We have demonstrated that there is a cuff of adipose tissue around the origin of nutrient arterioles, isolated from cremaster muscles from obese Zucker rats [83,125]. Using a variety of insulin signaling pathway inhibitors, we have shown that in these animals, the PI3K insulin signaling pathway is impaired, and NO production is suppressed [83]. This has led us to propose that

in states of obesity, perivascular fat may signal to the vessel wall, both learn more locally (paracrine) and downstream (vasocrine), through outside-to-inside signaling [125]. Perivascular fat around nutrient arterioles may inhibit the effects of systemic insulin on local vasodilatation, with consequent inhibition of nutritive blood flow and insulin action. Recently, some evidence has been published in support of the hypothesis that obesity-related changes

in adipose tissue have direct effects on the vasoactive properties of perivascular adipose tissue [35]. Small arteries with and without perivascular adipose tissue were taken from subcutaneous gluteal fat biopsy samples and studied with wire myography and immunohistochemistry. It was demonstrated that healthy adipose tissue around human small arteries secretes adiponectin that influences vasodilatation by increasing NO bioavailability. https://www.selleckchem.com/products/CAL-101.html However, in perivascular fat from obese subjects with metabolic syndrome, the loss of this dilator effect was accompanied by an increase in adipocyte area and immunohistochemical evidence of inflammation, with increased activity of TNF-α. In isolated resistance arteries of the rat

cremaster muscle, we could demonstrate that adiponectin influences insulin signaling in the endothelium by activating AMPK in microvascular endothelium, and inhibiting insulin’s vasoconstrictor effects, leading to overall insulin-mediated vasodilatation [28]. In concordance with these findings, other preliminary data in mice suggest Tyrosine-protein kinase BLK that PVAT controls insulin-mediated vasodilatation in muscle arterioles by secreting adiponectin (abstract, 9th World Congress for Microcirculation, 2010). This mechanism is impaired in db/db mice, leading to impaired insulin-mediated vasodilatation. The possible origins and driving forces behind the deposition of PVAT are currently under investigation. In conclusion, elevated FFA and TNF-α concentrations and decreased adiponectin concentrations are likely candidates to link (perivascular) adipose tissue with defects in microvascular function, at least in part, by influencing insulin signaling and thereby insulin’s vascular effects. Obesity has been implicated in the rising prevalence of the metabolic syndrome, a cluster of risk factors including, hypertension, insulin resistance, which confer an increased risk for type 2 diabetes and CVD.

These Galunisertib solubility dmso results showed a shift of FEZ1 expression from dopamine neurones in sham-lesioned rats to astrocytes in PD rats. Parkinson’s disease is the second most prevalent age-related neurodegenerative disease and leads to a worldwide social burden. The aetiology and pathogenesis of PD have been extensively investigated for the past several decades, and although genetic and epigenetic factors have been recognized to lead to the initiation and progression of PD, an effective treatment for the disease remains elusive

[36]. It has been shown that animal PD models, which simulate the clinical features of PD, are a useful way to examine the pathophysiology of PD, its treatment and the underlying molecular mechanism. A unilateral injection of 6-OHDA in the MFB simulates the progressive pathological process of PD [37, 38]. 6-OHDA has high affinity at the dopamine transporter, which carries the toxin into the dopaminergic neurones and selectively kills dopaminergic neurones by generating ROS, such as superoxide radicals selleck inhibitor [39]. The unilateral damage to the intrastriatal-nigrostriatal dopaminergic system by 6-OHDA injection is followed by a reduction of dopamine levels in striatum and an ipsilateral upregulation of dopaminergic

postsynaptic receptors. These changes produce a prominent functional and motor asymmetry that can be evaluated by dopaminergic agonists such as apomorphine [40, 41], and motor asymmetry is considered a reliable indicator of nigrostriatal dopamine depletion [42, 43]. The contralateral rotations experienced by the 6-OHDA-lesioned rats in our study demonstrated that the deficits in the dopaminergic

