Investigation on the association between inerleukin-10 -592C/A, 819C/T and -1082A/G gene polymorphisms and development of diabetic nephrophathy

We conducted a case-control study to investigate the association between interleukin (IL)-10-592C/A, -819C/T and -1082A/G polymorphisms and susceptibility to diabetic nephropathy. A hospital-based case-control study was taken in our study. A total of 172 patients with proven type 2 diabetes mellitus and 344 controls were recruited from the First Affiliated Hospital of Xinxiang Medical University between March 2012 and October 2014. Genotyping of IL-10 -592C/A, -819C/T and -1082A/G polymorphisms was done by done by PCR-RFLP methods. By the χ² test, the distributions of the GG, GA and AA genotypes in IL-10 -1082A/G were significantly different between patients with diabetic nephropathy and control subjects (χ² = 8.09, P = 0.02). By conditional logistic regression analysis, we found that the AA genotype of IL-10 -1082A/G was associated with an elevated risk of diabetic nephropathy compared to the GG genotype in codominant model, and the adjusted OR (95% CI) was 2.38 (1.23-4.57). In dominant model, the GA+AA genotype was associated with a significantly increased risk of diabetic nephropathy compared to the GG genotype in dominant model (OR = 1.47, 95% CI = 1.05-2.16). In recessive model, the AA genotype could influence the susceptibility to diabetic nephropathy when compared with the GG+GA in recessive model (OR = 2.08, 95% CI = 1.12-3.85). In conclusion, we suggested that IL-10 -1082A/G gene polymorphism was correlated with development of diabetic nephropathy, but no association was observed between IL-10 -819T/C and -592A/C and risk of diabetic nephropathy.     

Porphyromonas gingivalis Fimbriae Use β2 Integrin (CD11/CD18) on Mouse Peritoneal Macrophages as a Cellular Receptor, and the CD18 β Chain Plays a Functional Role in Fimbrial Signaling

In this study, we demonstrate that Porphyromonas gingivalis fimbriae use molecules of β₂ integrin (CD11/CD18) on mouse peritoneal macrophages as cellular receptors and also show that the β chain (CD18) may play a functional role in signalling for the fimbria-induced expression of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) genes in the cells. Using a binding assay with ¹²⁵I-labeled fimbriae, we observed that fimbrial binding to the macrophages was inhibited by treatment with CD11a, CD11b, CD11c, or CD18 antibody but not by that with CD29 antibody. Western blot assays showed that the fimbriae bound to molecules of β₂ integrin (CD11/CD18) on the macrophages. Furthermore, Northern blot analyses showed that the fimbria-induced expression of IL-1β and TNF-α genes in the cells was inhibited strongly by CD18 antibody treatment and slightly by CD11a, CD11b, or CD11c antibody treatment. Interestingly, intracellular adhesion molecule 1 (ICAM-1), a ligand of CD11/CD18, inhibited fimbrial binding to the cells in a dose-dependent manner. In addition, ICAM-1 clearly inhibited the fimbria-induced expression of IL-1β and TNF-α genes in the cells. However, such inhibitory action was not observed with laminin treatment. These results suggest the importance of β₂ integrin (CD11/CD18) as a cellular receptor of P. gingivalis fimbriae in the initiation stage of the pathogenic mechanism of the organism in periodontal disease.     

The PPAR gamma 2 Pro12Ala variant predicts ESRD and mortality in patients with type 1 diabetes and diabetic nephropathy

The Pro12Ala polymorphism in the peroxisome proliferator-activated receptor-γ2 gene is suggested to associate with diabetic nephropathy and cardiovascular disease in type 2 diabetes. The aim of this study was to investigate the polymorphism in relation to diabetic nephropathy, end-stage renal disease (ESRD), mortality and cardiovascular (CVD) events in type 1 diabetic patients.This prospective observational follow-up study included 415 type 1 diabetic patients with overt diabetic nephropathy (252 men; age 42.2 ± 10.4 years [mean ± SD], duration of diabetes 28.3 ± 8.8 years, GFR 66 ± 8.8 ml/min) and 428 patients with longstanding type 1 diabetes and persistent normoalbuminuria (230 men; age 45.4 ± 11.6 years, duration of diabetes 27.8 ± 10.1 years). Follow-up: 8.1 (0.0–12.8) years (median [range]).There where no significant differences between cases and controls in genotype (p = 0.51) or allele frequencies (p = 0.25). Cox regression analysis revealed a covariate-adjusted hazard ratio (HR) for all-cause mortality in patients with the Ala/Ala genotype of 2.44 (1.23–4.84). The Pro12Ala polymorphism did not predict CVD events. However, the Ala/Ala genotype predicts ESRD (covariate-adjusted HR 2.60 (1.11–6.07)). Furthermore, Carriers of the Ala-allele had a higher rate of decline in GFR (p = 0.040).In conclusion, the Pro12Ala polymorphism is not associated with type 1 diabetic nephropathy. The Ala-allele is associated with enhanced decline in GFR and predicts ESRD and all-cause mortality in patients with nephropathy.     

