In contrast, the antigens present in RD1, RD5, RD7 and RD9 may be involved in protection by inducing preferential secretion of the protective cytokine IFN-γ, Ivacaftor cell line and none, or very little, IL-10. Previous studies have identified the major Th1-stimulating antigens of RD1 (ESXA, ESXB and PPE68), RD7 (ESXO and ESXP) and RD9 (ESXV and ESXW) (8, 29, 59). Among these antigens, ESXA and ESXB have been shown to have vaccine potential in animal models of TB (58, 60). However, ESXA and ESXB are already in use for specific diagnosis of latent and active TB (17, 18), and these antigens cannot be used for both vaccination and diagnosis of infection with M. tuberculosis.

Therefore, further work should be carried out to determine the vaccine potential of other Th1-stimulating antigens of RD1, RD5, RD7 and RD9 by testing them in animal models before they are included in future antigen cocktails for use as vaccine(s) against TB. In conclusion, the results presented in this paper suggest that complex mycobacterial antigens selleck screening library and proteins encoded by various RDs of M. tuberculosis have opposing effects in cell mediated immunity assays, that is, Th1 versus Th2. Culture filtrate and RD1, RD5, RD7 and RD9 are strong

Th1 inducers whereas whole cells, cell walls, RD12, RD13 and RD15 are strong Th2 inducers. Therefore, in new vaccine design against TB, the antigens of the former group may be more relevant. This work was supported by the Kuwait Foundation for the Advancement of Sciences (KFAS) grant no. 2002-1302-04. “
“Autoimmunity may contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). Studies have identified disease-specific learn more autoantibodies (DSAAbs) in COPD

patients, but natural autoantibodies (NAAbs) may also play a role. Previous studies have concentrated on circulating autoantibodies, but lung-associated autoantibodies may be most important. Our aim was to investigate NAAbs and DSAAbs in the circulation and lungs of COPD smoking (CS) patients compared to smokers (S) without airway obstruction and subjects who have never smoked (NS). IgG antibodies that bind to lung tissue components were significantly lower in the circulation of CS patients than NS (with intermediate levels in S), as detected by ELISA. The levels of antibodies to collagen-1 (the major lung collagen) detected by ELISA were also signifcantly reduced in CS patients’ sera compared to NS. The detection of these antibodies in NS subjects indicates that they are NAAbs. The occurrence of DSAAbs in some CS patients and S subjects was indicated by high levels of serum IgG antibodies to cytokeratin-18 and collagen-5; furthermore, antibodies to collagen-5 eluted from homogenised lung tissue exposed to low pH (0.1M glycine, pH 2.8) were signifcantly raised in CS compared to S and NS. Thus, this study supports a role in COPD for both NAAbs and DSAAbs.

The aim of this study was to investigate whether diabetes and insulin resistance affect B-1 cells and their production of natural IgM. We found that diabetic db/db mice have Talazoparib nmr lower levels of peritoneal B-1a cells and a decreased

IgM response to pneumococcal immunization and TLR-4 activation. Furthermore, our in-vitro studies showed that glucose in high concentrations reduces B-1 cell IgM secretion and differentiation into antibody-producing cells concurrent with proliferation arrest and increased apoptosis. Specific pathogen-free C57BL/6 mice were purchased from Taconic (Skensved, Denmark). For isolation of peritoneal B-1 cells, male and female C57BL/6 mice were fed a normal chow diet. As a model for insulin resistance, 8-week-old male C57BL/6 mice were assigned randomly to a low glycaemic control diet or a high-fat diet (Harlan

Laboratories, Madison, WI, USA) for 12 weeks. On a caloric basis, the low glycaemic control diet contained 16·8% fat, 60·9% carbohydrate and 22·3% protein (3·3 Kcal/g), whereas the high-fat diet contained 60·3% fat, 21·3% carbohydrate and 18·4% protein (5·1 Kcal/g). The diets contained comparable amounts of vitamins and minerals. Male db/db mice and control mice (+/+ or +/db) on a C57BL/6 background from Jackson Laboratories (Bar Harbor, ME, USA), and db/db and wild-type controls (+/+) on a BKS background from Taconic, were maintained on a normal chow diet. For in-vivo assessment Tamoxifen mw of the effect of TLR-4 agonist, 10–12-week-old db/db mice (on a C57BL/6 background) and controls Axenfeld syndrome were injected intraperitoneally with 0·34 mg/kg of the TLR-4 agonist Kdo2-Lipid A (Avanti Polar Lipids, Inc., Alabaster, AL, USA) or vehicle. For immunization studies, 10–12-week-old db/db mice and controls (on a C57BL/6 or BKS background) and C57BL/6 mice maintained on diets for 3 months were injected intraperitoneally with 11·5 μg of a 23-valent vaccine (Pneumovax; Sanofi Pasteur MSD, Lyon, France), containing 0·5 μg each of 23 types of polysaccharides from S. pneumoniae

