It has not been proved formally that changes in T cell function observed with advancing age are completely disconnected from the consequences of modification in the peripheral T cell pool, as events such as proliferation induced by the homeostatic milieu of the ageing organism may contribute to the reduced functional capacity of T cells [11]. A potential driver of age-related check details changes in the peripheral T cell pool is atrophy of the thymus. A reduction in thymic activity is a feature of ageing in mammals. In humans, fat accumulates in the thymus throughout

life [12] reducing the active areas of thymopoiesis, and this contributes to a decline in the output of T cells. Measurement of this decline in previous studies has produced different views on the kinetics of this process. Some studies indicate an exponential decline [13] with T cell output beginning early in life and estimated to terminate at approximately

75 years of age [14]. Others suggest that the thymus atrophies in a biphasic manner [15] with the initial phase beginning early in life, at least as early as the first year and proceeding at a rate of 3% per year until middle age. Thereafter the rate changes to a constant rate of 1% per year, leading to the estimated total loss of thymic tissue by 105 years of age [16,17]. Recent work shows that the reversal of thymic atrophy is a viable option, but the timing of when selleck kinase inhibitor Paclitaxel cost such a procedure should begin would be critically dependent upon determining the period when thymic output ceases. In order to provide more information about the decrease in thymic output later in life we analysed samples collected from 215 healthy elderly individuals, with ages ranging from 60 to 100 years, and to reduce any bias related to environmental factors and/or lifestyle we obtained samples from participating centres across five European countries

(France, Germany, Greece, Italy and Poland)) [18]. We quantified changes in thymic output using signal-joint T cell receptor excision circles (sjTRECs) per T cells measured by real-time polymerase chain reaction (PCR), as described previously [19]. Peripheral blood (PB) samples were collected from healthy elderly individuals from participating centres across five European countries (France, Germany, Greece, Italy and Poland) [18]. Informed consent was obtained from healthy adult volunteers, with ages ranging from 58–104 years. Peripheral blood mononuclear cells (PBMC) were isolated and the samples were stored at −140°C until required for analysis. Frozen PBMC were thawed and an aliquot containing 1 × 105 cells stained with phycoerythrin (PE)-conjugated anti-CD3 (BD Bioscience, Oxford, UK) according to the manufacturer’s instructions.

These events include phosphorylation of the CD3ζ chain, ZAP70, and LAT 37. Moreover, the Scr-family kinase LCK is inhibited 38, 39 which leads to a modulation of the calcium signaling 39. Therefore, while the inhibition of LFA-1

accumulation by dexamethasone is probably mediated by the inhibition of L-plastin phosphorylation, the additional defective accumulation of the TCR/CD3 complex in dexamethasone-treated T cells might be due to the inhibition of TCR/CD3-induced tyrosine phosphorylation and calcium signaling by dexamethasone. In contrast to other actin-binding proteins, such as cofilin or Arp2/3, the expression of L-plastin is restricted to leukocytes and certain tumors 47, potentially making it a valuable target for immunosuppression. Supporting this assumption, Wang et al. 46 demonstrated that LPL−/− mice showed a less severe experimental autoimmune encephalomyelitis (EAE). Moreover, they found that BTK inhibitor L-plastin expression has an important role in delayed, but not immediate

allograft rejection in the murine system. Therefore, interference with L-plastin phosphorylation and/or functions may be a sophisticated approach to modulate T-cell immune responses in order to prevent transplant rejection or to treat T-cell-mediated autoimmune diseases in humans. Abs employed were specific for the following markers: CD3 (mouse mAks, clone OKT3 or SK3), CD2 (mouse mAb, clone 3PT2H9, kindly provided by S. F. Schlossman, Dana Farber Cancer Institute, Boston, MA, USA), CXCR4 (R&D Systems, Wiesbaden-Nordenstadt, Germany) CD28 (CD28.2), and CD3-PerCP, LFA-1 (CD18-FITC, CD18-PE or CD11a-FITC), Rucaparib CD28-PE (mouse mAb, BD Biosciences, Heidelberg, Germany). The CD3-PeTxR Ab was purchased from Caltag (Buckingham, UK) and the actin antiserum from Sigma-Aldrich (Hamburg, Germany). The GFP Ab was from Clontech. Unconjugated anti-mouse and horseradish peroxidase-conjugated anti-rabbit Abs were purchased from Dianova (Hamburg,

