Losartan was administered orally in the above studies, but in our study, was continuously administered subcutaneously using an osmotic pump. In the rat, the concentrations

of losartan in the blood and tissue vary widely among individuals after oral administration, because the absorption of losartan is visibly affected by the timing of administration, such as before or after eating feed.31 In addition, oral treatment is also affected by first-pass metabolism, and bioavailability is low. Therefore, the concentration of losartan in bladder tissue stabilizes after 14-day repeated oral administration.32 The continuous subcutaneous infusion is assumed to produce stable concentrations of the drug in the blood and tissue at an early stage of treatment. Losartan blood concentrations were not monitored in the two studies PLX4032 cited above, or in our study. However, the dose and method of administration of losartan that we used in our study was reported to prevent injury progression after myocardial infarction in rats.30 In addition, because the contractile response of bladder strips to 0.1 µM AngII disappeared in the losartan group, it is believed that the signaling from the AT1s that were expressed in the bladder was sufficiently blocked. The histological characteristics of the hypertrophic bladder growth in BOO rats, as revealed

by Elastica-Masson staining, included a marked increase in collagen fibers in the bladder smooth muscle layer and resulting muscle division. C59 wnt ic50 out Losartan treatment decreased these hypertrophic characteristics, and the collagen-to-muscle ratio also decreased

to sham levels. Consistent with these results, HB-EGF mRNA levels that were increased in obstructed bladders were reduced by losartan treatment. In a previous study, HB-EGF mRNA and protein levels were reported to increase in murine bladder tissue in response to urethral ligation, and these increases in HB-EGF mRNA were mainly confined to the bladder muscle layer.33 In cardiovascular studies, locally overexpressed HB-EGF after myocardial infarction increased the level of fibrosis through a mitogenic effect on fibroblasts and exacerbated remodeling at the subacute and chronic stages post-myocardial infarction.34 The combined observations suggest that AT1s are activated by BOO, at least partially, and this activation upregulates HB-EGF and induces fibrosis through induction of the proliferation of fibroblasts in the bladder muscle layer. The urodynamic findings of our study; a shortened micturition interval, a decrease in urine volume per void volume and development of residual urine, indicated that BOO decreased bladder function. However, bladder capacity is greater in the losartan group than in the sham group. The reason for this may be due to bladder hypertrophy induced as a compensatory response to obstruction before treatment with losartan.

Neuroblastoma cells expressing mSOD1 had increased cytoplasmic calcium levels and a significant decrease in mitochondrial membrane potential [85]. Studies of brain,

RG7204 cost spinal cord and liver mitochondria isolated from mSOD1 transgenic mice demonstrated an early decrease in the calcium buffering capacity of the mitochondria from the brain and spinal cord, leading to reduced membrane potential and dysfunctional mitochondria [60]. After challenge with calcium, mitochondria underwent less efficient repolarization, consistent with defective calcium buffering in the presence of mSOD1, which could sensitize motor neurones to excitotoxic stress and eventual death [60]. G93A mice crossed with mice genetically modified to have a decreased calcium permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the spinal motor neurones showed a significant delay in the onset of the ALS phenotype [86]. The trigger for this early increase in calcium levels in SCH772984 mouse motor neurones requires resolution. In SALS, it could potentially be attributed to decreased expression of the glutamate transporter, Excitatory Amino Acid Transporter 2 (EAAT2) [87,88]. Additionally, motor neurones normally have a low expression of GluR2 and thus a higher percentage of calcium permeable