or saline. As indicated for each experiment, body weight, plasma insulin, glucose and antibody titres were followed in longitudinal blood samples. Before blood sampling, mice were fasted for 4 h. Plasma glucose in blood samples from fasted, non-anaesthetized animals was determined with a glucose dehydrogenase method by using HemoCue® B-glucose microcuvettes (HemoCue®, Ängelholm, Sweden) and insulin was determined by a mouse insulin enzyme-linked immunosorbent assay (ELISA) (Mercodia, Uppsala, Sweden). Plasma triglycerides and cholesterol were measured using Konelab 20 Autoanalyzer (Thermo Electron Corporation, Vantaa, Finland). All mice were housed in a controlled environment and all experimental protocols were approved by the animal ethical committee in Gothenburg.

Twenty-one patients whose diagnosis had been made between 1 and 3 months before the commencement of dialysis was excluded from the analysis. The main clinical features of the late diagnosis group at presentation were dyspnoea/pulmonary oedema (41%), severe hypertension (26%),

severe asthenia (22%) and apathy/mental changes (8%). The rate of pulmonary infections (17.9% vs 5.1%, P < 0.01) and mean systolic blood pressure (172 ± 4 mmHg vs 161 ± 4 mmHg) were significantly higher in the late diagnosis group. All patients in the late diagnosis group required a CVC for initiation of dialysis. In the early diagnosis group, 33% of patients had a vascular access created electively. Creatinine clearance at the time of initiation of dialysis was significantly lower in the late dialysis group (4.4 ± 0.5 mL/min vs 6.4 ± 0.5 mL/min, P < 0.01). Smoothened Agonist mouse Survival at 6 months was significantly decreased (69% vs 87%, P < 0.01) and the risk of death was 2.77 times higher in the late dialysis group. In multivariate

analysis, the most significant predictors of poor outcome were age, intercurrent pulmonary infection and low serum albumin at the commencement of dialysis. In Ratcliffe et al.’s retrospective review of characteristics of all patients accepted for dialysis in the Oxford Unit in 1981, criteria for commencement of dialysis were uraemic symptoms associated with a creatinine clearance PD98059 less than 6 mL/min.31 Thirty-two patients were referred >1 month (early diagnosis Cetuximab group) and 23 patients were referred <1 month (late diagnosis group) before the commencement of dialysis. In the early referral group, 91% of patients commenced dialysis electively, 72% had a functioning fistula at the time of initiation of dialysis and 22% were commenced on continuous ambulatory peritoneal dialysis. Only two patients required initiation of dialysis via a CVC. In the late referral group, 39%

of patients commenced haemodialysis via a CVC. ‘Serious complications’, which significantly prolonged the length of stay in hospital, were significantly more frequent in the late diagnosis group (70% vs 9%, P < 0.001). Jungers et al. retrospectively reviewed records of 250 patients who commenced dialysis at the Necker Hospital between January 1988 and December 1990.32 The records of patients who required emergency dialysis and who had been referred within 4 weeks of commencing dialysis were identified. Of the total cohort, 25% were in this late referral category. From these patients, 20 records were randomly selected and compared with a control group of 20 age- and sex-matched patients who had been regularly followed up at the renal clinics for at least 6 months prior to the commencement of dialysis.

