In three groups a nerve defect of 20 mm was bridged with a vein graft. Our first experimental group comprized

an empty venous graft, in group II the venous nerve graft was filled with saline where as in group III the venous nerve graft was filled with BMSC. The animals were tested for functional recovery up to 3 months post repair. Our results show that the BMSC filled venous graft resulted in significantly better regeneration of the nerve defect compared to controls, as confirmed by the functional recovery measured by somatosensory evoked potentials, toe spread, pin prick, and gastrocnemius muscle index. Conclusively, the results confirm that the vein graft supported with BMSC is associated with better functional Selleckchem PD0325901 nerve regeneration. © 2010 Wiley-Liss, Inc. Microsurgery, 2010. “
“The aim of this study was to

evaluate the effect of Platelet Rich Plasma (PRP) and Platelet Romidepsin supplier Rich Fibrin (PRF) on peripheral nerve repair. Thirty-two Wistar rats were randomly divided into four equal treatments groups: autologous nerve grafts (ANG), silicon tube plus saline solution (SS), silicon tube plus PRP, and silicon tube plus PRF. In ANG group, 10 mm segment from sciatic nerve was excised and reimplanted between the nerve stumps. In the SS, PRP, and PRF groups, 5 mm segment from sciatic nerve was excised and bridged with a 12 mm silicone conduit to create a 10 mm nerve gap. The conduit was filled in accordance with the different treatments. Walking track analysis was performed periodically and on the 90th post-operative day histomorphometric analysis was performed. The ANG, PRF, and PRP groups presented a significant functional improvement

in relation to the SS group (P = 0.001) on 90 days after surgery. Histomorphometric analysis demonstrated that the Immune system ANG group achieved a larger nerve fiber diameter in proximal stump while comparing with the SS group (P =0.037) and showed larger fiber diameter in median stump in comparison to the PRP group (P = 0.002) and PRF group (P = 0.001). Axonal diameter and myelin sheath thickness showed no statistical significant difference between the groups in the three stumps (P ≥ 0.05). This study suggests that PRP and PRF have positive effects on the functional nerve recovery; however, these groups don’t achieve a significant improvement on the histomorphometric analysis. © 2013 Wiley Periodicals, Inc. Microsurgery 33:383–390, 2013. “
“Among possible causes of a condition of immunodeficiency, we have to consider the presence of a serious chylous dysplasia, due to the great loss of proteins through the intestinal lumen. A 20-year-old male, suffered from diarrhoea (2–4 times a day), weight loss (8 kilos in 5 years), and malnutrition (hypogammaglobulinemia, hypoalbuminemia, leukocytopenia with lymphocytopenia).

5), the number of cycles at which fluorescence was reached the threshold line was 31.09 on the lipase gene and 5.09 on 16S rRNA, respectively.

In contrast, when we used RNA sample from the cells cultured in NB (3.0) the number of cycles was 28.00 on the lipase gene and 4.98 on 16S rRNA. Consequently, we estimated by the ΔΔCt method that the relative transcriptional level of lipase gene in 3% NaCl is 7.8 times higher signaling pathway than that in 0.5% NaCl. Moreover, as shown in Figure 8, the densities of the samples recovered at 12 and 24  hrs from the culture in NB (3.0) were certainly higher than those from the culture in NB (0.5), showing that the gene for the lipase is well transcribed in A. sobria under the condition of 3.0% NaCl. Transcription of the lipase gene by A. sobria in NB (3.0) at 12 and 24  hrs was more active than in NB (0.5). As shown, the amount of lipase in the culture supernatant from culturing in NB (3.0) was low compared with that in NB (0.5) (Fig. 1); however, transcription of the lipase gene by A. sobria was not suppressed in 3.0% NaCl and the mRNA of the lipase gene was produced well (Fig.  8). We therefore considered that the posttranscriptional process to become mature lipase had been disrupted in NB

