In short, 100 ng of total RNA was transcribed into cDNA using T7-(N)6 primers. Anti-sense RNA is generated by in vitro transcription of the cDNA, which is used as a template to generate 2′-deoxyuridine, 5′-triphosphate (dUTP)-containing sense cDNA fragments. Following the removal of RNA fragments, the sense cDNA fragments were terminal-labelled with biotin. The labelled samples were hybridized to the Human Gene 1·0 gene ST array (Affymetrix). The arrays were washed and stained with phycoerythrin-conjugated streptavidin (SAPE) using

the Affymetrix Fluidics Station® 450, and the arrays were scanned in the Affymetrix GeneArray® 3000 scanner to generate fluorescent images, as described in the Affymetrix GeneChip® protocol. Group sensitization rates were compared using a χ2 test and logistic regression analyses. The challenge outcome, Autophagy Compound Library mw whether positive or negative, was used as

the dependent variable and skin status, sex and age as the independent variables. Results were expressed as odds ratios (OR) with 95% confidence intervals (CI). The strength of reactions in the three groups was analysed with both the visual scores and ultrasound check details data. The Mann–Whitney two-sample rank sum test was used to compare the sum clinical scores in the groups. Using the ultrasound data, linear regression analyses of the elicitation responses were calculated for each individual and the means of the slopes and the intercepts, used as parameters for strength of the elicitation reaction, were compared

for each group using a Mann–Whitney U-test, as recommended for serial measurements. P-value < 0·05 was considered to be statistically significant. All data analysis was performed using spss version 12 (SPSS, Inc., Chicago, IL, USA). The degree of infiltration of positively stained cells was scored semi-quantitatively using a five-point scale: 0 = none, 1 = few positive, 2 = some positive, 3 = many positive and 4 = highly positive. To detect significant differences between the group mean values, the Mann–Whitney U-test was applied. Significance was determined with a P-value < 0·05. All data analysis was performed using spss version 12 (SPSS, Inc.). Analysis Edoxaban the of microarray data followed the overall strategy described previously [12]. Briefly, a single log2 scale expression measure for each probe set was attained from the low-level data files (CEL files), using the robust multi-array analysis procedure with quantile normalization [13] implemented in the Affymetrix library for the r statistical environment. Principle component analysis (PCA) was carried out with the prcomp r function. The 2·5% of the probe sets with the most extreme positive or negative loading values for the first two principal components were extracted and used for analysis of over-represented GO terms using Fisher’s exact test for proportions with Bonferroni’s correction for multiple testing.

In order to further investigate the mechanism of podocyte protection, we here examined

the effect of nicorandil in another model with podocyte injury, the puromycin aminonucleoside induced nephrosis (PAN). Methods: PAN nephrosis was induced by a single intraperitoneal injection of PAN (10 mg/100 g body weight). Rats were divided into three groups: Normal control rats (CONT), PAN model group (PAN), PAN rats treated with nicorandil 30 mg/kg/day (NICO). Blood and urine samples were measured for examining kidney function and proteinuria. 9 days later, the rats were sacrificed and obtained kidney specimens Smoothened Agonist concentration were subjected ro pathological investigation with light microscopy, immunohistochemistry and electron microscopy. Results: Proteinuria

was significantly ameliorated by nicorandil compared with PAN rats at 9 days. PAN rats revealed significantly lowered number of WT-1-positive cells and reduced podocin immunoreactivity while both findings were prevented in NICO rats. In addition, electron microscopy documented that the number of filtration slits in podocyte was reduced in PAN rats whereas such alteration was BGB324 molecular weight significantly restored by nicorandil. Conclusion: Nicorandil reduces proteinuria and ameliorates podocyte injury in PAN nephrosis. Nicorandil may warrant a novel candidate for future treatment of diseases involving podocyte injury. KIM SEJOONG1, LEE JEONGHWAN2, HEO NAM JU3, NA KI YOUNG3, HAN JIN SUK3 1Internal Medicine, Seoul National University Bundang Hospital, Seongnam; 2Internal Medicine, Hangang Sacred Heart Hospital, Seoul; 3Internal Medicine, Seoul PI-1840 National University College of Medicine, Seoul Introduction: In the kidney with unilateral ureteral obstruction (UUO), alteration of cytoskeleton can induce apoptosis. Colchicine, which inhibits microtubule polymerization, may reduce tissue injury.

