aeruginosa and those isolated from chronic INK 128 cell line skin wounds with respect to the production of virulence determinants such as pyocyanin and extracellular protease. The six strains fell into three categories:

the first included the two type strains as well as one of the clinical isolates (PAO1, NCTC 6750 and 15159), the second contained the clinical isolates 23:1 and 27:1 and, finally, strain 14:2 (also a clinical isolate) formed a group on its own. In the first group, all strains expressed pyocyanin, elastase and alkaline proteinase, and two of the three produced the quorum-sensing molecule C4-HSL, while the second group showed no expression of C4-HSL or elastase. Interestingly, strain 14:2 was negative for the expression of C4-HSL, pyocyanin and the proteases. A similar spread in the expression of virulence factors and quorum-sensing molecules among P. aeruginosa strains has been described by others, for instance, Luzar & Montie (1985) and Lee et al. (2005), who investigated chronically

infected cystic fibrosis patients. Both studies showed not only variations between strains isolated from different patients but also changes associated with disease progression. Isolates from patients with more advanced disease showed lower pyocyanin and protease production, suggesting that the evolution of P. aeruginosa strains towards a less virulent phenotype may confer a survival advantage during chronic infection. Thus, in our study, the clinical isolate 14:2, which had Roxadustat the greatest inhibitory effect on biofilm formation by S. epidermidis and lacked the production of C4-HSL, pyocyanin and proteases, may represent a less virulent strain that has become adapted to enhance its persistence

in the chronic sore environment (Lee et al., 2005). In a recent study by Qin et al. (2009), extracellular products from P. aeruginosa were shown to disrupt S. epidermidis biofilms and it was suggested that extracellular polysaccharide could be responsible for the effect. Thus, the authors proposed that extracellular polysaccharides from P. aeruginosa may represent a novel target for the development of agents to control S. epidermidis biofilms at sites of infection. Mannose- and galactose-containing extracellular polysaccharides were detected in biofilms of all the strains of P. aeruginosa tested here, and thus the inhibition of S. epidermidis ZD1839 biofilm formation seen in our study may occur through a mechanism similar to that proposed by Qin and colleagues for biofilm dispersal. Expression of the two extracellular polysaccharides, Pel and Psl, is known to vary according to the strain and environmental conditions (Branda et al., 2005). Although 14:2 did not appear to produce higher levels of these polysaccharides than the other strains, which could account for its enhanced effect on S. epidermidis biofilms, it is possible that, for instance, differences in their relative expression may play an important role.

There has been growing evidence of distinct properties of synovial membrane-derived human mesenchymal stromal cells [41-43]. Some studies suggest that S-MSC may be discriminated from B-MSC by their transcriptional profiles [44]. Apart from a negative CD146 expression in S-MSCs, there were no differences regarding the surface markers between B-MSC and S-MSC in our

study. Also, no differences in plastic adherence or differentiation Saracatinib nmr potential between these cells were observed in our experiments. While the synovium is located in the centre of joint inflammation associated with OA [5, 7], this did not seem to have an influence on the immunomodulatory properties of MSCs in our experiments. The elevated IL-6 production, however, suggests that S-MSC from OA patients exert distinct properties in this particular setting. The question of whether or not the higher IL-6 secretion by S-MSCs is caused by the inflammatory conditions in the joint cannot be answered from our data, but it must be an aim of future experiments to link the degree of synovial inflammation to IL-6 secretion and MSC immunomodulatory potential. We have used an in-vitro model in our experiments; thus, it is difficult to draw any conclusions regarding the in-vivo situation.