system were progressive from 2 to 5 weeks after lesion. Most investigations into the aetiology and pathogenesis of PD have focused on the degeneration of dopamine neurones. However, it has been gradually recognized that astrocyte activation and hyperplasia are important and easily overlooked phenomena in PD pathogenesis [8]. Activated astrocytes have high expression levels of GFAP, HSP90 have enhanced metabolism, release a series of cytokines, and increase cell processes that envelope damaged and degenerating neurones. Furthermore, astrocytes seem to be involved in the formation of synapses and in modulating synaptic function through bidirectional communication with neurones [44]. It caused the activation of astrocytes with increased levels of GFAP in striatum and substantia nigra of PD models. Similarly, our results showed that GFAP expression levels were elevated at 2–5 weeks in the PD group compared with GFAP expression levels in the sham group. Emerging evidence suggests that FEZ1 is closely related to dopaminergic neurone differentiation and dopamine release, but it is still unclear what role FEZ1 plays in PD.

1; [12, 21, 22]). The role of IRFs in regulating IFN-β and IL-6 expression following CpG stimulation Selleckchem AZD3965 of CAL-1 cells was examined by nuclear translocation assays

and transient knockdown experiments (Fig. 2 and 4). Previous reports showed that IRFs 3 and 7 were the main inducers of type I IFN following virus infection of human pDCs [1, 17, 41, 48]. Yet, neither of those IRFs was involved in the gene activation induced by “K” ODN (Fig. 4). Rather, “K” ODN induced the rapid translocation of IRF-5 from the cytoplasm to the nucleus, followed several hours later by the translocation of IRF-1 (Fig. 2A and B). siRNA-mediated knockdown studies confirmed that IRF-5 but not IRF-1 played a central role in regulating “K” ODN mediated IFN-β and IL-6 mRNA expression (Fig. 4). Experiments involving IRF-5 KO mice showed that the induction of IL-6 but not type I IFN was impaired in CpG-stimulated pDCs [15]. Yet, Paun et al. [45] reported Inhibitor Library that IFN-β mRNA declined when DCs from IRF-5 KO mice were stimulated with “K” ODN. Due to differences in the splice patterns of murine versus human IRF-5, it was unclear whether the murine results would be applicable to human

pDCs [47]. Current findings clarify that IRF-5 plays a critical role in the upregulation of IFN-β and IL-6 in CpG-stimulated human pDCs. Evidence that MyD88 associates with IRF-5 in the cytoplasm was previously provided by studies involving murine HEK293T cells that overexpressed both proteins [15]. The current work examined this

issue by transfecting CAL-1 cells with HA-tagged MyD88. Immunoprecipitation using anti-HA Ab provided the first evidence that endogenous IRF-5 as well as IRF-7 physically interacted with MyD88 under physiologic conditions in human pDC-like cells. Importantly, “K” ODN stimulation led to a significant decline in the amount of IRF-5 that co-precipitated with MyD88 (Fig. 5). This observation is consistent with the data showing that IRF-5 (but not IRF-7) translocates from the cytoplasm to the nucleus of “K” ODN activated CAL-1 cells (Fig. 2 A and B). Controversy exists regarding Silibinin the role of IRF-1 in CpG-mediated gene activation [16, 49]. Schmitz et al. [16] observed that cytokine production was impaired in CpG-treated DCs from IRF-1 KO mice and concluded that IRF-1 contributed to the subsequent upregulation of IFN-β. In contrast, Liu et al. [49] reported that “K” ODN actively inhibited the binding of IRF-1 to the IFN-β promoter of murine DCs, thereby preventing the upregulation of type I IFN. Current findings indicate that IRF-1 accumulates in the nucleus of CpG-stimulated CAL-1 cells, but that this is a relatively late event (Fig. 2A and B) mediated by an increase in mRNA influenced by type 1 IFN feedback (Fig. 2C). In this context, the knockdown of IRF-1 had no impact on early or late IFN-β and IL-6 expression (Fig. 4B and C). Thus, current findings lead to a reinterpretation of the results of Schmitz et al. and Liu et al.