This pathway may also regulate the analogous processes of neurite extension and tumor cell invasion. “
“Please cite this paper as: Nunez, Trach, Burnett, Handa, Dyke, Callahan, and Smith (2011). Vasoactive Properties of Keratin-Derived Compounds. Microcirculation18(8), 663–669. Objective:  Keratin proteins have been utilized as biomaterials for decades, and are currently under investigation for a variety of tissue regeneration and trauma applications. It has been suggested that

certain keratins may have the capacity to act as a colloid in fluid resuscitation applications, providing viscosity and oncotic properties that www.selleckchem.com/products/forskolin.html may be beneficial during acute ischemic events. Oxidized Selleckchem Kinase Inhibitor Library keratin derivatives, also known as keratoses, show good blood and cardiovascular compatibility and thus are the subject of this study. Methods:  The effects of keratose compounds will be assessed using a topload i.v. infusion model and

observation of changes in the microvasculature of the cremaster muscle of rats. Results:  Keratose resuscitation fluid (KRF) administration resulted in significant vasodilation in the cremaster muscle. This effect was blocked with pretreatment of l-NA to inhibit NO. Another keratin fraction, alpha-keratose, which is the primary viscosic compound, was not found to induce vasodilation. Conclusions:  The apparent mechanism of vasodilation was found to be NO-mediated and isolated to a particular purified fraction, the KAP. “
“Microcirculation (2010) 17, 1–10. doi: 10.1111/j.1549-8719.2009.00010.x Objectives:  Knowledge of glomerular structural and hemodynamic changes in vivo is still limited under diabetic conditions. In this study, we examined the alterations in glomerular structure and permeability of macromolecules and the effects of telmisartan using a confocal laser microscope. Methods:  Diabetes was induced by injecting streptozotocin. After 4 and 8 weeks, the filtration and

permeability of differently sized compounds across the glomerular capillaries were visualized using a confocal laser microscope by injecting 500-kilodalton and 40-kilodalton dextran. At 7 weeks, some diabetic rats were treated either with telmisartan for 1 week. The permeation of the 40-kilodalton dextran across the glomerular capillaries into Bowman’s space was quantified. Glomerular volume, diameters of the afferent and efferent arterioles, and glomerular permeability were compared. Results:  Glomerular volume was significantly increased in the diabetic rats, and there was heterogeneity in the glomerular volumes. The diameter ratio of the afferent to efferent arterioles significantly increased, and there was increased glomerular permeability in the diabetic rats compared with the control rats. Telmisartan treatment reduced glomerular permeability without affecting glomerular volume.

The release of TGF-β1 by live DC upon apoptotic DC uptake was regulated at the translational level, as no upregulation of TGF-β1 mRNA was observed. In order to investigate the underlying mechanism, we looked at the role of the mammalian target of rapamycin

(mTOR). mTOR, a serine/threonine protein kinase, is a regulator of translation and its major substrates this website include p70S60K serine/threonine kinase and 4E-binding protein (4EBP-1). Live DC were co-cultured with apoptotic DC in the presence of rapamycin, a known inhibitor of mTOR pathway. Next, we looked at the levels of total and active TGF-β1 released in the media (Fig. 8A). Our findings indicate that pre-treatment with rapamycin resulted in significant reduction of both total and active TGF-β1 released in media, indicating a role of mTOR in the observed TGF-β1 release upon uptake of apoptotic DC by viable DC.

Furthermore, TGF-β1 secretion in response to LPS stimulation of viable DC that Metformin had taken apoptotic DC was also suppressed in the presence of rapamycin (Fig. 8B). Taken together, our results show that the impact of dying DC on the immune system is dependent on the manner in which DC die. If DC undergo apoptosis and viable DC take them up, then viable DC transform into tolerogenic DC. These tolerogenic Carnitine dehydrogenase DC are resistant to stimuli-induced maturation, secrete TGF-β1, which is dependent on mTOR pathway and induce generation of Foxp3+ Treg. Surprisingly, our findings show that necrotic DC, irrespective of their maturation status are not immunostimulatory, which may be due to the paucity of the presence of certain immunosuppressive factors in primary DC, rendering them

non-immunogenic even after the cellular contents are released into the extracellular milieu. However, such factors still need to be identified. Studies have shown that DC can take up antigen from dying cells and cross-present the antigenic material onto both MHC I and MHC II 20, 21. However, these studies relied on the use of mature DC to phagocytose apoptotic cells. We can speculate that perhaps in a physiological setting, if the causative agent of DC apoptosis is an infection, then it is usually the semi-mature or mature viable DC in close proximity that take up apoptotic DC. Thereby, these viable DC can cross-present the antigen and then prime a T-cell response rather than induction of tolerance, as seen in our study. Previous studies have indicated that phosphatidylserine, an anionic aminophospholipid, which is exposed to cell surface as cells undergo apoptosis, plays an important role in the recognition and clearance of apoptotic cells by macrophages.

