, Montgomery, TX, USA) for 30 min on ice and finally washed with 1% BSA–PBS. Multi-colour flow cytometry was performed on a fluorescence activated cell sorter (FACS)Canto,

interfaced check details to a FacsDiva software (BD Biosciences, San Jose, CA, USA) and analysed through Flow-Jo software version 8·8·3 (Three Star Inc., Ashland, OR, USA). The binding of the antibody to the cells incubated with the different plasma samples was measured and the percentage of binding-inhibition calculated according to the background staining (cells incubated without plasma). A cartoon showing the principles of the assay is presented in Fig. 1. Purified PBMCs were thawed and stained with the following conjugated monoclonal antibodies: CD19-Alexa 488, interleukin (IL)-21R-phycoerythrin (PE), CD27-peridinin chlorophyll-cyanin 5·5 (PerCP-Cy5·5), Ibrutinib supplier CD21-allophycocyanin (APC), IgD-H7 (all from BD Biosciences) and the CD10-PE-Cy7 (Biolegend, San Diego, CA, USA). The frequencies of MA (defined as CD10–CD21–) and DN (defined as CD27–IgD–) B cell subpopulations were calculated from total CD19+ B cells. Multi-colour flow cytometry was performed on a FACSCanto,

interfaced to a FacsDiva software (BD Biosciences) and analysed through Flow-Jo software version 8·8·3 (Three Star Inc.). Plasma IL-21 titres were measured using the human IL-21 platinum ELISA kit (eBioscience, San Diego, CA, USA), following the manufacturer’s instructions. The Mann–Whitney U-test and Spearman’s correlation were used for all analyses. A P-value <0·05 was considered statistically significant. GraphPad Prism software for Windows was used to perform the analyses. The ALA titres before and after flu vaccination were quantitated as described in the Materials and methods and in Fig. 1. Before vaccination, significantly lower ALA titres were found in the

HIV group compared to KT and HC Silibinin (P < 0·0001) (Fig. 2a), while no significant difference was found between the KT and the HC groups (P > 0·05) (Fig. 2a). Interestingly, after vaccination individuals in both the HIV and KT groups increased ALA titres substantially compared to HC (P = 0·0001 and P = 0·0002, respectively) (Fig. 2b). Between HIV and KT, the biggest increase was recorded in the HIV group (P = 0·0008) (Fig. 2c). HC increased ALA titres only slightly compared to HIV and KT (P = 0·0001 and P = 0·0003, respectively (Fig. 2c). Fifteen per cent of the HIV-1-infected individuals (10 of 65) were having a viraemic blip at the time of vaccination (Table 1). However, this did not relate to any of the parameters analysed as confirmed by Spearman’s correlation (P > 0·05). Moreover, the CD4+ T cell counts were similar in the viramic and aviraemic patients (P > 0·05).

The DWT at an emptied bladder was 4.73 ± 0.97 mm at anterior wall, 3.83 ± 1.06 mm at posterior wall, 4.67 ± 1.12 mm at bladder base and 9.10 ± 2.11 mm at the bladder neck.87 When we measured the DWT of the same group of patients from

lower abdomen using an 8 MHz trans-abdominal sonographic probe (8C, GE, model LOGIQ P5/A5), the DWT was 0.926 ± 0.287 mm at a bladder volume of 250 mL, 0.739 ± 0.232 at the bladder capacity, and 0.925 ± 0.257 mm after the bladder capacity was corrected to 250 mL. Putting these data together, it is clear that DWT changes with bladder volume and varies greatly when measuring through different scanning route. Therefore, it is necessary to standardize the technique and scanning frequency in measurement of DWT if we try to compare Inhibitor Library purchase selleck DWT between different bladder disorder subgroups or performing a longitudinal study for DWT as biomarker of assessing OAB. The differences in the values of DWT obtained in various previous studies may have been caused by the use of different ultrasound probes with different frequency as well as to differences in the resolution of images. Review of previous reports found that studies using a higher frequency probe (7.5 MHz) reported a DWT of around 1–2 mm,80,81,83 whereas those using a low-frequency probe (2–5 MHz) reported a greater DWT of around 4–5 mm.77,82,88,89