However, the effect of colchine on renal apoptosis in UUO has not been explored. Methods: UUO was induced in C57BL/6 mice and colchicine (60 μg/kg, intraperitoneally, everyday) or vehicle was administered for 7 days. Results: UUO mice showed increased alpha-tubulin and renal apoptosis. Colchine inhibited the expression of alpha-tubulin and decreased renal apoptosis 7 days after UUO. In colchicines treated UUO mice, the expression of phopho-glycogen synthase kinase-3β and phospho-p38-mitogen-activated protein kinase was decreased, while the expression of Akt and B-cell lymphoma-extra large was increased. Caspase-9 expression was also decreased. Interstitial fibrosis scores on Masson’s trichrome stain were not different between vehicle and colchicines treated UUO mice. Expression of alpha-smooth muscle actin, vimentin, collagen type 4 and fibronectin was not different between the two groups. Conclusion: These data suggest that colchicine may have anti-apoptotic effect but lack of anti-fibrotic effect on obstructive kidney models.

The problem is compounded when the biofilm is associated with tissue, which itself also needs to be digested to release bacteria that may be attached within surface convolutions or have invaded the tissue itself. We have found that the physical disruption of tissue by bead beating, followed by digestion with lysis buffer (Qiagen AL) and proteinase K (Invitrogen), yielded more consistent results than the use of lysozyme alone, which under-represented Gram-positive bacteria relative to Gram-negative bacteria (data not shown). Once nucleic acids are extracted and purified, short nucleic acid primers are used to PCR amplify specific FK506 ic50 DNA sequences. Notably, sequences of the 16S ribosomal

DNA that encode the 16S rRNA gene are used because 16S rRNA gene is universal to prokaryotes and is widely used as a phylogenetic ‘fingerprint’

to identify organisms at the species, genus or phylum level. Other genes of interest such as virulence genes selleck products may be probed to identify antibiotic resistance (i.e. mecA for MRSA) or sets of genes can be probed for multilocus strain typing, although this is usually done on single isolates. After PCR, the resulting amplicon should contain enough material for analysis. The presence and, in some cases the relative abundance, of amplified gene sequences can be measured using a number of techniques including gel electrophoresis and ionizing spray mass spectroscopy. Quantitative real-time PCR can be used to quantify the starting amounts of DNA by monitoring the amplification during PDK4 the amplification step. In the case of looking for mRNA to demonstrate not only the presence of a bacterial species but

also activity, the mRNA is converted to cDNA by reverse transcriptase before PCR amplification. It is helpful to visualize a giant forest of mixed bacterial and host DNA that has been extracted from the sample within which small primers seek out corresponding sequences of bases and, when they locate and hybridize with them, produce very large numbers of identical amplicons. The repeated cycling of this process produces very large numbers of identical target sequences termed amplimers or amplicons. The strategies for deciding which genes to amplify, and for selecting methods for the analysis of the amplicons that are produced, have been driven by practical considerations. If one is involved in a leisurely world cruise to study the microbial ecology of the oceans (Ivars-Martinezet al., 2008), speed is not of the essence, and the amplicons can be frozen and analyzed by pyrosequencing over a period of months or years. If one manages a wastewater treatment plant, and is only interested in the detection and identification of a particular invidious bacterium that blocks phosphate removal (Crocettiet al., 2000), a simple and rapid PCR for that particular organism will suffice.

S2c). FcγRIIIB was expressed by a smaller percentage of CD4+ T cells (Fig. S2). The examination of three independent fields of cells expanded using anti-CD3 and anti-CD28 showed that a total of 49% of cells expressed FcγRIIIA, 27% expressed FcγRIIIB and 22% stained for MRs. Treatment of the cells with TCC, ICs purified from SLE patients (SLE–ICs) or TCC together with ICs did not alter the

protein pattern of immunoprecipitates 17-AAG generated using anti-FcγRIIIA/B (Fig. S7). Western analysis of immunoprecipitates obtained using monoclonal anti-FcγRIIIA/B from naive CD4+ T (CD45RA+) cells showed protein bands migrating at the molecular weights of 26–29 kD that correspond to a previously reported molecular mass for FcγRIIIA and B

