Islets were isolated from C57BL/6 mouse pancreas using the collag

Islets were isolated from C57BL/6 mouse pancreas using the collagenase digestion method. Briefly, Ivacaftor chemical structure the organs were minced into smaller pieces and subsequently incubated with collagenase

type V solution (1 mg/ml; Sigma, St Louis, MO, USA) in Hanks’s balanced salt solution (HBSS) at 37°C for 10–15 min with vigorous shaking. Following cold HBSS addition to stop the digestion, the islets were handpicked and seeded into 96-well flat-bottomed plates (30/well) in culture medium (RPMI-1640 + 0·5% FCS). After overnight rest, pancreatic islets were treated with apoTf (25 µg/ml) and added to the proinflammatory cytokine cocktail for the next 24 h to be then analysed for cell viability. The viability of RINm5F cells, as well as of pancreatic

islets, was assessed using the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to formazan. Cells were washed with phosphate-buffered saline (PBS) to remove non-adherent dead cells, and MTT (0·5 mg/ml) was added to the remaining adherent cells. Pancreatic islets were then collected, centrifuged learn more and the pellets dissolved in MTT solution for 60 min at 37°C. After incubation, dimethyl sulphoxide (DMSO) was added to the adherent insulinoma cells, or pellets of pancreatic islets to dissolve the formazan crystals. Absorbance was finally measured at 570 nm wavelength, with a correction at 690 nm, using an automated microplate reader (LKB 5060-006; LKB, Vienna, Austria). The results of the MTT assay are presented as the proportion of control values obtained in untreated cell cultures. Data are expressed as the mean ± standard deviation (s.d.) of values obtained in at least five, three and seven individual experiments for mouse pancreatic islets or RINm5F cells, respectively. All animal experiments were

conducted in accordance with national and local regulations regarding animal welfare, and with the approval of the institutional animal care and use committee (IACUC). Female non-obese diabetic (NOD) C59 mice (8–9 weeks old) and male diabetes-prone (DP) BB rats (5–6 weeks old) were purchased from Charles River, Milano (Italy). Rodents were housed under standard conditions with ad libitum food and water at the University of Catania (Italy). All animals were housed for 1 week prior to study initiation and then randomized into five per cage corresponding to one specific experimental condition. NOD mice and DP-BB rats were screened for glycosuria twice per week starting at the ages of 8–9 and 5–6 weeks, respectively. Two animal models of type 1 diabetes were used for these experiments. First, male DP-BB rats (36–45 days of age) were divided into four groups (n = 14 per group) to be treated with human apoTf at different concentrations (1·25, 2·5 or 5 mg/kg) or PBS for 7 consecutive weeks.

Activated B cells also infiltrate into the rheumatoid synovium [2

Activated B cells also infiltrate into the rheumatoid synovium [26]. In this study, we found that the frequency of CD19+IgD+CD27− naive B cells in RA patients was significantly higher than that in the HC, while

the percentages of preswitch CD19+IgD+CD27+ B memory cells in RA patients were significantly lower than that in the HC. Our findings were consistent with a previous report that showed a higher frequency of naive B cells, but lower percentages of memory B cells in patients with new-onset RA [27]. selleck chemicals llc This suggests that antigen stimulation may promote the redistribution of naive B cells from lymph tissues to circulation. Souto-Carneiro et al. [28] found that the percentages of circulating preswitch CD19+IgD+CD27+ memory B cells decreased in RA patients, while the frequency of preswitch CD19+IgD+CD27+ memory B and post-switch CD19+IgD−CD27+ memory B cells increased in the synovial membrane. It is possible that circulating CD19+IgD+CD27+ B cells could migrate and accumulate in the synovium of RA patients. However, a previous study has suggested that there may be an accumulation of post-switch CD19+IgD−CD27+ memory B cells, whereas the CD19+IgD+CD27+ memory B cells are reported

in RA patients with long-standing disease [29]. The disparities between our data and the results of previous selleck chemical studies may be due to a number of factors, including varying genetic backgrounds, disease duration, Buspirone HCl cohort size and therapy. Activated B cells increased the expression levels of certain activation markers, such as CD86 and CD95 [30, 31]. CD86 is a critical co-stimulatory molecule for B cell activation and CD95 is

