form of NADPH to meet cellular needs for reductive biosynthesis and maintenance of the cellular redox status [84]. NADPH is the electron donor used by the NADPH buy MI-503 oxidase to reduce the molecular diglyceride oxygen to superoxide. Gene mutations affecting G6PD are found on the distal long arm of the X chromosome (OMIM # 305900). Notably, the G6PD and NEMO genes are encoded in opposite directions on the X chromosome and share the same promoter. The diversity of point mutations and possible interactions with other

genes account for the phenotypic heterogeneity of G6PD deficiency [85]; over 400 biochemical variants have been reported [86]. The level of G6PD activity in affected erythrocytes is generally much lower than in other cells [87], as most mutations affect protein stability rather than function, and anucleate erythrocytes cannot synthesize more enzymes. G6PD-deficient persons are predisposed to the development of sepsis and complications related to sepsis after a severe injury [88]. Patients with sufficiently severe G6PD deficiency to affect leucocyte enzyme levels may demonstrate low NADPH oxidase activity because of impaired substrate supply and suffer recurrent infections, mimicking the phenotype of CGD [89]. Agudelo-Florez et al. [90] reported an unusual association of X-linked CGD and the usually mild African variant of G6PD deficiency in a boy with recurrent respiratory infections, chronic lung disease and anaemia [91].

40,57 For example, increased expression of NGAL was found in kidney epithelial cells during ischaemic injury.47,50 Of the aforementioned Trametinib biomarkers, none has met all of these criteria. While TEC biomarkers await further validation by assessing in consecutive series of patients with multiple aetiologies and longitudinal studies, urinary tubular biomarkers that can be

measured non-invasively may be useful as a preliminary screening assay (Table 1). Patients testing positive for certain biomarkers could then be considered for allograft biopsy to determine the nature of the injury. For example, CXCL-10, NGAL or HLA-DR ELISA assays which showed >80% specificity may facilitate in selecting true-positives (i.e. high risk for allograft rejection) patients for biopsy while ruling out false positives,57 limiting unnecessary biopsy procedures. Moreover, tubular

biomarkers that are induced during AR or acute injury such as NGAL and KIM-1 have been shown in different studies to improve the sensitivity for early detection of postoperative kidney injury compared with the routine measurement of serum creatinine,52,57 which is a relatively late manifestation of graft dysfunction.64–66 Alternatively, these tests may also be applied in the setting of delayed graft function, where there is a persistently elevated serum creatinine. In conclusion, non-invasive measurements of urinary tubular biomarkers can provide information of the microenvironment of the allograft in transplant recipients. MAPK Inhibitor Library cell line Monitoring their response to host immune system may reveal early state of injury and thus allow the clinician to provide timely intervention. Future advancements in modulating the expression of these biomarkers on tubular cells may also potentially aid in identifying new therapeutic targets. Our hope is that the completion of multicentre, large cohort studies using a range of biomarker assays will ensure uptake of these new tests for routine clinical

monitoring of renal transplant patients in the near future. YT would like to thank the University of Otago for a publishing bursary. “
“Autosomal dominant polycystic kidney disease (ADPKD) is a highly prevalent inherited disorder and results in the progressive development of cysts in both kidneys. In recent studies, several cytokines and growth factors Avelestat (AZD9668) secreted by the cyst-lining epithelia were identified to be upregulated and promote cyst growth. According to our previous study, chemokines with a similar amino acid sequence as human interleukin-8 (IL-8) are highly expressed in a rodent model with renal cysts. Therefore, in this study, we focused on whether IL-8 signalling is associated with renal cyst formation, and tested the possibility of IL-8 as a new therapeutic target for ADPKD. Expression of IL-8 and its receptor were screened either by enzyme linked immunosorbent assay (ELISA) or Western blot.

Due to differences in dietary fats in the western world in the United States versus Europe [22], it is likely that the diet-induced changes in intestinal microbiota composition could partly explain the controversy regarding, e.g. the Firmicutes/Bacteroidetes ratio in humans [4, 14]. Nevertheless, Hydroxychloroquine solubility dmso it is now accepted that intestinal microbiota are involved in obesity, as germ-free ob/ob mice on both normal chow and high-fat diets remain

significantly leaner than conventionally raised mice, despite a significantly higher food intake [23]. In line with this, metagenomic sequencing of the caecum microbiome of these ob/ob mice revealed that an enrichment of genes was involved in the breakdown

