The heparinized FDA approved Drug Library research buy blood was layered carefully onto Ficoll (density 1·077 g/ml; Fresenius Kabi Norge AS for Axis-Shield PoC AS, Oslo, Norway) and centrifuged at 800 g for 30 min without brake to obtain a density gradient separation. After centrifugation, the mononuclear cell layer was recovered and washed twice with PBS; Sigma). Human CD4+ T cells were isolated from the PBMCs by positive selection using the Midi MACS CD4+ T cells magnetic isolation kit (Milteny Biotec), according to the manufacturer’s instructions. In order to evaluate the immunosuppressive activity of MSCs, these cells were isolated from both HC and SSc and plated in triplicate into 12-well plates. HC–PBMCs resuspended in 2 ml of RPMI-1640 (Invitrogen,

Cergy, France) supplemented selleck compound with 10% inactivated human serum (from human male AB plasma; Sigma) were added to wells in a 1:1 ratio with BM–MSCs and cultured in the presence of 4 ug/ml phytohaemagglutinin (PHA) for 5 days, as described previously [20]. After PHA stimulation, PBMCs were pulsed with 1 uCi/well of [3H]-thymidine ([3H]-TdR)

(Amersham Pharmacia) for 18 h. Cells were harvested and thymidine incorporated in DNA was recovered on filters. [3H]-TdR incorporation was measured using a scintillation counter (KLB Wallac, Gaithersburg, MD, USA). Lymphocyte proliferation was quantified by means of an 18-h pulse with 1 mCi/well ([3H]-TdR) (Amersham, Bucks, UK) and expressed as counts per minute (cpm). CD4+ T cells were isolated from SSc and HC PBMCs, resuspended in 2 ml RPMI-1640 (Invitrogen) supplemented with 10% inactivated FBS (Gibco) and co-cultured with HC– and SSc–MSCs at a 1:5 ratio. To evaluate the role of MSCs and CD4+ T cells in our system, we planned a set of experiments in autologous and heterologous conditions: (i) HC–MSCs+HC–CD4; (ii) SSc–MSCs+SSc–CD4; (iii) HC–MSCs+SSc–CD4; and (iv) SSc–MSCs+HC–CD4,

to assess the specific activity of each cell subset. After 5 days, CD4+ cells were harvested and analysed for the expression of specific surface antigens by monoclonal antibody directed against CD3, CD4, CD25 (Beckman-Coulter), FoxP3 (BioLegend) and CD69 (Miltenyi Biotec, Ltd, Bisley, Surrey, UK). CD4+CD25brightFoxP3+ and CD4+CD25brightFoxP3+CD69+ cells were quantified by cytofluorimetric analysis (Cytomics FC500; Beckman-Coulter) within an initial fraction Interleukin-2 receptor of 1 × 106 CD4+ cells. Tregs were isolated further from each experimental culture by CD25 microbeads (Miltenyi Biotec). The suppressive capacity was established as follows: CD4+ cells were cultured in 96-well plates with PHA (4 μg/ml) alone and in the presence of enriched Tregs (the CD4+ T cell/Treg cell ratio was 10:1). After 4 days of co-culture, [3H]-TdR was added for a further 24 h. Cells were harvested into glass fibre filters and [3H]-TdR incorporation was assessed by a beta scintillation counter. The concentrations of both IL-6 and TGF-β released in the culture supernatants were measured by a specific ELISA.

5 One technique

to increase the number of cells available and to develop clonal populations of cells which should in theory be homogeneous and stable is to transform the cells with an oncogene. The transforming Nutlin-3 price gene usually used is SV40, a monkey-derived gene which promotes unregulated proliferation of the cells into which it is transfected. Sraer and colleagues in Paris produced an SV40-transformed human podocyte cell line6,7 and they generously shared this reagent with other workers including us. We found that this cell line was easy to propagate and we rapidly accumulated large numbers of cells for in vitro experiments. However, again the cells did not develop the phenotype of differentiated podocytes and we felt that newer more representative cell lines were needed. In 1997, Peter Mundel and colleagues reported8 the characterization of a mouse podocyte cell line derived from the