associated with apoptosis. To assess activated B cells further in RA patients, we analysed the frequency of CD86+ or CD95+ B cells and found that the percentages of CD86+CD19+ and CD95+CD19+ B cells were significantly higher in the RA patients than that in the HC, consistent with a previous report [32, 33]. These data indicated more activated B cells in RA patients. Given that CD95 is a death receptor, the higher frequency of CD95+ B cells in RA patients suggests that those activated B cells may be susceptible to spontaneous apoptosis, diminishing the total number of activated B cells in RA patients. Moreover, it is possible that the relatively higher frequency of naive B cells may stem from high differentiation of bone marrow stem cells due to the continuous loss of memory B cells, and this feedback regulation will help in maintaining B cell homeostasis in RA patients. O’Neill et al. [34] found that the expression of CD80/CD86 co-stimulatory molecules on B cells was critical for inducing autoreactive T cell activation and autoimmunity during the development of arthritis. In our study the percentages of CD86+CD19+ B cells in the RA patients were correlated positively with the DAS28 scores, suggesting that activated B cells might be major players in the pathogenesis of RA.

4%) directed antifungal therapy Mostly, systemic antifungal ther

4%) directed antifungal therapy. Mostly, systemic antifungal therapy was administered empirically or pre-emptively. Twenty-nine per cent of cases receiving systemic antifungal treatment met the international consensus criteria of mostly possible IFI, whereas 71% did not. Proven invasive fungal infections

were rare. “
“Candidaemia is associated with high mortality. BEZ235 datasheet Despite the fact that Candida species account for close to 10% of all nosocomial bloodstream infections, relatively few studies have investigated the management of candidaemia in hospitals. Our objective was to find out how candidaemia is managed in hospitals. Data relating to all episodes of candidaemia for the year 2008 were retrospectively collected Alvelestat cost in five centres in Scotland and Wales. A total of 96 candidaemic episodes were recorded in the year 2008, yielding 103 isolates of Candida. Fifty candidaemic episodes were caused by Candida albicans. Fluconazole was the most common agent prescribed for the treatment of candidaemia. There was great variation in the prescribed dose of fluconazole.

Forty per cent of patients who survived received <2 weeks of systemic antifungal therapy. Central venous catheters (CVC) were removed in 57% of patients. CVC removal was not associated with better survival. The overall mortality was 40.4%. Management of candidaemia varies between the UK centres and is often inadequate. There is need to have consensus on the dosages of antifungal agents and the duration of therapy. The current guidance on removal of CVC in all cases of candidaemia should be reviewed. "
“Although photodynamic therapy (PDT) has shown great promise for the inactivation of Candida species, its effectiveness against azole-resistant

pathogens remains poorly documented. This in vitro study describes the association of Photogem® (Photogem, Moscow, Russia) with LED (light emitting diode) light for the photoinactivation of fluconazole-resistant (FR) and American Type Culture Collection (ATCC) strains of Candida albicans and Candida glabrata. Suspensions of each Candida strain were treated with five Photogem® concentrations and exposed to four LED light fluences (14, 24, 34 or 50 min of illumination). Rho After incubation (48 h at 37 °C), colonies were counted (CFU ml−1). Single-species biofilms were generated on cellulose membrane filters, treated with 25.0 mg l−1 of Photogem® and illuminated at 37.5 J cm−2. The biofilms were then disrupted and the viable yeast cells present were determined. Planktonic suspensions of FR strains were effectively killed after PDT. It was observed that the fungicidal effect of PDT was strain-dependent. Significant decreases in biofilm viability were observed for three strains of C. albicans and for two strains of C. glabrata.

The classification is updated regularly, according to the classif

The classification is updated regularly, according to the classification

of the International Union of Immunological Societies (IUIS) [1] and progress in research. The technical structure of the ESID online database has been described in detail previously [17]. The database is used as a data collection platform by several national registries, including France, the Netherlands, Germany, Switzerland, Austria and the Czech Republic. In addition, data are imported on a regular basis from other national and local databases that operate separately. These include the national registries of Spain (REDIP; and Italy (ipinet;, and local hospital databases at University College London, NSC 683864 cell line Newcastle General Hospital and University Medical Center Freiburg. Most of the participating centres are located in Europe, but there are also centres in Egypt. selleck kinase inhibitor The complete list of documenting centres is available at Data are generally collected via electronic case report forms. The database has an inbuilt automatic quality assurance system, including field type,

range and plausibility checks. In addition, data sets are checked regularly for plausibility, completeness and double entries. As of 13 July 2011, a total of 13 708 patients had been registered in the ESID database. These had been entered by 102 documenting centres and national registries from 30 countries between 2004 and 2011. Some centres also diagnose or treat patients from abroad, so patients were from a total of 41 countries (including North Africa and the Middle East). The number of documented patients in relation to the total population varied considerably between countries.