AMPA receptors compared to other neuronal groups, and reduction in the normal editing of the GluR2 subunit may further increase AMPA receptor calcium permeability in motor neurones in ALS [89]. Thus, excessive glutamate stimulation of the calcium-permeable AMPA receptor occurs, emphasizing the need for efficient calcium buffering in motor neurones. In FALS, studies in mice have revealed that mSOD1 interacts with AMPA receptors, altering both their expression patterns and function, rendering them more permeable to calcium [90]. Furthermore, the presence of mSOD1 leads to selective loss of EAAT2 expression, specifically in areas of neurodegeneration [91]. In mSOD1 mice, excessive glutamate application was found to be toxic to Progesterone the neurones, consistent with decreased calcium buffering in motor neurones [74,78,92]. Motor neurones also have reduced expression of cytosolic calcium

buffers, such as parvalbumin and calbindin; thus, motor neurone mitochondria may play a more pivotal role in the buffering of cytosolic calcium [5,44,93]. Although not sufficient in itself to induce excitotoxic cell death, in the presence of mSOD1, any physiological calcium influx will serve to exacerbate mitochondrial dysfunction in the cell, resulting in the eventual degeneration of the motor neurone [5]. Furthermore, at the neuromuscular junction, mitochondria in the synapse of motor neurones show greater membrane potential depolarization in G85R and G93A mice compared to controls [94]. This is linked to a reduced capacity of the ETC to limit depolarization and correlates with onset and progression of ALS symptoms at the motor neurone terminals.

Biopsied tissues were evalueted by light microscopy & indirect immunofluresence study. Reports were demanded within 24 hours time. The initial reports were validated by subsequent clinical course &/or re-biopsy. Results: Amongst 623 renal transplants performed during the study period (jan 2010 to jan 2013), 72 patients had primary graft dysfunction & were biopsied. Biopsy results on day 1 were as shown

in table no.1. Conclusion: Per-operative graft biopsy of non-functioning kidney is a safe procedure with high diagnostic yield & considerable specificity. AN JUNG NAM1, HWANG JIN HO1, JEON HEE JUNG2, JUNG IN MOK1, PARK SU-KIL3, KIM YON SU2, KIM YOUNG HOON3, OH YUN KYU1, LIM CHUN SOO1, LEE JUNG PYO1 1Seoul National University Boramae Medical Center; 2Seoul National University College see more of Medicine; 3Asan Medical Center and University of Ulsan College of Medicine Introduction: Cardiovascular

disease is a leading cause of mortality in patients with end-stage renal disease, even undergoing PXD101 in vivo transplantation. Left ventricular hypertrophy (LVH) is the most common feature and an independent risk factor for cardiac complications in kidney transplant recipients. The aim of this study was to identify cardiac alteration after kidney transplantation and analyze predictors of the post-transplant LVH. Methods: Among 2957 kidney transplant recipients in a multicenter cohort from 1997 to 2012, a total of 206 patients who conducted echocardiography before and one year after transplantation were enrolled in this study. Echocardiographic findings and clinical

parameters were evaluated. Results: Kidney transplantation Tideglusib significantly reduced mean left ventricular mass index (LVMI) from 128.8 ± 47.2 g/m2 to 106.4 ± 33.0 g/m2 (p < 0.001) [Figure 1]. The ejection fraction was improved (59.4 ± 8.0% vs. 62.1 ± 6.7%, p < 0.001). The prevalence of LVH by echocardiography significantly decreased (62.6% vs. 46.1%, p = 0.001). The prevalence of diastolic dysfunction, mitral and tricuspid regurgitation, and pulmonary hypertension also decreased. Pre-transplant lower hemoglobin level (OR 0.74, 95% CI 0.56–0.96, p = 0.026) and pre-transplant higher LVMI (OR 1.02, 95% CI 1.01–1.02, p < 0.001) were independently associated with persistent LVH after kidney transplantation. On the other hand, ejection fraction, diastolic dysfunction, underlying renal disease, albumin or cholesterol level, blood pressure, rejection, and allograft function were not correlated with post-transplant LVH. Conclusions: Cardiac morphology and function were significantly improved by kidney transplantation. Treatment of anemia might be crucial in regression and prevention of persistent LVH in kidney transplant recipients.