(Fig. S6) [29]. In naive CD4+ T cells, an additional band at approximately 34 kD was also observed (Fig. S6). The FcγRIIIA consists of 254 amino acids with a predicted molecular mass of 29 kD (Accession no. P08637-1) and FcRIIIB consists of 233 amino acids with a predicted molecular mass of 26 kD (Accession no. P75015-1). In addition to the light and heavy chains of immnoglobulins, faint protein bands at 72, 98 and 130 kD were also observed. These proteins were also observed in the immunoprecipitates prepared from Jurkat cells. Jurkat cells are used traditionally to study T cell activation (Fig. S6). To further confirm the presence of FcγRIIIA/B in the CD4+ T cells, we analysed the presence of RNA Flucloronide transcripts by RT–PCR. The RT–PCR analysis of the total RNA isolated from both see more peripheral CD4+ T cells and naive CD4+ T cells using a primer set designed from the gene ID NM_001127596·1 (FCGRA) and a second primer set published recently [27] showed the presence of appropriate-sized products for the FcγRIII gene. These FcγRIII transcripts were

also amplified from the total leucocyte RNA. Negative controls without the template RNA did not show the PCR amplification product. Both CD4+ T cells (not shown) and naive CD4+ T cells showed transcripts for the FcγRIIIA/B gene. Jurkat cells also demonstrated these RNA transcripts (Fig. 4). The sequencing of PCR-amplified cDNA confirmed it to be the FcγRIIIA/B gene product. The staining pattern of FcRγ chain in T cells showed them to be present in microclusters, a pattern that is observed for TCR signalling proteins in activated CD4+ T cells (Fig. 3a). The treatment of cells with purified ICs triggered the microclusters to move towards one side of the cell due to capping (Fig. 3a). The presence of TCC during IC treatment further enhanced staining for the FcRγ chain. We observed that the ICs and TCC treatment triggered migration of these receptors into MRs (Figs 5 and S5). We have observed previously that the assembly of non-lytic C5b-9 using purified C5b-6, C7, C8 and C9 labelled with AlexaFluor® 594 trigger MR aggregation beneath C5b-9 deposits (Fig. S4). In quiescent cells, both FcγRIIIB and the FcγRIIIA were not observed in the MRs.

41,42 Many studies have demonstrated that complement activation contributes to kidney RG7204 price IRI.43–45 The mechanisms by which complement is triggered during IR and the effectors that are responsible for renal IRI remains to be fully elucidated, but loss or reduced function of complement regulators are likely to play a role. Accordingly, patients with one or more of their regulators deficient or defective may be at increased risk

of suffering from IRI. In a study of mice deficient in DAF and CD59, either alone or in combination, Yamada et al. have shown that both regulators are important in preventing catastrophic renal IRI.46 Thus, although DAF-deficient, but not CD59-deficient, mice were significantly more susceptible to renal IRI than wild-type mice, DAF/CD59 double deficiency caused a much greater degree of renal pathology

and functional impairment, suggesting that CD59 deficiency in the context of DAF deficiency exacerbated renal injury even though CD59 deficiency alone was inconsequential.46 One of the consequences of ischaemia may be cell membrane disruption, resulting in the transient SCH772984 loss of membrane regulators such as DAF and CD59. Both of these proteins attach to the cell membrane via a GPI anchor and are known to be capable of shedding from and reincorporating into the lipid bilayer of the cell membrane.47 Positional and functional disruption of transmembrane regulators may also occur as has been shown for mouse Crry during renal IR.48 It has been demonstrated that Crry, normally found on the basolateral side of

tubular cells along the basement membrane, was sequestered in the tubular lumen upon ischaemic insult, allowing increased complement deposition and injury on these cells.48 Additionally, changes in the cell membrane structural integrity and exposure of neoepitopes may alter the binding kinetics of the fluid-phase complement regulator fH, which can also impact on complement activation and renal IRI.49,50 Although both classical and lectin pathways have been implicated in IRI of other organs, likely through binding of natural antibodies and MBL to neoepitopes exposed on ischaemic cells, most animal modelling Progesterone studies in mice have suggested that renal IRI is mediated by the AP.43 Nevertheless, there is evidence that CP and MBL activation may be important contributors to IRI in some cases of transplant rejection as renal biopsies from these patients showed numerous deposits of C3d and C4d.51,52 Clinical studies have also shown that while injury can decrease complement regulation in some cells, there are cases where inhibitor expression actually increases in response to injury, which can offer enhanced protection from complement-mediated injury.53–56 A recent study with patients experiencing allograft rejection presented evidence that increased DAF expression correlated with increased allograft survival.