In addition, the documentation in some countries is biased towards certain diseases because of centres specialized in a particular disease. This is, for example, the case in Hungary: of 367 reported cases, 130 (35·4%) were patients with hereditary angioedema, while the proportion of this Rho disease in the total study population is a mere 3·5%. In our analyses, we focused on eight countries (core countries) with a high documentation rate, a large number of reporting centres and a disease distribution that does not diverge strongly from the total distribution. These were France (3240), Spain (1662), Turkey (1486), United Kingdom (1148), Germany (1126), Italy (1083), Poland (508) and the Netherlands (433) (number of reported living patients given in brackets). Furthermore, we restricted some of our analyses to the most frequent diseases (core diseases).

Membrane vesicles, bound to SF proteins in a calcium-dependent ma

Membrane vesicles, bound to SF proteins in a calcium-dependent manner, were washed twice using this buffer in order to eliminate unspecifically bound proteins. The

specifically bound proteins were released from membrane by including 1 mM EGTA minus calcium-containing buffer by centrifugation at 28 000 g for 30 min at 4°C. The supernatant containing NAP was dialysed and purified further by size exclusion chromatography using Sephadex G-100, after which its identity was determined by peptide mass fingerprinting and N-terminal protein sequencing. The purified fraction was assayed for proangiogenic activity using human umbilical vein endothelial click here cells INCB024360 (HUVECs) for tube

formation [21]. Purified NAP was used to produce monoclonal antibody. Briefly, BALB/c mice were immunized four times over a 2-month period with 50 μg of purified NAP with Freund’s adjuvant. Serum samples were collected 2 weeks after the second, third and fourth immunizations and screened for anti-NAP antibody using indirect ELISA. Spleen from mice that displayed high antibody titres were used subsequently to generate hybridomas using standard spleen cell/myeloma fusion. Briefly, NAP-primed B cell 1 × 108 (splenocytes) from mouse producing high-titre neutralizing antibodies were fused with logarithmically growing Sp2/0 myeloma cells (1 × 107), using polyethyleneglycol-1500. Hybridoma selection was carried out in hypoxanthine–aminopterin–thymidine (HAT) medium. The resulting monoclonal hybridomas were grown to confluency and the cell supernatant from a single clone was collected as a source of anti-NAP mAb, verified using

ELISA in which NAP was used for capture of the anti-NAP mAb, and purified by protein-A agarose affinity column chromatography. Further immunodetection Verteporfin mouse of anti-NAP mAb was carried out by Western blot analysis. Arthritis was induced in Wistar rats by subcutaneous (s.c.) injection of NAP or ovalbumin (OVA; Sigma, St Louis, MO, USA), as described previously [22]. There were five groups containing six animals, each in duplicate, as follows: group 1, controls; group 2, positive control [OVA-induced arthritis (AIA; untreated)]; group 3, NIA untreated; groups 4 and 5 served as test (AIA DMRD-treated and NIA mAb-treated), respectively. All rats except controls were sensitized twice during a 6-week period with 2 mg/ml of OVA or 50 μg/ml NAP emulsified in complete Freund’s adjuvant (CFA) (Sigma) and administered s.c. At the end of 6 weeks, animals received an intra-articular injection of 2 mg/ml of OVA or 50 μg/ml NAP in CFA in order to induce arthritis. The control rats were injected only with Freund’s adjuvant. Arthritis was achieved in 6–7 days post-IA injections and was considered as day ‘0’.

Although, as described by the authors and in our own analyses, th

Although, as described by the authors and in our own analyses, there are rare populations of CD16+CD8α− NK cells in the peripheral blood of chimpanzees, the data we present here indicate that these populations are often likely to be contaminated by phenotypically FDA approved Drug Library chemical structure and functionally defined CD16+ mDCs. Fresh chimpanzee blood samples were obtained from captive chimpanzees housed at the Yerkes National Primate Research Center, Emory University (supported by NIH grant RR000165). These studies were approved by the