Quantitative analysis was performed after densitometric scanning, and the results were normalized to internal control GAPDH. Immunofluorescence

staining of STIM1 translocation in RPMCs was performed as described previously [22]. Briefly, after fixation, permeabilization and blocking, the cells were incubated with rabbit anti-rat STIM1 antibody (1:100 dilution) at 4 °C overnight. Subsequently after three washes with PBS, the cells were incubated with FITC-conjugated secondary antibody (goat anti-rabbit IgG, 1:1000) for 1 h at room temperature. Signals were then detected by Olympus 1000 confocal microscope (Olympus, Japan). Control staining was carried out with non-immune IgG used at the selleck chemicals same concentration as the primary antibody. Six randomly selected fields in each sample in an individual experiment were scored, and at least three independent experiments were performed. The contents of tumour necrosis factor-α (TNFα), interleukin-4 (IL-4), interleukin-10 (IL-10), interferon-g (IFNγ) and histamine in rat peritoneal lavage solution (RPLS) and serum were assayed by commercial ELISA kits using paired antibodies according to

the manufacturer’s instructions. The kits for detecting TNF-α, IL-4, IL-10 and IFN-γ were bought from eBioscience (USA), and the kit for detecting histamine was bought from selleck screening library R&D Inc. (Minneapolis, MN, Minneapolis, MN, USA). Serum IgE levels were also detected using a commercial ELISA kit (BD Biosciences Pharmingen,

San Jose, CA, USA), following the manufacturer’s Lenvatinib chemical structure instructions. Data are presented as means ± SD. When two comparisons were obtained, Student’s unpaired two-tailed t test was used. When multiple comparisons were obtained, the analyses consisted of one-way anova for repeated measures and Student–Newman–Keuls multiple comparison test. A value of P < 0.05 was considered to be statistically significant. In the present study, we used OVA oral sensitization to establish food-allergic model in Brown-Norway rats as previously reported [17]. The cytokine levels in RPLS were measured by ELISA. The results showed that type Th2 cytokines (IL-4, 11.8 ± 1.52 pg/ml; IL-10, 101.3 ± 15.37 pg/ml) were significantly higher than those in control groups (IL-4, 3.73 ± 0.18 pg/ml;IL-10, 61.66 ± 8.33 pg/ml; Fig. 1A). However, the concentrations of type Th1 cytokines, including IL-2 and IFNγ, were similar to those in control group. The above results indicate that the ratio of Th1/Th2 was decreased, and the balance of Th1/Th2 was skewed in OVA-induced food-allergic model. ELISA analysis showed that the concentrations of OVA-specific IgE in both serum and RPLS were significantly higher (0.23 ± 0.03 versus ctrl 0.16 ± 0.01 μg/ml in serum; 0.45 ± 0.04 versus ctrl 0.37 ± 0.01 μg/ml in RPLS) in OVA-induced food-allergic group (Fig. 1B).

An even more pronounced age-inappropriate decline of newly generated T cells associates with rheumatoid arthritis suggesting that

premature decline of thymic activity might be a common feature in these and other autoimmune disorders 7. The cytokine interleukin-7 (IL-7), a pleiotropic hematopoietic growth factor, is known to stimulate the thymus and to promote the differentiation and maintenance of naïve T cells including Treg 8–10. Signaling from IL-7 occurs through the heterodimeric IL-7 receptor (IL-7R), which is expressed on lymphocytes and consists of the α-chain subunit (IL-7Rα) and the common cytokine γ-chain. The importance of this pathway for naïve T-cell homeostasis is underlined by several recent studies showing that expression levels of membrane-bound IL-7Rα www.selleckchem.com/products/bay80-6946.html (CD127) on conventional CD4+ T cells correlate