In our previous studies, we used an 8 MHz high-frequency probe to measure the DWT either by TAU or TVU.85,86 Because the resolution power was able to differentiate the detrusor wall Mirabegron from the posterior rectus fascia, the measured DWT tended to be much less than would have been obtained using a 2–5 MHz low-frequency probe. Careful identification of the true bladder wall and accurate placement of cursors to measure the landmarks of DWT require experience. TVU assessment of mean BWT has been postulated to be a sensitive screening tool to detect DO in women with equivocal laboratory urodynamics. In women who have no evidence

of genuine SUI on laboratory studies, a cut-off of 6 mm of BWT by TVU has been highly suggested of having DO.89 Serati M et al. compared the ultrasound measurement of BWT in women with different urodynamic diagnosis and to correlate BWT to the different urodynamic findings of DO.90 They found that women with DO had a significantly higher BWT value. The measured BWT was 5.22 ± 1.17 mm in DO, 4.09 ± 0.86 mm in USI, 4.73 ± 1.27 mm in mixed incontinence, and 4.19 ± 1.14 mm in normal urodynamics. A cut-off of 6.5 mm for BWT had a positive predictive value of 100% for all DO. Although the ultrasound BWT showed a highly significant association with DO, data show a high level of overlap and it is only reliable in women with DO with a BWT cut-off value of >6.5 mm. The authors concluded that TVU-BWT cannot currently replace urodynamic testing.

In both humans and mice (Fig. 2), one of the two syncytins (human syncytin 2 and mouse syncytin-B) is immunosuppressive and, rather unexpectedly, the other (human syncytin 1 and mouse syncytin-A) is not although both are able to induce cell–cell fusion.33 Syncytin-A plays an important biological role in syncytiotrophoblast

development, because syncytin-A null mice die in utero because of the failure of trophoblast cells to fuse and form one of GPCR & G Protein inhibitor the two syncytiotrophoblast layers present in the mouse placenta39 that play a key role in transport of nutrients for the developing conceptus.29 Given that two syncytins are immunosuppressive, they may play a role in maternofetal tolerance, although this concept has not been mechanistically tested in vivo.33 Recently, Heidmann et al.24 identified an env gene of retroviral origin in the rabbit Oryctolagus cuniculus, termed syncytin-Ory1, with the characteristic features of human syncytin (Fig. 2). An in silico search for full-length env genes with an uninterrupted open reading frame within the rabbit genome resulted in the identification of an env gene with placenta-specific expression and belonging to a family Kinase Inhibitor Library cell assay of endogenous retroelements present at a limited copy number in the rabbit genome. The placenta-expressed env gene demonstrated fusogenic activity

in an ex vivo cell–cell fusion assay. Interestingly, the receptor for the rabbit syncytin-Ory1 was found to be the same as that for human syncytin 1, i.e. the previously identified sodium-dependent neutral amino acid transporter type 2 (SLC1A5). Syncytin-Ory1 mRNA was specifically present at the level of the junctional zone of the placenta, where the invading

syncytial fetal tissue contacts the maternal decidua to form the labyrinth, consistent with a role in the formation of the syncytiotrophoblast. The identification of a novel syncytin gene within a third order of mammals displaying syncytiotrophoblast formation during placentation strongly supports the notion that on several occasions, retroviral infections have resulted in the independent capture of genes that were positively selected for a convergent physiological role in development of the placenta.24 Domestic sheep have at least either 27 copies of ERVs in their genome, termed enJSRVs (Fig. 1), because they are highly related to the exogenous and pathogenic JSRV.6,40 JSRV is the causative agent of ovine pulmonary adenocarcinoma, a transmissible lung cancer of sheep.41 A unique feature of JSRV among oncogenic retroviruses is that its Env glycoprotein is the main determinant of cell transformation both in vitro and in vivo.42–48 Expression of the JSRV Env alone is able to transform a variety of cell lines in vitro, including mouse, rat, and chicken fibroblasts as well as human bronchial, canine, and rat epithelial cells.

Conclusions: Pollution of community and seaways are serious considerations. So are diversion High Content Screening of funds otherwise available for healthy food alternatives, excess empty calories, obesity, diabetes, metabolic syndrome, cardiovascular risk and tooth decay. Furthermore, dehydration

and sugar excess probably facilitate the growing multicentric global epidemic of CKD of unknown etiology, and might well be renal toxic per se. An exacerbating role in Aboriginal renal disease cannot be excluded. It is time to act. 228 ESTABLISHING A NEPHROLOGY NEWSLETTER J WOON1, E MACKNAMARA1, AM WALKER1, J KAUSMAN1,2, C QUINLAN1,2 1The Royal Children’s Hospital, Melbourne, Victoria; 2The Murdoch Children’s Research Institute, Melbourne, Victoria, Australia