of complex dietary polysaccharides [18]. Similar alterations showing enriched bacterial genes involved in carbohydrate sensing and degradation have also been observed in obese humans [24]. Studying intestinal microbial composition in well-phenotyped human subjects enrolled in relatively large metagenome-wide association studies (MGWAS) in both Chinese and European populations has further increased our understanding of the gut microbiota in the development of obesity and insulin resistance [25-27]. Karlsson et al. detected an enrichment of L. gasseri and S. mutans (both Palbociclib in vitro commensal bacteria in the mouth and upper intestinal tract) to predict development of insulin resistance in their cohort of postmenopausal obese Caucasian females [26]. Conversely, Qin et al.’s Chinese T2DM cohort demonstrated that Escherichia coli, a Gram-negative Cediranib (AZD2171) bacterium which is associated with development of low-grade endotoxaemia, was more abundant. Moreover, clusters of genomic sequences acted as the database signatures for specific groups of bacteria and both studies found independently that subjects with T2DM were characterized by decreased

short chain fatty acid (SCFA) butyrate-producing Clostridiales bacteria (Roseburia and F. prausnitzii), and greater amounts of non-butyrate producing Clostridiales and pathogens such as C. clostridioforme, underscoring a potential unifying pathophysiological mechanism. It has long been recognized that insulin resistance and development of type 2 diabetes are characterized by systemic and adipose inflammation [19, 28]. The lipopolysaccharides (LPS) produced in the intestine due to the lysis of Gram-negative bacteria triggers proinflammatory cytokines that result in insulin resistance both in mice [5] and humans [29]. A more causal role was defined when germ-free mice were colonized with E. coli, as this promoted macrophage accumulation and up-regulation of proinflammatory cytokines resulting in low-grade inflammation [30].

All experiments were performed in triplicate. Percentage of cytotoxicity was calculated as follows: [experimental counts per minute (cpm) −  spontaneous cpm]/[total cpm − spontaneous cpm] × 100. Freshly isolated PBMCs were stimulated with 25 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich) at 37°C in humidified 7% CO2 for 4 h. To block cytokine

secretion, brefeldin A (Sigma) [27] was added to a final concentration of 10 µg/ml. After addition of stimuli, the surface staining was performed with anti-CD4-PC5 (13B8·2), anti-CD8-PerCP (SK1) and anti-CD56-PC5 Caspase inhibitor (N901) (Beckman Coulter). Maraviroc clinical trial Subsequently, the cells were permeabilized, stained for intracellular IFN-γ and IL-4 using the FastImmuneTM system (BD Pharmingen), resuspended in phosphate-buffered saline (PBS) containing 1% paraformaldehyde (PFA),

and analysed on a flow cytometer (≈ 10 000 gated events acquired per sample). ELISPOT assays were performed as described previously with the following modifications [28–30]. HLA-A24 restricted peptide epitopes, squamous cell carcinoma antigen recognized by T cells 2 (SART2)899 (SYTRLFLIL), SART3109 (VYDYNCHVDL), multi-drug resistance protein 3 (MRP3)765 (VYSDADIFL), MRP3503 (LYAWEPSFL), MRP3692 (AYVPQQAWI), alpha-fetoprotein (AFP)403 (KYIQESQAL), AFP434 (AYTKKAPQL), AFP357 (EYSRRHPQL), human telomerase reverse transcriptase (hTERT)167 (AYQVCGPPL) (unpublished), hTERT461 (VYGFVRACL) and hTERT324 (VYAETKHFL) were used in this study. Negative controls consisted of an HIV envelope-derived peptide (HIVenv584). Positive controls consisted of 10 ng/ml PMA (Sigma) or a CMV pp65-derived

peptide (CMVpp65328). The coloured spots were counted with a KS ELISPOT Reader (Zeiss, Tokyo, Japan). The number of specific spots was determined by subtracting the number of spots in the absence of antigen from the number of spots in its presence. Responses were considered positive if more than 10 specific spots were detected Clomifene and if the number of spots in the presence of antigen was at least twofold greater than the number of spots in the absence of antigen. Serum cytokine and chemokine levels were measured using the Bioplex assay (Bio-Rad, Hercules, CA, USA). Briefly, frozen serum samples were thawed at room temperature, diluted 1:4 in sample diluents, and 50 µl aliquots of diluted sample were added in duplicate to the wells of a 96-well microtitre plate containing the coated beads for a validated panel of 27 human cytokines and chemokines (cytokine 27-plex antibody bead kit) according to the manufacturer’s instructions.

