1%) and sensitivity (88 5%), in both Parkinson’s disease and deme

1%) and sensitivity (88.5%), in both Parkinson’s disease and dementia with Lewy bodies, suggesting that this finding can be a useful hallmark of Lewy body-related disorders. “
“Pseudopolyneuritic form of ALS is a subtype of ALS characterized by distal weakness of the unilateral lower limb and absence of Achilles tendon reflex (ATR) at disease onset. Recognition of this form of ALS is important for clinicians because the combination of distal weakness of the lower limb and absence of ATR usually suggests peripheral neuropathy. We reviewed the clinical records of 42 autopsy-proven sporadic ALS cases

and found three cases that showed onset of weakness of the unilateral lower limb with distal dominance and absence of ATR. The disease duration in the three cases was 2, 3 and 19 years, respectively.

The clinical features of the patient with a course of 19 years had been restricted to lower motor neuron signs. Histopathologically, consistent findings learn more in the three cases were severe motor neuron loss throughout the whole spinal cord, with relative preservation of the hypoglossal nucleus. Reflecting this finding, TDP-43-positive neuronal cytoplasmic inclusions in the spinal cord were sparse in two cases, and absent in a third. In the patient showing a clinical course of 19 years, mild corticospinal tract degeneration appeared to correspond to the absence of upper motor neuron signs and prolonged disease duration. In this case only, Bunina bodies were not demonstrated. In https://www.selleckchem.com/products/gdc-0068.html this study, we clarified the clinical and pathological heterogeneity of this form of ALS. “
“Prader-Willi syndrome (PWS) is caused by

the absence of paternally contributed genes in chromosome 15, and is characterized by hypotonia, feeding difficulty, mental retardation, growth failure, hypogonadism and severe obesity. To elucidate the pathogenesis of neurological disorders, we immunohistochemically examined the L-NAME HCl γ-aminobutyric acid (GABA)ergic interneurons (GABAis) in the cerebral cortex and acetylcholine neurons (AchNs) in the nucleus basalis of Meynert (MyN) and pedunculopontine tegmental nucleus pars compacta (PPNc) in an autopsy case of one PWS patient with a deletion in the 15q11-q12 region and three control patients. The GABAis in the cerebral cortex and AchNs in the MyN were well preserved in the PWS patient. The AchNs in the PPNc in the PWS patient were severely reduced in comparison with those in controls, whereas catecholaminergic neurons and GABAis were preserved. The selective loss of AchNs in the PPNc may be involved in hypotonia and/or REM sleep abnormalities in PWS patients. “
“Extraventricular neurocytoma (EVN) shares histological features with central neurocytoma, but has a wide morphological spectrum. Little is known regarding its clinicopathologic nature, biological behavior and genetic abnormalities. The aim of this study is to examine the diagnostic criteria, genetic abnormalities and biological behavior of EVN.

Studies have demonstrated that developing haematopoietic cells ex

Studies have demonstrated that developing haematopoietic cells express TLRs7,25 and Temsirolimus order therefore would be expected to be sensitive to stimulation with their ligands. Our experiments indicate that the presence of TLR4 or TLR9 ligands (LPS and CpG ODN, respectively) during the generation of BMDCs in the presence of GM-CSF inhibits the differentiation of cells with the phenotype of BMDCs. This is in agreement with other studies which show that LPS or CpG ODN inhibit in vitro differentiation of DCs.26–28 Bartz et al.28 demonstrated that the generation of myeloid DCs from murine bone marrow was impaired by stimulation

with LPS or CpG ODN. The cells generated exhibited reduced expression of CD11c and MHCII and a reduced ability to activate T lymphocytes. In humans, LPS stimulation has been shown to influence both early and late monocyte differentiation by blocking their ability to differentiate