The following experiments were performed after monocyte incubation for 18–24 hr, but total shaving could be observed as early as 1–2 hr after monocyte–B-cell co-culture (data not shown). Interestingly, in-vitro-generated monocyte-derived dendritic cells also induced RTX shaving (Fig. 1g). B find more cells were also viable after 24 hr of co-culture, but when testing for CDC, addition of activated autologous serum

to co-cultures resulted in some induction of B-cell apoptosis, which seemed to vary between donors (data not shown). Hence, as complement-mediated killing does not seem to be the only effector function of RTX, monocyte-mediated shaving could be an important problem both in leukaemic and non-leukaemic applications, as it renders target cells less sensitive for natural killer (NK) cell-mediated killing. We next investigated the mechanisms resulting in monocyte-mediated shaving. We used a modified RTX where the Fc part was deleted and demonstrated that interaction with the Fc part of the antibody was pivotal for monocyte-mediated shaving (Fig. 2). However, using another approach to test for Fc dependency, addition of pooled human IgG or anti-CD64 antibody,

to block Fc receptors, only resulted in a minor inhibition of RTX shaving, which could reflect the relatively long co-culture period learn more used. We then tested whether the mechanisms for cleavage of the RTX complex could be the result of simple endocytosis, but as addition of hyperosmolar

sucrose did not inhibit RTX shaving this does not seem likely (Fig. 3). To investigate the involvement of proteases in the mafosfamide shaving reaction, 10 mm EDTA was added to the B-cell–monocyte co-culture and this led to a partial inhibition of the shaving reaction (Fig. 4). Protease inhibitors can be divided into aspartic protease inhibitors, cysteine protease inhibitors, metalloproteinase inhibitors and serine protease inhibitors. Here, the serine protease inhibitor PMSF caused a partial decline in shaving activity (Fig. 5), whereas aprotinin did not. Also, the metalloproteinase inhibitors bestatin hydrochloride 1,10-phenanthroline monohydrate and phosphoramidon disodium salt did not have any effect (data not shown). The endoprotease inhibitor α2-macroglobulin, which also acts as a cysteine protease inhibitor, serine protease inhibitor, metalloproteinase inhibitor and aspartic protease inhibitor, also did not have any effect. PMSF does also have cysteine protease inhibitor activity and phosphoramidon disodium salt has metalloproteinase inhibitor activity. Next, we tested a panel of alternative type I and type II anti-CD20 antibodies to identify possible anti-CD20 antibodies with reduced effect on monocyte-mediated shaving. First, a series of mouse antibodies was tested.

Finally, inhaled house dust mite extracts have been shown to induce the recruitment to MLNs of FcγRI+ inflammatory type DCs that appeared to be necessary PD-0332991 purchase and sufficient, as APCs, for the development of Th2-type inflammation. This observation clarifies a controversy regarding the role of DCs versus basophils in Th2 priming [25-27] and suggests that basophils may amplify, rather than induce, Th2 immunity to house dust mite allergen [28]. The observations discussed in the previous section suggest that, in some conditions (when alum is used

as an adjuvant or upon intranasal administration of house dust mite antigen), inflammatory DCs may induce Th2-type immune responses. However, inflammatory DCs also appear to be critical for host resistance in several

infectious models where Th1-type responses are protective. In particular, oral infection with the enteric pathogen Toxoplasma gondii has been shown to provoke the recruitment of CCR2+ inflammatory monocytes, a process that was associated with the control of infection. These inflammatory monocytes homed to the lamina propria where they expressed IL-12, TNF-α, and iNOS, but not CD11c. These observations indirectly suggest that inflammatory monocytes may gain the capacity to trigger Th1 immunity. The analysis of plt mice clearly demonstrated that inflammatory DCs can potently stimulate Th1 responses. These mice display the “paucity of GABA Receptor lymph node T cell” mutation, that is, deletion of the Ccl19 and Ccl21 genes [29]. Surprisingly, although these mice have strongly reduced migration of CYC202 cell line T cells and DCs, these mice have increased numbers of antigen-specific T cells and increased delayed-type hypersensitivity responses [30]. Nakano et al. reported that the DC-subset composition was altered in plt LNs: the frequency

of CD11bhiGr-1+ inflammatory DCs was higher in resting LNs and increased considerably after immunization or viral infection, as compared with the frequencies in WT mice [30]. These CD11bhiGr-1+ inflammatory DCs produced IL-12p70 upon stimulation in vitro and stimulated T-cell production of IFN-γ; their paucity in CCR2−/− mice correlated with much lower IFN-γ production, suggesting that blood-derived inflammatory DCs were critical for the development of Th1 responses [30]. Using an anti-mouse DC-SIGN mAb to distinguish monocyte-derived DCs from conventional DCs in tissues, Cheong et al. [31] reported that LPS rapidly recruited, to the T-cell area of LNs, DC-SIGN+ cells that were distinct from other DCs and were derived from monocytes. These cells efficiently presented proteins and bacteria captured in vivo to T cells, and had the capacity to induce strong production of IFN-γ and IL-2 by CD4+ T cells in vitro. Iijima et al.