Differential Regulation of Proinflammatory Cytokine Expression by Mitogen-Activated Protein Kinases in Macrophages in Response to Intestinal Parasite Infection

Blastocystis is a common enteric protistan parasite that can cause acute, as well as chronic, infection and is associated with irritable bowel syndrome (IBS). However, the pathogenic status of Blastocystis infection remains unclear. In this study, we found that Blastocystis antigens induced abundant expression of proinflammatory cytokines, including interleukin 1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), in mouse intestinal explants, in mouse colitis colon, and in macrophages. Further investigation utilizing RAW264.7 murine macrophages showed that Blastocystis treatment in RAW264.7 macrophages induced the activation of ERK, JNK, and p38, the three major groups of mammalian mitogen-activated protein (MAP) kinases that play essential roles in the expression of proinflammatory cytokines. ERK inhibition in macrophages significantly suppressed both mRNA and protein expression of IL-6 and TNF-α and mRNA expression of IL-1β. On the other hand, JNK inhibition resulted in reductions in both c-Jun and ERK activation and significant suppression of all three proinflammatory cytokines at both the mRNA and protein levels. Inhibition of p38 suppressed only IL-6 protein expression with no effect on the expression of IL-1β and TNF-α. Furthermore, we found that serine proteases produced by Blastocystis play an important role in the induction of ERK activation and proinflammatory cytokine expression by macrophages. Our study thus demonstrated for the first time that Blastocystis could induce the expression of various proinflammatory cytokines via the activation of MAP kinases and that infection with Blastocystis may contribute to the pathogenesis of inflammatory intestinal diseases through the activation of inflammatory pathways in host immune cells, such as macrophages.     

The association of Pro12Ala polymorphism in the peroxisome proliferator-activated receptor-gamma2 gene with the metabolic characteristics in Chinese women with polycystic ovary syndrome

Background: The Pro12Ala polymorphism in the peroxisome Proliferator-activated receptor-gamma2 PPARγ2) gene that account for metabolic dysfunction in women with polycystic ovary syndrome (PCOS) remain elusive. Aim: To explore the association between PPARγ2 gene pro12ala polymorphism and the metabolic characteristics in Chinese women with PCOS. Methods: PPARγ2 gene Pro12Ala polymorphism was assayed by PCR/RFLP methods in 120 Chinese women with PCOS and 118 normal subjects. All subjects were examined by anthropometry, lipid profile, sex hormone, oral glucose tolerance tests and insulin tolerance tests. Results: In PCOS patients, women with the non-Pro/Pro genotypes of the PPARγ2 gene Pro12Ala polymorphism showed statistically significantly higher fasting triglycerides (TG) levels and WHR value than those with the Pro/Pro genotype (P=.006 for both). There was no significant difference with PPARγ2 Pro12Ala polymorphism distributions between Chinese Han women with PCOS and controls. Conclusion: PPARγ2 gene Pro12Ala polymorphism was not supposed to be susceptible genes in PCOS. However, in PCOS patients, the PPAR-gamma Pro12Ala polymorphism may modulate the concentrations of serum fasting TG levels and fat-deposition in abdomen, respectively.     

