In humans remission of Crohn’s disease patients was observed after human immunodeficiency virus (HIV) infection [6] and thymectomy was demonstrated to prevent relapse in ulcerative colitis (UC) patients [7].

In addition, a case study described cure of UC by excision of an invasive thymoma [8]. T lymphocytes are generated from haematopoietic stem cells in the bone marrow and become immunocompetent through a maturation process in the thymus, during which they are termed thymocytes. In the thymus they undergo negative selection, deleting self-reactive thymocytes Ku-0059436 datasheet by apoptosis, thereby generating central tolerance. Our previous studies on the Gαi2-deficient mouse model of colitis, as well as mice with dextran sodium sulphate (DSS)-induced colitis, demonstrated aberrant thymocyte development with reduced frequencies of immature and increased frequencies of mature thymocytes before and during onset of colitis, as well as reduced migration towards intrathymic PD0332991 chemokines [9,10]. We therefore hypothesized that

similar abnormalities might also be present in human IBD. Due to the very limited access of thymic tissue from IBD patients, we used the technique of T cell receptor excision circle (TREC) analysis to investigate the relative abundance of recent thymic emigrants (RTE) in the periphery. Upon entrance into the thymus the thymocytes undergo rearrangement of their TCR genes, along with intense proliferation. T lymphocytes have four sets of TCR genes that will form either of two types of heterodimers: αβTCRs which are expressed by the majority of peripheral T cells, or γδTCRs, expressed by a subset of T cells mainly in the skin and intestinal epithelium [11]. The great diversity in the antigen-recognizing domains of the TCR molecules are generated by random combinations of multiple variable (V), diversity (D) and joining (J) gene segments (TCR δ and β chains), or V and J gene segments (TCR γ OSBPL9 and α chains). V(D)J recombination

is initiated by the recognition of recombination signal sequences (RSSs) that flank the coding segments, and during this process the DNA located between the two RSS regions is circularized, forming an extrachromosomal circular excision product containing the two ligated RSS regions [11]. These so-called TRECs are stable and are not duplicated during mitosis, and are thus diluted-out with each cell division [12]. The levels of TRECs in naive T cells in peripheral blood are therefore a good measurement of thymic output. The method has been used extensively to study T cell reconstitution in highly active antiretroviral therapy (HAART)-treated HIV-patients [13] as well as after bone marrow transplantation following, e.g. myeloablative therapy for leukaemia [14].

In particular, it is now apparent that probiotic feeding can influence immune responses in the respiratory tract and improve protection against bacterial and viral pathogens (6–11). In this Aloxistatin ic50 regard, we previously showed that the immunomodulatory probiotic strain Lc431 is able to improve

immunity in the respiratory tract in both immunocompetent and immunocompromised hosts (7, 8). In these studies, we observed that mice orally treated with the optimal dose with adjuvant effect of Lc431 had a higher resistance to challenge with the respiratory pathogen Streptococcus pneumoniae (7, 8). In addition, our laboratory has isolated different lactobacilli strains from goat milk and studied their ability to stimulate host defenses. We selected two of the strains evaluated, Lr1505 and Lr1506, because of their capacity to improve intestinal immunity and increase resistance against Salmonella typhimurium (12). In addition, our studies MLN0128 solubility dmso have demonstrated that oral administration of Lr1505 is also able to improve resistance against pneumococcal infection (12). In order to improve understanding of the mechanisms through which certain probiotic

strains exert their immunomodulatory effect at sites distant from the gut, in this study we evaluated the influence of oral treatment with Lc431, Lr1505 or Lr1506 on the activity of macrophages at sites distant from the gastrointestinal tract. In particular, we studied the effect of these treatments on the phagocytic and microbicidal activity of alveolar and peritoneal macrophages. Male 6-week-old Swiss albino mice were obtained from the closed colony at CERELA. Farnesyltransferase They

were housed in plastic cages and their environmental conditions kept constant, in agreement with the standards for animal housing. The Ethical Committee for Animal Care at CERELA approved the experimental protocols. Lc431, Lr1505 and Lr1506 were obtained from the CERELA culture collection. Bacteria were cultured for 8 hr at 37°C (final log phase) in Man-Rogosa-Sharpe broth (Oxoid, Cambridge, UK), then harvested by centrifugation at 3000 g for 10 min, washed three times with sterile 0.01M PBS, pH 7.2, and finally resuspended in NFM at appropriate concentrations for administration to the mice. Lc431 was administered by the oral route for 2 consecutive days at dose of 109 cells/mouse/day, which is the optimal dose able to achieve stimulation of respiratory immunity (8, 9). Lr1505 and Lr1506 were administered by the oral route for 5 consecutive days at doses of 108 cells/mouse/day (12). Lactic acid bacteria were suspended in 5 mL sterile 10% NFM and added to the drinking water (20% v/v). The control group received sterile NFM under the same conditions. All mice were fed a conventional balanced diet ad libitum. Cytokine concentrations were measured in serum and intestinal and BAL fluids.