‘Immortomouse’ whose cells all express SV40 transforming gene under the control of a gamma-interferon response element. Thus, cells from this mouse can be induced to express higher levels of SV40 by treatment in vitro with gamma-interferon. The original mouse podocyte cell Selleckchem BGJ398 line, which in time came to be known colloquially as ‘Mundelocytes’, was shown to express markers of mature podocytes Methocarbamol and was generously shared with other researchers, becoming very widely used for understanding podocyte biology. In collaboration with Peter Mundel, we9 applied a similar principle to the development of a human podocyte cell line: this time

the SV40 had to be supplied to the cells in vitro after isolation of the cells of interest. The SV40 construct that we used is temperature-sensitive, giving us control of its expression in vitro: at 33°C the transgene is expressed, allowing the cells to be transformed and to proliferate vigorously. When the cells are moved to a culture temperature of 37°C, akin to the normal physiological body temperature, the transgene is silenced and the cells become differentiated, ceasing to proliferate. This approach had been previously used by our collaborator Mike O’Hare in other cell types10 and the original normal human podocyte cell line, known colloquially as ‘Saleemocytes’, has now been widely shared and studied by numerous groups worldwide. The next section gives more details of the techniques required for the generation of these cells.

The primary limitation of studies utilizing biomarkers identified in amniotic fluid is that they require an invasive and sometimes risky procedure (i.e., amniocentesis) in order to determine the in-utero environment. For a biomarker to be incorporated into routine practice, the information needs to be obtainable in a non-invasive manner. However, there are several challenges using non-invasive sampling to predict intrauterine environment including: what is the best non-invasive sampling site that can predict a specific intrauterine

immune compartment? For example, is it the urine or the blood sample that has the best biomarkers predictive of placental immune environment? Is this non-invasive sample also predictive of other compartments’ immune environment such as amniotic fluid? Can Doxorubicin mw we combine several biomarkers from different non-invasive samples to predict for example placental immune environment? To begin

to answer these questions, we conducted a pilot study comparing inflammatory mediators from non-invasive samples (maternal blood, urine, saliva, vaginal, or cervical secretions) with traditional gold standard invasive samples (amniotic fluid and placenta samples).[14] Term, non-laboring patients without major maternal, or fetal complications Smad inhibitor undergoing Cesarean delivery were recruited (n = 20). We obtained fluid samples from different maternal and fetal compartments and determine the inflammatory mediator expression in each. These mediators include cytokines, chemokines, and growth factors that again were measured via the Bio-Plex™ Suspension Array system. The results indicated that different intrauterine compartments are mostly immunologically distinct with few compartments showing similar cytokine expression (Table 1). This finding provides important insight into what has been shown in other studies. For example, in the placenta, low IL-10 has been linked to preterm labor;[15] however, high IL-10 and high pro-inflammatory mediators were observed in amniotic fluid samples associated with preterm labor.[11] Although this finding might appear contradictory, it may indicate a primary deficiency of placental IL-10 production (the pathology) that

triggers intrauterine inflammatory environment and increased new production of pro-inflammatory mediators. Such inflammatory environment will initiate a feedback up-regulation of anti-inflammatory molecules such as IL-10 in amniotic fluids (the response). Not surprisingly, in our study, there was significant correlation between vaginal and cervical samples. The data indicated there are several potential cytokines in non-invasive samples that can be targeted as a biomarker reflecting their expression in the intrauterine environment. Significantly, the study demonstrates that a specific correlation of an intrauterine cytokine may be reflected in one non-invasive site but not another, depending upon the type of cytokine, and the compartment from which it is secreted.

However, membrane-bound TNF is also capable of Torin 1 research buy binding to TNF receptors and initiating a signal transduction pathway through cell–cell interactions.

At this time, a number of possible steps in transcription, translation or secretion ranging from diminished synthesis of mRNA or protein to increased destruction of either macromolecule may also be possible targets for the action of doxycycline. In this regard, preliminary data in our lab indicate that doxycycline [and even more so, 6-demethyl 6-deoxy 4-de-(dimethylamino) tetracycline (CMT-3), a chemically modified tetracycline analog that has been shown to be more potent than doxycycline, although it results in more side effects] can decrease nuclear factor-κB Z-VAD-FMK concentration phosphorylation/activation, which could decrease cytokine and MMP expression (Gu et al., 2009). Doxycycline has been found to inhibit the protein and RNA expression of MMP-8 induced by TNF-α and phorbol myristic acetate in human rheumatoid and gingival fibroblasts (Hanemaaijer et al., 1997). Doxycycline

also inhibited type I and II collagenolytic activity in these fibroblasts (Hanemaaijer et al., 1997). Using the ECM system, we demonstrated that monocyte-derived macrophages can directly mediate ECM degradation, and doxycycline protected the ECM degradation possibly by reducing the activities and/or the levels of MMPs through reducing Tyrosine-protein kinase BLK extracellular cytokine levels. Doxycycline and nonantimicrobial tetracyclines have been shown by Golub’s group to inhibit MMP activity, connective tissue breakdown and modulate host responses. One of these compounds, CMT-3, was found to be superior to the other CMTs as an MMP inhibitor. In separate preliminary studies, we evaluated the effect of CMT-3 on the extracellular levels of the cytokines, TNF-α and IL-1β, and on MMP-9. CMT-3 (0.1 μM) inhibited 56% of the extracellular TNF-α level, while 1 and 10 μM CMT-3 reduced the extracellular levels of TNF-α by 70% and 71%, respectively (Y.