Aim: To evaluate the views of nephrology patients and their families in a regular nephrology newsletter and to establish the preferred format CHIR-99021 cost and content. Background: The importance of regular education and support for nephrology patients and their families is pivotal in their overall care while providing a forum for interaction between families and recruitment to research studies. Method: A pilot survey was distributed amongst 10 adults at The Royal Children’s Hospital (RCH), including doctors, nurses, cleaning staff and volunteers. Following their comments, the survey was amended and then distributed to patients and family in clinic, wards and in the haemodialysis unit. Results: 15 patients responded to the survey; 3 female, 12 male, mean age 13 ± 2.9 years. 10 (66%) patients were interested or very interested in receiving a newsletter from Erlotinib supplier the department. 11 patients would prefer a paper based newsletter, 4 patients stated that they would be not interested in facebook. 34 family members responded to the survey; 8 fathers, 23 mothers, 2 grandmothers and 1 aunt, mean age 43 ± 12 years. 28 individuals were interested or very interested

in receiving a newsletter. 22 (64%) individuals would prefer a paper based newsletter, 20 (58%) individuals were interested in an emailed newsletter and 15 (44%) individuals were interested in a facebook-based newsletter. There was broad enthusiasm for all suggested content, including community activities and reminders, with the favourite topics including community activities, patient profiles and research. In free text family members expressed interest in community websites or support groups, menu ideas, and the latest research. Conclusion: Based on this project we have introduced “Nephrology News” as a paper based quarterly newsletter. 229 RELATIONSHIP DIABETES MELLITUS TYPE 2 AND INCIDENT WITH CHRONIC KIDNEY DISEASE IN THE HOSPITAL DR.

On day 9, culture medium was replaced by fresh medium containing anti-CD3 antibody (1 μg/mL). On day 10, lymphocytes were harvested and used for phenotypic, functional analyses or co-culture assays. CD4+CD25− T

cells were fluorescently labeled using carboxyfluorescein diacetate succinimidyl ester (CFSE) according to manufacturer’s instructions (CellTrace from Molecular Probes/Invitrogen). iTreg cells and autologous CFSE-labeled CD4+ T cells were co-cultured at different ratios for 5 days in the presence of plate-bound anti-CD3 (1 μg/mL), anti-CD28 (2 μg/mL), and IL-2 (100 IU/mL). Proliferation of CFSE-labeled CD4+ T cells was assessed by flow cytometry and data analysis was performed using Diva6 software. For phenotypic analysis, cultured cells were harvested, washed, and incubated in PBS (supplemented with 3% human serum and 0.1% sodium azide) containing optimal dilution selleck chemicals llc of each fluorochrome-conjugated mAb for 25 min at 4°C in the dark. Cells were subsequently washed and fixed with 2% v/v paraformaldehyde (PFA) or proceeded to intracellular staining for TGF-β or IL-10. For intracellular

staining, washed cells were incubated with BD Cytofix/Cytoperm solution for 30 min at 4°C in the dark, washed twice with BD Perm/Wash buffer, and incubated with fluorescent-labeled mAbs diluted in the same buffer for 25 min at this website 4°C in the dark. All mAbs used were pre-titrated on fresh PBMCs Adenosine triphosphate to determine their optimal working dilutions.

Respective isotype controls were used in all experiments. After staining, cells were immediately subjected to measurement in a FACS Canto II flow cytometer. The collected data were analyzed with Diva6 software (both from Becton Dickinson, Heidelberg, Germany). NK cells were activated with IL-2 (100 IU/mL) and co-cultured with control iTreg cells, nTreg cells, or CD4 cells at a ratio of 1:2 (NK cell/T cell). After 36 h, supernatants and cells were harvested. Surface expression of NKG2D and NKp44 on NK cells was assessed, and supernatants were analyzed for IFN-γ by ELISA according to manufacturer’s guidelines (R&D Systems). In some experiments, rh-TGF-β (1 ng/mL) was added to IL-2-activated NK cells. NK cells were activated or not with IL-2 (100 IU/mL) and co-cultured with autologous nTreg cells, iTreg cells, or with TGF-β for 18 h. CD107a FITC and in some experiments Colo699 tumor cells were added for further 6 h to the co-culture system. During the last 5 h, the BD Golgi-Stop was present in the co-culture. At the end, NK cells were stained with CD56-PE as described in the section Flow cytometry and analyzed by flow cytometry. CD107a (LAMP-1) is a marker for NK degranulation and its level of expression correlates with NK cytotoxicity 45. Cytotoxicity was determined in a standard 6-h chromium release assay.