Various chemokine receptors, cytokine receptors, and pattern recognition receptors are expressed by γδ T cells and these receptors have been shown to be involved in the activation of γδ T cells, especially for the induction of IL-17 (Fig. 1). IL-1, IL-6, IL-18, IL-23, and transforming growth factor beta

1 (TGF-β) have each been implicated in promoting IL-17 production by γδ T cells. Furthermore, activation via Toll-like receptor 2 (TLR2) and DC-associated C-type lectin 1 (dectin 1), as well as the internal receptor aryl hydrocarbon receptor (AhR), has also been associated with IL-17 production by www.selleckchem.com/products/BI6727-Volasertib.html γδ T cells [30]. However, highly purified γδ T cells do not appear to produce IL-17 following stimulation with TLR agonists in the absence of exogenous cytokines (Sutton, Mielke, and Mills, unpublished data). Furthermore, γδ T cells from IL-6−/−

mice produce IL-17 AP24534 research buy at comparable levels to wild-type mice [31], while ablation of TGF-β leads to a reduction but not a total loss of IL-17, suggesting that there may be a non-essential role for these cytokines in promoting IL-17 production by γδ T cells. In contrast, γδ T cells in a spleen cell preparation from IL-1 type I receptor-defective (IL-1RI−/−) mice fail to secrete IL-17 in response to IL-23 and/or TLR agonists (Sutton and Mills unpublished data). Furthermore, IL-1α or IL-1β in synergy with IL-23, has been shown to play a crucial role in the induction of IL-17 from γδ T cells in both mice and humans [6, 25, 32, 33]. Interestingly, γδ T cells express IL-1RI and have high levels of IL-18R on their cell surface, and it has recently been demonstrated that IL-18 can synergize with IL-23 to promote IL-17 production by γδ T cells [29]. It appears that the activation

of the inflammasome in DCs and macrophages, and the consequent processing of the cytokines IL-1β and IL-18, from an inactive precursor to an active form as a result Thymidine kinase of inflammasome-triggered pathways, is important for the generation of IL-17-secreting γδ T cells [29]. A defect in the response of IL-17+ γδ T cells, but not IFN-γ+ γδ T cells, to malaria infection has been reported in MyD88-deficient mice [34]. This provides further evidence that activation of TLR (and hence MyD88) signaling and the consequent production of inflammatory cytokines, such as IL-1 (that also signals via MyD88), IL-23, and IL-6, are important steps in driving IL-17 production from γδ T cells. CCR6, the chemokine receptor for CCL20, has been shown to be associated with IL-17+ RORγt+ CD4+ T cells and has also been shown to be present on IL-17+ γδ T cells [30]. IL-2, which has been shown to constrain Th17-cell differentiation [35], appears to have a role in inducing IL-17 production from γδ T cells.

[32] In the postnatal period in the pig, NOS activity is greatest in the pre-glomerular

resistance vasculature of the newborn kidney immediately after birth, but decreases as maturation progresses.[33] Furthermore, the different isoforms of NOS differentially regulate renal vasodilatation during the neonatal period compared with the adult. Expression of the neuronal isoform of NOS (nNOS) in the renal resistance vasculature is greatest in the newborn pig, but expression of endothelial NOS (eNOS) is greatest in the adult.[32] In buy FK506 alignment with this, nNOS predominantly contributes to renal blood flow in the postnatal period but CP-690550 nmr eNOS contributes to renal blood flow in the adult.[32]

Importantly, expression of nNOS has been shown to be greatest in the macula densa of the developing kidney of the pig,[32] a site important in modulating TGF activity. An increase in NO production has been shown to decrease the sensitivity of TGF.[34] Thus, it can be inferred that NO produced by nNOS facilitates the decrease in afferent arteriolar resistance in the postnatal period by decreasing the sensitivity of TGF. Although nNOS appears to be important in the resetting of TGF, it is not necessary in the long term since nNOS knockout mice have a normal TGF response.[29] This is supported by the fact that nNOS expression declines but expression of eNOS increases during the postnatal period.[32] Presumably this increase in eNOS expression compensates for the decline in expression of nNOS and in the long term, eNOS maintains basal renal haemodynamics. Nevertheless, it appears that the high expression

of nNOS at birth[32] is necessary to reset the sensitivity of TGF and promote afferent vascular dilatation. Normal postnatal maturation of the kidney is characterized by Nintedanib (BIBF 1120) both functional and structural adaptations of the glomerulus and tubules. The following sections of this review will focus firstly on both the structural and functional adaptations to nephron loss. We will then put forward a hypothesis regarding mechanisms via which compensatory renal growth may be implicated in the onset of hypertension and chronic kidney disease. Compensatory renal growth also occurs following surgical reduction in renal mass (uninephrectomy or sub-total nephrectomy) and is associated with significant hypertrophy of the tubules and the glomeruli. In the rat kidney, the increase in length of proximal tubules can be as much as 70–90%[10, 35, 36] with a more modest (17–40%) increase in length occurring in the distal tubules.