(3.0). As shown in Figure 4, lipase expresses esterolytic Selleckchem MG-132 activity; we therefore examined the esterolytic activity of the culture supernatant, using pNpp Bcl-w as the substrate. The supernatant

from the 24  hr culture in NB (0.5) expressed esterolytic activity, but that from the culture in NB (3.0) did not (Fig. 9). These findings suggest that the three-dimensional structure of the lipase differs from that of the active form when A. sobria is cultured in NB (3.0), and that the lipase produced in NB (3.0) is degraded by bacterial intracellular proteases. This explains why the amount of lipase in the supernatant from culture in NB (3.0) was low compared with that in NB (0.5). In this study, we found a protein of A. sobria whose production in the milieu was suppressed by NaCl in the medium. Analysis revealed that this protein is lipase; it degraded tributyrin, and expressed esterase activity against pNp-fatty acyl esters. We then cloned the lipase gene and determined the nucleotide sequence. The lipase substrate binding signature sequence (GLKVHFLGHSLGA) was contained in the sequence (28), supporting the contention that the protein is a lipase. The amino acid sequence deduced from the nucleotide sequence had 78.6% identity with the lipase of A. hydrophila AH-3 (11). Merino et al. have examined the substrate specificity of the lipase from A. hydrophila AH-3 (11). They found that E.

rPWV may add detailed insights into early microvascular pathophysiology, potentially beyond microalbuminuria. “
“Twin infants tend to have LBW and microvascular alterations but do not appear to have an increase in cardiovascular mortality later www.selleckchem.com/products/Gefitinib.html in life as singleton infants. We hypothesized that twin infants born to normotensive mothers would not have capillary rarefaction at birth. We studied 26 dizygotic

twin infants and compared them with 115 consecutive singleton infants to normotensive mothers. We used orthogonal polarized spectroscopy to measure basal (i.e., functional) and maximal (i.e., structural) skin capillary density according to a well-standardized protocol. Twin infants have significantly higher BCD (mean difference 4.3 capillaries/mm2, 95% CI: 0.4, 8.1, p = 0.03) and have marginally significantly higher MCD (mean difference 3.9 capillaries/mm2, 95% CI: −0.6, 8.3, p = 0.086) compared to singleton infants.

Birth weight was significantly associated with SB203580 molecular weight BCD and MCD (p = 0.003 and 0.006). Twin infants with low and NBWs tend to have higher functional and structural capillary densities compared to singleton infants. Further longitudinal studies of skin capillary density and of retinal vascular parameters commencing from birth to various stages in early childhood are essential to identify the dynamics and the exact timing, if any, of the remodeling of microcirculation in these individuals. LBW is now considered an independent risk factor for adult cardiovascular Phosphatidylinositol diacylglycerol-lyase disease as both clinical and epidemiological studies have shown an association with cardiovascular risk factors such as essential hypertension, dyslipidemia, diabetes mellitus, and insulin resistance in later life [7, 8]. Although the exact mechanism for this association is not as yet fully elucidated, several studies have suggested that microcirculatory abnormalities may be implicated [10, 15, 18, 25, 34]. LBW is known to be associated with several structural and functional microvascular abnormalities including

reduction in microvascular density or rarefaction [9, 11, 26, 34, 37]. Rarefaction of arterioles and capillaries is an early hallmark of essential hypertension [5, 30, 36] and we have previously shown that individuals with borderline intermittent essential hypertension, and normotensive individuals with familial predisposition to essential hypertension have significant capillary rarefaction [3, 4]. Twin infants are very interesting to study because as a group they tend to have LBW and significant microvascular alterations including narrower retinal arterioles [37] but do not appear to have an increase in cardiovascular mortality or morbidity later in life as singleton infants [13, 40].

“
“Systemic lupus erythematosus (SLE) and lupus nephritis (LN) have strong concomitance with cardiovascular disease that cannot fully be explained by typical risk factors. We examined the possibility that serum or urine expression of adipokines may act as biomarkers for LN, since these proteins have previously been associated with cardiovascular disease as well as SLE. Antibody arrays were performed on serum and urine from lupus patients and matched controls using a cross-sectional study design. From the initial array-based screening data of 15 adipokines, adiponectin, leptin, and resistin were selected for validation by ELISA. Correlations were determined between

adipokine expression levels and measures of disease activity or lupus nephritis. Expression of adiponectin and resistin were increased in both sera and urine from LN patients, while leptin was increased