with frequencies of recent thymic emigrant (RTE)-CD4+ T cells in healthy individuals and HIV-infected patients as well as in patients with MS 11, 12. IL-7Rα is also a component of the receptor for thymic stromal lymphopoietin (TSLP). The secretion of TSLP by Hassall’s corpuscles, structures composed of epithelial cells in the thymic medulla, has been demonstrated to condition CD11c+ myeloid dendritic cells (MDCs) to induce the differentiation of thymocytes into Treg 13. Accordingly, signals from the IL-7 receptor are required for Treg development Selleckchem GSK126 as shown in IL-7Rα knockout mice 14. Of note, a single nucleotide polymorphism (rs6897932-SNP) within the gene encoding the IL-7Rα chain (IL-7RA) has shown genetic association with human

autoimmunity and was found to be associated with MS, type 1 diabetes and chronic inflammatory arthropathies 15–19. Carnitine palmitoyltransferase II This SNP causes a change from threonine to isoleucine at amino acid position 244 that modifies the ratio of membrane-bound to soluble IL-7R 15, 20. In this study, we attempted to decipher in more detail the impact of IL-7/IL-7R signaling components on Treg homeostasis and Treg-suppressive function. We used peripheral blood and plasma samples from 56 treatment-naïve patients with relapsing remitting MS (RRMS) and 33 healthy individuals (HC) to analyze IL-7Rα-expression on total CD4+CD25−/lowCD127+FOXP3− conventional T cells (Tconv) and Tconv subsets together with plasma concentrations of soluble IL-7Rα (sIL-7Rα) and IL-7 as well as genotype screening for rs6897932-SNP. In parallel, we determined frequencies, phenotypes and suppressive activities of donor and patient-derived Treg. Treg obtained from both cohorts were further characterized as to quantities of cells harboring two T-cell receptor (TCR) Vα chains. Cells expressing TCRs with dual specificity on their surface are enriched in the Treg compartment and as this feature is acquired during T-cell maturation in the thymus, their proportions among total Treg should roughly correlate with the natural Treg lineage 21.

Treg cells have been implicated in infectious diseases, particularly in chronic or persistent infections 34, 35, but check details discordant results were found ex vivo in terms of Treg expansion during active TB disease, with some authors reporting an increase of CD4+ CD25+FoxP3+ T cells, and other reported the absence of modulation of this T-cell subset 36–40. Moreover, a recent study found that depletion of CD4+ CD25highCD39+ increased M. tuberculosis-specific responses, as well as other recall antigens responses, indicating that Treg broadly modulate antigen-specific immunity 41. In conclusion, this

study shows that active TB disease is associated with an increase in the proportion of 3+ “multifunctional” CD4+ T lymphocytes capable of simultaneously producing IFN-γ, IL-2 and TNF-α, but a relative paucity of CD4+ T cells that produce either both IFN-γ and IL-2, or IFN-γ alone, when compared with the pattern of cytokine produced by CD4+ T cells from LTBI subjects. Strikingly, this pattern of cytokine production seems to be associated with bacterial loads and disease

activity as it reverses 6 months after therapy. These different functional signatures of CD4+ T cells could be used as immunological markers of mycobacterial load to monitor the response to treatment, to evaluate new therapies selleck for active tuberculosis and the efficacy of new vaccines in clinical trials where new biomarkers are needed. Moreover, phenotypic and functional signatures of CD4+ T cells could also be used to monitor individuals LTBI at a high risk of progression to active TB, such as those with HIV coinfection or on anti-TNF therapy. Peripheral blood was obtained from 20 adults with TB disease (11 men, 9 women, age range 46–55 years) from the Dipartimento

di Medicina Clinica e delle Patologie Emergenti, University Hospital, Palermo, and Monaldi Hospital, Naples, Italy, 18 LTBI subjects (10 men, 8 women, age range 38–52 years) and 15 tuberculin (PPD)-negative healthy subjects (8 men and 7 women, age range 41–55 years). Morin Hydrate TB-infected patients had clinical and radiological findings consistent with active pulmonary TB 42. Diagnosis was confirmed by bacteriological isolation of M. tuberculosis in 18 patients. Two further patients were classified as having highly probable pulmonary TB on the basis of clinical and radiological features that were highly suggestive of TB and unlikely to be caused by any other disease; the decision was made by the attending physician to initiate anti-TB chemotherapy, which resulted in an appropriate response to therapy. All patients were treated in accordance with Italian guidelines and received therapy for 6 months. Treatment was successful in all participants all of whom completed the full course of anti-TB chemotherapy, as evidenced by the absence of any clinical or radiographic evidence of recurrent disease and sterile mycobacterial cultures. Peripheral blood was collected before (TB-0) and after completion of chemotherapy (TB-6).