5×106 DCs. Thirty days after EAE-induction, spleens were removed for restimulation and seeded out as triplicates of 4×105 cells 5-Fluoracil ic50 per well in a flat-bottomed 96-well plate (Greiner) in the presence of graded concentrations of MOG35–55 peptide. After 72 h of restimulation, supernatant was harvested and analyzed for

its cytokine content by ELISA. BALB/C mice were sensitized by i.p. injections of 10 μg OVA protein (Hyglos) mixed in aluminum hydroxide at days 0 and 14 of asthma induction. Mice treated as negative controls received injections of aluminum hydroxide only. DCs were injected at day −7, −5, and −3 before asthma induction in the tail-vein of mice and for a total of 2–2.5×106 cells. Then, mice were challenged by intranasal administrations of 100 μg OVA protein in 50 μL PBS at days 22, 23, and 24 of asthma induction. Six days after the last OVA challenge, mice were lethally anesthetized followed by bleeding of the axillary veins for serum immunoglobulin analysis. Blood was coagulated for 2 h at room temperature and centrifuged on 3000×g for 5 min to recover the serum. Circulating H 89 OVA-specific IgG subclasses were determined by ELISA. For this, 96-well plates (♯353279; BD) were coated overnight at 4°C with OVA protein (Sigma; 100 μg/mL) in 0.1 M NaHCO3 coating buffer. Sera were loaded as serial dilutions in 1% FCS in PBS. OVA-bound

Abs in the sera were detected by horseradish peroxidase-conjugated mouse heavy chain-specific Abs: anti-mouse IgG1-HRP (Serotec), or IgE-biotin and streptavidin-HRP (BD) followed by the substrate tetramethylbenzidine (BD). Absorbance was detected using an ELISA microplate reader (Vmax; Molecular Devices). Serum titers were calculated from the serial dilution, which was 1.5-fold increased compared with baseline (optical density

of negative control mice). BAL was performed by flushing the lungs through an opening in the trachea with PBS from a Ribonucleotide reductase syringe. Differential cell count of the BAL was determined by recording total cell amount and spinning cells on microscope glass slides using a Cytospin Universal centrifuge (Hettich, Germany). Cytospins were stained with hematoxylin-eosin solution (Diff-Quick staining set; Medion Diagnostic) and cells were classified using standard morphologic criteria. Data are represented as mean data±SD. Statistical significance was analyzed with GraphPad Prism software using one-way ANOVA followed by Bonferroni post-testing and significance accepted if p<0.05. Data of EAE and asthma experiments were validated using Kruskal–Wallis test followed by Dunn’s post-test and considered as significant if p<0.05. This work was supported by the German Research Council (DFG) through the Sonderforschungsbereich SFB581, International Research Training Grant IRTG1522 and the Transregio Collaborative Research Centre TR52. The authors thank A. Gessner for providing the C3H/HeJ and TLR4/MyD88−/− mice.

Moroni et al.[2] reported that death-censored graft survival at 15 years was approximately 10% lower in IgAN patients than in controls, suggesting that the culprit is IgAN recurrence. In fact, IgAN is one of the most common recurrent glomerulonephritis. The majority learn more of recurrent IgAN is not clinical but pathological. Factors deciding the activity of recurrent IgAN remain unclear. It is unpredictable when IgAN recurs

and why these recurrences occur immediately after transplantation. We report a case of active IgAN that recurred 19 days after transplantation. A 23-year-old man with ESRD underwent living-related ABO-identical pre-emptive kidney transplantation (PEKT) from his 57-year-old mother. At the age of 18, his urinalysis was normal. However, at the age of 19, 3+ proteinuria and 2+ occult blood were detected by annual physical examination. The abnormality on his urinalysis was diagnosed as IgAN by renal