L. P. R.: Contributed to the article and design of the study. S.S., M.H., T.W. and S.J.L.: Delivered PLX3397 mouse patient and donor material, performed the statistical data analysis and contributed to the manuscript. K.M.: Planned and designed the project

and established the collaboration. Participated in data analysis and drafted the manuscript. “
“Leishmaniasis is a group of important parasitic diseases affecting millions worldwide. To understand more clearly the quality of T helper type 1 (Th1) response stimulated after Leishmania infection, we applied a multiparametric flow cytometry protocol to evaluate multifunctional T cells induced by crude antigen extracts obtained from promastigotes of Leishmania braziliensis (LbAg) and Leishmania amazonensis (LaAg) in peripheral blood mononuclear

cells from healed cutaneous leishmaniasis patients. Although no significant difference was detected in the percentage of total interferon (IFN)-γ-producing CD4+T cells induced by both antigens, multiparametric flow cytometry analysis revealed clear differences in the quality of Th1 responses. LbAg induced an important proportion of multifunctional CD4+ T cells (28% of the total Th1 response evaluated), whereas LaAg induced predominantly single-positive cells (68%), and 57% of those were IFN-γ single-positives. Ipilimumab price Multifunctional CD4+T cells showed the highest mean fluorescence intensity (MFI) for the three Th1 cytokines assessed and MFIs for IFN-γ and interleukin-2 from those cells stimulated with LbAg were significantly higher than those

obtained after LaAg stimulation. These major differences observed in the generation of multifunctional CD4+ T cells suggest that the quality of the Th1 response induced by L. amazonensis antigens can be involved in the mechanisms responsible for the high susceptibility observed in L. amazonensis-infected individuals. Ultimately, our results call attention to O-methylated flavonoid the importance of studying a Th1 response regarding its quality, not just its magnitude, and indicate that this kind of evaluation might help understanding of the complex and diverse immunopathogenesis of American tegumentary leishmaniasis. Leishmaniasis is a group of sandfly-transmitted diseases caused by different species of protozoan parasites from the genus Leishmania, affecting 88 countries around the world [1]. The diverse clinical presentations depend upon which Leishmania species is involved and also upon host-related factors. American tegumentary leishmaniasis (ATL) is endemic in widespread areas of Latin America, and the main causative agents include species from the subgenus Viannia (Leishmania (Viannia) braziliensis, L. (V.) guyanensis, L. (V.) panamensis) and the subgenus Leishmania (Leishmania (Leishmania) amazonensis, L. (L.) mexicana) [1]. In addition to being a public health problem in the New World, ATL is a risk for those who travel to Latin America [2].

[60] However, these mice also lack CD8 T cells which were shown to be pathogenic in obesity,[50] and their loss alone improves obesity, and so clear conclusions could not

be drawn. To address this issue, Mantell et al. used CD1d−/− mice with normal CD8 levels to measure the contribution of iNKT cells to obesity.[61] Like other studies, Mantell et al. found that iNKT cells were decreased in the liver of obese mice but found an increase in adipose iNKT cells in obesity, unlike the majority of other studies. One caveat is that this study used NK1.1+ CD3+ as surrogate markers for iNKT cells, which also detects other T cells that are not iNKT cells, particularly in adipose tissue where NK1.1 is not expressed on a substantial proportion of iNKT cells. It may also detect non-invariant type II NKT cells, or MHC-restricted T cells, which check details have been shown to up-regulate NK markers when activated,[62] and these could be possible selleck chemicals llc explanations for an increase in NK1.1+ T cells in obesity. Nevertheless, in this study, obese CD1d−/− mice were not significantly different from obese wild-type mice in terms of weight gain and metabolic parameters.[61] Boes and colleagues found that iNKT-deficient mice had increased adipocyte size