selleck inhibitor in LN patient sera, as compared to matched controls. Serum resistin, but not urine resistin, was correlated with measures of renal dysfunction in LN. Serum resistin expression may be useful as a marker of renal dysfunction in patients with LN though longitudinal studies are warranted. Further Saracatinib datasheet studies are necessary to determine if resistin has functional consequences in LN. “
“Oestradiol and the selective oestrogen receptor modulator (SERM) raloxifene have been shown to ameliorate collagen-induced arthritis (CIA) in rats and in mice. One aim was to investigate if raloxifene exerts its anti-arthritic and anti-osteoporotic effects during the induction or effector phase of arthritis. A second aim was to analyse if raloxifene activates the oestrogen response element (ERE) to produce its immune-modulator effects. CIA or collagen–antibody-induced arthritis (CAIA) was induced in ovariectomized Dipeptidyl peptidase DBA/1-mice. CIA was used for evaluation of treatment during the induction, and CAIA for the effector phase of arthritis and osteoporosis development. Raloxifene, oestradiol or vehicle was administered 5 days/week. The clinical disease was evaluated continuously. Bone marrow density (BMD) was analysed with peripheral quantitative computer tomography, paws were collected for histological examination, and sera

were analysed for markers of bone and cartilage turnover and proinflammatory cytokines. Transgenic luciferase (Luc)-ERE mice were immunized with collagen (CII), and after 10 days injected once with raloxifene, oestradiol or vehicle before termination. Spleens were analysed for luciferase activity to measure ERE activation. Treatment with oestradiol or raloxifene during the induction phase of CIA failed to affect arthritis. Raloxifene did not hamper disease activity in CAIA, whereas oestradiol delayed the onset and ameliorated the severity. Both raloxifene and oestradiol preserved BMD in CAIA. CII-immunization increased the oestradiol-induced ERE activation in spleen, and raloxifene activated the ERE at about 25% the intensity of oestradiol.

5B). Next, we analyzed CCR2 and MCP-1 expression in the thymi of IL-12 + IL-18 cDNA-treated mice. We observed a significant increment in CCR2 mRNA expression in the bulk thymocyte population of IL-12 + IL-18 cDNA-treated mice (Fig. 5C). Moreover, thymocytes from IL-12 + IL-18 cDNA-treated mice cultured ex vivo, spontaneously produced much larger amounts of MCP-1 than thymocytes from control mice (Fig. 5D). Interestingly, an important boost in MCP-1 expression is observed in thymocytes from IL-12 + IL-18 cDNA-treated mice when rIL-12 and rIL-18 are added to the cultures but not in thymocytes from control mice, suggesting that rIL-12 and rIL-18 are able to drive MCP-1 expression only from thymocytes that

have been exposed to IL-12 and IL-18 in vivo (Fig. 5D). Based on these data, we next speculated if T cells entering the thymus expressed a particular Small molecule library mouse TCR or if it is a general polyclonal process. To evaluate whether T-cell recruitment depends on the TCR, we administered T. cruzi infection in OT-I mice that express a transgenic TCR specific for OVA peptide in CD8+ T cells, an antigen not expressed by the parasite T. cruzi. Similarly to what we observed

in WT mice, when CFSE splenocytes from OT-I T. cruzi selleck infected mice are adoptively transferred to T. cruzi infected WT mice, only B cells and CD4+ and CD8+ T cells are able to enter the organ (Supporting Information Fig. 2). Importantly, we observed that all CFSE+CD8+ splenocytes from OT-I-infected mice that enter the thymus of WT-infected mice express the TCR Vβ5 chain (OVA specific), demonstrating that those clones are probably activated during the infection in a bystander way and then acquire the capacity to reenter the thymus (Supporting Information

Fig. 2). The entrance of peripheral mature T cells has been described in mouse [6, 8], rat [9, 33], and pig [34] models, especially after T-cell activation by an Ag [6, 8, 10, 16]. In the case of B cells, recruitment of a low number of these cells to the thymus seems to be a normal process, however it could highly increase in certain pathological next situations such as thymic lymphoma [11] and certain autoimmune-prone mouse strains [12]. To examine this concept in greater detail, we report here that entrance of mature peripheral B cells as well as T cells is a common feature that occurs during an acute Th1 inflammatory/infectious process. There is one report that demonstrates the entrance of T cells to the thymus during a viral infection, but in this case it is the consequence of peripheral CD8+ T cells entrance in order to eliminate infected cells in the thymus [35]. In this article, we demonstrate that entrance of peripheral cells to the thymus during inflammatory/infectious disease processes is more a consequence of a bystander activation of certain peripheral B and T cells that express CD62L, CD44, and CCR2, thus allowing them to ingress the thymus due to local production of MCP-1.