Institutional Animal care and Use Committee of Emory University. The YNPRC is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Cryopreserved samples were analyzed from chimpanzees

originally housed at the Laboratory for Experimental Medicine and Surgery in Primates, New York University, the Coulston Foundation, Alamogordo, New Mexico in biosafety level 2 facilities in accordance with institutional guidelines and Animal Welfare Act guidelines. The protocol was approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Chimpanzee PBMCs were isolated from EDTA-treated venous blood by density gradient centrifugation over LSM (MP Biomedicals, Solon, OH, USA) and contaminating red blood cells were lysed using a buy MG-132 hypotonic ammonium chloride solution. After isolation all cells were washed and resuspended in PBS supplemented with 2% FCS (Sigma-Aldrich, St. Louis, MO, USA) for subsequent assays or frozen in a 90% FCS/10% DMSO solution. Cell surface staining was carried out using standard protocols Interleukin-2 receptor for our laboratory as described previously 2 using antibodies listed in Table 1. Intracellular staining for perforin was done using Caltag Fix & Perm (Invitrogen) according to the manufacturer’s recommended protocol. All acquisitions were made on an LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). To further confirm the identity

of NK cells and mDCs, we examined their functional responses to NK- and DC-specific ligands ex vivo. PBMCs were resuspended in RPMI 1640 (Sigma-Aldrich) containing 10% FBS and stimulated at an E/T ratio of 2.5:1 with 721.221 cells; PMA (50 ng/mL) and ionomycin (1 μg/mL); poly I:C (100 μg/mL); or medium alone. Anti-CD107a was added directly to each of the tubes at a concentration of 20 μL/mL and Golgiplug (brefeldin A) and Golgistop (monensin) were added at final concentrations of 6 μg/mL, then all samples were cultured for 12 h at 37°C in 5% CO2. After culture, samples were surface-stained using markers to delineate NK cells (CD3, CD8, CD16) and mDCs (HLA-DR, CD11c) as shown in Fig. 1. Cells were then permeabilized using Caltag Fix & Perm and intracellular cytokine staining was performed for IFN-γ, IL-12, and TNF-α. All statistical and graphical analyses were done using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA).

In a more recent study, ADCC responses can

induce epitope

In a more recent study, ADCC responses can

induce epitope-specific escape mutations as early as 50 days after T0.[26] Taken together, these studies suggest that the first functional antibody responses to Env appear almost MK-1775 solubility dmso concomitantly with binding antibodies, which is approximately 50 days before the emergence of the first detectable neutralizing antibodies against autologous viruses. It goes without saying that antibodies must be present at the time of acquisition to block it and this can only be accomplished by active or passive immunization. In recent years, a good picture of the early events during acquisition after vaginal exposure has emerged (reviewed in refs [21, 22, 36, 37]). Figure 3 summarizes the virological events that occur during the eclipse phase where the ‘window of opportunity’ is key for blocking acquisition. Passive immunization studies in NHPs

using neutralizing antibodies suggest that the window of opportunity is 24 hr at most.[38, 39] Transmission across the mucosal epithelium is thought to occur within hours of exposure and results in infectious virus reaching susceptible CD4+ target cells. Transmission CH5424802 across the mucosal barrier can be passive through epithelial breaks but an active transport mechanism is also known.[40] The nature of the first infected type of CD4+ cell has been debated over the years but recent acute transmission studies strongly suggest that it is a CD4+ CCR5+ memory T cell.[41, 42] Strikingly, most HIV infections are due to a single founder virus,[41, 42] which is also true for model AIDS viruses in NHPs.[41] It takes approximately 24 hr for an infected CD4+ cell to produce infectious virus,[43]

so it is likely that the earliest time that HIV can start to spread to other CD4+ CCR5+ T cells is within the first 24–28 hr, a small number of local infected founder cells 2–3 days after exposure[44, 45] (Fig. 3). Local expansion of the infected founder cells occurs around days 4 to 5 post-exposure,[44, 45] likely aided by an innate response of the mucosal epithelium that attracts additional Evodiamine target cells to the site (ref. [46] and discussed in ref. [36]). Virus or virus-infected cells from the local expansion spread via afferent lymphatics to the draining lymph node, which is rich in additional CD4+ CCR5+ target cells. From there, virus and infected cells spread systemically via the thoracic duct leading to distal and propagating infections in the gut and spleen by haematogenous flow and finally back to lymph nodes. Once the infection spreads from the local focus, it is very likely that the window of opportunity is closed because of the establishment of viral reservoirs and protective niches in distal tissues. The systemic spread of infection ultimately leads to plasma viral loads that cross the 100 copy limit of sensitivity (i.e.