into DCs in vitro.25 The addition of LPS to cultures of monocytes containing GM-CSF and IL-4 X-396 supplier reduced the cell yields, altered the morphology and phenotype of the cells generated, and compromised their capacity to present antigen.27,28 We did not explore the antigen-presentation function of the cells generated, but their phenotype, CD11clo/MHCIIlo, suggests a reduced antigen-presentation capacity because of the crucial role of MHCII in this process. Taken together, these findings confirm the inhibitory effects of LPS and CpG ODN stimulation during DC generation. Our experiments indicate that TLR stimulation during the development of BMDCs in vitro inhibited the differentiation of CD11c+/MHCII+ cells while simultaneously enhancing the production of CD11clo/MHCIIlo cells. Experiments with knockout mice revealed that TLR4 (data not shown) and MyD88 were required to generate both of these effects. TLR4 and MyD88

have been shown to be expressed by developing haematopoietic cells,5 and this study demonstrated that MyD88-dependent signalling promoted myeloid lineage differentiation from HSC-enriched cultures stimulated Tau-protein kinase with LPS in serum-free, stromal cell-free conditions. The differentiation potential of lymphoid progenitors has also been shown to be influenced by TLR9 ligation in a MyD88-dependent manner,29 and CpG ODN-induced inhibition of BMDC production is known to require TLR9.28 Although signalling via TLRs on granulocyte and macrophage progenitors has been shown to obviate the need for growth and differentiation factors to direct the differentiation of haematopoietic cells in vitro7 it was likely that the effects we observed were mediated by cytokines produced in response to TLR stimulation. This suggestion is supported by several reports which indicate that cytokines provide differentiation cues for developing haematopoietic cells.30–33 Tumour necrosis factors have been shown to inhibit haematopoiesis in vitro.

We next conducted a cellular analysis to evaluate the degree of l

We next conducted a cellular analysis to evaluate the degree of lymphocyte activation in both groups of disease-free mice (Fig. 3). Consistent with the adjuvanticity

of LPS, treated mice displayed overall increased lymphocyte number and activity in the spleen (Sp) and, more notably, in the lymph nodes draining the pancreas (pLN) (Fig. 3A). Gross subdivision of lineages as CD8 or CD4 T cells, B cells or Dendritic cells did not reveal preferential expansion (not shown and Fig. S2). Splenic B lymphocytes showed an activated phenotype as indicated by elevated MHC Class II expression (Fig. S2). Moreover, seric IgG and IgM titres were readily increased (not shown). Despite the systemic effect of long-term LPS treatment, effector CD4 T cells were not significantly affected as measured by the frequency of CD69+ or CD44high cells in spleen and pLN. Noteworthy, CD4+CD69+CD44hi cells, previously

described as primed cells enriched in diabetogenic selleck kinase inhibitor effectors [10], were found in the spleen and pLN of both groups of mice (Figs. 3B and S3). Similarly, while T lymphocyte differentiation into IFN-γ-producing helper cells is essential for diabetes establishment in NOD mice [50], LPS-treated and healthy controls displayed similar frequency of CD4+IFN-γ+ cells in spleen and pLN (Figs. 3C and S3). Infiltration of CD4+IFN-γ+ selleck chemical cells was also detected in pancreas of healthy and LPS-protected animals (Fig. S3). Taken together these analyses indicate that LPS treatment while preventing

disease development did not induce immune paralysis, nor impaired Th1 differentiation, an effector class essential for diabetes establishment in NOD mice [50]. We conclude that LPS-induced protection in NOD mice, similarly to spontaneous protection, operated by a mechanism that did not impede pancreatic islet infiltration and effector CD4+ cell activation. We Ureohydrolase next assessed whether Treg undergo phenotypic modifications in vivo upon LPS treatment by analysing the frequency and numbers (Figs. 4, 5 and S4–S6) of specific CD4 cell subsets. Historically, Treg were first defined as CD4+CD25+ T cells [51]. Yet, activated conventional T cells also express CD25 in a transient manner upon activation. LPS treatment in NOD mice did not increase the frequency and number of CD4 cells expressing CD25 (Fig. 4A). While CD4+CD25+ cells expressing high levels of l-selectin have been shown to be particularly potent in preventing diabetes occurrence [18], the frequencies of CD62LhiCD4+CD25+ splenocytes in each experimental group were not significantly different (data not shown). CD103-expressing CD4+CD25+ cells display increased regulatory function in vivo [52, 53] and CD103 expression is likely a molecular signature of pancreatic Treg [10]. Strikingly, LPS-protected mice presented an increased frequency and number of CD103+CD4+CD25+ cells, both in the spleen and in pLN (Figs. 4B and S4), when compared with healthy controls.