Low and moderate doses of ionizing radiation up to 2 Gy modulate transmigration and chemotaxis of activated macrophages,provoke an anti-inflammatory cytokine milieu,but do not impact upon viability and phagocytic function

Benign painful and inflammatory diseases have been treated for decades with low/moderate doses of ionizing radiation (LD-X-irradiation). Tissue macrophages regulate initiation and resolution of inflammation by the secretion of cytokines and by acting as professional phagocytes. Having these pivotal functions, we were interested in how activated macrophages are modulated by LD-X-irradiation, also with regard to radiation protection issues and carcinogenesis. We set up an ex-vivo model in which lipopolysaccharide pre-activated peritoneal macrophages (pMΦ) of radiosensitive BALB/c mice, mimicking activated macrophages under inflammatory conditions, were exposed to X-irradiation from 0·01 Gy up to 2 Gy. Afterwards, the viability of the pMΦ, their transmigration and chemotaxis, the phagocytic behaviour, the secretion of inflammatory cytokines and underlying signalling pathways were determined. Exposure of pMΦ up to a single dose of 2 Gy did not influence their viability and phagocytic function, an important fact regarding radiation protection. However, significantly reduced migration, but increased chemotaxis of pMΦ after exposure to 0·1 or 0·5 Gy, was detected. Both might relate to the resolution of inflammation. Cytokine analyses revealed that, in particular, the moderate dose of 0·5 Gy applied in low-dose radiotherapy for inflammatory diseases results in an anti-inflammatory cytokine microenvironment of pMΦ, as the secretion of the proinflammatory cytokine interleukin (IL)-1β was reduced and that of the anti-inflammatory cytokine transforming growth factor (TGF)-β increased. Further, the reduced secretion of IL-1β correlated with reduced nuclear translocation of nuclear factor (NF)-κB p65, starting at exposure of pMΦ to 0·5 Gy of X-irradiation. We conclude that inflammation is modulated by LD-X-irradiation via changing the inflammatory phenotype of macrophages.     

Macrophage-elicited osteoclastogenesis in response to bacterial stimulation requires Toll-like receptor 2-dependent tumor necrosis factor-alpha production

The receptor activator of NF-κB ligand (RANKL) and the proinflammatory cytokines are believed to play important roles in osteoclastogenesis. We recently reported that the innate immune recognition receptor, Toll-like receptor 2 (TLR2), is crucial for inflammatory bone loss in response to infection by Porphyromonas gingivalis, the primary organism associated with chronic inflammatory periodontal disease. However, the contribution of macrophage-expressed TLRs to osteoclastogenesis has not been defined. In this study, we defined a requirement for TLR2 in tumor necrosis factor-alpha (TNF-α)-elicited osteoclastogenesis in response to exposure to P. gingivalis. Culture supernatant (CS) fluids from P. gingivalis-stimulated macrophages induced bone marrow macrophage-derived osteoclastogenesis. This activity was dependent on TNF-α and occurred independently of RANKL, interleukin-1β (IL-1β), and IL-6. CS fluids from P. gingivalis-stimulated TLR2^−/− macrophages failed to express TNF-α, and these fluids induced significantly less osteoclast formation compared with that of the wild-type or the TLR4^−/− macrophages. In addition, P. gingivalis exposure induced up-regulation of TLR2 expression on the cell surface of macrophages, which was demonstrated to functionally react to reexposure to P. gingivalis, as measured by a further increase in TNF-α production. These results demonstrate that macrophage-dependent TLR2 signaling is crucial for TNF-α-dependent/RANKL-independent osteoclastogenesis in response to P. gingivalis infection. Furthermore, the ability of P. gingivalis to induce the cell surface expression of TLR2 may contribute to the chronic inflammatory state induced by this pathogen.     

TNF-α-308G/A polymorphism associated with TNF-α protein expression in patients with diabetic nephropathy

Aims: This study was to clarify the regulated effects of TNF-α -308G/A polymorphism on TNF-α and investigate the relationship of -308G/A polymorphisms with diabetic nephropathy (DN) susceptibility. Methods: 86 DN patients and 94 healthy individuals were enrolled in our study. Polymerase chain reaction-sequence specific primer (PCR-SSP) detection technology was used to testify single nucleotide polymorphism (SNP) of TNF-α gene. Enzyme-linked immunosorbent assay (ELISA) was used to measure the content of TNF-α protein. Odds ratio (OR) with 95% CI were used to evaluate the association of TNF-α -308G/A polymorphism and DN susceptibility. Results: The level of TNF-α protein was much higher in DN patients compared to that of controls (P < 0.05). For TNF-α -308G/A, G/A genotype could increase the risk for DN (OR = 2.15, 95% CI = 1.08-4.30). Moreover, a allele frequency was found higher in cases compared with controls, which suggested that A allele served as an genetic-susceptibility factor for DN (OR = 1.89, 95% CI = 1.10-3.26). Further analysis indicated that level of TNF-α for individuals with mutant genotype (GA and AA) were higher than that of individuals with wild genotype (P < 0.05). However, AA genotype showed no effects on DN susceptibility (OR = 2.08, 95% CI = 0.56-7.33). Conclusion: TNF-α-308G/A polymorphism was associated with expression level of TNF-α and served as an genetic-susceptibility factor for DN.     