Anderson and co-workers established an innovative approach that allows the detection Selleckchem PD 332991 of gluten-specific T cells in the peripheral blood of CD patients after a short period of gluten-containing food consumption [4,5]. Basically, gluten-sensitized

CD4+ T cells, normally scarcely detectable in the blood of coeliac patients, circulate transiently in the peripheral blood after 3 days of wheat challenge, and can be detected by a sensitive interferon (IFN)-γ enzyme-linked inmmunospot (ELISPOT) assay. Using this in-vivo procedure, the authors further screened large libraries of prolamin peptides and assessed the hierarchy and immunodominance of gluten T cell epitopes [6]. More recently, T cells reactive to DQ2-α-I and DQ2-α-II epitopes were monitored in the peripheral blood of bread-challenged coeliacs with specific DQ2-tetramer constructs [7,8]. Of Selleckchem ALK inhibitor note, both Australian and Norwegian studies enrolled adult coeliac volunteers, with an average age of 43 years. To our knowledge, no information is available on the responsiveness to short gluten challenge in very young coeliac patients. Furthermore, very little is known about the in-vivo challenge reproducibility

in the same subject cohort, with the exception of a few cases of coeliacs who underwent two separate gluten consumptions described in the above-mentioned studies [7,8]. In the present study we have validated the in-vivo short gluten challenge in a cohort of

14 young CD patients of Italian origin. In particular, we analysed the peripheral blood response against whole gliadin and the immunodominant 33-mer peptide (α-gliadin 57–89). We also assessed the feasibility of exposing the patient cohort to a second in-vivo challenge after a period of 3–10 months of wash-out, in order to estimate the reproducibility of the procedure in the same study population and the intra-individual variations. If replicated successfully in other studies, the short wheat challenge could Amrubicin represent a strategic tool to evaluate non-invasively the individual’s response to gluten, and could be applied to intervention studies. In fact, the evaluation of small bowel mucosa damage after long-term wheat challenge has been used since the 1950s to assess cereal toxicity or to define the toxic peptides [9–11]. Such studies required repeated endoscopies, before and after treatment, which are always not well accepted by participants. To detect a dysregulated response to gluten, other functional markers, such as faecal fat and xylose malabsorption, resulted in low specificity and sensitivity [12–15]. Furthermore, recent studies have indicated that a short gluten challenge could be used to support diagnosis in doubtful cases of CD [16–18]. Fourteen DQ2-positive volunteers with CD (mean age 18·6, range 15–24 years) participated in the study (Table 1).

At the age of 22, she suffered from akinesia, resting tremor, and rigidity. At the age of 28, she was admitted to our hospital because of worsening parkinsonism and dementia. Within several years, she developed akinetic mutism. At the age of 49, she died of bleeding from a tracheostomy. Autopsy revealed a severely atrophic brain weighing 460 g. Histologically, there were iron deposits in the globus pallidus and substantia nigra pars reticulata, and numerous axonal spheroids in the subthalamic nuclei.

selleck chemical Neurofibrillary tangles were abundant in the hippocampus, cerebral neocortex, basal ganglia, and brain stem. Neuritic plaques and amyloid deposits were absent. Lewy bodies and Lewy neurites, which are immunolabeled by anti-α-synuclein, were absent. We also observed the presence

of TDP-43-positive neuronal perinuclear cytoplasmic inclusions, with variable frequency in the dentate gyrus granular cells, frontal and temporal cortices, and basal ganglia. TDP-43-positive glial cytoplasmic inclusions were also found with variable frequency in the frontal and temporal lobes and basal ganglia. The present case was diagnosed with adult-onset NBIA-1 with typical histological findings in the basal ganglia and brainstem. However, in this case, tau and TDP-43 pathology was exceedingly more abundant than α-synuclein pathology. This case contributes to the increasing evidence for the heterogeneity of NBIA-1. “
“Department of Clinical Neuroscience and Therapeutics, see more Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima We performed clinicopathological analyses of two amyotrophic lateral sclerosis (ALS) patients with homozygous Q398X optineurin (OPTN) mutation. Clinically, both patients presented signs of upper and lower motor neuron degeneration, but only Patient 1 showed gradual frontal dysfunction and extrapyramidal signs, and temporal lobe and motor cortex atrophy. Neuropathological examination of Patient 1 revealed extensive cortical and spinal motor neuron degeneration and widespread degeneration of the basal ganglia. Bilateral corticospinal tracts exhibited