Cells from spinal cord were restimulated in vitro with MOG peptid

Cells from spinal cord were restimulated in vitro with MOG peptide and stained intracellularly for IL-17 and IFN-γ. As shown in Fig. 4, MOG-specific T cells from inflamed Olaparib cell line spinal cords belonged to Th1, Th17, and Th17/Th1 subsets. However, the percentage of CD4+ T cells from LFA-1+/+ and LFA-1−/− mice producing these cytokines was absolutely comparable on the level of antigen-specific cells. In addition, the amount of cytokines

produced did not differ (MFI for IL-17: 20 020±1457 (LFA-1+/+) versus 21 460±1080 (LFA-1−/−), p=0.436; MFI for IFN-γ: 15 436±2127 (LFA-1+/+) versus 14 940±804 (LFA-1−/−), p=0.832). The same results were obtained for IL-2 and TNF-α. However, it is important to note that the increased total number of antigen-specific cells finally results in a higher absolute number of cytokine-producing CD4+ T cells. Interestingly, there was also no correlation between EAE score of an individual animal and cytokine production on the single cell level. Again, only the number of infiltrating CD4+ T cells correlates with disease severity (see above). Polyfunctional Th1 cells producing multiple effector cytokines at the same time are believed to be particularly

destructive in inflammation 12. Therefore, we also analyzed whether the frequency of IL-2, IL-17, IFN-γ double U0126 purchase or triple producers was altered between WT and KO mice, but did not find any significant differences (data not shown). Alternatively, a change in Th2 or anti-inflammatory cytokines could influence the severity of disease. Therefore, we tested for the production of IL-4 and IL-10. Only

very few (<2%) antigen-specific CD4+ T cells in the spinal cord produced these two cytokines. However, Phosphoprotein phosphatase we did not observe any significant differences between LFA-1+/+ and LFA-1−/− T cells (data not shown). To analyze the general capacity of T cells to produce certain cytokines, we additionally used an antigen-independent stimulation with PMA and ionomycin. Also, with this kind of stimulation, none of the analyzed cytokines differed between KO and WT mice (data not shown). Taken together, these results clearly show that loss of LFA-1 does not alter the cytokine pattern of autoreactive CD4+ T cells. Therefore, only the increased total number of antigen-specific, cytokine-producing cells in LFA-1−/− mice can be accounted for the increased severity of EAE. Treg play an important role for the suppression of chronic inflammation 8, 13. They control the expansion as well as the function of autoreactive effector T cells. Utilizing intracellular staining for the lineage-specific transcription factor FoxP3, we analyzed Treg in the spinal cord of LFA-1−/− and LFA-1+/+ mice after EAE induction. The absolute number of Treg was the same in both groups (Fig. 5).

The importance of type I IFN and TLR-7 signalling in aggravating

The importance of type I IFN and TLR-7 signalling in aggravating kidney injury was established in mice that overexpress TLR-7 (Y-linked autoimmune accelerating locus mice – Yaa mice) or that were treated with pristane.[93-95] In a pristane-induced mouse model of SLE, it was shown that an intact type I IFN signalling pathway is prerequisite to the upregulation of TLR-7 receptors in B cells and effective activation through TLR-7 and TLR-9 of B cells to produce lupus-specific autoantibodies.[96]