[3] As there are multiple mechanistic possibilities, there may also be multiple targets for therapy. This article aims to review the evidence for pharmacological and non-pharmacological therapies that may reduce the Anti-infection Compound Library ic50 risk of SCD, specifically in haemodialysis patients. An overactive sympathetic nervous system predisposes to malignant arrhythmia. In a prospective study of 196 asymptomatic maintenance

haemodialysis patients with left ventricular hypertrophy (LVH), heart rate variability (a measure of autonomic function) was assessed between dialysis sessions. After a mean follow-up of 4.5 ± 1.9 years, there were 23 SCD, here defined as sudden death in a patient who was well 24 h earlier. SCD-free survival rate at 5 years was 29.4% in patients who had cardiac sympathetic over-activity at baseline (demonstrated as a heart rate variability of low frequency/high frequency ratio (LF/HF) > 1.9) compared

with 98.1% in those without (LF/HF < 1.9).[4] In dialysis patients, there are numerous observational data suggesting beneficial effect of β-blockade, but limited trial evidence. In a retrospective study of 316 haemodialysis patients followed up for 4.88 ± 1.88 years, patients using β-blockers had a lower rate of SCD. There were 3/80 SCD events in the β-blocker group in comparison with 27/236 in patients not prescribed β-blockers, P = 0.047.[5] buy MLN8237 Similarly from Dialysis Outcomes and Practice Patterns Study (DOPPS), an analysis of 9046 deaths in haemodialysis patients, after multivariate analysis adjusting for comorbidities, blood results and dialysis parameters, β-blockers were associated with a lower risk of sudden death (hazard ratio, HR = 0.88, 95% confidence interval, 95% CI = 0.78–0.99, P = 0.33).[6] One randomized before controlled trial (RCT) investigated survival benefits of β-blockade versus placebo in haemodialysis patients with left ventricular systolic dysfunction. One hundred fourteen haemodialysis patients with New York Heart Association class II–III for >1 year and a left ventricular ejection fraction, LVEF, <35%, were randomized to either carvedilol treatment or placebo.[7]

After 2 years follow-up, there was a reduction in cardiovascular deaths in the carvedilol arm versus placebo (29.3% vs 67.9%, relative risk reduction, 43.7%). The study lacked power to show any statistical significance in SCD due to a low SCD event rate (6/56 (10.6%) in the placebo arm vs 2/58 (3.4%) in the treatment arm; HR = 0.76, 95% CI = 0.52–1.13, P = 0.12). Recently, an RCT of 200 haemodialysis patients investigated the effect of lisinopril or atenolol three times a week after dialysis on LVH.[8] Baseline and subsequent blood pressure improvements were comparable in both groups. The study was terminated early because there was an increased incidence of serious adverse events in the lisinopril-treated group.

The role of the microcirculation in the etiopathogenesis of vascular disease has been highlighted in a series of epidemiological studies over the last century. We currently recognize selleck kinase inhibitor the independent morbidity of microvascular disease and the prognostic role this carries for future disease. Current epidemiological studies are focusing on attempting to untangle the interrelationship between risk factors and pathological mechanisms to attempt to determine whether these represent therapeutic targets or simple markers of unmeasured risk. These studies have produced a paradigm

shift in the understanding of vascular disease, have triggered many mechanistic studies, and provide evidence to support clinical monitoring of microvascular function in the future. The importance of the microcirculation is increasingly recognized in the aetiopathogenesis of vascular disease and premature mortality. Currently, however, the only therapies used to treat microcirculatory dysfunction are exploiting so called “pleiotropic”? effects of antihypertensive agents, such ACE-inhibitors, angiotensin receptor antagonists and R788 supplier direct renin inhibitors. As we understand better the mechanisms that lead through microcirculatory dysfunction or dysregulation to cardiovascular disease, novel agents may be developed

to specifically target the microcirculation. Further, a better knowledge of the steps that lead to target organ damage may allow better risk stratification and earlier targeting of individuals at higher risk with appropriate risk modification, while providing reassurance to those at low risk. We acknowledge support of the Peninsula NIHR Clinical Research Facility. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR, or the