However, these are only initial findings stating that the interaction of MSCs and regulatory T cells may play a role in the osteoarthritic joint in vivo, as has been suggested for numerous other diseases, including rheumatoid HSP inhibitor arthritis [2, 27]. Future experiments will need to determine whether these findings MycoClean Mycoplasma Removal Kit will allow the application of some of the therapeutic strategies for rheumatoid arthritis locally to the OA-affected joint. IL-6 was the predominant cytokine in the co-cultures, which is why we chose to supplement Treg-enriched lymphocyte cultures with this cytokine. Our data suggest that IL-6 plays a role in S-MSC- and B-MSC-mediated

immunomodulation, as supplementation of IL-6 to the culture media was shown to partially reproduce the MSC-mediated Treg maintenance. To our knowledge, this is the first study to report that MSC from OA patients may exert some of their effects via IL-6 and thus may play an important role in shifting the balance of regulatory and effector T cells in OA. The full effect of Treg maintenance by MSCs, as seen in the MSC–lymphocyte co-cultures, was observed in the group supplemented with MSC supernatants. In our opinion, MSC–Treg interaction therefore seems to be based on paracrine effects rather than on cell–cell interaction. However, other soluble factors, that remain to be detected, appear to be involved in these processes, although none of the other cytokines analysed in our experiments seem to be of major importance in this particular in-vitro setting.

The mean number of lymph nodes in quadrants I–IV were 3.3 ± 1.6, 2.0 ± 1.2, 1.5 ± 1.3, and 1.9 ± 1.4, respectively. The difference between the four quadrants was statistically significant (P < 0.001). In quadrant I, the appearance rate of SCIA was 100% while SIEA was 6.6%. In quadrant II, no SCIA was observed but the

appearance rate of SIEA was 78.0%. There were neither SCIA nor SIEA observed in quadrants III and IV. The superior lateral quadrant of the groin region was found to have the most lymph nodes. The superficial circumflex iliac vessels are the major sources for blood supply to this region. The findings from this study provide evidence for the clinical design of the lymph node flap from the groin area. © 2014 Wiley Periodicals, Inc. Microsurgery 34:558–561, 2014. “
“Background: Vascular thrombosis with

www.selleckchem.com/products/U0126.html flap loss is the most dreaded complication of microvascular free tissue transfer. Thrombolytic agents such as tissue plasminogen activator have been used clinically for free flap salvage in cases of pedicle Selleck CH5424802 thrombosis. Yet, there is a paucity of data in the literature validating the benefit of their use. Methods: A retrospective review of the breast reconstruction free flap database was performed at a single institution between the years of 1991–2010. The incidence of vascular complications (arterial and/or venous thrombosis) was examined to determine the role of adjuvant thrombolytic therapy in flap salvage. Pathologic examination was used to determine the incidence of fat necrosis after secondary revision procedures. Results: filipin Seventy-four cases were identified during the study period.

In 41 cases, revision of the anastamoses was performed alone without thrombolytics with 38 cases of successful flap salvage (92.7%). In 33 cases, anastamotic revision was performed with adjuvant thrombolytic therapy, and successful flap salvage occurred in 28of these cases (84.8%). Thrombolysis did not appear to significantly affect flap salvage. Interestingly, only two of the salvaged flaps that had received thrombolysis developed fat necrosis, whereas 11 of the nonthrombolysed flaps developed some amount fat necrosis (7.1% vs. 28.9%, P < 0.05). Conclusions: The decreased incidence of fat necrosis may be attributable to dissolution of thrombi in the microvasculature with the administration of thrombolytics. Although the use of adjuvant thrombolytic therapy does not appear to impact the rate of flap salvage, their use may have secondary benefits on overall flap outcomes. © 2011 Wiley-Liss, Inc. Microsurgery 2011. "
“It is important to preserve the length, appropriate durable skin, and sensation of the stump when performing below-knee amputation to achieve functional ambulation with a prosthesis.