5). Case 5. IgA nephropathy A 50-year-old man presented with significant proteinuria, 5 years post diagnosis of T2DM. His medical history included obesity, hypertension and hyperlipidaemia. Urinary protein excretion was 11 g/day, with normal eGFR and active urinary sediment. HbA1C was

8%. Renal biopsy showed features of mesangial proliferative IgA nephropathy www.selleckchem.com/products/acalabrutinib.html with chronic tubulointerstitial damage and nephrosclerosis (Fig. 6). Case 6. Membranous nephropathy and anti-GBM disease7 A 22-year-old male with T1DM presented with nephrotic syndrome (urinary protein excretion 14 g/day, serum albumin 23 g/L), acute kidney injury (serum creatinine 387 μmol/L) and active urinary sediment (>1000 × 106/L dysmorphic erythrocytes). Renal biopsy showed focal segmental necrotizing glomerulonephritis on a background of moderate nodular mesangial expansion and hypercellularity with several showing Kimmelstiel–Wilson nodules (Fig. 7). Immunofluorescence showed strong linear GBM staining for IgG. Electron microscopy showed Stage 1 membranous nephropathy with small subepithelial electron dense ‘immune-type’ deposits with GBM membrane spike formation. The earliest clinical evidence of classical DKD is the appearance of microalbuminuria

GDC973 (≥ 30 mg/day or 20 μg/min). Without specific interventions, up to 80% of T1DM patients with sustained microalbuminuria develop overt proteinuria (≥300 mg/day or ≥200 μg/min) over 10–15 years.[8-10] ESRD develops in 50% of T1DM patients with overt proteinuria within 10 years and in >75% by 20 years. A higher proportion of T2DM individuals are found to have established proteinuria at the time of diagnosis of their diabetes due to the delay in the diagnosis of diabetes. Without specific interventions, up to 40% of T2DM patients Amino acid with

microalbuminuria progress to overt nephropathy, but by 20 years after onset of overt nephropathy, only approximately 20% will progress to ESRD.[11] The exact reasons why an individual with diabetes will progress to develop DKD and then subsequently develop ESRD still remain to be fully defined. Despite this, there is most likely a strong genetic determinant for the risk of developing DKD and ESRD. Indeed, recent genomic-wide linkage studies have described the localization of quantitative trait loci that influence GFR in diabetes.[12, 13] These findings may help to further elucidate the genetic susceptibility to the development of advanced DKD. The spectrum of histologic changes seen in DKD is variable. In 2010, a new pathological classification of DKD was proposed for patients with diabetes,[14] based on glomerular features: Class I: Glomerular basement membrane (GBM) thickening, diagnosed by transmission electron microscopy. Class II: Mesangial expansion – A: mild; B: severe. Class III: Nodular glomerulosclerosis (Kimmelstiel–Wilson lesion). Class IV: Advanced diabetic glomerulosclerosis (>50% global glomerulosclerosis).

In addition, we have noted increased venous KV2.1, an important player in the HPV response, in FGR [11]; however, whether altered expression is a cause or effect of disease remains unclear. The lack of an obvious “K+ channelopathy” in FGR suggests the latter is the more likely, but this requires confirmation. Application of KATP channel activators, potent vasodilators

of chorionic plate Dabrafenib order arteries and chorionic plate veins, in vessels obtained from pathological pregnancies will be of especial interest. In the pregnancy complication PE (late pregnancy hypertension and proteinuria), adenosine, a nucleoside suggested to modify vascular tone via modified KATP channel function and nitric oxide release, is increased in umbilical venous blood [74]. This may represent a physiological response to maintain a high-flow/low-resistance fetoplacental circulation.