Germany). The L-plastin polyclonal antiserum was produced against recombinant L-plastin protein 8. Phalloidin-AlexaFluor647 and Hoechst33342 was from Invitrogen (Darmstadt, Germany). Dexamethasone was purchased from Calbiochem (Bad Soden, Germany) and Ru486 (mifepristone) was from Sigma-Aldrich. All inhibitors and drugs were reconstituted Tideglusib in DMSO. Thus, the respective controls in the experiments were performed as solvent controls with the relevant concentration of DMSO. In the titration experiment, the highest concentration of DMSO was used as solvent control. Human PBMCs were obtained by Ficoll-Hypaque (Linaris, Wertheim-Bettingen, Germany) density gradient centrifugation of heparinized blood from healthy volunteers upon approval by the local ethics committee. T cells were subsequently isolated with magnetic associated cell sorting using pan T-cell negative isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) 5.

As seen in Figure 1, five RCTs involving 263 patients reported Ccr, the meta-analysis showed that the Ccr level was significantly higher in the groups receiving calcium disodium EDTA as compared with the groups receiving placebo (SMD,

0.68; 95% CI, 0.43 to 0.93; P < 0.00001). These results suggest that calcium disodium EDTA chelation therapy has a significant benefit of retarding the progression BMN 673 mw of chronic kidney disease in patients with measurable body lead burdens. However, the pooled estimate showed that the calcium disodium EDTA treatment group did not have a significant decrease in urine protein level compared with that of the control group (SMD, −0.17; 95% CI, −0.46 to 0.11; P = 0.24, Fig. 1). The first reported case of nephrotoxicity associated with lead was described more than a hundred years ago. From then on, exposure to lead has been thought of as a risk factor for kidney injury. Because blood lead levels indicate only recent exposure to lead, MG-132 the level of body lead burden is considered as a more accurate indicator

reflecting the lead load in the human body, and urinary lead excretion <600 μg/72 h after calcium disodium EDTA chelation therapy is considered as a normal body lead burden. However, it was found that lead has a direct toxic effect on the kidney even at ‘normal or acceptable’ levels.[2, 3] The pathogenesis of nephrotoxic effects of lead is mainly related to direct toxicity, inflammation and oxidative stress.[2,

13, 14] A growing body of research has shown that lead exposure is a reversible risk factor for CKD progression,[4-9] nonetheless, the optimal strategy to treat lead nephrotoxicity remains uncertain. Chelating agents such as calcium disodium EDTA are widely used to remove toxic metals, and this therapy could exert long-term antioxidant, anti-inflammatory effects.[15] However, lead chelation is controversial owing to the potential of its use in lieu of exposure reduction. In addition, cases of acute tubular necrosis have been reported following early clinical use of calcium disodium EDTA that involved very large doses.[16] Fortunately, adverse renal effects have not been observed science at low levels of exposure such as in the trials included in our meta-analysis. The main finding of the current meta-analysis is that calcium disodium EDTA chelation therapy could effectively delay the progression of chronic kidney disease among patients with measurable body lead burdens by increasing the levels of GFR and Ccr. There is no conclusive evidence that chelation therapy with calcium disodium EDTA reduces proteinuria. However, our findings indicate the need to be confirmed by more larger randomized clinical trials. There are several important potential study limitations to this meta-analysis. First, most of the included studies were small-scale studies that may have had patient selection and treatment bias. Second, most studies were not blinded.

[73] The C protein of human parainfluenza virus type 1 impedes the nuclear translocation of STAT1 by

physically retaining it in the cytoplasm in perinuclear aggregates associated with late endosomal markers.[74] RSV NS-1 and NS-2 prevent the Protein Tyrosine Kinase inhibitor phosphorylation and nuclear translocation of STAT1 and STAT2 after IFN-β treatment of bone-marrow-derived DCs,[75] whereas in the respiratory epithelium, NS2 causes the degradation of STAT2.[76, 77] Viral interferon regulatory factor 2 (vIRF2) from KSHV decreases STAT1 and IRF9 levels to impair ISGF3 function.[78] HSV-2 causes the selective loss of STAT2 transcripts and proteins in some cell types, whereas in others, STAT2 levels remain constant but its phosphorylation and nuclear translocation are inhibited.[79] The papain-like