Department of Health. David Strain, BSc (Hons), MB.ChB, MD, Clinical Senior Lecturer, Peninsula College of Medicine and Dentistry Tyrosine-protein kinase BLK and Hon Consultant in general internal medicine and medicine for the elderly, Royal Devon and Exeter Foundation NHS Hospital Trust. After graduating from Liverpool University, David attained his MD from Imperial College London in 2001. His thesis on the ethnic difference in the effects of insulin resistance on the microvasculature described a novel abnormality of microcirculatory autoregulatory function and its links to left ventricular hypertrophy, urinary albumin excretion and coronary atherosclerotic load. In 2007 he moved to Peninsula College of Medicine and Dentistry and in 2010 was awarded a prestigious HEFCE clinical senior fellowship. He is the clinical lead of a research team exploring the role of the microcirculation in the aetiopathogenic mechanisms of a diverse range of vascular disease, from stroke to diabetic cardiomyopathy.

The cytokines were measured in the cell culture supernatants; TNF-α after 4 h incubation at 37°C and IL-6, IL-10 and IL-12 after 18 h incubation at 37°C using commercial ELISA kits (Milenyi Biotec Thermo Scientific). The cytokine responses were measured without (spontaneous) and after stimulation and the values of the spontaneous secretion were deducted from the challenge values to allow for comparison with other, similar studies (e.g. [20]). Counting of white blood cells showed only minor differences in the monocyte/lymphocyte ratio between controls

and subjects with sarcoidosis. The participants Belinostat clinical trial were supplied with a pump and filter holders preloaded with cellulose acetate filters (Mixed Cellulose Esters, 25 mm PCM Casettes, 0·8 µm pore size; Zeflon International Inc., Ocala, FL, USA). The subjects turned on the pump and sampling was performed for about 4 h. The exact volume sampled was read from a volume meter attached to the pump and was usually

LDE225 mouse about 2·5 m3. The filters were analysed for their content of N-acetylhexosaminidase (NAHA) using an enzyme technique [10,21]. One ml of a fluorogenic enzyme substrate (4-methylumbelliferyl N-acetyl-β-D-glucosaminide; Mycometer A/S, Copenhagen, Denmark) was added to the filter, followed by 2 ml of a developer after an incubation period of around 30 min, determined by the room temperature. The liquid was sucked out through the filter and placed in a cuvette. The fluorescence in the cuvette was read in a fluorometer (Picofluor; Turner Designs, Sunnyvale, CA, USA). To decrease the variance induced by methodological variations in the analysis technique, the fluorescence values were divided by 10 and rounded off to the nearest whole number to express NAHA activity in units Phosphoribosylglycinamide formyltransferase (NAHA U/m3). Values in the different groups were calculated using spss version 17–W7 and expressed as mean and standard error of the mean. Differences between groups were evaluated using the Mann–Whitney

test and the intercorrelations assessed using Spearman’s-test. A P-value of < 0·05 was considered statistically significant. The spontaneous secretion of different cytokines from PBMC is reported in Table 1. The spontaneous secretion of all cytokines was higher from PBMC from subjects with sarcoidosis with significant differences for TNF-α and IL-12. Table 2 reports the secretion of cytokines after incubation with different FCWA and LPS. After stimulation with S-glucan the secretion of TNF-α was significantly higher among subjects with sarcoidosis, but there were no differences for the other cytokines. Stimulation with P-glucan caused a high secretion of all cytokines, which was significantly higher among subjects with sarcoidosis. Chitin was a comparatively weak inducer of cytokines in both groups except for IL-6, and no differences were found between controls and sarcoidosis.