and insulin resistance, and their depletion increased insulin resistance. They also found that CD1d−/− mice on an HFD had increased insulin resistance, but they did not observe any difference in weight gain between wild-type and NKT-deficient mice on an HFD. However, Kotas et al. found that iNKT-deficient mice

were metabolically normal on a standard diet, but CD1d−/− mice had mildly but significantly impaired glucose tolerance and increased insulin resistance on an HFD.[63] This study particularly focused on hepatic iNKT cells, which they found were reduced in obesity, and mice lacking iNKT cells had increased liver triglycerides and worse hepatic steatosis. However, many of these features were not seen in Ja18−/− mice on HFD, suggesting that either Abiraterone other CD1d-restricted T cells are responsible for the worsened metabolism and steatosis, or that Ja18−/− mice displayed some compensation for the iNKT cell deficiency. Hams et al. also found that while iNKT cells positively regulated obesity and metabolism and were depleted in obese mice, they did not find any differences in weight or glucose homeostasis in CD1d−/− or Ja18−/− mice.[64] This series of studies suggests that deficiency in iNKT cells from birth does not alter weight gain and metabolism, yet iNKT deficiency from birth resulted in excess weight and impaired metabolism on a normal diet. On the other hand, our laboratory, Qi and colleagues, and Kim and colleagues all found that mice deficient in iNKT cells had accelerated obesity and had significantly more severe glucose impairment and insulin resistance on an HFD.

aureus and S. pneumoniae, resulting in elevated TNF and IL-10 secretion and diminished IL-12 levels (Fig. 1C). Since IRAK4 is a key signaling adaptor in the TLR pathway but whole pathogens represent complex mixtures of multiple PRR ligands we sought to perform experiments

with defined TLR ligands to better assess the role of IRAK4. We therefore analyzed cytokine secretion in response to synthetic TLR2 ligand Pam3CSK4 and TLR4 agonist LPS. Consistent with the observations made in monocytes of IRAK4-deficient patients [23], down-regulation of IRAK4 lead to a reduction of TNF secretion levels in response to LPS (Fig. 2A). Similarly, LPS-induced production of IL-12 (Fig. 2A), PLX4032 IL-6, and IL-1β (not shown) was diminished in IRAK4-deficient cells. Similarly, secretion of TNF and IL-12 in IRAK4-silenced cells was markedly

decreased after Pam3CSK4 stimulation selleck compound (Fig. 2B). Of note, differences in cytokine concentrations were not statistically significant in all cases, but, despite donor-dependent variation in the cytokine levels, the trend was clear in all donors and experiments shown. To further confirm the specificity of our siRNA knockdown, we next studied TLR-induced TNF production in the presence or absence of a commercially available IRAK1/4 inhibitor. As expected, both LPS and Pam3CSK4-induced TNF secretion was reduced under IRAK1/4 inhibition (Fig. 2C). Finally, we analyzed activation of NF-κB subunits p50 and p65. These transcription factors form part of the classical NF-κB pathway and are activated upon TLR

stimulation. Confirming our earlier observations LPS-triggered induction of p50 as well as p65 was decreased in IRAK4-knockdown Parvulin cells when compared with that in cells transfected with unspecific control siRNA (Fig. 2D), thus highlighting the key role of IRAK4 in mediating NF-κB-dependent pro-inflammatory cytokine secretion. Having confirmed that TLR-triggered pro-inflammatory cytokine production is decreased under IRAK4 knockdown conditions we analyzed the release of anti-inflammatory IL-10. As already observed using live bacteria (Fig. 1C), we found that IL-10 levels were markedly increased after LPS and Pam3CSK4 stimulation in IRAK4-deficient cells (Fig. 3A). Elevated IL-10 secretion and specificity of the knockdown was again confirmed with the IRAK1/4 inhibitor (Fig. 3B). Further analysis demonstrated increased IL-10 mRNA expression under IRAK4-silencing conditions (Fig. 3C), thus indicating that increases in IL-10 protein levels are due to enhanced gene transcription. Not surprisingly, elevated IL-10 levels were accompanied by increased mRNA expression of the IL-10-dependent genes socs3 and tnfr2 (Fig. 3C) while that of others such as stat3 or CREB-dependent cox2 was unaffected (data not shown).