protease from FK228 SARS-CoV has a complex mechanism of interference: it is a de-ubiquitinating enzyme that up-regulates the expression of ubiquitin-conjugating enzyme E2-25k, leading to the ubiquitin-dependent proteasomal degradation of extracellular signal-regulated kinase (ERK) 1, which interferes with ERK1-mediated STAT1 phosphorylation.[80] Interestingly, adenovirus stabilizes tyrosine-phosphorylated, activated STAT1, sequestering it at viral replication centres, potentially through binding with viral DNA.[81] Adenovirus also impairs the dephosphorylation of STAT1 by obstructing its interaction with the protein tyrosine phosphatase TC45.[81] Once activated, ISGF3 binds the promoters of ISGs, leading to their transcriptional activation.[70] While investigating how the human adenovirus protein Anacetrapib E1A evades the type I IFN response, Fonseca et al.[82] furthered our understanding of this process, demonstrating how studying the virus leads to a better understanding of the host. They found that IFN-mediated antiviral activity requires the mono-ubiquitination of histone 2B (H2B) at lysine 120, a post-translational modification associated with transcriptionally active chromatin, in both the transcribed regions and the promoters of ISGs.

This finding is a novel and unexpected aspect of antiviral signalling. Additionally, they found that E1A disrupts the hBre1 complex responsible for H2B mono-ubiquitination, preventing the expression of ISGs, and allowing viral escape of antiviral signalling.[82] In another elegant study, Marazzi et al.[83] demonstrated how viruses exploit epigenetic signalling to regulate antiviral gene expression. They found that the NS1 protein of influenza A strain H3N2 contains a short sequence that mimics the histone H3 tail. This permits histone-modifying enzymes to act on NS1; accordingly, NS1 is both acetylated and methylated in infected cells.[83] Modified NS1 associates with the human PAF1 transcription elongation complex, allowing the virus to hijack the host transcriptional elongation machinery. NS1 also disrupts transcriptional elongation at sites of active antiviral gene transcription, selectively impairing the expression of ISGs).

Proposals about the rules of generalization have been a central topic of discussion among learning theorists since the time of Pavlov (1927) and Skinner (1938). A more modern treatment of generalization in the context of statistical learning comes from the work of Marcus, Vijayan, BandiRao, and Vishton find more (1999). In a variant of the syllables-of-speech design of Saffran et al. (1996), Marcus et al. presented 9-month-olds with 3-syllable strings separated by pauses rather than with continuous streams devoid

of pauses. These 3-syllable strings were composed from a set of eight consonant-vowel syllables into one of three different patterns defined by the repetition of one of the syllables, thereby forming AAB, ABA, or ABB “rules”. After exposure to multiple repetitions of the 16 3-syllable strings, infants heard two types of test trials, both of which were composed of entirely new CV syllables. One type of test trial conformed to the familiar “rule” and the other did not.

Infants showed a novelty preference—they listened longer to the unfamiliar rule. These results led Marcus et al. to propose that there are two different learning mechanisms: (1) statistical learning that is limited to extracting “surface” patterns embedded in the input to which the infant is exposed, and (2) rule learning that goes beyond the exposure materials to generate “abstract” patterns. Although this proposed dichotomy between statistical learning and rule learning seems compelling, Selleck RGFP966 there are reasons to suggest an alternative hypothesis. Gerken (2006) conducted a follow-up experiment to Marcus et al. (1999) in which separate groups of infants were familiarized to slightly different families of 3-syllable strings. As shown in Table 1, both groups of infants heard a subset of the 16 strings used in Marcus et al.

However, one group heard four strings that each ended in a different syllable, and the other group heard four strings that ended in the same syllable. Importantly, the four strings presented to both groups had an AAB pattern. But for the group whose four strings ended in the same syllable, an alternative to the AAB www.selleck.co.jp/products/Neratinib(HKI-272).html “rule” is a rule that is more restrictive—the first two syllables are the same, followed by the syllable/di/. For this group of infants, when presented with test strings that conformed to the AAB rule but not the “ends in/di/” rule, they did not generalize (i.e., they showed a novelty response). In contrast, for the group of infants presented with the set of AAB strings that ended in four different syllables, they formed a broader generalization that accommodated novel syllables even in the final-syllable position. This latter group performed as the infants in the Marcus et al. study by forming an “abstract” rule (i.e., AAB), whereas the former group exhibited a more restrictive rule even though AAB was a plausible inference from the strings presented during familiarization.