Gu, H.-M. Lee and L.M. Golub unpublished data). 0.1, 1 and 10 μM CMT-3 also reduced MMP-9 levels by 12%, 20% and 53% (data not shown). CMT-3 at different concentrations exhibited greater capacity in reducing TNF-α and MMP-9 levels, but not IL-1β levels. Future experiments will compare the potency of CMT-3 relative to doxycycline as MMP and cytokine inhibitors, especially as both subantimicrobial formulations of doxycycline or nonantibiotic CMT-3 have been tested in humans with conditions ranging from chronic inflammatory diseases such as periodontitis, rheumatoid arthritis and acne and rosacea (Ryan et al., 1996; Grenier et al., 2002; Bikowski, 2003), to a fatal lung disease (Moses et al., 2006), and a type of cancer, Kaposi’s sarcoma (Dezube et al., 2006).

The exclusion criteria were patient’s refusal, inability to complete questionnaire, amputation of both legs, and severe illness. Biochemical laboratory data were collected and, in the meanwhile

ankle-brachial index (ABI) was measured. Results: There were 171 patients included in the study. The prevalence of PAOD was 29.8%. The odds ration (OR) of amputation in patients with PAOD www.selleckchem.com/products/Trichostatin-A.html was 12.6 (95% C.I. = 2.6∼60.9). The patients with PAOD had significantly older age, more diabetes, higher serum glucose, hemoglobin (Hb), white blood cell counts (WBC), and lower creatinine, albumin, and sodium. Logistic regression analysis showed age (OR = 1.06, 95% C.I. = 1.03∼1.09, p < 0.001), diabetes (OR = 4.71, 95% C.I. = 2.24∼9.89, p < 0.001), serum glucose (OR = 1.006, 95% C.I. = 1.002∼1.01, p = 0.001), hemoglobin (OR = 1.39, 95% C.I. = 1.06∼1.80, p < 0.016), white blood cell counts (OR = 1.35, 95% C.I. = 1.13∼1.60, p = 0.001), and sodium (OR = 0.85, 95% C.I. = 0.77∼0.94, p = 0.002). Conclusion: In hemodialysis patients, age, DM, serum glucose, Hb, and WBC were positively correlated with PAOD. Apoptosis inhibitor Serum creatinine, albumin, and sodium were negatively correlated with PAOD. TSUJI AKIRA, OOSHIMA KOUJIROU Department of Blood Purification, National Defense Medical College Hospital Introduction: We have more than

60 hemodialysis (HD) introduction patients, and about 35% of those have cardiovascular complications (CVC) a year in National Defense Medical College Hospital. We often

experience hypotension due to hypovolemic or overhydration states during dialysis therapy, but might cause a poor result in HD introduction Phloretin patients with CVC. The aim of this study is to assess appropriate quantity of ultrafiltration in HD introduction patients with CVC by body composition using a bioelectrical impedance analysis (BIA) and ultrasonic inferior vena cava diameter (IVCD). Methods: Sixty-three HD introduction patients (45 male and 18 female, average age 64 ± 14 years old, 261 dialysis sessions before dry weight being decided, 43 planned and 20 urgent) were divided into two groups with CVA (CVA+, 22 patients, 135 dialysis sessions) or without CVA (CVA-, 41 patients, 126 dialysis sessions) for a year in 2013. Total body water (TBW), intracellular water (ICW), extracellular water (ECW), ECW/TBW on BIA (MLT-550N®, SK Medical, Japan), IVCDe (expiration), IVCDi (inspiration) and collapsibility index (CI) were measured before and after HD. Quantity of ultrafiltration was calculated for each dialysis treatment. Brain natriuretic peptide (BNP) and cardio thoracic ratio (CTR) were measured before HD at the time of need. Results: In CVC+ group, there was a significant correlation between quantity of ultrafiltration and CI (r = −0.4058, p < 0.0001), IVCDe (r = 0.3548, p < 0.0001), IVCDi (r = 0.41, p < 0.0001), TBW (r = 0.6606, p < 0.0001), ICW (r = 0.3658, p < 0.0001), ECW (r = 0.7009, p < 0.0001) and ECW/TCW (r = 0.4537, p < 0.0001).