The median

age of all participants was 37 years (IQR 35–48 years) and most were men (81%). No difference in gender distribution was observed between the groups for the leprosy and co-infected groups. Most patients had paucibacillary presentation at the time of diagnosis for both leprosy groups. Our results demonstrated that healthy controls had higher CD4+ T-cell counts (median 917 cells/mm3, IQR 687–1170) when compared with HIV-1-infected patients (median 391 cells/mm3, IQR 272–536) and co-infected patients www.selleckchem.com/products/abt-199.html (median 285 cells/mm3, IQR 235–480), P < 0.001. Leprosy patients had higher numbers of CD4+ T cells (median 733 cells mm3, IQR 699–870) when compared with co-infected patients (P < 0.001). For CD8+ T-cell counts, healthy controls (median 556 cells/mm3, IQR 376–735) had lower numbers when compared with co-infected patients (median 806 cells/mm3, IQR 578–1548), P < 0.05 (Table 1). The NKT cells represent a subset of lymphocytes, defined operationally as bearing both the T-cell receptor and the NK cell marker CD161 (NK1.1 in mice).19 We defined selleck NKT cells as those with the CD3+ Vα24+ Vβ11+ phenotype (Fig. 1a), and further subdivided NKT cell subsets using CD4, CD161 and HLA-DR. The gating strategy enabled

delineation of CD4+ NKT subsets (Fig. 1b). Because of the variability of NKT cell frequencies and limitations of available PBMC, data

were included in this study if > 30 events were collected within the NKT gate. Berzins et al.20 reported an NKT cell frequency in adult blood ranging from 0.006 to 0.78%. Farnesyltransferase Our results demonstrated that the healthy controls had more NKT cells in the peripheral blood (median 0.077%, IQR 0.032–0.405) than co-infected patients (median 0.022%, IQR 0.007–0.051), P < 0.01. Co-infected patients also had fewer NKT cells when compared with HIV-1-infected patients (median 0.072%, IQR 0.030–0.160), P < 0.05 (Fig. 2a). The CD4 molecule distinguishes two phenotypic and functionally distinct subsets of NKT cells. CD4+ NKT cells were found to produce both T helper type 1 and type 2 cytokines, whereas CD4− NKT cells mainly produce T helper type 1 cytokines.21,22 In peripheral blood from healthy adult volunteers, close to 50% of NKT cells are CD4− with no, or low, expression of CD8.23 We observed that leprosy patients have more CD4+ CD161+ HLA-DR– NKT cells (median 21.40%, IQR 3.65–59.95) compared with HIV-1-infected patients (median 0.375, IQR 0.00–19.30), P < 0.05 (Fig. 2b), but this was not statistically different from healthy controls or co-infected patients. We used CD161 and HLA-DR as activation markers to determine the activation profile of NKT cells.

2A). However, the number of antigen-specific cells recovered at day 5 was not different between CpG-treated and control mice. Co-injection

of poly(I:C), LPS, or imiquimod did not modify the number of tetramer+ cells recovered from the spleens or LN at these early time points compared with mice immunized with peptide alone (data not shown), demonstrating a selective potency of CpG to enhance the early expansion of CD8+ T cells in response to soluble peptide in vivo. Consistent with the increased clonal population size at day 3 post-immunization, tetramer+ cells recovered from mice treated with CpG displayed a more robust proliferation profile compared with control mice that received peptide alone, as indicated by CFSE dilution (Fig. 2B). The effects of CpG were not as striking PLX4032 mouse in the spleen, though similar trends were observed. By day 5, however, there was no accumulation of CFSElo cells regardless of CpG treatment, with proliferation profiles similar to those observed previously at day 5 in all groups (data not shown). Further, the numbers of tetramer+ cells recovered from the spleens of immunized mice 10 days post-immunization were

not changed by treatment with any TLR, including CpG (Fig. 2C). Thus, in spite of inducing more robust early proliferative activity, CpG treatment could not modify the widespread cell death observed after peptide immunization. Addition of MHC class II-restricted peptides to the Daporinad concentration inoculum to elicit help from CD4+ T cells did not enhance the survival of the peptide-stimulated CD8+ T cells, even in the presence of CpG (Supporting Information Fig. 2). In mice that were immunized with peptide alone, we could not detect antigen-specific T cells by ELISPOT, suggesting that they were unable