These findings suggest that NKT cells can differentially regulate immune responses through the use of appropriate strategies depending on the local inflammatory environment 38. The differentiated IFN-γ-producing cells observed in experimental autoimmune encephalitis and uveitis may also play an important pathogenic role, buy KPT-330 as the transfer of effector Th1 cells has revealed distinct disease patterns 17, 39. The presence of cells producing both IL-17 and IFN-γ in encephalitis 3 and experimental uveitis (our unpublished data)

also suggests that Th17 and Th1 cells are not mutually antagonistic and are representative of different aspects of pathogenesis in autoimmune disease. Human autoimmune diseases, including encephalitis and uveitis, have diverse spectrums of clinical diseases that are composed of various aspects of the immune response 40, 41. Therefore, CD1d-dependent invariant NKT cell-mediated regulation of different Th effector cells could provide a more ideal strategy for the control of human autoimmune disease caused by diverse pathogenic profiles. OT-II TCR transgenic mice, which express a TCR specific for OVA peptide (amino acid residues 323–339) in the context of I-Ab, were purchased from Jackson Laboratory (Bar Harbor, ME, USA).

CD1d−/− mice on a C57/B6 (B6) background have been described previously 20. Jα18−/− mice on a BL6 background were obtained from Dr. Masaru Taniguchi (RIKEN Research Center). IL-4−/−, IL-10−/−, and IFN-γ−/− mice on B6 background and B6 and B6.Thy1.1 IWR-1 purchase mice were purchased from Jackson Laboratory. All mice were bred

and maintained in specific pathogen-free conditions at the animal facility of Seoul National University College of Medicine. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at Seoul National University. Human IRBP peptide1–20 (GPTHLFQPSLVLDMAKVLLD) was synthesized by Peptron (Korea). Purified pertussis toxin and incomplete Freund’s adjuvant were purchased from Sigma (St. Louis, MO, USA). Mycobacterium tuberculosis this website strain H37RA was purchased from Difco (Detroit, MI, USA). α-Galcer was synthesized as described previously 20 and resuspended in 0.5% Tween-20 in PBS at a concentration of 220 μg/mL. OT-II mice were depleted of NK1.1+ cells by i.p. injection of an anti-NK1.1 antibody (PK136) 5 days and 2 days before being euthanized for the experiment (100 μg each day). Lymph node cells from OT-II mice (5×105) were stimulated with 0.2 μM OVA peptide in the presence of FACS-purified NKT cells (2×104). Th17 differentiation was initiated by the addition of 10 ng/mL of recombinant mouse IL-6 and 5 ng/mL of human TGF-β to the culture. NK1.1+ TCR+ cells were purified from hepatic MNC using a FACSAria (Becton Dickinson, USA).

The removal of biofilms made up of two or more bacterial communities is thus critical to decrease the incidences of gene transfer between bacteria. This may significantly decrease the formation of new multiple antibiotic-resistant strains (Johnson et al.,

2006). Based on the present study, we show the ability of A. baumannii isolates obtained from UTI to adhere to different abiotic surfaces under experimental conditions. The role of plasmids with antibiotic-resistant characteristics in gene transfer and resistance towards antibiotics in biofilm-forming strains check details has been established. Finally, biofilm formation as well as the potential ability of spreading the antibiotic-resistant markers to other pathogens has been highlighted. The authors would like to acknowledge Dr R.B. Patwardhan, Professor K.B. Niphalkar, Mrs M.G. Satpute, Miss N.V. Telang and Miss A. Engineer for their constant help. D.H.D. would like to acknowledge

Bhabha Atomic STAT inhibitor Research Centre – University of Pune collaborative research programme for senior research fellowship (SRF). “
“There is increasing evidence that inflammation in the synovium plays a major role in the progression of osteoarthritis (OA). However, the immunogenic properties of mesenchymal stromal cells (MSCs), which are considered to regulate immunity in various diseases, remain largely unknown in OA. The purpose of this study was to determine the influence of MSCs from OA patients on regulatory T cells (Tregs) in an allogeneic co-culture model. Bone marrow (BM) and synovial membrane (SM) were harvested from hip joints of OA patients and co-cultured with lymphocytes enriched in CD4+CD25+CD127– regulatory T cells (Treg+LC) from healthy donors. Treg proportions and MSC markers were assessed by flow cytometry. Cytokine levels Protein kinase N1 were assessed after 2 and 5 days of co-cultivation. Additionally, Treg+LC cultures were analysed in the presence of interleukin (IL)-6 and MSC-supernatant complemented medium. B-MSCs and S-MSCs were able to retain

the Treg proportion compared to lymphocyte monocultures. T cell–MSC co-cultures showed a significant increase of IL-6 compared to MSC cultures. S-MSCs produced higher amounts of IL-6 compared to B-MSCs, both in single and T cell co-cultures. The effect of retaining the Treg percentage could be reproduced partially by IL-6 addition to the medium, but could only be observed fully when using MSC culture supernatants. Our data demonstrate that retaining the Treg phenotype in MSC–T cell co-cultures can be mediated by MSC derived from OA patients. IL-6 plays an important role in mediating these processes. To our knowledge, this study is the first describing the interaction of MSCs from OA patients and Tregs in an allogeneic co-culture model.