Malaria remains one of the main global infectious diseases and cerebral malaria is a major complication, often fatal in Plasmodium falciparum-infected children and young adults [1]. Cerebral malaria pathophysiology is still poorly understood, combining cerebral vascular obstruction, and exacerbated immune responses. Indeed, investigations

in humans and mice documented STA-9090 the sequestration of erythrocytes, parasitized or not, platelets and leucocytes in cerebral blood vessels with an increased proinflammatory cytokine expression [1-3]. The specific role of T cells in cerebral malaria pathogenesis has been difficult to address in humans. In mice however, T-cell sequestration and activation in the brain are crucial steps for experimental cerebral malaria (ECM) development after Plasmodium berghei ANKA (PbA) infection [4-7]. In particular, αβ-CD8+

T cells sequestrated in the brain play a pathogenic, effector role for ECM development [6], and we showed recently a role for protein kinase C-θ (PKC-θ) in PbA-induced ECM pathogenesis [8]. Besides being a critical regulator of TCR signaling and T-cell activation, PKC-θ is involved in interferon type I/II signaling in human T cells [9]. Type II IFN-γ is essential Lenvatinib mw for PbA-induced ECM development [10-12], promoting CD8+ T-cell accumulation in the brain [7, 12-14]. Type I IFNs are induced during viral infection but they also contribute Terminal deoxynucleotidyl transferase to the antibacterial immune response. In Mycobacterium tuberculosis infection, types I and II IFNs play nonredundant protective roles [15], while type I IFNs inhibit IFN-γ hyper-responsiveness by repressing IFN-γ receptor expression in a Listeria monocytogenes infectious model [16]. Moreover, type I IFNs role in central nervous system (CNS) chronic inflammation is ambiguous [17].

IFN-β has proinflammatory properties and contributes to some auto-immune CNS diseases, while IFN-β administration is routinely used in relapsing-remitting multiple sclerosis treatment, characterized by inflammatory cell infiltration to the CNS, including Th1 and Th17 [17]. Crossregulations between type I and type II IFNs have been documented [18-21], they can have similar or antagonistic effects, and type I IFN-α/β precise role in ECM development after sporozoite or merozoite infection remains unclear. Here, we addressed the role of IFN-α/β pathway in ECM development in response to hepatic or blood-stage PbA infection, using mice deficient for types I or II IFN receptors. Unlike IFN-γR1−/− mice that were fully resistant to ECM, we show that IFNAR1−/− mice are partially protected after sporozoite or merozoite infection. Magnetic resonance imaging (MRI) and angiography (MRA) confirmed the reduced microvascular pathology and brain morphologic changes in the absence of type I IFNs signaling.

0–16.1). There was considerable heterogeneity due to differences in the definition of late referral (regarded as ‘management that could have been improved by earlier contact’) ranging

from <1/1 month to 1/1 year. The authors recommend concordance with the Kidney Disease Outcomes Quality Initiative guideline of referral at CKD stage IV (GFR <30 mL/min per 1.73 m2). Abderrahim et al. studied 299 Tunisian diabetic patients.2 One-third initiated dialysis as an emergency and 91% of all patients commenced with temporary venous access. Survival at 1 year was 68.4%, at 2 years 59.6%, and at 4 years it was 45.3%. Nearly 27% of patients died in the first 3 months, mainly from infection or cardiovascular disease. Age, comorbidity (hypertension and Type I diabetes) and urgent initiation of dialysis were independent risk factors for death. Astor et al. in the CHOICE study examined a cohort of 356 patients.3 Those that had been seen by a nephrologist BMN 673 manufacturer at least 1 month prior to initiation of dialysis were more likely to start dialysis

with an arteriovenous (AV) fistula or graft than those referred later (39% vs 10%). Late referrals had a more prolonged period of catheter use. Furthermore, patients referred earlier than 4 months were more likely to use an AV fistula rather than an AV graft as their first AV access than those referred later (45% vs 31%). Bhan et al. studied 93 consecutive patients commencing dialysis over a 1-year period.4 Patients referred late (<90 days) were more Selleck Palbociclib likely not to have a functioning fistula (48%). However, most of the late referrals were due to acute disease, rather than true late referrals