05). In contrast, both ligands increased the VEGF levels (Fig. 3D). Previous studies have suggested 5-Fluoracil price the possible involvement of Gal-3 in diverse physiological and pathological processes, including pre-mRNA splicing, neoplastic transformation and immune response [18]. Gal-3 is also reported to play a negative role in T-cell activation, a process that requires clustering of a threshold number of T-cell receptor at the site of antigen presentation [19, 20]. Based on these early findings, we investigated the potential effect of Gal-3 gene silencing in MSC on T-cell proliferation to alloantigens. To identify

effective siRNA against Gal-3, we visually examined the sequence of Gal-3 mRNA and selected 3 targeting sites. The silencing potency of the designed siRNA was tested in freshly isolated human monocytes (Fig. 4A). All the 3 siRNA inhibited Gal-3 expression with siRNA-3 being the most effective. At a concentration of 2 μg, the silencing efficiency was around 99% when compared to control cells. Having demonstrated that siRNA-3 is effective in human monocytes, next we assessed its silencing potency in MSC (Fig. 4B and C). The designed siRNA resulted in nearly 94% (±3%) reduction in intracellular protein levels, and around 95% (±4%) reduction in the secreted protein when compared to cells transfected with control siRNA. In contrast, depletion of Gal-3 has no

significant Bortezomib heptaminol effect on either β actin or VEGF expression, thus confirming the specificity of the designed siRNA-3. To uncover the potential effects of Gal-3 knockdown on MSC function, we asked whether MSC-expressing Gal-3 could have an effect on the proliferation of lymphocytes in response to alloantigens. To this end, we first determined the cell concentration that gave a significant inhibition and found that suppression can be achieved after the addition of approximately 10–50 000 MSC to mixed lymphocyte cultures. Second, we tested lymphocyte response in the presence of 30 000 allogeneic MSC that have been transfected with either siRNA-3 against Gal-3

or control siRNA. In these experiments, peripheral blood mononuclear cells from donor 1 (PBMC1) were incubated with PBMC from a responder donor 2 (PBMC2) in the absence or presence of irradiated “third-party” MSC. In contrast to Gal-3 expressing MSC, knockdown of Gal-3 resulted in less immunosuppressive effect on T-cell proliferation (Fig. 4D, P < 0.05, as a representative example). In addition to the expression of certain TLR, this study shows that MSC also express NOD-1. Unlike TLR, NLR consist of soluble proteins that survey the cytoplasm for signs that advertise the presence of intracellular invaders [15]. By screening the expression profiles in response to NOD-1 and TLR-2 synthetic ligands, we have identified a set of genes that were altered subsequent to overnight activation of MSC.

Image analysis (substratum coverage) was carried out using the function ‘Cell Counting-Batch’ in the software package bioimage_l (Chávez de Paz, 2009). For the preparation of biofilm supernatants, mid-exponential growth-phase cultures (corresponding to 109 CFU mL−1) of the P. aeruginosa strains (NCTC 6750, PAO1, 14:2, 23:1, DNA Damage inhibitor 27:1 and 15159) in TH medium were inoculated into tissue culture flasks and allowed to grow in biofilms under static conditions for 24 h (5% CO2, 37 °C). Culture supernatants

were collected and subjected to centrifugation (10 min, 3000 g), sterile filtered (0.20 μm) and stored at −20 °C until use. Six-hour S. epidermidis biofilms were exposed to P. aeruginosa biofilm supernatants for 1 h and then visualized using 16S rRNA FISH with the STA3 probe and examined using CSLM. Selleckchem Luminespib The viability of the attached cells was investigated in parallel biofilm cultures using the BacLight LIVE/DEAD stain according to the manufacturer’s instructions. To investigate the viability of dispersed cells of S. epidermidis, aliquots of the spent medium were cultured on 110 agar or stained using BacLight LIVE/DEAD staining. Two independent experiments were performed. The production of N-butanoyl-l-homoserine lactone (C4-HSL) was

studied with a well-diffusion assay using the reporter strain Chromobacterium violaceum CV026 as described by Ravn et al. (2001). Culture supernatants from 24-h biofilms were extracted twice with equal volumes of ethyl acetate Isotretinoin acidified with 0.5% formic acid. The combined extracts were then vacuum-dried and the residues were dissolved in 0.5 mL