An even more pronounced age-inappropriate decline of newly generated T cells associates with rheumatoid arthritis suggesting that

premature decline of thymic activity might be a common feature in these and other autoimmune disorders 7. The cytokine interleukin-7 (IL-7), a pleiotropic hematopoietic growth factor, is known to stimulate the thymus and to promote the differentiation and maintenance of naïve T cells including Treg 8–10. Signaling from IL-7 occurs through the heterodimeric IL-7 receptor (IL-7R), which is expressed on lymphocytes and consists of the α-chain subunit (IL-7Rα) and the common cytokine γ-chain. The importance of this pathway for naïve T-cell homeostasis is underlined by several recent studies showing that expression levels of membrane-bound IL-7Rα MK-2206 research buy (CD127) on conventional CD4+ T cells correlate

with frequencies of recent thymic emigrant (RTE)-CD4+ T cells in healthy individuals and HIV-infected patients as well as in patients with MS 11, 12. IL-7Rα is also a component of the receptor for thymic stromal lymphopoietin (TSLP). The secretion of TSLP by Hassall’s corpuscles, structures composed of epithelial cells in the thymic medulla, has been demonstrated to condition CD11c+ myeloid dendritic cells (MDCs) to induce the differentiation of thymocytes into Treg 13. Accordingly, signals from the IL-7 receptor are required for Treg development RG7204 chemical structure as shown in IL-7Rα knockout mice 14. Of note, a single nucleotide polymorphism (rs6897932-SNP) within the gene encoding the IL-7Rα chain (IL-7RA) has shown genetic association with human

autoimmunity and was found to be associated with MS, type 1 diabetes and chronic inflammatory arthropathies 15–19. Forskolin This SNP causes a change from threonine to isoleucine at amino acid position 244 that modifies the ratio of membrane-bound to soluble IL-7R 15, 20. In this study, we attempted to decipher in more detail the impact of IL-7/IL-7R signaling components on Treg homeostasis and Treg-suppressive function. We used peripheral blood and plasma samples from 56 treatment-naïve patients with relapsing remitting MS (RRMS) and 33 healthy individuals (HC) to analyze IL-7Rα-expression on total CD4+CD25−/lowCD127+FOXP3− conventional T cells (Tconv) and Tconv subsets together with plasma concentrations of soluble IL-7Rα (sIL-7Rα) and IL-7 as well as genotype screening for rs6897932-SNP. In parallel, we determined frequencies, phenotypes and suppressive activities of donor and patient-derived Treg. Treg obtained from both cohorts were further characterized as to quantities of cells harboring two T-cell receptor (TCR) Vα chains. Cells expressing TCRs with dual specificity on their surface are enriched in the Treg compartment and as this feature is acquired during T-cell maturation in the thymus, their proportions among total Treg should roughly correlate with the natural Treg lineage 21.

However, IL-8 was found in all intestinal samples from the pigs infected with Salmonella. The flagellin of this bacterial species is its main inducer [44]. As a flagellated bacterium, EcN also induces IL-8 in enterocytes [45,46] and this could be one of the mechanisms by which it protects against Salmonella infection [25,43]. High plasma levels of IL-10 were observed in piglets infected with Salmonella alone or in piglets colonized with bifidobacteria and infected with Salmonella. IL-10 levels Saracatinib cost correlated with TNF-α levels and with the presence of Salmonella in blood, suggesting an interplay between both cytokines,

or more generally the interplay between pro- and anti-inflammatory reactions. In contrast, IL-10 was absent in the blood of piglets colonized with EcN and subsequently infected with Salmonella. Blood IL-10 levels increase in several septic states, including E. coli sepsis [47] and a swine model of shock caused by heat-killed Neisseria meningitis[48]. The continued presence of IL-10 in blood 24 h after infection of gnotobiotic pigs with S. Typhimurium seems to be a prognostic marker of poor survival in infected animals [43]. Levels of IL-10 also reflect the severity of Salmonella infection in mice [49]. In contrast, increased levels of IL-10 in blood coincided with recovery from experimentally induced swine dysentery [50]. In this study, IL-10 was not