These findings suggested that type I IFN is upstream ubiquitin-Proteasome system of TLR signalling in the activation of autoreactive B cells in SLE. Furthermore, in lupus-prone mice, severe nephritis can be induced by the activation of TLR-9 signalling pathway through CpG-rich DNA.[97] These observations were supported BMN 673 nmr by a study that tested a dual inhibitor of TLR-7 and TLR-9 (known to inhibit IFN-α production by PDC) in lupus-prone mice. The inhibition of TLR-7 and TLR-9 would lead to a significant improvement of proteinuria, glomerulonephritis, and survival as well as decreased nucleic acid-specific autoantibodies.[98] Elevation of type I IFN in lupus patients was one of the first described

cytokine abnormalities in autoimmune diseases. The link between IFN levels and disease activity, anti-dsDNA levels and clinical manifestations backs the role of IFN in SLE pathogenesis.[99] In lupus patients, PDC was detected in the dermal lesions and are responsible for sustained IFN release, although their circulating number is lower in the peripheral blood.[100] Migration of PDC to the glomeruli is observed in patients with lupus nephritis and this movement Tobramycin is thought to be influenced by IL-18.[101] In patients with cerebral lupus, autoantibodies with the capacity to form very potent interferonogenic immune complexes together with RNA-containing auto-antigens were detected in the cerebrospinal fluid.[102] Gene expression profiling showed that SLE patients expressed IFN-inducible genes in PBMC and the expression correlated

with disease activities.[78] These findings revealed that raised IFN levels are capable of altering gene expression in active lupus patients and supported the pathogenic role of type I IFN in human lupus. Data derived by the genetic studies had further delineated the causal role of IFN in SLE. Transcription factor IRF5 was the first identified gene directly involved in IFN production and was associated with heightened risk of SLE.[103] Lupus patients with a risk haplotype of IRF5 showed more intense serum IFN activity when compared with patients lacking this risk genotype and the effect was most obvious in patients with autoantibodies against either RNA-binding proteins or double-stranded DNA.[104] Another example is the signal transducer and activator of transcription 4 (STAT4) which interacts with the cytoplasmic part of the IFNAR and variants of STAT4 have been shown to be strongly associated with lupus.

2% in Australia A low awareness rate in contrast to high prevale

2% in Australia. A low awareness rate in contrast to high prevalence of CKD is MK-8669 order a serious public health problem in Taiwan. Hsu et al. 8 reported an overall awareness rate of CKD of 9.7% in contrast to 6.9% prevalence rate for CKD stage 3–5. Awareness rates for each stage of CKD were 8.0% (stage 3), 25.0% (stage 4) and 71.4% (stage 5). In Wen’s report,13 the overall awareness of CKD stage 1–5 was only 3.5%. Awareness rates for each stage of CKD are 2.66% (stage 1), 2.68% (stage 2), 4.10%

(stage 3), 23.67% (stage 4) and 52.40% (stage 5). Notably, low awareness in contrast to high prevalence of CKD is especially more common in subjects of low socioeconomic and educational status. This fact raises the importance of promoting awareness of CKD through patient education and an intensive screening program. For example, World Kidney Day and a public media campaign have been implemented in Taiwan since 2007. More importantly, continuing medical education is crucially needed for each level of medical physician in all specialties. We must foster the health-care professionals to learn the new concept of CKD definition AZD3965 price and classification4 and to provide the rational care for this rapidly growing population of CKD. Taiwan has the highest incidence and prevalence rate of ESRD based on international comparisons of the USRDS report.11 Based on the

National Dialysis Registry by the Taiwan Society of Nephrology (TSN), Yang et al. reported that from 1990 to 2001 incidence and prevalence rates of ESRD patients increased 2.6 times from 126 to 331/million populations (pmp) and 3.46 times from 382 to 1322/pmp, respectively, from 1990 to 2001.27 Recent data from the Dialysis Registration of the TSN in 2007 reported 48 072 haemodialysis