Among the five peptides that failed to elicit a response in any subject, GAD201–220 and GAD369–388 were previously shown to be processed and presented by autologous monocytes. T cells that recognize these epitopes are apparently not prevalent or these epitopes are

not processed efficiently. Since none of our experimental results suggest that GAD1–20, GAD73–92 and GAD473–492 are able to be processed and presented, these may simply be cryptic epitopes that are not particularly relevant in GAD65 responses. The results summarized in Fig. 4(b) suggested that both healthy donors and subjects with T1D have GAD65-specific T-cell repertoires that recognize multiple epitopes. We wondered whether having a susceptible PF2341066 class II HLA such as DR0401 is sufficient to generate a diverse repertoire of GAD65-specific T cells. To address this question, we examined responses to each of the 15 putative GAD65 epitopes in 11 healthy DR0401 donors and six subjects with T1D diabetes using tetramers. Since our goal for these experiments was to examine the GAD-specific repertoire, irrespective of disease status, CD25+ T cells were depleted as previously described to remove selleck chemicals llc regulatory T cells.[19] A summary of the tetramer staining results for all of the subjects tested is shown in Table 2. In these experiments we used

more samples from healthy donors than from subjects with T1D, anticipating that a higher fraction of the healthy subjects might lack detectable T-cell RAS p21 protein activator 1 responses to GAD65. However, the positive response rates were not statistically different (9/11 for healthy versus 5/6 for T1D, P = 0·73 Fisher’s exact test). This lack of difference in response rate suggests that depletion of CD25+ cells enabled us to observe the repertoires of both healthy donors and subjects with T1D as intended. Not surprisingly, the number of epitopes detected in each subject varied. The number of responses to GAD65 epitopes

ranged from 0 to 5 in healthy donors, and from 0 to 3 in diabetic subjects (Table 2). There was no statistically significant difference in the number of epitopes detected in these two groups (unpaired Student;s t-test, P = 0·74). This would suggest that GAD65-specific repertoires were equally broad in subjects with T1D and healthy controls. The most commonly observed epitopes included GAD433–452 (six subjects), GAD553–572 (five subjects) and GAD305–324 (four subjects). Additional epitopes, such as GAD473–492, GAD265–284 and GAD113–132, were also positive in multiple subjects. The GAD65 T-cell repertoires selected by healthy and diabetic subjects appear to be similar. However, it has been previously documented that only patients with T1D have expanded memory populations of T cells that recognize β-cell antigens.[20] Therefore, GAD-specific T-cell responses in healthy and diabetic subjects could still differ significantly.

Co-transfection experiments designed to validate the miR-155 binding site present in the 3′UTR of SOCS-1 were also performed using FA-associated liposomes. Two hundred microlitres of FA-lipoplexes, containing pmiR-155 and a plasmid encoding the reporter gene luciferase and the 3′UTR of SOCS-1 (pSOCS-1 3′UTR) were delivered to N9 cells to obtain a final plasmid concentration www.selleckchem.com/products/nutlin-3a.html of 1 μg/well for each plasmid. In parallel experiments, plasmid (p) GFP was used in addition to pSOCS-1

3′UTR to serve as a control. In all transfection protocols, after 4 hr of incubation, the medium was replaced by new RPMI-1640 medium and N9 microglia cells were incubated for different periods of time, before further analysis. Luciferase expression following

co-transfection of pSOCS-1 3′UTR and pmiR-155 or pGFP was evaluated by assessing luciferase activity. Briefly, 48 hr after transfection, cells were washed twice with PBS and 100 μl lysis buffer (1 mm dithiothreitol, 1 mm EDTA, 25 mm Tris–phosphate, 8 mm MgCl2, 15% glycerol, 1% [volume/volume (v/v)] Triton X-100, pH 7·8) were added to each well. After cell lysis at MAPK inhibitor −80°, 50 μl of each lysate were incubated with luciferin and ATP and light production was determined in a luminometer (Lmax II384; Molecular Devices, San Jose, CA). The protein content of the lysates was evaluated through the DC Protein Assay reagent (Bio-Rad, Hercules, CA), using BSA as the standard.