This is consistent with molecular diagnostics increasingly being applied to microbial detection and identification in the microbiology laboratory for many putative infections that are either not able to be cultured (viruses) or are fastidious or slow-growing. Several molecular selleck techniques are now used routinely to either augment existing culture results (for bacteria)

or to detect and identify pathogens in the absence of culture (primarily for virus detection). The most widespread molecular methods are nucleic acid (NA) amplification techniques such as the polymerase chain reaction (PCR). Advantages of PCR include: high sensitivity that may detect very few microorganisms, availability of primer/probe sets for most common pathogens, routine extraction protocols for nucleic acid extraction, and the

development of automated systems and readouts for higher throughput of samples. Quantitative Kinase Inhibitor Library price PCR can also provide quantitative data on the relative abundance of microorganisms that are present. Disadvantages include: disassociation of the sample prevents microscopic evaluation of aggregated microorganisms, the detection sensitivity may not necessarily correspond to diagnostic sensitivity, potential sample contamination, complex samples containing inhibitors of PCR (such as eukaryotic DNA), and the potential amplification of DNA from nonviable microorganisms. Thus, PCR is a powerful approach that needs to be interpreted

in the context of other diagnostic approaches and clinical data (Hall-Stoodley et al., 2006; Larsen et al., 2008; Rudkjøbing et al., 2011; Wolff et al., 2011). FISH is another sensitive and specific approach, which is particularly well suited to the either study of complex tissue samples and evaluation of the presence of microbial aggregates. FISH relies on hybridization of a fluorescently labeled probe to the 16S or 23S ribosomal RNA in bacteria or the 18S or 26S ribosomal subunits in eukaryotic microorganisms such as dimorphic fungal and protozoan pathogens. These molecular regions are specific to species level in microorganisms, and with careful optimization and use of controls, this approach can give robust in situ evidence of pathogens in a sample (Fig. 1). Advantages of FISH include: culture-independent evidence of specific pathogens as spatially organized aggregates, in situ localization in the tissue and co-localization with other cell types (such as PMNs if used in conjunction with other NA probes or stains) (Fig. 2), or other microbial members of a biofilm (such as in polymicrobial communities in dental biofilms), and demonstration of rRNA content specific to microorganisms indicating recent metabolic activity.

We note, however, that expression NVP-BKM120 mouse of RORγ and Runx1, two factors that are essential for NKT cell differentiation 43, was normal in Bcl11bdp−/− mice, indicating that Bcl11b controls NKT cell development independently of these factors. Our expression profiling analyses suggest that Bcl11b is required to prevent premature and inappropriate expression of many genes specifically expressed in mature CD4+ and/or CD8+ T cells. We speculate that Bcl11b may serve as a timing

factor that holds cells in the immature, DP state until a constellation of factors is in place to support SP differentiation. It is likely that the premature SP gene expression program that is induced in the Bcl11b-deficient DP cells reflects both the direct loss of Bcl11b-dependent repression, and the precocious activity of SP-specific transcription factors (such as Klf2, Zbtb7b, Runx3, and Id2). Therefore, our data suggest that correct regulation of SP cell differentiation

involves mechanisms not only to induce cell-specific gene expression programs, but also to prevent these programs from being inappropriately expressed in immature cells. Mechanisms that prevent early expression of differentiation-associated genes have also been described in other systems. For instance, Polycomb-dependent repression has recently been shown to prevent the premature expression of structural genes in differentiating keratinocytes 44. It is of particular interest that that loss of Bcl11b in DP cells expressing low levels of CD3 results in the induction of genes encoding Zbtb7b and selleck kinase inhibitor Runx3, which are required for, and strongly upregulated during, CD4 and CD8 SP differentiation programs, respectively 45, 46. We found that Bcl11b bound to sequences in the regulatory regions of these genes, suggesting that Bcl11b directly represses

expression of Zbtb7b and Runx3 in immature T cells. The regulation of Zbtb7b has been intensively investigated in recent Vasopressin Receptor years. Induction of Zbtb7b expression occurs downstream of TCR signaling and requires activation of GATA3 expression 47, whereas Runx3 contributes to Zbtb7b repression in CD8-committed cells 19. The mechanisms that render Zbtb7b silent prior to TCR signaling are less well understood but may in part involve repression by Runx complexes 19. Our present data suggest an essential role for Bcl11b in this early silencing, and thus identify another key player in the regulatory network controlling the dynamic regulation of Zbtb7b during T-cell differentiation. However, our results also raise several questions about how Bcl11b participates in Zbtb7b regulation. It will be important to identify activators responsible for Zbtb7b expression in Bcl11b-deficient DP cells, and determine how Bcl11b antagonizes these activators at the transcriptional level in WT cells.

g. CVDs, less manageable diabetes) associated with this and other local diseases. Chronic periodontitis (CP) is one of (if not) the most common chronic inflammatory diseases known to mankind. It is not only the most common cause of tooth loss in adults but has also been associated, in a number of studies, with an increased risk for various ABT-263 research buy medical disorders including cardiovascular disease

(CVD) (Genco & Stamm, 1998; Kuula et al., 2009), reduced diabetic control (Mealey & Ocampo, 2007), preterm delivery (Radnai et al., 2009) and osteoporosis (Golub et al., 2008). Destructive CP is initiated by infection with specific bacterial species, particularly anaerobic gram-negative microorganisms such as Porphyromonas gingivalis, but the breakdown and loss of the periodontal connective tissues, including bone, are primarily the result of the host response, particularly the production of inflammatory mediators (prostanoids, cytokines, nitric oxide), and neutral proteinases, particularly the matrix metalloproteinases (MMPs; e.g. collagenases and gelatinases) and serine proteinases (e.g. elastases) (Ryan, 2002; Lamster et al., 2008; Persson & Persson, 2008).