found in any intestinal sample. This may be caused by the absence of cells capable of producing it, e.g. by the paucity and immaturity of T lymphocytes in intestinal villi of germ-free pigs. High levels of TNF-α were found in plasma and ileum of piglets infected https://www.selleckchem.com/products/17-AAG(Geldanamycin).html with Salmonella alone or in MycoClean Mycoplasma Removal Kit piglets pre-colonized with bifidobacteria before this infection. The statistically significant reduction in TNF-α in pigs di-associated with EcN and LT2 correlated with the ability of EcN to interfere with Salmonella in the ileum and ultimately stop translocation to the mesenteric lymph nodes.

The levels of TNF-α are markers of inflammation and high levels are found in bacteraemia. Rapid turnover of TNF-α in blood of pigs challenged by living or heat-killed bacteria or bacterial lipopolysaccharide has been described [47,48]. Prolonged presence of TNF-α in blood circulation was seen in our experimental gnotobiotic piglets which, together with IL-10 levels, correlated with increased lethality. Decreasing levels or neutralization of TNF-α in blood can be one method of protection against the lethal sequelae of bacteraemia [51]. Preliminary association of germ-free piglets with EcN significantly reduced levels of TNF-α in Salmonella-infected piglets compared to animals infected with Salmonella alone. Unlike conventional animals, the germ-free animals show no resistance to colonization [3], and a single dose of bacteria suffices for the prolonged colonization of their gastrointestinal tract.

4–8 Therefore, it is thought that FFA might play find more a role in the pathogenesis of the tubulointerstitial damage in various kidney diseases Free fatty acids loaded into the human proximal tubules are bound to the 14 kDa renal liver-type fatty acid binding protein (L-FABP) and transported to mitochondria or peroxisomes, where they are metabolized by β-oxidization.9 Expression of the L-FABP gene is induced by FFA, and

this protein may regulate the metabolism of FFA and be a key regulator of FFA homeostasis in the cytoplasm.10 Moreover, L-FABP has a high affinity and capacity to bind long-chain fatty acid oxidation products, and may be an effective endogenous antioxidant.11 However, until now, renal L-FABP had not been investigated under pathological conditions of the kidney. Recent development of the method for measuring urinary human L-FABP (hL-FABP), using

a two-step sandwich enzyme linked immunosorbent assay (ELISA) procedure (CMIC, Tokyo, Japan),12 and the establishment of a transgenic (Tg) mouse model harbouring the hL-FABP chromosomal gene have enabled deeper insights into this protein.13 This review is mainly focused on the pathophysiological roles and dynamics of hL-FABP as revealed by Tg animal models of kidney disease. Deterioration of kidney disease is determined to a large extent by the degree of tubulointerstitial Selleck Alisertib changes rather than by the extent of histological changes in the glomeruli.3 Therefore, a tubular marker that accurately reflects CHIR-99021 purchase the tubulointerstitial damage may be an excellent biomarker for early detection or prediction of kidney disease. Although the importance and necessity of measuring clinical parameters in serum or urine of the patients with CKD are emphasized, there are few clinical markers

to predict and monitor the progression of CKD. Urinary protein is widely accepted to help physicians predict the risk of disease progression and the risk of dialysis for individual patients.14,15 However, in patients with nephrosclerosis, renal dysfunction deteriorates in spite of the low levels of urinary protein levels. In order to clarify the clinical relevance of urinary excretion of hL-FABP, urinary hL-FABP levels in 120 nondiabetic adult patients were measured.12 As a result, urinary hL-FABP was shown to reflect the progression rate of kidney disease, as determined by significantly higher hL-FABP levels in patients with deteriorating renal function as opposed to low levels in those with stable renal function. Moreover, in order to confirm the clinical usefulness of urinary hL-FABP as a maker for the monitoring of CKD, a multicenter trial was carried out.16 In this study, urinary hL-FABP was demonstrated to be more sensitive than urinary protein in predicting the progression of CKD.