Urine phosphate concentration (uPi) and creatinine concentration measurements were performed on spot and 24-hour

urine collections. Pearson’s correlation coefficients, multiple regression analysis and Bland-Altman plots were used to assess agreement between spot uPiCr and UPE. Results: 65 CKD patients (49 male) were studied, median age 67 years (IQR 53–74) and mean (± SD) serum creatinine 182 (± 84) μmol/L. Mean (± SD) spot uPi, spot uPiCr and total UPE were 12.6 (± 6.2) mmol/L, 1.58 (± 0.55) mmol/mmol and 24.5 (± 11.7) mmol/d respectively. There was no significant correlation between spot uPiCr and UPE (r = 0.116, learn more P = 0.336). Spot uPi correlated with 24-hour UPE significantly (r = 0.306, P = 0.019). Bland-Altman analysis of 24-hour versus spot uPi showed acceptable agreement with bias +0.2 mmol/L (95%CI −1.2284–1.6508). Multiple regression analysis was undertaken to predict UPE from gender, sPi, spot uPi and eGFR. Apart from eGFR, these variables significantly predicted UPE, F(3,51) = 5.321, P = 0.003, R2 = 0.238. Gender, sPi and spot uPi added significantly to the prediction, P < 0.05. Conclusion: This

see more study suggests that normalisation of uPi to uCr on spot urine samples may not be appropriate when evaluating urinary phosphate excretion in adults with CKD. 179 SYSTEMIC MICROVASCULAR/HYPERTENSIVE DISEASE IS INCREASED IN PATIENTS WITH OBSTRUCTIVE SLEEP APNEA (OSA): A CROSS-SECTIONAL OBSERVATIONAL STUDY N TAN1, C CHOY1, S CHEW1, D COLVILLE1, A HUTCHINSON1, P CANTY1, E LAMOUREUX2, TY WONG2, J SAVIGE1 1The University

of Melbourne, Northern Health and Melbourne Health, Australia; 2Singapore Eye Research Institute, University of Singapore, Singapore Aim: This study used retinal examination to compare the prevalence of microvascular disease (severity of changes and calibre) in patients with obstructive sleep apnoea (OSA), chronic obstructive pulmonary disease (COPD) and other hospital patients. Background: Microvascular MEK inhibitor abnormalities in the retina reflect systemic small vessel disease. Methods: Patients were recruited from a single hospital clinic and ward. OSA was diagnosed on an overnight sleep study (apnoea: hypopnoea index >5), and COPD with a forced expiratory ratio (FER) <70%. Participants underwent retinal photography using a non-mydriatic camera (KOWA, Japan). Images were graded for microvascular/hypertensive retinopathy (Wong and Mitchell classification), and sent to the Centre for Eye Research Australia for computer-assisted measurement of the retinal arteriole and venular calibre using Knudtson’s revised version of the Parr-Hubbard formula. Statistical analysis was performed using Stata version 11.2 software (Stata Corp). Results: Patients with OSA alone (n = 79) were younger, had a higher BMI, higher mean arterial pressure, and more dyslipidemia than those with COPD (n = 132) or other hospital patients (n = 143). They were less likely to be smokers.

Successful fluorochrome incorporation was confirmed by native polyacrylamide gel electrophoresis generating a single band at about 600 kDa corresponding to toxin A protein dimer [29, 30] under non-denaturing

conditions, which exhibited fluorescence during illumination with UV light. Toxin A488 was also shown to induce morphological changes in Vero cells and Caco-2 cells identical to that seen for unlabelled toxin A (treated as per labelled toxin A without the addition of Syk inhibitor the label), confirming that labelling had not compromised receptor-binding ability. To confirm that fluorescence in flow cytometry was because of toxin A488 only, without any contribution from free label that may have either not been removed following the labelling procedure or become detached from the toxin during storage or binding studies, toxin A488 was preincubated with PCG-4-conjugated beads prior to incubation with Caco-2 cells. A complete loss of Caco-2 cell-associated fluorescence was seen after incubation with the toxin A-depleted preparation (Fig. 1), confirming that all fluorescence was toxin A specific. In initial studies, following incubation of PBMNCs with toxin A488 at 37 °C for up to 24 h, monocytes were distinguished from lymphocytes by their forward- and side-scatter characteristics. In contrast to toxin A488-exposed lymphocytes, toxin