VCAM-1/α4β1 Integrin Interaction Is Crucial for Prompt Recruitment of Immune T Cells into the Brain during the Early Stage of Reactivation of Chronic Infection with Toxoplasma gondii To Prevent Toxoplasmic Encephalitis

Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4β1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8β, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4⁺ and CD8⁺ T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.     

Sulfasalazine prevents T-helper 1 immune response by suppressing interleukin-12 production in macrophages

Interleukin-12 (IL-12) plays a pivotal role in the development of T-helper 1 (Th1) immune response, which may be involved in the pathogenesis of chronic inflammatory autoimmune disorders. In this study we investigated the effects of sulfasalazine, a drug for treating inflammatory bowel disease and rheumatoid arthritis, on the production of IL-12 from mouse macrophages stimulated with lipopolysaccharide (LPS). Sulfasalazine potently inhibited the production of IL-12 in a dose-dependent manner, in part through the down-regulation of nuclear factor κB (NFκB) activation in IL-12 p40 gene. Activation of macrophages by LPS resulted in markedly enhanced binding activities to the κB site, which significantly decreased upon addition of sulfasalazine as demonstrated by an electrophoretic gel shift assay. Importantly, macrophages pretreated with sulfasalazine either in vitro or in vivo reduced their ability to induce interferon-γ (IFN-γ) and increased the ability to induce IL-4 in antigen-primed CD4⁺ T cells. From these results, sulfasalazine may induce the Th2 cytokine profile in CD4⁺ T cells by suppressing IL-12 production in macrophages, and sulfasalazine-induced inhibition of IL-12 production in macrophages may explain some of the known biological effects of sulfasalazine.     

Porphyromonas gingivalis Lipopolysaccharide Weakly Activates M1 and M2 Polarized Mouse Macrophages but Induces Inflammatory Cytokines

Porphyromonas gingivalis is associated with chronic periodontitis, an inflammatory disease of the tooth''s supporting tissues. Macrophages are important in chronic inflammatory conditions, infiltrating tissue and becoming polarized to an M1 or M2 phenotype. As responses to stimuli differ between these phenotypes, we investigated the effect of P. gingivalis lipopolysaccharide (LPS) on M1 and M2 macrophages. M1 and M2 polarized macrophages were produced from murine bone marrow macrophages (BMMϕ) primed with gamma interferon (IFN-γ) or interleukin-4 (IL-4), respectively, and incubated with a low or high dose of P. gingivalis LPS or control TLR2 and TLR4 ligands. In M1-Mϕ, the high dose of P. gingivalis LPS (10 μg/ml) significantly increased the expression of CD40, CD86, inducible nitric oxide synthase, and nitric oxide secretion. The low dose of P. gingivalis LPS (10 ng/ml) did not induce costimulatory or antibacterial molecules but did increase the secretion of IL-1α, IL-6, IL-12p40, IL-12p70, and tumor necrosis factor alpha (TNF-α). P. gingivalis LPS marginally increased the expression of CD206 and YM-1, but it did enhance arginase expression by M2-Mϕ. Furthermore, the secretion of the chemokines KC, RANTES, eotaxin, and MCP-1 from M1, M2, and nonpolarized Mϕ was enhanced by P. gingivalis LPS. TLR2/4 knockout macrophages combined with the TLR activation assays indicated that TLR2 is the main activating receptor for P. gingivalis LPS and whole cells. In conclusion, although P. gingivalis LPS weakly activated M1-Mϕ or M2-Mϕ compared to control TLR ligands, it induced the secretion of inflammatory cytokines, particularly TNF-α from M1-Mϕ and IL-10 from M2-Mϕ, as well as chemotactic chemokines from polarized macrophages.     