biopsy, which showed mild to moderate mesangial hypercellularity, with fibrocellular crescent in 1 of 10 glomeruli (Fig. 1a). Subsequently, tonsillectomy was performed and steroid pulse therapy was initiated. However, unfortunately, nephrotic-range proteinuria still developed. The patient then progressed to ESRD regardless of the treatment with steroid pulse therapy, cyclosporine (CyA), prednisolone, a renin–angiotensin system inhibitor, an antiplatelet agent, and anticoagulation therapy. He was referred to our hospital to undergo PEKT from his mother. Laboratory data revealed ESRD with a serum creatinine concentration histone deacetylase activity of 8.53 mg/dL, haemoglobin of 8.8 g/dL and serum albumin of 3.54 g/dL. Urinalysis showed 3+ proteinuria and microscopic haematuria. PEKT was performed when the patient was 23

years old. Initial immunosuppressive therapy consisted of prednisolone, mycophenolate mofetil, CyA and basiliximab, and the transplantation procedure was successful. Allograft biopsy performed 1 h after transplantation revealed normal glomeruli and an immunofluorescence study showed negativity for IgA and C3. The second biopsy was performed 19 days after transplantation to elucidate the cause of the increasing proteinuria (1.1 g/day) and serum creatinine (1.80 mg/dL). This biopsy showed segmental tuft necrosis with fibrin (Fig. 2a), and an immunofluorescence during study showed strong positivity for IgA and C3 (Fig. 2b). Neither acute rejection nor CyA nephrotoxicity was observed. These pathological findings and negative results for MPO-ANCA and PR3-ANCA completed the diagnosis of recurrent IgAN. The patient received steroid pulse therapy, double filtration plasmapheresis (DFPP), and anticoagulation therapy with warfarin. However, he developed nephrotic-range proteinuria and the serum creatinine concentration increased gradually. A cytomegalovirus infection made it inevitable to reduce the immunosuppressive agents.

These results also suggest that Mel-18

can function as conventional transcriptional repressor in Th cells in a gene-dependent U0126 solubility dmso context. We did not notice any changes in the expression levels of Ifng or Il4 mRNAs as a result of Mel-18 or Ezh2 knockdown in Th17 cells (Fig. 2G and H). We neither found any changes in the expression levels of the two Gata3 transcripts 71 (data not shown). The mRNA level of Tbx21, encoding T-bet, was increased in some experiments following Ezh2 knockdown (data not shown), but this result was inconsistent. In summary, our results show that PcG proteins positively regulate the expression of Il17a, Il17f and Rorc in restimulated Th17 cells. Considering the binding pattern of PcG proteins at the promoter of Il17a, a direct transcriptional regulation is suggested, but the involvement of additional indirect regulatory pathways is

also possible. The inducible binding activity of Mel-18 and Ezh2 at the Il17a promoter was regulated by factors downstream to the TCR (Fig. 1). However, since PcG proteins are expressed non-differentially in Th1, Th2 and PI3K inhibitor Th17 cells (here and 66), the lineage selectivity of their binding pattern is probably instructed by the polarizing cytokines. We aimed therefore to determine whether the presence of the polarizing cytokines is required for the binding activity of PcG proteins at the Il17a promoter in differentiated Th17 cells. First, we wanted to examine the requirement of these cytokines to maintain Th17 phenotype under our experimental conditions. Freshly purified CD4+ T cells were differentiated under Th17 conditions

Leukocyte receptor tyrosine kinase for 6 days (TGF-β and IL-6 including IL-23) and then were restimulated with PMA and ionomycin for 2 h in either the presence of Th17 skewing cytokines, without cytokines or in the presence of the Th1 polarizing cytokine IL-12 (data not shown). We did not observe statistically significant changes in the expression levels of the mRNAs of Rorc, Rora, Il17a and Il17f. Similar results were observed when the cells were restimulated 2 h with anti-CD3 and anti-CD28 antibodies (data not shown). Therefore, shortly after restimulation Th17 cells maintain their ability to express the specific cytokines and transcription factors in the absence of polarizing cytokines. Next we wanted to determine whether a continuous presence of the polarizing cytokines is necessary to maintain the Th17 transcriptional program during a longer restimulation. Freshly purified CD4+ T cells were differentiated with TGF-β, IL-6 and IL-23 for 6 days and then were restimulated with anti-CD3 and anti-CD28 antibodies for 18 h in the presence of different cytokines as indicated in Fig. 3A.