of chronic disease. On multivariate analysis, peripheral vascular disease and tuclazepam rapid deterioration of GFR were negative predictive factors for a fistula. Caskey et al. examined the quality of life of patients by a visual analogue scale (262 patients) and the SF-36 (226 patients) and showed that a planned first dialysis rather than early referral per se was associated with better quality of life at 8 weeks following initiation of dialysis.5 Two interesting studies using data from the ANZDATA Registry database have been published by Cass et al.6 All patients with end-stage kidney disease (ESKD) commencing dialysis over a period of 45 months from 1 April 1995 to 31 December 1998 were studied. Patients who either died or were transplanted in the first year were excluded from the analysis. Of the 4243 patients (26.9%), 1141 were referred late – defined as commencing dialysis within 3 months of referral to a nephrologist. The late referral group had more comorbidity. These patients not only were less likely to receive a transplant (adjusted RR 0.78, 95% CI: 0.64–0.95), but were more likely to die after the first year on dialysis (adjusted HR 1.19, 95% CI: 1.04–1.35). Dialysis modality and creatinine clearance at the time of dialysis initiation did not affect these results.

This study has several limitations: Bone remodeling increases once it has been subjected to weight bearing and bone-to-bone contact.[20, 21] In this study, a heterotopic model was used in which bone remodeling was identified by fluorescent labeling, and bone Venetoclax datasheet remodeling was observed in all samples. In future research, we aim to

apply orthotopic transplantation in larger animal models to determine iso- and allograft cell lineage, which will be one step further toward the study of physiologic cell lineage. Second, bone remodeling areas contain osteoblasts, osteocytes, and osteoclasts. We did not determine specific cell amounts or biologic activity as it was the aim of this study to determine the overall lineage of cells in specific bone remodeling areas using a new technique to selectively acquire fluorescent labeled bone remodeling areas with the laser capture microdissection procedure. However, it will be interesting to correlate quantitative bone remodeling data in selected cortical remodeling areas with cell lineage data in

future research. Furthermore, the effect of Tacrolimus immunosuppression on bone remodeling has been studied by Voggenreiter check details et al. in a fracture model.[22] They found no significant effects of Tacrolimus on biomechanical or histological properties. However, they did observe increased bone remodeling with net bone loss in trabecular bone. While remodeling was observed throughout the cortex in both isotransplants and allotransplants in this study, the effect of Tacrolimus on bone remodeling was not quantified. Third, while little significant differences were

found in this study, likely due to a small number of animals per group, we do present interesting descriptive data of cell heritage Ribose-5-phosphate isomerase within selected bone forming areas. In future research, we will therefore use larger groups with longer term analysis to acquire further insight into bone transplantation cell lineage. We describe the cell lineage within vascularized isotransplants and allotransplants accurately with a new combination of methodology consisting of fluorescent labeling, selective laser capture microdissection, and quantitative RT-PCR. The changes over time and differences between remodeling areas offer a distinct insight into cellular movement within the transplant and provide more knowledge of bone transplanting biology and transplant chimerism specifically. “
“Background: Resections of oromandibular squamous cell carcinoma involving lateral mandible, oral cavity, and the skin, lead to composite oromandibular defects that can be approached in several ways depending on the extension of the bone defect, of the soft tissue and cutaneous resection, the patient’s general status and the prognosis.

For example, Treg cells that express the Th1 lineage defining transcription factor T-bet expand with Th1 effector CD4+ T cells following Th1 stimulation conditions, whereas the ablation of T-bet specifically in Foxp3+ cells results in uncontrolled Th1 inflammation and autoimmunity.62 Similarly, Foxp3+ cell expression of the transcription factors signal transducers and activators of transcription (STAT)-3, interferon regulatory factor (IRF)-4, B cell lymphoma protein (BCL)-6 and GATA-3 have each been shown to suppress

other specialized effector CD4+ T-cell subsets that Talazoparib would otherwise cause unchecked self-reactive inflammation.63–67 Importantly, the specialization and dynamic regulation among these various Treg-cell subsets also play important roles in coordinating and fine-tuning immune responses

after infection. For example, under Th1 inflammatory conditions LDK378 solubility dmso triggered by M. tuberculosis, T-bet-expressing Treg cells and effector T cells both expand and are recruited into the sites of infection creating a balanced response that facilitates pathogen control, but not eradication.62 On the other hand, under Th2 inflammatory conditions triggered by pulmonary thymic stromal lymphopoietin or intestinal Heligmosomoides polygyrus infection, T-bet+ Treg cells fail to accumulate and are instead replaced by Treg cells enriched for the Th2 promoting transcription factor GATA-3.62,67 Interestingly, although the ablation of Foxp3+ Treg cells early after H. polygyrus infection augments parasite-specific effector Th2 responses and intestinal inflammation, no significant impacts