Data were expressed as relative light units of luciferase per mg of total cell protein and presented as fold change with respect to control (untransfected cells). Total RNA, including small RNA species, was extracted from N9 microglia cells or primary microglia cultures using the miRCURY™ Isolation Kit – Cells (Exiqon), according to the manufacturer’s recommendations for cultured cells. Briefly, after cell lysis, the total RNA was adsorbed to a silica matrix, washed with the recommended buffers and eluted with 35 μl RNase-free water by centrifugation. After RNA quantification, cDNA conversion for miRNA quantification was performed Mannose-binding protein-associated serine protease using the Universal cDNA Synthesis Kit (Exiqon). For each sample, cDNA for miRNA detection was produced from 20 ng total RNA according to the following protocol: 60 min at 42° followed by heat-inactivation of the reverse transcriptase for 5 min at 95°. The cDNA was diluted 80 × with RNase-free water before quantification by qRT-PCR. Synthesis of cDNA for mRNA quantification was performed using the iScript cDNA Synthesis Kit (Bio-Rad) and employing 1 μg total RNA for each reaction, by applying the following protocol: 5 min at 25°, 30 min at 42° and 5 min at 85°. Finally, the cDNA was diluted 1 : 4 with RNase free water. Quantitative PCR was performed in an iQ5 thermocycler (Bio-Rad) using 96-well microtitre plates.

2 cells, using protein G columns according to standard protocols. Soluble TNFR1 fusion protein (sTNFR1-Ig) was a kind gift from Geoff Hale (Therapeutic Antibody Group, University of Oxford, UK). All fluorochrome-conjugated anti-mouse mAbs and secondary detection reagents used were purchased from BD Biosciences (Oxford, UK). Biotinylated anti-CD3ζ was from Upstate (Watford, UK), and purified polyclonal rabbit anti-mouse EP1, EP2, EP3 and EP4 were from Cayman Chemicals (Ann Arbor, MI). Bone marrow (BM) Mϕ were generated using a method adapted from Munder et al.21 Briefly, bone marrow cells were resuspended at 5 × 105

cells/ml selleckchem in complete media supplemented with 5% v/v horse serum (Invitrogen), and 50 pg/ml macrophage colony-stimulating factor. The cell suspension was transferred to hydrophobic PTFE-coated tissue culture

bags (supplied by Dr M. Munder, University of Heidelberg, Heidelberg, Germany) and incubated for 8 days at 37° in 5% v/v CO2. Single-cell splenocyte suspensions were generated by grinding spleens through a 70-μm cell strainer (BD Biosciences) with a syringe plunger. When used as APCs, splenocytes were irradiated with 3000 Rads using a caesuim-137 source (Gravatom, Hants, UK). The OT-II CD4+ T cells were prepared by enriching CD4+ cells from single cell suspensions of C57BL/6 OT-II splenocytes, using anti-CD4 microbeads (Miltenyi Biotech, Bisley, UK) according to the manufacturer’s instructions. B cells were prepared from spleens using anti-B220 microbeads (Miltenyi Biotech). selleck Dendritic cells were generated from cultures of bone marrow cells as previously described.22 The 1 × 105 APCs were co-cultured with CD4+ T cells at ratio of 1 : 1 in round-bottom 96-well plates in complete media. The OVA peptide was added at the indicated concentrations. To some cultures the arginine analogue, l-NG-monomethyl arginine, the NO donor S-nitroso-N-acetyl-l,l-penicillamine,

or the cyclo-oxygenase (COX) inhibitor indomethacin (all from Sigma) was added. In some experiments, recombinant IFN-γ (Peprotech, London, UK), or PGE2 (Sigma) was added. Cells were cultured in a humidified Adenosine environment at 37°, 5% v/v CO2. Proliferation was measured by pulsing with 18·5 kBq [3H]thymidine (GE Healthcare, Bucks, UK) per well for the final 8 hr of culture and determining thymidine uptake [measured in counts per minute (c.p.m.)]. Accumulated NO production was measured after 64 hr in culture supernatants using Griess reagent (Sigma) as previously described.23 Production of IFN-γ was assessed using a murine T helper type 1 (Th1)/Th2 Flow cytomix 10plex kit (Bender Medsystems, Vienna, Austria) according to the manufacturer’s instructions. Concentration of PGE2 was measured using an enzyme immunoassay competition enzyme-linked immunosorbent assay kit (Caymen Chemical, Ann Arbor, MI) according to the manufacturer’s instructions.