of ethyl acetate acidified with 0.5% formic acid and stored at −20 °C until use. Luria–Bertani (LB) agar seeded with C. violaceum CV026 (cultured overnight in LB broth supplemented with 20 μg mL−1 kanamycin, 28 °C) was poured onto prewarmed LB agar and allowed to solidify (10 μL C. violaceum culture mL−1 LB). Wells punched into the agar were filled with 50 μL of the solvent extracts and incubated for 24 h at 28 °C. Synthetic C4-HSL (Sigma) (1 mM) and TH medium were used as positive and negative controls, respectively. The presence of purple pigmentation around the wells indicated violacein production by C. violaceum CV026 in response to C4- to C8-HSL (McClean et al., 1997). Pyocyanin production was investigated by inoculating Pseudomonas medium A agar (Atlas & Parks, 1993) with the P. aeruginosa strains and incubating for 24 and 48 h in 5% CO2 at 37 °C. The production of the phenazine pigment pyocyanin was indicated by the presence of green colour around the CFU. Protease expression in biofilms of the different strains was determined by electrophoresis on Novex Zymogram gels (Invitrogen) according to the manufacturer’s instructions.

Continued and extensive progress in stem cell research in both basic and pre-clinical settings should support the hope for development of NSC-based therapies for neurodegenerative diseases. This review focuses on the utility of stem cells, particularly NSCs, as substrates for structural and functional repair GSK458 cost of the diseased or injured brain. Parkinson’s disease, characterized by an extensive loss of dopamine (DA) neurons in the substantia nigra pars compacta and their terminals in the striatum,

affects more than 500 000 people in the US and about 50 000 new cases are reported annually.[20, 21] While the etiology of idiopathic PD is not known, several predisposing factors for the dopamine depletion associated with the disease have been suggested, including programmed cell death, viral infection, and environmental toxins. As an effective treatment for

PD, patients have been given L-dihydroxyphenyl alanine (L-DOPA), a precursor of dopamine, but long-term administration of L-DOPA consequently produces grave side effects.[22, 23] More recently, surgical deep brain stimulation has been adopted as a successful treatment for PD patients.[24] Since the late 1980s, transplantation of human fetal ventral mesencephalic tissues into the striatum of PD patients has been used as a successful therapy for patients with advanced disease.[25-28] However, this fetal tissue transplantation has serious problems associated Selleckchem MLN0128 with ethical and religious questions and logistics of acquiring fetal tissues. In addition, recent reports have indicated that the survival Erastin research buy of transplanted fetal mesencephalic cells in the patients’ brain was very low and it was difficult to obtain enough fetal tissues needed for transplantation.[29] To circumvent these difficulties, utilization of neurons with dopaminergic (DA) phenotype generated from ESCs, iPSCs, MSCs or NSCs could serve as a practical and effective alternative for the fetal brain tissues

for transplantation. DA neurons were generated from mouse ESCs after treatment with fibroblast growth factor 8 (FGF8) and sonic hedgehog,[30, 31] over-expression of Nurr1[32, 33] or Bcl-XL,[34] or co-culture with a mouse bone marrow stromal cell line.[35] Neurons with DA phenotype have been generated from monkey ESCs by co-culturing with mouse bone marrow stromal cells and behavioral improvement was seen in MPTP-lesioned monkeys following intra-striatal transplantation of these cells.[36] DA neurons were also generated from neural progenitor cells derived from fetal brain and induced functional recovery following brain transplantation in parkinsonian monkeys.[37] Transplantation of NSCs in the brain attenuates anatomic or functional deficits associated with injury or disease in the CNS via cell replacement, the release of specific neurotransmitters, and the production of neurotrophic factors that protect injured neurons and promote neuronal growth.

Of the 47 patients who received anakinra (25 anakinra with dexamethasone), progression-free survival ensued for more than three years and in 8 patients for

more than 4 years 100. Patients with a decrease in serum CRP of 15% or greater after 6 months of anakinra monotherapy resulted in progression-free survival times greater than 3 years as compared with 6 months in patients with less than a 15% fall during anakinra therapy (p<0.002). Thus, an effective reduction in IL-1β activity using CRP as the marker for IL-1β-induced IL-6 halts progression to active myeloma. Anakinra results in resolution of all signs and symptoms within hours after the first injection. However, approximately 20% of patients with Schnitzler's syndrome develop a lymphoproliferative disorder, mostly lymphoma or Waldenstrom