In placentas from pregnancies complicated by diabetes mellitus [4], KATP function is also impaired. Unfortunately, the application of KATP channel modulators to stimulate arterial/venous vasodilatation has not been documented PD-0332991 ic50 in PE, FGR, or diabetes mellitus. A more likely trigger leading to abnormal K+ channel activity in FGR is via production of ROS. ROS regulate K+ channel physiological function [48, 24], and increased ROS generation contributes to systemic cardiovascular pathology (e.g., coronary atherosclerosis) [24]. Mills et al. noted acute/chronic ROS-induced modification of isolated fetoplacental vessel reactivity [46]; similar processes are therefore apparent in the placenta. It is well known that oxidative stress/ROS are increased in PE/FGR, [64] and therefore the activity of K+ channels present in

the placental vasculature could be altered by increased ROS; unfortunately this tenet has not been directly assessed. Future placental vascular function studies should focus on: (i) demonstrating whether K+ channels’ responses to applied ROS are altered in pathological samples and; (ii) assessing if exposure to pharmacological and/or dietary antioxidant treatments modifies K+ channel activity. Putative K+ channel modulators application to vessels from PE/FGR placentas would also be extremely informative. In summary, these findings highlight the lambrolizumab need for future studies of placental vascular K+ channels to include data from compromised pregnancies to confirm/negate the role of these channels as the primary pathogenic stimulus. Our knowledge of how human fetoplacental blood flow is controlled is rudimentary compared with our understanding of systemic and pulmonary vascular beds. Local factors such as tissue oxygenation are thought to play key roles. Indeed, HFPV has been suggested but not definitively demonstrated. Inconsistent findings in isolated vessel studies have failed to resolve this controversy. K+ channels are expressed in human fetoplacental vascular tissues.

Interaction with non-pathogenic E. coli HB101 did not induce localization of TLR5 on the cell surface (Figure S2). These results are consistent with the FACS experiments, where almost all TLR5 was located in intracellular compartments. In contrast, in cells infected with EPEC strains, E2348/69 and E22, TLR5 was clearly detected on the cell

surface (Figure S2). These results confirmed that EPEC infection induces TLR5 re-localization towards the cell surface. Infection with any of the E22 mutant was unable to provoke TLR5 detection on the epithelial cell surface (Figure S2). These results indicate that EPEC T3SS and flagellum participate in the re-localization of TLR5 towards the cellular surface. Notably, in these assays intimin appeared to be necessary KU-57788 for the re-localization of TLR5, a more obvious result than the one obtained with FACS. To know if the localization of another receptor besides TLR5 is altered during EPEC infection, we inquired about TLR4 subcellular

distribution in non-infected cells and in cells infected with E2348/69 during 4 h by examination of immunofluorescent preparations (Figure S3). In mock cells, we found TLR4 equivalent signal intensity and distribution selleck chemicals in permeabilized and in non-permeabilized cells (total and surface TLR4). This indicates that TLR4 is mainly located at the surface of HT-29 cells, which was also true for E2348/69 cells. Therefore, EPEC infection does not affect TLR4 distribution, unlike TLR5 recruitment to the cell surface that was induced by EPEC infection. ERK1/2 signalling pathway (phosphorylation and nuclear translocation) is an important activator of cellular proinflammatory responses. ERK1/2 phosphorylation during EPEC infection (at 2 or 4 h) was detected by WB. Phosphorylated ERK1/2 was not detected in mock-treated cells (normalized band intensity value of 0.026 ± 0.045). HB101 interaction