Chronic inflammatory conditions including CP are characterized by a local accumulation of leukocytes, predominantly (70%) mononuclear cells. Endotoxin derived from P. gingivalis, a virulent periodontal pathogen, can induce the production of proinflammatory cytokines in monocytes. These mediators exert autocrine and/or paracrine Autophagy inhibitor cell line activities by upregulating the expression of various proteinases including MMPs, resulting in the destruction of connective tissue including periodontal tissues. Because recent studies have also linked this oral infection with an increased risk for developing www.selleck.co.jp/products/cobimetinib-gdc-0973-rg7420.html a number of systemic disorders including CVD (Genco & Stamm, 1998; Kuula et al., 2009), it is essential to optimally

control this oral disease and maintain periodontal health. In our lab, we have repeatedly shown that tetracycline derivatives, some with no antimicrobial activity, can reduce inflammatory tissue damage (Ryan et al., 1996). We have previously shown that the activities of the polymorphonuclear leukocyte MMPs, MMP-8 and MMP-9, can be inhibited by therapeutically relevant doses of chemically modified nonantibiotic tetracyclines (Golub et al., 1995). In the current study, we used a complete interstitial extracellular matrix (ECM) secreted by R22 smooth muscle cells as a model system (Gu et al., 2005) to determine whether doxycycline (a tetracycline antibiotic) can inhibit inflammatory cytokines and MMPs in mononuclear cells, thereby preventing connective tissue breakdown. All chemical reagents, lipopolysaccharide and doxycyline were purchased from Sigma-Aldrich Co. (St. Louis, MO).

[9] Stimulation indices (SI) were calculated

as proliferative response in the presence of antigen divided by response in the absence of antigen. Brains and spinal cords were fixed in 5% formalin saline and processed for routine histology. Sections, 5 μm thick, were cut and stained with haematoxylin & eosin to evaluate inflammatory infiltrates or Luxol fast blue/cresyl fast violet (LFB/CFV) to assess the degree of demyelination. Data were analysed using Graphpad prism and expressed as mean ± standard error of the mean (SEM). The EAE clinical scores were assessed by Mann–Whitney U-test and day of onset and disease incidence were analysed by Kaplan–Meier using sigmastat software (SPSS Inc., Chicago, IL). Group EAE score represents the maximum neurological deficit in all animals within the group and mean EAE score represents the maximum neurological deficit developed by mice, which exhibited EAE, as

previously described Bortezomib solubility dmso and the mean day of onset of signs.[3, 16] P-values < 0·05 were considered significant. To identify the immunodominant B-cell epitopes C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice, which will lack any immune tolerance and deficits in their immune repertoire to MOG, were immunized with rmMOG corresponding to MOG sequence 1–116. On day 20, plasma was collected and examined using ELISA to identify responses to 23 mer overlapping peptides (Table S3). No differences were observed between the responses of MOG+/+ and MOG−/− mice to rmMOG on day 20 (Fig. 1). Similarly, antibody responses to peptides in both PRKD3 https://www.selleckchem.com/products/a-769662.html WT and MOG−/− knockout mice were restricted to sequences below residues 82 and dominant responses to epitopes within residues MOG45–67 and MOG50–72 (Fig. 1a).

Similar to responses to MOG35–55 (see ref. [9]) antibody responses to the 23 mer peptide MOG35–57, encompassing the encephalitogenic peptide MOG35–55, were not dominant. As expected, no responses were found in peptides above residues 116 (Fig. 1a). To examine antibody responses in more detail, C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice (n = 5) were immunized with a pool of 15 mer peptides and recall responses on day 20 to individual peptides were examined using ELISA. We identified immunodominant epitopes with residues MOG113–127 and MOG148–162 (Fig. 1b) in C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice. No responses were observed to any other peptide or in mice immunized with complete Freund’s adjuvant only. No differences were observed between responses in C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice (Fig. 1). Next, to identify the immunogenic T-cell epitopes within mouse MOG, mice were immunized with the overlapping peptide spanning the mouse MOG sequences. On day 10 responses were examined using a thymine incorporation assay as described previously.[9] This study revealed that while a T-cell response to MOG36–50 (SI = 3·90) was detectable (Fig. 2) a stronger response to peptide MOG183–197 (SI = 5·2) was also induced.