to produce IFN-γ (Fig. 2D). However, antigen-specific Parvulin cells from the dLN of mice treated with CpG and peptide were readily detected by IFN-γ ELISPOT. These differences were not merely due to differences in frequency, as there was a ten-fold increase in tetramer+ cells measured by FACS, but there were greater than 300-fold differences in the number of IFN-γ-producing cells. Curiously, antigen-specific IFN-γ secreting T cells were not detected in the spleen when immunizing mice with either peptide alone or CpG with peptide. CpG clearly modulates the CD8+ T-cell response to soluble peptide by promoting cell division and clonal expansion, as well as supporting IFN-γ production. However, CpG could not induce T-cell survival, as there was no significant increase in the final magnitude of the CD8+ T cell after the contraction phase. Since CpG has been shown to have many effects on the immune system 21 that may change over time, we modified the timing of the CpG administration relative to the peptide to investigate whether there were temporal effects of the CpG that could enhance T-cell survival.

Interestingly, not a single surface-associated protein was identified as PD98059 mw being solely expressed in sessile or planktonic cells. Nineteen proteins were significantly overexpressed in C. albicans

biofilms grown in 24-well microtiter plates, compared with planktonic cultures, and in contrast to the results obtained by Thomas et al. (2006), ENO1 was twofold underexpressed. Highly significant overexpression was observed for citrate synthase (14.45-fold), and several proteins involved in oxidative stress, including alkyl hydroperoxide reductase AHP1 and several other reductases (GRP2, MCR1, TSA1, PST1 and TRX1), were also overexpressed. Proteomics has also been used for a three-way comparison of planktonic yeast cells, planktonic hyphae and sessile cells (Martinez-Gomariz PI3K Inhibitor Library manufacturer et al., 2009). One hundred and seventy-five cytoplasmic and 70 cell surface-associated proteins were differentially expressed between sessile and planktonic yeast cells, while these numbers were 218 and 51, respectively, when sessile cells were compared with planktonic hyphae. The fold over- or underexpression varied considerably depending on the comparison

made. For example, MET15 was downregulated in biofilms when compared with planktonic yeast cells, but upregulated when biofilms were compared with planktonic hyphae, confirming that morphology is an important factor. Further complicating the comparison of protein expression is the presence of various isoforms of the same protein. For example six

isoforms of pyruvate decarboxylase were identified by Martinez-Gomariz and colleagues: isoforms 1, 2, 5 and 6 are underexpressed in biofilms compared with planktonic yeast cells, while isoforms 3 and 4 are overexpressed. A detailed analysis of the results obtained in the studies summarized PTK6 above reveals that, although generally representatives of particular classes of genes are differentially expressed between planktonic and sessile cells (Fig. 1), there is very little overlap between C. albicans genes identified as differentially expressed in different studies and the same is true for other microorganisms. The observation that the experimental conditions for culturing the cells before RNA extraction are often variable (Table 1) offers a first explanation. There are a wide range of biofilm model systems available, and few studies have used the same model system. Similarly, planktonic cells are cultured in a variety of ways (Table 1).

It remains unknown whether RGMa plays a role in the neurodegenerative process of Alzheimer’s disease (AD). We hypothesize that RGMa, if it is concentrated on amyloid plaques, might contribute to a regenerative failure of degenerating axons in AD brains. Methods: By immunohistochemistry, we studied RGMa and neogenin (NEO1) expression in the frontal cortex and the hippocampus of 6 AD and 12 control cases. The levels of RGMa expression were determined by qRT-PCR and Western blot in cultured human astrocytes following exposure

to cytokines and amyloid beta (Aβ) peptides. Results: In AD brains, an intense RGMa immunoreactivity was identified on amyloid plaques Copanlisib cost and in the glial scar. In the control brains, the glial scar and vascular foot processes of astrocytes expressed RGMa immunoreactivity, while oligodendrocytes and microglia were negative for RGMa. In AD brains, a small subset of amyloid plaques expressed a weak NEO1 immunoreactivity, while some reactive astrocytes in both AD and control brains showed INCB024360 datasheet an intense NEO1 immunoreactivity. In human astrocytes, transforming growth factor beta-1 (TGFβ1), Aβ1–40 or Aβ1–42 markedly elevated the levels of RGMa, and TGFβ1 also increased its own levels. Coimmunoprecipitation analysis validated the molecular interaction between RGMa and