disease, which is similar to patients with IgM MGUS. This latter point and its consequences have been already been addressed in the Decitabine purchase literature 101. Blocking IL-1β may reduce the progression to a lymphoproliferative disorder in patients with Schnitzler’s syndrome. Similar to smoldering myeloma, the concept that IL-1β drives IL-6 production was tested in a patient with another lymphoproliferative disorder, Castleman’s selleck compound disease, which is usually treated with anti-IL-6 receptor antibodies 102. The patient failed to respond to cladribine, rituximab, steroids, etanercept and anti-IL-6 antibody but within 1 wk of anakinra treatment, the constitutional symptoms markedly improved, and anemia, thrombocytosis, leukocytosis, and elevated markers of systemic inflammation reverted to normality 103. In cytokine biology as applied to the treatment of disease, associations of elevated circulating levels of a particular cytokine with a disease do not allow for a conclusion of causation by that cytokine for the pathological process. Rather, only specific

blockade or neutralization provides the evidence. This is especially 3-mercaptopyruvate sulfurtransferase the case with IL-1β, as circulating levels, even in severe systemic inflammatory diseases, are undetectable and yet the disease manifestations are dramatically reduced upon blockade of IL-1 activity. This commonly observed therapeutic response is due to the high specific activity of IL-1β, which can be in the picomolar range in humans. Therefore, establishing a role for IL-1β in inflammatory diseases has succeeded by using short-term IL-1β-blockade and its role and usefulness will likely increase with clinical testing, facilitated by the safety of short-acting anakinra and the availability of neutralizing anti-IL-1β antibodies. Supported by NIH Grants AI-15614, CA-04 6934 and JDRF 26-2008-893. The author thanks Antonio Abbate, Mihai Netea, Leo Joosten, Anna Simon and Jos van der Meer for many helpful suggestions in the preparation of this MS. Conflict of interest: The author declares no financial or commercial conflict of interest. This article is editorially independent of Novartis.

We postulate that the affinity of this mAb for the canine epitope is low, a view supported by a recent study in which a specific anti-canine CD25 MI-503 in vivo mAb was developed in mice.55 A proportion of the ACT-1-negative cells may therefore be CD25+, which would reconcile this apparent anomaly with the observation that the majority of Foxp3/FOXP3+ T cells in both rodents and humans are CD25+. Stimulation of mononuclear cells derived from peripheral LNs with Con A for up to 120 hr elicited a significant increase in percentage and MFI of FOXP3 expression by both CD4+

and CD8+ T cells (Fig. 2). This phenomenon occurred in the absence of exogenous IL-2 or TGF-β, though the addition of low concentrations of IL-2 augmented CD25 and FOXP3 expression (Fig. 3a). Robust increases in CD25 expression were also observed in a recent study of CD4+ T cells derived from PB stimulated with Con A, yielding parallel increases in FOXP3 expression.64 However, similar experiments performed in an earlier study failed to elicit significant increases in the proportions of FOXP3+ CD4+ T cells without the addition of IL-2 and TGF-β,49 ABT-888 nmr presumably reflecting differences in experimental conditions. Interestingly, in our study removal of the stimulus and continued culture disclosed a FOXP3high

population of lymphocytes that was IFN-γ− and predominantly CD4+ (Fig. 2d). Both the high level of FOXP383,84 and the lack of IFN-γ expression – Foxp3 directly represses the Ifng gene85,86 – suggested that this population was regulatory in nature, supported by our subsequent functional studies in vitro (Fig. 3d). Two alternative, non-mutually exclusive explanations for the increased proportion and absolute numbers of FOXP3+ T cells with polyclonal stimulation were considered – namely, up-regulation of FOXP3 in cells

that were originally either FOXP3intermediate or FOXP3−, or proliferation of pre-existing FOXP3+ T cells. The impressive increase in MFI of FOXP3 suggested that up-regulation of this molecule had occurred in individual cells, but parallel proliferation of pre-existing Treg cells could not be excluded. Reasoning that in both mice and humans Helios expression is restricted to nTreg cells and is not Galeterone induced by stimulation, even in the presence of TGF-β, we explored the expression of Helios in cells that had been stimulated in an identical manner to those for the functional studies. We observed an impressive increase in the number of FOXP3+ Helios+ cells with Con A stimulation, arguing for the proliferation of pre-existing nTreg cells. However, Helios expression was not limited to the FOXP3high population, which we speculated were Treg cells on the basis of their IFN-γ− phenotype in earlier studies (Fig. 2d).