(HD) and 4465 peritoneal cases, corresponding to a prevalence of 2288/pmp and incidence of 415/pmp, respectively.11 The heavy burden of renal replacement therapy by dialysis was managed by a total of 1081 board-certificated nephrologists, 534 dialysis centres and 14 502 HD machines. Moreover, the domestic renal transplant patients from 1997–2007 were 2054 cases based on the data of the Bureau of National Health Insurance (BNHI). However, it was estimated that another 50% of patients received NADPH-cytochrome-c2 reductase off-shore renal transplantation, mainly from China. There are several possible explanations for the high incidence and prevalence of ESRD in Taiwan. First, a major reason is that the launching of the NHI in 1995 provided free coverage for dialysis therapy without co-payment.28 The universal coverage facilitates the utilizations of renal replacement therapy and further accelerates the inflow of dialysis patients. Second, the better health-care system may improve the survival rate of chronic diseases patients and increase the overall life expectancy. This reason is supported by the evidence that the increased ESRD population consisted of mainly elderly (>65 years) and diabetic patients in Taiwan.

(31) The results were expressed as reactivity index (RI = OD sam

(31). The results were expressed as reactivity index (RI = OD sample/cut-off), and graphs were made using the software GraphPad Prism® version 3·0 (GraphPad Softward Inc., San Diego, CA, USA). Leishmaniasis is a great problem of public health in several countries worldwide. In South America, visceral leishmaniasis is mainly caused by L. Chagasi. As dogs Romidepsin cost are the main reservoirs of this protozoan parasite in these regions along with the fact that they live

close to humans in urban and rural areas, it is necessary to control the level of canine infection. In this work, two L. chagasi recombinant proteins produced in E. coli, rLci2B and rLci1A, referred to parasite kinesins and heat shock proteins, respectively, were previously selected from a parasite cDNA library. They were expressed and purified by column liquid chromatography after bacteria cell disruption. click here For the purification of the histidine-tagged protein rLci2B, two chromatographic steps were employed, whereas the rLci1A

protein, expressed as an inclusion body, required urea dissolution before column fractionation. The purified recombinant proteins were used in the development of an enzyme immunoassay for leishmaniasis diagnostic. The proteins rLci2B and rLci1A were expressed in E. coli with a yield of approximately 105 and 225 mg/L bacterial culture, respectively, according to modified

Folin–Lowry quantification methodology. The bacterial crude protein extracts (I and II) analysed by denaturing gel electrophoresis showed, in both cases, one predominant electrophoretic band whose molecular mass was comprised between 36 and 52 kDa ifenprodil (extract I) and 52 and 95 kDa (extract II) (Figure 1, panels a and b). The rLci2B purification performed by nickel affinity chromatography followed by Superdex™ 200 gel chromatography (Figure 2, panel a) recovered 10·5 mg of protein. The rLci1A protein recovery was 18·0 mg after Poros® HQ fractionation (Figure 2, panel b). The homogeneities of rLci2B and rLci1A isolated preparations were determined by methodologies based on isoelectric point (IEF-PAGE), molecular weight (SDS-PAGE) and immunological characterization (Western blot). Estimated molecular mass and isoelectric point were 46·37 kDa and 5·91 for rLci2B (Figure 2, panel c, lane 2) and 88·40 kDa and 6·01 for rLci1A (Figure 2, panel d, lane 2), respectively. Preliminary ELISA studies were performed to establish methodology standardization.

To clarify conformational differences between these two isoforms,

To clarify conformational differences between these two isoforms, PrP-deficient mice were immunized with brain homogenates of normal and scrapie-infected

animals. All mice generated anti-PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrPSc consisting of QGSPGGN (PrP41–47) at the N-terminus. This study demonstrated a conformational dissimilarity at the N-terminus between PrPSc and PrPC, a finding that may provide novel information about conformational features of PrPSc. “
“Although NKT cells have been implicated in diverse immunomodulatory responses, the effector mechanisms underlying the NKT cell-mediated regulation of pathogenic T helper cells are not well understood. Here, we show that invariant NKT cells inhibited the