, 2005). Among the positive clinical samples, 68.9% (31/45) were cutaneous biopsies, 17.8% (8/45) were cutaneous swabs, 4.4% (2/45) were total blood samples and 8.9% (4/45) were serum samples. The identification of rickettsial infections using cutaneous swab specimens and PCR testing has recently been reported (Bechah et al., 2011; Mouffok et al., 2011); based on these preliminary results, we collected cutaneous swabs from patients rather selleck screening library than cutaneous biopsies. Our retrospective analysis recovered eight positive cutaneous eschar swabs from different patients, confirming that these provide a rapid and simple means method that can be performed easily without the

risk of the side effects related to biopsy collection in patients who display an inoculation eschar and/or a vesicular rash (Mouffok et al., 2011). In conclusion, the widespread use of qPCR is less expensive than conventional PCR and reduces delay in the diagnosis of rickettsial infections. The development of qPCR strategies in the diagnosis of rickettsioses has previously Ganetespib been proposed (Stenos et al., 2005). Our 2 years of experience of rickettsial diagnosis using qPCR suggests that these molecular tools improve the efficiency of the management of patients with suspected cases of rickettsiosis. These qPCR assays could therefore

be easily implemented in laboratories with molecular facilities and may be added to existing molecular tools as a point-of-care strategy (Holland & Kiechle, 2005). “
“Semen is the primary medium for sexual transmission of HIV-1 and contains high concentrations of TGF-β1,

but its role in regulating HIV-mediated immune activation is unclear. TGF-β1 and sCD14 were compared in blood plasma (BP) and seminal plasma (SP) from HIV-uninfected and infected, antiretroviral therapy (ART)-naive and ART-treated men and in THP-1 nearly cells following exposure to HIV-1. The relationship between TGF-β1 and sCD14 was determined by Spearman correlation. Active and latent forms of TGF-β1 were compartmentalized between BP and SP. Highest active TGF-β1 levels were present in SP of ART-naïve chronic-infected men and decreased following ART treatment. Latent TGF-β1 was upregulated in BP following HIV infection, and highest levels were observed in BP of acute-infected men. Similar expression trends were observed between latent TGF-β1 and sCD14 in BP. A significant negative correlation was observed between active TGF-β1, sCD14, and semen viral load in ART-naive men. TGF-β1 is compartmentalized between blood and semen, possibly co-expressed with sCD14 by activated monocytes/macrophages in BP as a result of HIV infection. Conversion of latent TGF-β1 into its active form could contribute to regulation of viral load and immune activation in the male genital tract, but depends on the stage of infection.

All animal procedures and experimental protocols were in accordance AZD6244 with the local Ethical Committee for Animal Research (CEEA – Protocol no. 212). NOD mice were distributed in three groups: non-immunized NOD mice (NOD); NOD mice immunized with BCG vaccine (BCG–NOD) and NOD

mice immunized with the prime-boost BCG/pVAXhsp65 (BCG/DNAhps65–NOD). Diabetes type 1 in male C57BL/6 mice was induced with STZ and animals were allocated into four groups: non-immunized, non-diabetic mice (control); non-immunized diabetic mice (STZ), mice immunized with BCG (BCG-STZ) and mice immunized with the prime-boost BCG/pVAX-hsp65 (BCG/DNAhps65–STZ). The vaccine pVAXhsp65 was derived from the pVAX vector (Invitrogen, selleck Carlsbad, CA, USA), digested previously with BamHI and NotI (Gibco BRL, Gaithersburg, MD, USA) by inserting a 3·3 kb fragment corresponding to the Mycobacterium leprae hsp65 gene