Although numerous studies have investigated the outcome of exogen

Although numerous studies have investigated the outcome of exogenous or endogenous IL-10 on a variety of infectious and inflammatory animal models, surprisingly few studies have directly addressed if and how IL-10 influences neutrophil responsiveness in vivo or ex vivo. Most in vivo studies, in fact, have overlooked the effects Gamma-secretase inhibitor of IL-10 in different models of inflammation-driven pathologies, including adjuvant- or crystal-induced arthritis 63, 64, zymosan-induced peritoneal inflammation

65, LPS-induced or IgG immune complex-induced acute lung injury 66–68, bacterial or fungal infections 69–71, myocardial- 72, hepatic- 73 or visceral- 74, 75 dependent ischemia-reperfusion injuries, BSA-induced delayed type of hypersensitivity 76, OVA-induced model of asthma 77 and hemorrhagic shock 78. Independent of the type or cause of injury, in all of these studies exogenous IL-10 (or IL-10 gene transfer) effectively reduced the severity of local or PLX3397 datasheet systemic inflammation, mainly by blocking cell trafficking, in particular the early influx of neutrophils to the injury site. The reduced accumulation of neutrophils in inflamed organs was ascribed to an IL-10-mediated inhibition of macrophage- or tissue-derived neutrophil chemoattractants 63–68 or, in a single instance, to an IL-10-mediated

increase in neutrophil apoptosis via unidentified mechanisms 79. Conversely, the exacerbated inflammatory reactions occurring in IL-10−/− mice following acute Akt inhibitor lung inflammation triggered by LPS 80, zymosan-induced

peritonitis 81 or liver injury 82, correlated with increased production of neutrophil chemoattractants and with augmented neutrophil infiltration at inflammatory sites. Additional evidence that IL-10 keeps inflammation under control in vivo by selectively inhibiting the recruitment of neutrophils derives from neutrophil depletion experiments performed in IL-10−/− mice; the combination of a lack of IL-10 and neutrophils decreased the severity of gastritis in Helicobacter felis-infected mice 83. Similarly, mice carrying neutrophil- and macrophage-specific conditional IL-10R1 gene targeting displayed increased sensitivity to LPS in an IL-10-dependent LPS model of endotoxemia 84; a result resembling that described in IL-10−/− mice 80–82 and, additionally, providing supporting for the crucial role of neutrophils (and macrophages) as direct IL-10 cellular targets in vivo. Interestingly, in a recent article, neutrophils were shown to play an important regulatory role during various murine microbial infections in vivo by secreting IL-10 85. In the same study, the authors mention (data not shown) that monocytes, but not neutrophils, from IL-10−/− mice showed a tenfold increase in the production of pro-inflammatory cytokines in response to BCG, indicating that (at least in mice) an autocrine IL-10 regulatory loop controls the monocyte response but does not inhibit pro-inflammatory cytokine production by neutrophils 85.

002) Furthermore, 36 4% of the C-allele carriers and none of the

002). Furthermore, 36.4% of the C-allele carriers and none of the patients with the TT genotype belonged to group B (P = 0.005). C-allele Galunisertib carriers also had a worse kidney survival in the Kaplan–Meier analysis (P = 0.027). Conclusion:  Our results indicate that aldosterone synthase gene C-344T polymorphism not only acts as a risk factor for the development of FSGS, but also may influence its pathologic appearance

and could serve as a marker of disease progression. “
“Autosomal dominant polycystic kidney disease (ADPKD) is a monogenetic disorder that leads to kidney failure. Our aim was to undertake a meta-analysis of randomized trials of interventions that have been hypothesized to reduce the progression of total kidney volume (TKV) and renal function in ADPKD. Relevant trials were identified, and outcomes were: change in TKV, total cyst volume (TCV), renal function and adverse events. Meta-analysis used random effects, with results expressed as mean difference and risk ratio both with 95% confidence intervals (CI). Eleven trials (2262 patients) were included. Compared with placebo, Target of Rapamycin complex 1 (TORC1) inhibitors (5 trials, n = 619), showed no significant change in TKV (P = 0.21), TCV (P = 0.06) or eGFR (P = 0.22). Somatostatin analogues (3 trials, n = 157) reduced TKV by 9% (95% CI −10.33 to −7.58%) but did not alter eGFR. The vasopressin receptor

antagonist (n = 1455) attenuated TKV increase to 3%/year (95% CI −3.48 to −2.52) and slowed kidney function Protease Inhibitor Library cell assay decline over a 3-year period. A single trial (n = 41) of eicosapentaenoic acid did not alter the progression of either TKV (P = 0.9) or renal dysfunction (P = 0.78). Adverse events were significant for interventions in all trials compared with placebo. These data suggest