the C-terminal fragment β of amyloid beta precursor protein (APP). Furthermore, recombinant RGMa protein interacted with amyloid clonidine plaques in situ. Conclusions: RGMa, produced by TGFβ-activated astrocytes and accumulated in amyloid plaques and the glial scar, could contribute to the regenerative failure of degenerating axons in AD brains. “
“Chronic granulomatous CNS infections may be caused by tuberculosis, fungi and rarely by free-living amoeba, especially in immunocompromised individuals. We report a rare, fatal case of granulomatous amoebic encephalitis in an immunocompetent patient mimicking CNS

tuberculosis, and review the imageological features and diagnostic tests. “
“A 57-year old man with chronic alcoholism presented with apraxia of speech and disturbance of consciousness. He had a history of gastrectomy and had been drinking alcohol. The symptoms improved with administration of thiamine, but he later developed diarrhea and delirium, and died approximately 40 days after the onset. Autopsy findings were consistent with Wernicke’s encephalopathy and pellagra encephalopathy. Furthermore, laminar cortical necrosis with vacuoles and astrocytosis was found in the second and third layers of the bilateral frontal cortices, suggesting Morel’s laminar sclerosis. The lesions were mainly located in the bilateral primary motor cortices. Involvement of the lower part of the left primary motor cortex may be associated with apraxia of speech in our case. “
“S. J. Crocker, R. Bajpai, C. S. Moore, R. F. Frausto, G. D. Brown, R. R. Pagarigan, J. L. Whitton and A. V.

The samples were then incubated with 50 µl/well of OVA-biotin (1 mg/ml; Sigma, St Louis, MO, USA) at room temperature for 1 h. Plate-bound antibody was detected by treatment

with 50 µl/well of streptavidin–horseradish peroxidase (1 : 10 000; Southern Biotechnology) for 1 h at room temperature. The colour reaction was developed by adding 100 µl/well of 200 pmol of OPD (Sigma) in pH 5·0 citrate phosphate buffer plus 0·04% H2O2 for 10 min and stopped with 50 µl of 5% sulphuric acid per well. The plates were read at 492 nm in an ELISA reader (Bio-Rad, Hercules, CA, USA). The lungs of five mice per group were removed and treated with 100 U/ml of collagenase from Clostridium histolyticum (Sigma) for 30 min at 37°C. Subsequently, the digested lung tissue was filtered through a 70 micrometre cell strainer and the red blood cells were lysed with ACK buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2; Invitrogen, CA,

USA). The cell Enzalutamide chemical structure suspension was washed twice in RPMI-1640 and adjusted to 1 × 106 cells per well for surface staining and to 2·5 × 106 cells for the intracellular cytokine experiment. For CD4 and forkhead box P3 (FoxP3) staining, the cells were generally blocked with anti-mouse CD16/CD32 monoclonal antibodies (mAbs) (Fc-block) and stained for surface marker using fluorescein isothiocyanate (FITC)-labelled anti-mouse CD4 (BD Bioscience) mAb or isotype control, which were incubated for 20 min at 4°C with antibody dilution learn more solution (PBS 0·15 M, 0·5% BSA, 2 mM NaN3). The cells were then washed with 0·15 M PBS and incubated with strepatavidin–phycoerythrin–cyanine 5 (PE-Cy5) (1 : 200) Tolmetin for an additional 20 min at 4°C. Surface-stained cells were washed twice with 0·15 M PBS and incubated with fixation/permeabilization buffer (eBioscience) for 30 min at

4°C. Anti-FoxP3-PE-labelled antibodies in permeabilization buffer (eBioscience) were added to cells and then incubated for 30 min at 4°C. Cells were washed twice with 150 µl of permeabilization buffer (eBioscience) and fixed with 2% paraformaldehyde. For IL-10 and FoxP3 intracellular staining, cells were cultured for 14 h in medium or OVA (25 µg/ml). After this stimulation period, 1 mg/ml of brefeldin A was added to the cell culture, which was incubated for an additional 4 h in a CO2 incubator at 37°C. Before CD4 staining, the cells were treated with anti-CD16/CD32 (Fc-block). Cell surface and intracellular staining were performed as described above for surface experiments; however, the cells were stained for CD4, IL-10 and FoxP3 using anti-CD4 FITC-labelled, anti-IL-10 PE-labelled, and anti-FoxP3 biotin-labelled plus streptavidin–PE-Cy5 antibodies. Data acquisition was performed using fluorescence activated cell sorter (FACScan) (Becton Dickinson, San Jose, CA, USA). Data analysis was performed using a FlowJO interface (Becton Dickinson). Statistical analysis was performed using the software GraphPad Prism (GraphPad Software, San Diego, CA, USA). The mean ± standard deviation (s.d.