Our data revealed that adding AFP resulted in inhibition of IL-12 production at the transcriptional level, not by decreased expression of TLRs. Although the regulation of transcription of IL-12p40 and IL-12p35

has been elucidated in various studies [23,24], the detailed mechanism of inhibition of IL-12 transcription by AFP remains unclear. Further study is needed to clarify the detailed mechanism of inhibition Selleckchem Proteasome inhibitor of IL-12 by AFP. Taken together, IL-12 might play a mainly essential role in the impairment of NK activity by AFP. To evaluate the possibility of involvement of other immunosuppressive cytokines inhibiting NK activity, we examined the IL-6 and IL-10 levels in the supernatants of the co-cultures of NK cells and AFP-DCs/Alb-DCs by specific ELISAs. IL-6 levels in the supernatants of AFP-DCs were similar to those of Alb-DCs, and IL-10 levels in the supernatants of

AFP-DCs were significantly lower than those of Alb-DCs (M. Yamamoto, unpublished data). These results suggest that the addition of AFP might impair the ability GSK458 manufacturer of cytokine production of DCs. In a previous report, Um et al. demonstrated that AFP impairs the function of dendritic cells and induces their apoptosis [13]. In their report, they used the commercially available human cord blood AFP. Thus, we used human cord blood AFP because this is the only commercially available AFP. The carbohydrates of AFP are heterogeneous, which is reflected by differences in the binding of individual Astemizole AFP molecules to lectins. Therefore, we also added the supernatants of Huh7 cells, AFP-producing HCC cells or control medium on the DCs and evaluated IL-12 production after LPS stimulation by specific ELISA. The supernatants of Huh7 cells contained AFP (1·76 µg/ml) and control medium contained no AFP. The IL-12 production of DCs co-cultured with the supernatants of Huh7 cells was significantly lower than that with control medium (M. Yamamoto, unpublished data). These results were consistent with the results using human cord blood AFP.

Although we cannot deny the possibility that unknown factors, except AFP, in the supernatants of Huh7 cell might affect the IL-12 production of DCs, these results suggest that another type of AFP might also have immunoregulatory ability on DCs. In this study, we demonstrate that AFP might down-regulate IL-12 production from DCs which inhibit NK activity. Zhang et al. demonstrated that IL-12 improves the cytotoxicity of NK cells via up-regulated expression of NKG2D on NK cells [25]. We have demonstrated previously that NKG2D expression on NK cells was down-regulated in the progression of chronic liver disease, including HCC [18], which suggested that NK activities were impaired in HCC patients. The expression of NKG2D on NK cells in HCC patients with high serum AFP was significantly lower than those in HCC patients with low serum AFP (M. Yamamoto, unpublished data).

2; P = 0.037) and CPSI (26.1 ± 5.0 vs 17.2 ± 8.3; P = 0.0016) scores improved from baseline to end of treatment. Incontinence episodes per day improved slightly (P = 0.042). When only those completing at least 8 weeks

of treatment were INK 128 mouse examined (n = 9), significant changes in CPSI, VAS, and PSQI were still observed. At the final visit, 8/9 (88.9%) men also reported some improvement in pain related to sex. Side-effects were generally mild and well tolerated. Conclusion: These results suggest that apremilast may improve CP/CPPS symptoms with only mild side-effects. However, placebo controlled studies are necessary to determine efficacy. “
“Over the past decade, the use of quality of life (QOL) questionnaires in the evaluation of pelvic organ prolapse (POP) has become a standard part of most clinical studies. Investigators have attempted to correlate QOL scores with objective findings and treatment efficacy and as outcome measures in comparing different treatment modalities. Many of the QOL questionnaires are available in short forms, making them easier to adapt to clinical settings. This article includes an overview of several validated QOL questionnaires and their application in studies whose results provide useful