that somatostatin analogues and vasopressin receptor antagonists attenuate TKV increase. The neutral effects of TORC1 inhibitors on TKV could be true, or due to heterogeneity in study population, drug efficacy and follow-up duration. In the future, further well-designed and powered trials of longer duration using new biomarkers or therapeutic agents with better tolerance are required. “
“We herein describe the unique case of a 59-year-old man who underwent living kidney transplantation for IgA nephropathy (IgAN) and developed progressive kidney failure associated with the appearance of proliferative glomerulonephritis. Ibrutinib concentration An early protocol biopsy revealed recurrent IgAN with mesangial IgA2 deposits restricted to a single immunoglobulin λ light-chain isotype. Despite treatment with tonsillectomy and rituximab, the patient eventually lost his allograft 31 months after transplantation. Serum electrophoresis showed a monoclonal IgA pattern. This case might share common pathological characteristics with the newly described entity referred to as proliferative glomerulonephritis with monoclonal IgG deposits. A 59-year-old Japanese man developed end-stage renal disease secondary to IgA nephropathy (IgAN).

2), purchased from BD Biosciences (Stockholm, Sweden), APC-Alexa

2), purchased from BD Biosciences (Stockholm, Sweden), APC-Alexa Fluor 700-conjugated anti-CD107a (H4A3), APC-conjugated anti-IL-7 receptor α-chain (IL-7Rα; R34.34), PE-Texas Red-conjugated anti-CD45RA (2H4), fluorescein isothiocyanate (FITC) -conjugated anti-CD8β chain (2ST8.5H7), purchased from Beckman Coulter (Marseille, France), and Pacific Blue-conjugated anti-CD4 (S3.5) purchased from Caltag Laboratories (Burlingame, CA). The lymphocytes were then washed in phosphate-buffered saline (PBS)

with 0·1% fetal bovine serum, and incubated at 4° for 15 min with the anti-CD27 (1A4CD27) antibody (Beckman Coulter) labelled with Pacific Orange using the Zenon Opaganib molecular weight Pacific Orange Mouse IgG1 Labeling Kit obtained from

Invitrogen (Stockholm, Sweden). Human samples were processed the same day, and NHP samples were processed on a different occasion, but also the same day. The median fluorescence intensity (MFI) of IL-7Rα expression therefore allows a comparison of the intensity of IL-7Rα expression on T cells within each species but not between humans and NHPs. Data acquisition was performed using a FACSAria Flow cytometer (BD Biosciences) and results were analysed with FlowJo software (Tree Star Inc., Ashland, OR). Cytokine production was analysed in frozen PBMCs, which were thawed, rested overnight and stimulated for 6 hr in the presence of brefeldin A (10 mg/ml) purchased from Sigma-Aldrich (Sweden AB, Stockholm, Sweden) either Dichloromethane dehalogenase with medium: RPMI-1640 containing l-glutamine

(2 mm), penicillin (100 IU/ml) and streptomycin selleck (10 mg/ml), 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen), or medium and phorbol 12-myristate 13-acetate (PMA)/ionomycin (25 ng/ml and 1 mg/ml, respectively; Sigma-Aldrich). Cells were then washed in PBS, and stained with cell surface marker antibodies: Pacific Blue-conjugated anti-CD3 (SP34-2), PerCP-Cy5.5 conjugated anti-CD4 (L200; BD Biosciences), APC-Cy7-conjugated anti-CD8α chain (SK1), and FITC-conjugated anti-CD8β chain (2ST8.5H7), in the presence of the live/dead fixable dead cell marker (Aqua LIVE/DEAD; Invitrogen), for 30 min at 4°. After washing with PBS, cells were fixed and permeabilized using the IntraPrep Fix/Perm Kit (Beckman Coulter) and incubated with antibodies specific for intracellular cytokines for 30 min at 4°: PE-conjugated anti-IL-2 (MQ1-17H12), PE-Cy7-conjugated anti-IFN-γ (B27), and APC-conjugated anti-TNF-α, all obtained from BD Biosciences. Cells were analysed using a BD FACSCanto flow cytometer (BD Biosciences) and data analysis was performed using FlowJo software. Human and NHP frozen PBMCs were thawed, rested overnight and distributed into 96-well plates (0·4 × 106 cells/well) coated with 50 μl anti-CD3 (OKT3, 1 μg/ml) and anti-CD28 (CD28.2; Beckman Coulter, 1 μg/ml) antibodies.