A488-exposed monocytes showed significant fluorescence at all time points up to 5 h, with Cell press a peak at 1 h (Fig. 2A). Drop

in monocyte-associated fluorescence from 1 h onwards after exposure to PD 332991 toxin A488 was associated with loss of events in the monocyte gate (Fig. 2B). The fluorescence level of toxin A488-exposed lymphocytes remained low, with no significant change (compared with control lymphocytes) over the 24 h period of study (Fig. 2A). Thus, at 24 h, there was no significant difference in fluorescence between lymphocytes incubated (at 37 °C) in control medium, compared with those cultured with toxin A488. In contrast to monocytes, the number of events in the lymphocyte gate (in toxin A488-exposed PBMNCs) did not change significantly from cells exposed to control medium over the 24 h period of study (Fig. 2B). When studied after 48-h incubation at 37 °C, fluorescence of toxin A488-exposed lymphocytes was marginally, but significantly greater than lymphocytes cultured with control medium (5.35 versus 4.97; P < 0.01). By contrast, following incubation at 4 °C, the difference in fluorescence between toxin A488-exposed and control lymphocytes fell short of statistical significance (5.0 versus 4.85; P = 0.07). The ability of trypan blue to quench fluorescence of monocytes exposed to toxin A488 at 37 and 4 °C was subsequently investigated. Initial studies, using PBMNCs labelled with anti-CD45 antibody, followed by labelling with Alexa Fluor 488-conjugated anti-mouse antibody, showed that trypan blue quenched 87.27 (±4.7)% of cell surface–associated fluorescence.

32 TLR agonists are therefore potent stimulants of IFN-I release by antigen-presenting cells.33 To mimic the immune response observed https://www.selleckchem.com/products/Paclitaxel(Taxol).html during viral infections, PBMC were treated overnight with poly(I:C) in order to induce endogenous production of IFN-I. In a preliminary study, we confirmed that poly(I:C) treatment of PBMC from several donors resulted in IFN-α secretion ranging between 30 and 200 pg/ml (data not shown). The addition of poly(I:C) 24 hr prior to anti-CD3 activation led to an average decrease of 40% (P = 0·007) in the production of aTregs (Fig. 4; for cell numbers see Fig. S2). However, in

contrast to IFN-α, poly(I:C) had an inconsistent effect on aTeffs (Figs 4 and S2), which may result from the effects of other cytokines (e.g. IFN-β) induced by TLR3 ligation. To further address the role of endogenously produced IFN-I in the suppression of

aTregs, these assays were also performed in the presence of an antibody that blocks binding of IFN-I to cellular receptors, as well as neutralizing antibodies against TNF-α and IL-6 (Figs 4 and S2). Blocking of IFNα/β receptor produced a significant (P = 0·0001) normalization of Treg activation, with an average recovery selleck products of 92% in Treg activation. In contrast, the presence of antibodies against TNF-α and IL-6 had a minimal effect on the suppression of Treg activation induced by poly(I:C). Taken together, these data suggest that innate signals that mimic the immune response to viral infections are able to suppress Treg activation, and that IFN-I probably plays a major role during this process. As IL-2 plays a critical role in Treg development and proliferation,34,35 and because it has previously been shown that IFN-α is a potent

inhibitor of IL-2 production,36 we addressed whether the reduced expansion of Tregs in the presence of IFN-α might result from a decrease in IL-2 production in the polyclonally stimulated PBMC cultures. To that end, IL-2 levels in the culture supernatants were measured by ELISA at 24 and 48 hr post anti-CD3 activation of PBMC in the absence or presence of exogenous IFN-α (1000 U/ml) Rutecarpine (Fig. 5). IFN-α reduced the production of IL-2 in polyclonally activated PBMC by an average of 45% in the first 24 hr (P = 0·01) and by an average of 55% after 48 hr (P = 0·05) (Fig. 5a). This reduction in IL-2 production correlated with a 66% (P = 0·04) reduction in the generation of aTregs (Fig. 5b). In order to address whether IL-2 inhibition by IFN-α could be reversed in activated PBMC, we tested whether suppression of Treg activation was reversed by exogenous IL-2 (100 Units/ml). Indeed, Treg activation in the presence of IFN-α was improved almost threefold (P = 0·01) by the addition of IL-2 (Fig. 5b), strongly suggesting that down-regulation of endogenous IL-2 production may play a critical role in IFN-α-mediated suppression of Treg activation.