guidelines for health care professionals who diagnose and manage women with POP. Pelvic organ prolapse (POP) is a condition that affects millions of women with a prevalence estimated in a clinical population to be 40% of parous women.[1] Age[2, 3] and parity[4] are well known risk factors for the development of POP, parity being the strongest risk factor PCI-32765 supplier with an adjusted risk ratio of 10.85.[4] Neurologic injury to the pelvic floor[5, 6] and underlying connective tissue disorders[7] have also been implicated. Other suspected predisposing factors include chronic conditions that increase abdominal pressure such as heavy lifting, chronic cough, bowel dysfunction, previous Etoposide ic50 hysterectomy, estrogen deficiency[8-10] as well as obesity in some[11, 12] but not all[13, 14] studies. The development of POP

in nulliparous women combined with its absence in many multiparous women suggests that genetics may also play a role.[15] Though POP and its associated disorders are rarely life threatening, they have a direct and profound impact on quality of life (QOL). Historically, objective evaluation of POP was commonly done by physical examination alone or in combination with instruments that addressed only a single organ, making it difficult to assess multi-organ involvement. Further, the absence of valid and reliable tools to measure QOL issues made the assessment of outcomes to various treatment modalities incomplete. Over the years, researchers and clinicians have recognized the need to develop (i) a comprehensive staging system that involved all pelvic organs and (ii) standardized quality of life assessment tools specifically designed for POP disorders that would better evaluate treatment efficacy.

2–18.3 (C6 of Qui3N), two HOCH2-C groups at δ 62.3 and 62.6 (C6 of Gal and GalN), one carboxyl group at δ 175.3 (C6 of GlcA), one N-acetyl group at δ 23.7 (CH3), and 176.2 (CO) as well as one N-formyl group at δ 167.0 and 169.6 (major and minor signals for the Z and E isomers, respectively). The 1H NMR spectrum showed signals for four anomeric protons at δ 4.49–5.37, a CH3-C group at δ 1.29–1.30 (H6 of Qui3N), one N-acetyl group δ 2.01 and one N-formyl group at δ 8.18 and 7.95 (Z and E isomers in the ratio 1.7 : 1, respectively). The NMR spectra showed structural heterogeneity, which could be due to the occurrence of the N-formyl group as the E and Z stereoisomers.

The 1H and 13C NMR spectra of the polysaccharide were assigned (Table 1) using a set of two-dimensional experiments, Ipatasertib including 1H,1H COSY, TOCSY, ROESY, H-detected 1H,13C HSQC (Fig. 2), and HMBC. The COSY and TOCSY spectra revealed spin systems for two sugar residues having the gluco configuration (Qui3N and GlcA) and two residues having the galacto

configuration (Gal and GalN). The β configuration of the glycosidic linkages of Qui3N, GlcA and GalN was established by J1,2 coupling constant values of 7.5–8.0 Hz. A relatively small J1,2 coupling constant (< 3 Hz, H1 signal was not resolved) showed that Gal is α-linked. Significant downfield displacements of the signals for C4 of β-Qui3N to δ 82.5 and 82.9, C3 of α-Gal, β-GlcA and β-GalN to 80.2, 83.1 and 81.8, respectively, EGFR inhibitor from PIK3C2G their positions in the corresponding nonsubstituted monosaccharides (L’vov et al., 1983; Jansson et al., 1989) revealed the substitution pattern of the monosaccharides in the O-unit. The absence of other signals in the region δ 80–88 indicated that all sugar residues are pyranosidic (Bock & Pedersen, 1983). The 1H,13C HMBC spectrum (Fig. 3) showed interresidue cross-peaks between the following anomeric protons and linkage carbons: β-Qui3N H1/α-Gal C3 at δ 4.74/80.2, α-Gal H1/β-GlcA C3 at δ 5.37/83.1, β-GlcA H1/β-GalN C3 at δ 4.57/81.8 and β-GalN H1/β-Qui3N C4 at δ 4.49/82.5 and 4.53/82.9. These