degeneration. Loss of spinal anterior horn cells (AHCs) and gliosis were observed, whereas posterior columns, Clarke’s columns, intermediate lateral Paclitaxel columns, and the Onuf’s nucleus were spared. In the brainstem, moderate neuronal loss and gliosis were noted in the hypoglossal and facial motor nuclei. No Bunina bodies were found in the surviving spinal and brainstem motor neurons. Transactivation response (TAR) DNA-binding protein 43 (TDP-43)-positive neuronal and glial cytoplasmic inclusions were observed throughout the central nervous system. The Golgi apparatus in motor neurons of the brainstem and spinal cord was often fragmented. Immunoreactivity for OPTN was not observed in the brain and spinal cord, consistent with nonsense-mediated mRNA decay of OPTN. The TDP-43 pathology of Q398X was similar to that of an autosomal dominant E478G mutation.

Recent evidence suggests that similar mechanisms may regulate the commitment of Thp between Treg and Th17. In human cells, FoxP3 exists in two separate but equally expressed isoforms: one (FoxP3), which is encoded by a full length mRNA and the other a truncated form lacking exon 2 (FoxP3Δ2), which is coded by a splice variant mRNA [104,109]. Tregs, perhaps unexpectedly, also express Th17-specifying transcription factors, notably RORα[110] and RORγt [111]. However, co-immunoprecipitation experiments have shown that FoxP3 binds to RORα and RORγt and inhibits their biological activity

in a dose-dependent fashion [110,111]. This interaction is mediated through a (LxxLL) motif in the FoxP3 second exon; as expected, the FoxP3Δ2 isoform is unable to bind RORα or LBH589 selleck chemical RORγt [110,111]. A similar interaction has subsequently been described, by the same group and others, in murine cells. Specifically, both FoxP3 and RORγt are co-expressed

in naive CD4+ T cells exposed to TGF-β, where FoxP3 inhibits RORγt directly through a physical interaction, repressing the Th17 programme [111]. In these experiments exposure of Thp to TGF-β leads to rapid induction of RORγt [92], but the binding of RORγt to the IL-17 promoter is suppressed by interaction with FoxP3 [112]. Upon addition of exogenous IL-6 or IL-21, the inhibitory effect of FoxP3 on IL-17 induction is circumvented [111] and FoxP3 levels are reduced [112]. The interaction between FoxP3 and RORγt

in murine cells is also dependent upon the second exon of FoxP3 [111,112]. These observations have also been confirmed by another, independent group [74]. These interactions can, in part, explain the conversion of Tregs to Th17, at least in mice. While TGF-β induces both FoxP3 and RORγt expression, IL-6 does not alter expression of RORγt but inhibits FoxP3. As a result, exposure of Tregs to IL-6 down-modulates FoxP3 preferentially and reduces the Dichloromethane dehalogenase physical inhibition of RORγt, permitting binding to the IL-17 gene promoter. In addition, very recent murine data suggest that IL-1 regulates expression of RORγt [79]. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is a receptor-coupled signal transduction mechanism linking cytokine–receptor interactions to gene expression. There are seven STAT (STAT1-4, 5A, 5B and 6) and four JAK [JAK1-3 and TYK2 (tyrosine kinase 2)] proteins in humans (reviewed in [113]). Specific JAKs are associated with the cytoplasmic tails of multimeric cytokine receptors, and are activated upon ligand-induced receptor oligomerization [113,114]. Activated JAKs phosphorylate specific tyrosine residues on cytoplasmic tails of their associated cytokine receptors, creating docking sites for the SH2 (Src-homology-2) domain of STAT proteins, and then activate the docked STATs through tyrosine phosphorylation.

abscessus (4–6). One of them, M. abscessus Group II strains, was reported as M. massiliense and M. bolletii (7). As a genetic identification method to differentiate M. massiliense from M. abscessus and other species recently became available, human infections caused by M. massiliense have been continuously

reported (8–12). Nearly half of the RGM isolates initially identified as M. abscessus, which is the species of RGM that is most frequently INCB024360 mouse isolated in Korea, are actually M. massiliense (7). So far, differentiation between M. abscessus and M. massiliense depended on sequence analysis of housekeeping genes (e.g. rpoB and hsp65) (7, 9). However, additional housekeeping genes were analyzed because of the discordant results between rpoB and hsp65 gene analysis (7, 13). Clarithromycin is a 14-membered ring macrolide that binds