induced phosphorylation of ERK1/2 (0.673 ± 0.108) but only until 4 h post-interaction. However, in EPEC-infected cells, p-ERK1/2 was clearly detected (Fig. 2A). At 2 h post-infection, both EPEC strains caused equivalent phosphorylation of ERK1/2 (0.737 ± 0.246 for E2348/69 and 0.741 ± 0.064 for E22 infection). However, at 4 h, p-ERK1/2 was stronger during E22 infection (E2348/69: 0.643 ± 0.089 and E22: SDHB 1.01 ± 0.126). Therefore, we confirmed that ERK1/2 phosphorylation in epithelial cells is caused by EPEC E2348/69 infection and found that it was also true for E22. To understand the role of EPEC virulence factors on the phosphorylation of ERK1/2, we performed WB analysis of lysates from cells infected for 4 h with the isogenic EPEC mutants E22 Δeae, ΔescN, ΔespA or ΔfliC (Fig. 2B). Cells infected with T3SS mutants induced ERK1/2 phosphorylation at levels not significantly different than the ones produced by WT infection (1.01 ± 0.126); normalized band intensity values were 1.186 ± 0.207 for E22ΔescN and 1.025 ± 0.209 for E22ΔespA.

The pro-tumorigenic property of the NLRP3 inflammasome may be more related to its pro-inflammatory activity associated with IL-1β and IL-18 release. Further studies will be required to clarify the exact function played by the NLRP3 inflammasome in the DDR pathway. The anti-proliferative and pro-apoptotic functions of NLRP3 have been reported both here and elsewhere [39, 40], but the proposed oncosuppressive activity of the NLRP3 inflammasome now requires confirmation at least in alternative models

of inflammation-induced cancer. In summary, we have shown that MSU-induced DNA damage activated the NLRP3 inflammasome in a priming-independent manner, supported oxidative stimulation of DDR, and promoted p53 activation and subsequent cell death. These new roles identified for the NLRP3 inflammasome in suppressing DNA repair www.selleckchem.com/products/epacadostat-incb024360.html and enhancing p53-mediated apoptosis of innate cells will open new avenues of research that clarify the role of NLRP3 in diseases associated with aberrant cell death. C57BL/6 mice were purchased from the Biological Resource Center (BRC, A*STAR, Singapore).

Nlrp3−/− mice were kindly provided by J. Tschopp (University of Lausanne, Switzerland) [7] and casp-1−/− mice were a generous gift from R. A. Flavell [41]. All experiments were conducted with age-matched mice (8–12 weeks of age), and all mutants were backcrossed to C57BL/6 background for at least ten generations. Animals were bred under specific pathogen-free conditions at the BRC (Singapore). Experiments were performed under the approval of the Institutional Animal Care & Use Committee in compliance with the Law and Guidelines Acalabrutinib mw for Animal Experiments Carnitine palmitoyltransferase II of the BRC, Singapore. BM-derived DCs (BMDCs) from 8- to 12-week-old C57BL/6 mice and Nlrp3−/− and casp-1−/− mice were prepared

as previously described [8]. Cells (1 × 106 cells/mL) were stimulated in complete medium (IMDM with 10% FBS) in 96-well plates (Corning) and exposed to MSU crystals (250 μg/mL, Alexis), silica (silicon dioxide, 250 μg/mL, Sigma), ultrapure LPS (1 μg/mL, Alexis), camptothecin (1 μM, Sigma), rotenone (10 μM, Sigma), and H2O2 (100 mM, Sigma) for the indicated times. MSU preparations were assayed using the limulus amebocyte lysate test and were endotoxin free. For radiation experiments, BMDCs were rested for 24 h before being subjected to 4 or 10 Gy of γ-radiation and harvested after 8 or 24 h. RNA was extracted from three biological replicates as previously described [8], and 8 μg total RNA was used for cRNA target preparation following the Affymetrix GeneChip expression analysis technical manual (Affymetrix, Santa Clara, CA, USA). Biotinylated cRNA (15 μg) was hybridized to 12 Affymetrix GeneChip Mouse Genome MOE430 2.0. using the one-cycle target-labeling kit according to the manufacturer’s instructions. Microarray analysis was performed using R language and Bioconductor software [42].