of pathogen burden or fitness were identified.68 Specialization among Treg cells during persistent 4-Aminobutyrate aminotransferase infection is not limited to expression of CD4+ T-cell lineage-defining transcription factors, but also extends to individual cell intrinsic molecules that probably mediate immune suppression. Foxp3+ Treg cells recovered from the pulmonary lymph node and lung selectively up-regulate expression of inducible T-cell co-stimulator (ICOS) and programmed death (PD)-1 at relatively early and late time point respectively, after aerosol M. tuberculosis infection whereas these shifts do not occur for Treg cells in lymph nodes that do not drain the site of infection.58 Similarly when the impacts of Treg-cell ablation are progressively reduced from early to late time-points after systemic Salmonella infection, Foxp3+ Treg cells in the spleen progressively lose CTLA-4 expression that is replaced by increased glucocorticoid-induced tumor necrosis factor receptor (GITR) expression.59 Hence, functionally distinct Treg-cell subsets that express unique combinations of cell intrinsic molecules accumulate and shift throughout the course of persistent infection.

(B) Western blot analysis of nuclear fractions of primary CD4+ lymphocytes from FoxP3-IRES-GFP reporter mice. Cells were treated 1 hour with 4 μg/ml of anti-CD3 and 1 μg/ml of anti-CD28 mTOR inhibitor antibodies. (A, B) Lamin B used as loading control. Data shown are representative of two experiments. C, D. Analysis of nuclear transcription factors and chromatin conformation at theTNF TSS in primary CD4+ T cells. Effect of Cyclosporine A (CsA), JNK inhibitor SP600125 (C) and protein synthesis inhibitor Anisomycin (D) on nuclear concentrations of NFATc2 and c-Jun (top) and chromatin conformation at TNF TSS (middle and bottom). (C) Cells were pretreated 1 hour with indicated concentrations

of CsA and SP600125 and treated 1 hour with 4 μg/ml of anti-CD3 and 1 μg/ml of anti-CD28 antibodies. (D) Cells were treated 1 hour with indicated concentrations of Anisomycin or with 4 μg/ml of anti-CD3 and 1 μg/ml of anti-CD28 antibodies. (C, D) Western blot analysis. Lamin B used as loading control and data shown are representative of two experiments. Extra lanes were deleted from the blot image (C) between lanes 2 and 3,

3 and 4 (top). Relative resistance to MNase digestion at the TNF TSS (amplicon -50+73) calculated and normalized to control MNase-digested genomic DNA and average of signals for amplicons +67+189 and +121+240. Data are shown as mean ± SD of five (C) or two (D) experiments. Statistical significance determined by Student’s T-test. E, F. Analysis of nuclear transcription factors and chromatin conformation at theTNF TSS in primary CD4+ T cells Effect of Cyclosporine A (CsA), JNK inhibitor SP600125 (E) and www.selleckchem.com/products/dabrafenib-gsk2118436.html protein synthesis inhibitor Anisomycin chromatin conformation at TNF TSS (F) (profile of MNase resistance around TNF TSS (-124 +240) normalized only to control MNase-digested genomic DNA). (E) Cells were pretreated 1 hour with indicated concentrations of CsA and SP600125 and treated 1 hour with 4 μg/ml of anti-CD3 and 1 μg/ml of anti-CD28 antibodies. (F) Cells were treated 1 hour with indicated concentrations of Anisomycin or with 4 μg/ml of anti-CD3

and 1 μg/ml of anti-CD28 else antibodies. (E, F) Relative resistance to MNase digestion at the TNF TSS (amplicon -50+73) calculated and primary data representative of five (E) or two (F) experiments are shown. Figure S6. Effect of CsA and SP600125 on chromatin conformation around TNF TSS (-124 +240) in quiescent polarized T cells Th2s and Th17s cells were polarized in the presence of soluble anti-CD3 antibodies, Th1i – in presence of immobilized anti-CD3 antibodies. After polarization cells were cultured in the medium without cytokines or antibodies for 12 hours with indicated concentrations of inhibitors. Examples of primary data normalized only to control MNase-digested genomic DNA are representative of two (Th2s) and three (Th1i and Th17s) experiments. Centers of amplicons covering TNF TSS are labeled with arrows. Figure S7.