to the large ribosomal subunit in the vicinity of the peptidyltransferase center and inhibits protein synthesis, which results in the arrest of bacterial growth (14). Clarithromycin is given orally, and is highly active against many species of NTM. Although M. massiliense shares many traits with M. abscessus and M. bolletii, M. massiliense can be differentiated by marked susceptibility to clarithromycin (2, 7, 11). Moreover, patterns of clarithromycin resistance differed between M. massiliense and M. abscessus (7), which led us to investigate another mechanism, involvement of erm. This is because the erm gene is frequently involved in macrolide resistance in human pathogens as with the 23 rRNA gene mutation. Acalabrutinib solubility dmso The erm gene encodes N6-mono or N6, N6-dimethyltransferases that cause specific methylation of nucleotide A2058 and/or neighboring nucleotides (A2057 and A2059; based on Escherichia coli numbering) in the 23S rRNA, which Carnitine palmitoyltransferase II results in resistance to macrolide. Because Mycobacterium species possess only one or two rrn operons, alteration of this specific site is critical to the development of resistance (25). Among the 33 erm genes that have

been reported and numbered to date, five innate erm genes [erm(37), erm(38), erm(39), erm(40) and erm(41)] have been identified within the genus Mycobacterium (15). Recently, three types of erm(41) of M. abscessus were reported. One M. massiliense clinical isolate was confirmed to have short erm(41) by PCR and was reported as one of the three erm(41) types without sequence analysis (16). Because quite different responses of M. massiliense compared to M. abscessus against clarithromycin were observed in our previous report (7), exact information on erm(41) of more clinical M. massiliense isolates, and their relevance to the susceptibility pattern of clarithromycin was needed. In the present study, the erm(41) sequences of M. massiliense, M. abscessus and M. bolletii isolates were investigated in relation with MIC to clarithromycin, and a simple erm(41) PCR to differentiate M. massiliense from closely related M. abscessus and M.

Different types of T-cell lineages exhibit independent and distinct gene expression and regulation signatures 6, 17. Based on our observations

that tumor-derived Th17 clones converted to T-cell populations with mixed Treg, Th1 and Th17–Th1 phenotypes Enzalutamide mouse following TCR stimulation and expansion, we reasoned that these phenotypic alterations could be the result of changed expression of lineage-restricted transcriptional regulators and regulatory cytokines that control and direct T-cell programming 7, 17, 21. To test this possibility, we first determined the gene expression of the key transcriptional factors, including RORγt and IRF-4 (Th17) as well as T-bet (Th1), GATA-3 (Th2) and FOXP3 (Treg), in the expanded Th17 clones using real-time PCR. As expected, we found that the primary (E0) and early expanded Th17 clones (E1) expressed higher levels of the Th17-specific transcriptional factors RORγt and IRF-4, and the expression levels dramatically decreased following subsequent unbiased expansion cycles (Fig. 5A). In addition, T-bet and FOXP3 expression gradually increased in Th17 clones with the expansions, whereas GATA-3 expression was at a relatively NVP-LDE225 low level in expanded Th17 cells (Fig. 5A). We then analyzed the mRNA expression of Th1, Th2 and Th17-associated cytokines and cytokine receptors in the expanded Th17 cells following

each round of expansion, using real-time PCR. As shown in Figs 1D and 5B, Th17 cells from primary or early expansion clones expressed high levels of IL-17A, IL-21 and IL-22, but the expression of these genes decreased markedly with subsequent expansions. This suggested that the isometheptene expanded Th17 clones had undergone down-regulation of autocrine cytokines and had decreased responsiveness to Th17-associated growth cytokines, such as IL-21. Unexpectedly, however, we found markedly

increased IL-23R expression in Th17 clones after subsequent expansions, which may be due to the increased T-bet expression in these expanded Th17 clones 46. In addition, we observed significantly increased IFN-γ mRNA expression in Th17 clones after the expansions, whereas there was no or minimal IL-4 gene expression in expanded Th17 clones. These results indicate that the phenotypic changes of Th17 clones induced by TCR stimulation and expansion result from the reprogramming of lineage-specific gene expression. Given the significantly decreased IL-17 production and RORγt expression, as well as increased FOXP3 demethylation and expression, and TGF-β production in the expanded Th17 clones, we next questioned whether repetitive in vitro TCR stimulation and expansion of Th17 cells altered their effector functions and induced suppressive activity towards other immune cells.