differentiation mTOR inhibitor of CD4+ T cells into Th17 cells both in vitro and in vivo. The number of IL-17-producing CD4+ T cells was reduced following co-culture with purified NK1.1+TCR+ cells from WT, but not from CD1d−/− or Jα18−/−, mice. Co-cultured NKT cells from either cytokine-deficient (IL-4−/−, IL-10−/−, or IFN-γ−/−) or WT mice efficiently inhibited Th17 differentiation. The contact-dependent mechanisms of NKT cell-mediated regulation of Th17 differentiation were confirmed using transwell co-culture experiments. On the contrary, the suppression of Th1 differentiation was dependent MK-1775 on IL-4 derived from the NKT cells. The in vivo regulatory capacity of NKT cells on Th17 cells was confirmed using an experimental autoimmune uveitis model induced with human IRBP1–20 (IRBP, interphotoreceptor retinoid-binding protein) peptide. NKT cell-deficient mice (CD1d−/− or Jα18−/−) demonstrated an increased disease severity, which was reversed by the transfer of WT or cytokine-deficient (IL-4−/−, IL-10−/−, or IFN-γ−/−) NKT cells. Our

results indicate that invariant NKT cells inhibited autoimmune uveitis predominantly through Oxalosuccinic acid the cytokine-independent inhibition of Th17 differentiation. The long-served Th1/Th2 hypothesis has been updated by the identification of a third subset, IL-17-producing CD4+ Th (Th17 cells) 1. Although Th1 cells mainly provide protection against intracellular microorganisms and Th2 cells protect against helminthes, Th17 cells have been implicated in the host defense against extracellular bacteria and fungi 2. Uncontrolled Th17 responses have been recently reported in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, and inflammatory bowel diseases in human and mouse models 1–3, which were formerly considered Th1-mediated diseases. In mice, naïve CD4+ T cells differentiate into Th17 cells in the presence of IL-6 and TGF-β 4. TGF-β is a pleiotropic cytokine with potent regulatory capacities that modulates the activation and homeostasis of effector T cells and induces Foxp3+ Tregs 5.

9 years; range: 17–84 years) with PTB from Shandong Chest Hospita

9 years; range: 17–84 years) with PTB from Shandong Chest Hospital, May 2010–June 2012. According to American Tuberculosis Panobinostat mw Society criteria, patients were diagnosed on the history, clinical symptoms and signs, chest X-ray, sputum smear test and tuberculin skin test. No extrapulmonary tuberculosis was detected. Peripheral blood was collected before antituberculosis therapy. Subjects with other infectious diseases, immunological or autoimmune diseases as well as other diseases may affect the immune system were excluded. According to sputum smear

test, we subdivided patients into smear positive group and negative group. Healthy control group.  A total of 200 unrelated healthy controls (107 men and 93 women; mean age: 37.1 years; range: 21–80 years) with the positive history of tuberculin skin test were recruited from Shandong Chest Hospital, May 2010–June

2012. X-ray did not reveal PTB and all have inoculated with Bacillus Calmette–Guerin vaccine. Patients and controls were matched for genders, ages and ethnicity. All the controls with other infectious diseases, immunological or autoimmune diseases as well as other diseases may affect the immune system were excluded. This study had ethical approval from the Hospital Ethics Committee, and an informed consent was obtained from each individual. ICG-001 datasheet Genomic DNA isolation.  According to the manufacturer’s instructions, genomic DNA was extracted from 5 ml ethylene diamine tetraacetic acid (EDTA) anticoagulated peripheral blood using TIANamp Blood DNA kit (Tiangen Biotech, Beijing, China) and stored at −20 °C. We determined the integrity and quantity of DNA samples using UV spectrophotometer and DNA concentration was adjusted to 50 ng/μl. KIR genotyping.  Genotyping of KIR was conducted by SSP–PCR method, which was performed to detect the presence or absence of 12 known KIR genes, including 2DL1-3, 2DL5, 2DS1-5, 3DL1, 3DS1 and 1D. All primers (Bo Ya Biotechnology Co. Ltd, Shanghai, China) were validated and confirmed. The primers of 2DL1-3, 2DL5, 2DS1-5, Docetaxel cost 3DL1 and 3DS1 were designed based on primer sites described by

Martin et al. [12], and the primers of 1D were described by Hsu et al. [13]; 0.5 μl of genomic DNA was amplified in a volume of approximately 20-μl system including 6 μl primers, 6.6 μl PCR loading dye mix (Takara, Kyoto, Japan), 6.9 μl RNase Free (Takara). PCR was performed on Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). After an initial denaturation step at 94 °C for 1 min, PCR was used to increase specificity of primers annealing during the first 10 cycles, consisting of a melting temperature of 94 °C for 30 s and an annealing temperature of 65 °C for 30 s, followed by 20 cycles were performed at a melting temperature of 94 °C for 30 s, an annealing temperature of 62 °C for 30 s and an extension temperature of 72 °C for 40 s. At last, an extra extension step was preformed at 72 °C.