data confirmed the glycosylation pattern and defined the monosaccharide sequence in the O-unit. The location of the N-acyl groups was unambiguously determined by the 1H,13C HMBC experiment, which showed correlations of the proton of the N-formyl group in the Z isomer with C3 of Qui3N at δ 8.18/56.0 and the CO of the N-acetyl group with H2 of GalN at δ 175.9/3.82. N-Acetylation of GalN was confirmed by TOCSY and ROESY experiments with a polysaccharide solution in a 9 : 1 H2O/D2O mixture, which showed a major correlation between CH3 of the N-acetyl group and NH of GalN at δ 2.01/8.36. The TOCSY spectrum also showed a minor signal for NH of GalN at δ 8.43, which was tentatively assigned to a terminal GalNAc residue of the polysaccharide chain.

Little is known about the role of the NF-κB family member c-Rel in the development and function of TH17 and Treg. In this study, we show that while conversion of naive CD4+ T cells into both iTreg and nTreg requires c-Rel, this transcription

factor is not required for differentiation of TH17 cells. While our manuscript was prepared, Gerondakis and colleagues have shown that c-Rel is essential for the development of CD4+Foxp3+ T cells in the thymus and peripheral lymphoid organs 31. These authors also demonstrated that despite their lower frequency, c-Rel-deficient check details Treg suppressed effector T-cell function at normal levels. We here confirm reduced frequencies of CD4+Foxp3+ T cells in thymus, spleen and LN of c-Rel-deficient mice. In addition, we mechanistically extend this novel finding by examining the effect of c-Rel deficiency on differentiation of iTreg in vitro and show that c-Rel directly mediates upregulation of IL-2 production which is a prerequisite for iTreg development. WT C57BL/6 mice were purchased from Jackson Laboratory

(Bar Harbor, USA). c-Rel−/− mice were bred at the animal facility of the Biomedical Research MAPK Inhibitor Library Center, University of Marburg (Marburg, Germany). CD4+ and naive CD4+CD62L+ TH were purified from WT and c-Rel−/− mice by disrupting spleens and LN of 8- to 12-wk-old mice. All cells were cultured in Clicks medium supplemented with 10% fetal bovine serum, 2 mM glutamine and 2 μM β-mercaptoethanol. CD4+ and naive CD4+CD62L+ T cells were enriched by magnetic cell sorting with a Mouse CD4+ Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated naive CD4+ T cells (purity routinely >95%) were activated by plate-bound

anti-CD3 (5 μg/mL; 145-2C11) and soluble anti-CD28 (1.5 μg/mL; 37.51) for 3 days (unless stated otherwise) and cultured either under neutral “TH0” conditions: with anti-IL-4 (10% culture supernatant of clone 11B11), anti-IFN-γ GPX6 (5 μg/mL, XMG1-2) in the presence of recombinant human IL-2 (50 U/mL, Novartis (Nürnberg, Germany)); under TH17 culture conditions: recombinant human TGF-β1(ng/mL, R&D Systems (Wiesbaden-Nordenstadt, Germany)), recombinant murine IL6 (10 ng/mL, Peprotech (Hamburg, Germany)), anti-IL-4, and anti-IFN-γ; under iTreg conditions: TGF-β1(2 ng/mL, R&D Systems), anti-IL-4, and anti-IFN-γ. Where indicated, human IL-2 (50 U/mL, Novartis) or anti-murine IL-2 (50 μg/mL, S4B6.1) was added to the cell culture. After 3 days in culture, the T cells were washed and restimulated with PMA (50 ng/mL, Sigma (München, Germany)) and ionomycin (750 ng/mL, Sigma (München, Germany)) in the presence of brefeldin A (10 μg/mL, Sigma) for 4 h. Stimulation was terminated by fixing cells with paraformaldehyde.