and the cytomegalovirus (CMV) intron A. DH5a Escherichia coli transformed with plasmid pVAX or the plasmid carrying the hsp65 gene (pVAXhsp65) were cultured in Luria-Bertani liquid medium (Gibco BRL) containing kanamycin (100 μg/ml). The plasmids were purified using the Concert High Purity Maxiprep System (Gibco BRL). Plasmid concentrations were determined by spectrophotometry at 260 and 280 nm by using the Gene Quant II apparatus (Pharmacia Biotech, Amersham, UK). BCG vaccine [50 μl containing around 105 colony-forming units (CFU)] was administered subcutaneously at the base of the tail when NOD mice were 7 weeks old and C57BL/6 mice were 4–6 weeks old. In the prime-boost group, animals were additionally injected with pVAXhsp65 (100 μg/100 μl) associated with 25% of saccharose by the intramuscular route (quadriceps muscle) 15 days after BCG immunization. NOD mice were monitored until their 29th week of life, whereas STZ groups were monitored for 21 days after diabetes induction. Body weight and blood glucose level were measured weekly and insulitis scores were measured only after euthanasia.

In addition, in the NOD mice, Ergoloid cytokine production by spleen cells and the presence of Treg cells in the spleen were analysed. In order to induce diabetes, male C57BL/6 mice were given intraperitoneal injections of STZ diluted in citrate buffer (40 mg/kg; Sigma-Aldrich, St Louis, MO, USA) for 5 consecutive days. Using this protocol, glycaemia was determined once before the first STZ dose and three times after the last dose. Non-fasted glucose concentration was determined in blood samples collected from the facial vein and measured using Prestige LX Smart System Test-strips (Home Diagnostic, Inc., Fort Lauderdale, FL, USA). NOD mice are known to develop hyperglycaemia around week 12 and, therefore, blood glucose concentration was measured from the 11th week onwards. Animals were considered diabetic when blood glucose levels were higher than 200 mg/dl during 2 consecutive weeks.

Western blot and flow cytometry were used to assay the LC3-II expressions. RNAi techniques including shRNA and siRNA were used to investigate the function of MFN1 and FIS1 in HK2 cells cultured in the presence or absence of glucose. Mitochondrial morphology were stained by mitotracker and analyzed by confocal microscopy. TUNEL assay was used to examine the cellular apoptosis in glucose treated wild type and MFN1-depleted HK2 cells. Results: HFHS diet led to vacuolization and thyroidisation of renal tubules, reduced expressions of Mfn1 and Mfn2 and enhanced expressions

of Drp1 and Fis1. Glucose caused mitochondrial fragmentation and apoptosis in HK2 cells. MFN1-depleted cells were more susceptible to glucose-induced mitochondrial fragmentation and cellular apoptosis. SiRNA targeting FIS1 was able to rescue the glucose-induced injuries in MFN1-depleted cells. TEM demonstrated the LEE011 formation of autophagosome in glucose-treated HK2 cells. LC3-II expression was greatly increased in MFN1-depleted cells. Upon silencing FIS1, the increased LC3-II

expression in MFN1-depleted cells was reduced to a comparable level to wild type cells. Conclusion: Our results suggested that glucose drives the mitochondria to fission which eventually leads to mitochondrial fragmentation and cellular apoptosis. Autophagy could be a protective mechanism for glucose-induced injuries in renal tubules. MFN1 also played a protective role in these injuries. Silencing of FIS1, could be a novel strategy to treat DKD. YANG SUNG-SEN1,2, JIANG SI-TSE3, YU I-SHING4, LIN SHU-WHA4, LIN SHIH-HUA1,2 1Division of Nephrology, Department of Medicine, Tri-Service General Hospital,Taipei, Selleck PFT�� Taiwan; 2Graduate Institute of Medical Sciences National Defense Medical Center,Taipei, Taiwan; 3National Laboratory Animal Centre, National Masitinib (AB1010) Applied Research Laboratories, Taipei, Taiwan;

4Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University,Taipei, Taiwan Introduction: Recently, it was shown that an ubiquitously expressed Cab39 protein could also stimulate Na-(K)-(2)Cl cotransporter [N(K)CC] through activating SPAK/OSR1 kinases mimicking WNK1/4 kinases in vitro study. Methods: We generated and analyzed both the kidney tubule-specific cadherin gene promoter driven flag-tagged mouse Cab39 (KSP-Flag-mCab39) transgenic (Tg) and WNK4 knockout mice. At age of 10–12 weeks fed with normal rodent chaw, phenotype including blood pressure as well as serum and urine electrolytes was measured in WT, Cab39 Tg, Wnk4 knockout and Cab39 TgxWnk4 knockout transgenic mice. The expression of WNK1/4, Cab39, SPAK/OSR1 and N(K)CC was evaluated by western blotting and immunofluorescence stain. Results: Offspring from Cab39 Tg mice with mildly overexpressed abundance of flag-Cab39 (25% ± 6%) were phenotypically normal but a slightly increased p-SPAK/OSR1, p-NKCC2 and p-NCC in the kidneys was found.

Where indicated, human cells were stimulated in the presence of human IFN-α (1000 U/ml; PBL Biomedical Laboratories, Piscataway, NJ) and rhesus cells with universal type I IFN (1000 U/ml; PBL Biomedical Laboratories). To support viability in the rhesus B-cell cultures, IL-2 (100 ng/ml, PeproTech, Rocky Hill, NJ) and B-cell activation Seliciclib cost factor of the tumour necrosis factor family (BAFF; 100 ng/ml, PeproTech) were added to the rhesus cultures in the experiments where differentiation and antibody

production were measured. Human and rhesus PBMCs were labelled with 0·25 μm CFSE (Molecular Probes, Eugene, OR) for 7 min at 37° and thoroughly washed with complete medium as described elsewhere.2,3 Using the conditions described above 2 × 106 cells/ml were cultured at 37° in polystyrene round-bottom tubes in complete medium. TLR ligands were used at 1 μg/ml (Poly I:C and TLR7/8-L) and 5 μg/ml (CpG classes), optimal concentrations of each ligand that caused peak B-cell activation. Proliferation was measured by flow cytometry and data were analysed using FlowJo software. Live cells were gated on by exclusion of propidium iodide staining. B cells were gated based on expression of CD20 and CD19 for rhesus and human B cells, respectively, and lack of CD3 and CD14. Alternatively, proliferation was measured

selleck chemicals by thymidine incorporation where PBMCs or B cells were cultured in 96-well plates and pulsed with [3H]thymidine (1 μCi/well, Amersham Bioscience, GE Healthcare Biosciences AB, Uppsala, Sweden) for 16 hr after 4 days of culture. The level of incorporation mafosfamide of [3H]thymidine was measured by a 1450 MicroBeta PLUS counter (Wallac, PerkinElmer Sverige AB, Upplands Väsby, Sweden) and expressed as counts per minute (c.p.m.). Human or rhesus PBMCs at 6 × 106 cells/ml

were exposed to the TLR7/8-L (1 μg/ml) or CpG ODN class C (5 μg/ml) for 1 hr at 37° in polystyrene round-bottom tubes, followed by an additional 10 hr in the presence of the secretion inhibitor Brefeldin A (10 μg/ml; Sigma-Aldrich) and then stained as described previously.33,34 Briefly, the cells were fixed and permeabilized for 15 min using a BD Cytofix/Cytoperm kit (BD Pharmingen). The cells were then washed twice and stained with antibodies specific for IFN-α (clone MMHA-11, PBL Biomedical Laboratories), CD3, CD14, CD20, CD123, HLA-DR (antibodies as described above). The cells were analysed by flow cytometry. In addition, IFN-α levels in the supernatants of cells exposed for 24 hr to the TLR ligands were measured by ELISA (Mabtech, Stockholm, Sweden) performed according to the manufacturer’s instructions. Phenotypic differentiation of B cells was assessed for up to 6 days of culture by flow cytometry using antibodies against CD20, CD27, IgG and IgM (all BD Pharmingen). Expression of IgG and IgM was assessed by intracellular staining using the BD Cytofix/Cytoperm kit before staining.