Livers were perfused with 10 ml of phosphate-buffered


Livers were perfused with 10 ml of phosphate-buffered

saline (PBS) via the portal vein to remove circulating lymphocytes. Liver and spleen single-cell HSP inhibitor suspensions were prepared from whole tissue by mechanical disruption in RPMI-1640/2% (v/v) fetal bovine serum (FBS). Bulk liver non-parenchymal cells (NPC) were enriched by density centrifugation using Histodenz (Sigma, St Louis, MO, USA). B cells were purified by CD19-positive selection using the magnetic affinity cell sorting (MACS) system (Miltenyi Biotec, Auburn, CA, USA). mDCs were purified as described [18]. Briefly, liver and spleen cells were depleted of NK1·1+, CD3+, CD19+ and/or plasmacytoid dendritic cell antigen-1 (PDCA-1)+ cells, followed by positive selection of CD11c+ cells using the MACS system (Miltenyi Biotec). B cells were isolated from wild-type mice 18 h after LPS [100 μg/kg intraperitoneally (i.p.); Alexis Biochemistry, San Diego, CA, USA] or PBS administration. In some experiments, mice were given poly I:C

(4 mg/kg, i.p.) for this website 18 h. The purity of mDCs and B cells was consistently > 90%. mDCs were isolated from wild-type and B cell-deficient μMT mice given the endogenous DC poietin fms-like tyrosine kinase 3 ligand (Flt3L) (10 μg/mouse/day; i.p. for 10 days; Amgen, Thousand Oaks, CA, USA), with either PBS or LPS (100 μg/kg, i.p.) treatment for the last 18 h. B cell-depleted liver NPCs were stimulated with LPS (10 ug/ml) for 48 h in the presence or absence of liver or spleen B cells. Activation of mDCs was determined by the level of expression of CD80, CD86 and programmed cell death 1 TCL ligand 1 (PD-L1) (B7-H1; CD274) on CD19–B220–CD11c+ cells. Single-cell suspensions were blocked for 10–15 min with anti-CD16/32 followed by staining with a fluorescent-tagged antibody mixture

directed against the cell surface markers CD1d, CD3, CD5, CD19, CD23, CD24, CD39, CD40, CD80, CD86, PD-L1, B220, CR1/2, immunoglobulin (Ig)M and IgD (BD PharMingen, Franklin Lakes, NJ, USA or BioLegend, San Diego, CA, USA). Data were acquired on a LSR II or LSR Fortessa (BD Bioscience, San Jose, CA, USA) and analysed with FlowJo software (Tree Star, Ashland, OR, USA). Purified B cells were cultured with or without 500 ng/ml phorbol myristate acetate (PMA), 1 μM ionomycin and 10 μg/ml LPS; purified mDCs were cultured with or without 10 μg/ml LPS. The cells were maintained for 48 h at 37°C in RPMI-1640 supplemented with 50 μM 2-mercaptoethanol (ME), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Supernatants were collected and cytokine production measured using a cytometric bead assay (CBA) Flex Set system (BD Bioscience) and analysed using FCAP Array Software (BD Bioscience). Bulk splenocytes and liver non-parenchymal cells (NPC) were activated for 5 h with 10 μg/ml LPS, 500 ng/ml PMA (Sigma) and 1 μM ionomycin (Sigma) in the presence of GolgiStop (BD Bioscience), followed by staining with fluorescent-labelled CD19 monoclonal antibody (mAb).