Protective effects of calcitriol on diabetic nephropathy are mediated by down regulation of TGF-β1 and CIP4 in diabetic nephropathy rat

Objective: To explore the protective effects of calcitriol on diabetic nephropathy by modulating the expressions of transforming growth factor-beta 1 (TGF-β1) and Cdc42 interacting protein-4 (CIP4). Methods: Streptozotocin-induced diabetic nephropathy rats (n=36) were randomly divided into control group (control-H, control-M, control-L) and calcitriol group (calcitriol-H, calcitriol-M, calcitriol-L). The expression of TGF-β1 gradually decreased in control-H, control-M and control-L subgroups by injection of different virus vectors. Peanut oil and calcitriol were given to control and calcitriol group, respectively. The expressions of TGF-β1 and CIP4 in kidney, the pathology, and the renal function and lipid profiles were compared between control and calcitriol treatment groups. Results: In the control group, the higher level of TGF-β1 was associated with more severe glomerular pathology (P<0.05). There is a positive correlation between the expression of CIP4 and TGF-β1. Control-H subgroup had significant more severe kidney disease, higher levels of cholesterol, triglyceride, blood glucose, blood urea nitrogen (BUN) and creatinine (Cr) than control-M and control-L subgroups. After calcitriol treatment, the expression of TGF-β1 and CIP4 were significantly decreased compared to the corresponding control subgroups (all P<0.05). Renal fibrosis and pathological changes were markedly improved. The levels of cholesterol, triglyceride, blood glucose, BUN and Cr were significantly reduced (P<0.05). Conclusion: Calcitriol may protect diabetic nephropathy from fibrosis via inhibition of TGF-β1 and CIP4.     

Requirement for TLR2 in the development of albuminuria,inflammation and fibrosis in experimental diabetic nephropathy

Inflammation and fibrosis are essential elements of diabetic nephropathy (DN). We tested the hypothesis that these elements are dependent upon Toll-like receptor 2 (TLR2) signalling by examining WT and TLR2^-/- mice in an experimental model of DN. Diabetes was induced in WT and TLR2^-/- mice by i.p. injection of streptozotocin. Kidney injury was assessed at 6, 12 and 24 weeks after induction of diabetes. Gene expression of TLR2, its endogenous ligands and downstream cytokines, chemokines and fibrogenic molecules were upregulated in kidneys from WT mice with streptozotocin diabetes. TLR2^-/- mice were protected against the development of DN, exhibiting less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic WT controls. Marked reductions in interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) were also evident in TLR2 deficient mice. Consistent with our in vivo results, high glucose directly promoted TLR2 activation in podocytes and tubular epithelial cells (TECs) in vitro, resulting in NF-κB activation, inflammation and TGF-β production. We conclude that TLR2 was required for the full development of inflammation, kidney damage and fibrosis in this model of DN. As TLR2 is known to be expressed by intrinsic kidney cells and as high concentration glucose stimulated podocytes and TECs in vitro to express TLR2 and TLR2 ligands, pro-inflammatory and pro-fibrotic cytokines in a TLR2 dependent manner in the present study, it appears likely that TLR2 signalling in intrinsic kidney cells contributes to the pathogenesis of diabetic nephropathy.     

Pre-treatment with IL-1β enhances the efficacy of MSC transplantation in DSS-induced colitis

Mesenchymal stem cells (MSCs) have been used experimentally for treating inflammatory disorders, partly due to their immunosuppressive properties. Although interleukin-1β (IL-1β) is one of the most important inflammatory mediators, growing evidence indicates that IL-1β signaling elicits the immunosuppressive properties of MSCs. However, it remains unclear how IL-1β signaling accomplishes this activity. Here, we focus on the therapeutic efficacy of IL-1β-primed MSCs in the dextran sulfate sodium (DSS)-induced colitis model, in addition to the underlining mechanisms. We first found that IL-1β-primed MSCs, without any observable phenotype change in vitro, significantly attenuated the development of DSS-induced murine colitis. Moreover, IL-1β-primed MSCs modulated the balance of immune cells in the spleen and the mesenteric lymph nodes (MLNs) through elevating cyclooxygenase-2 (COX-2), IL-6 and IL-8 expression and influencing the polarization of peritoneal macrophages. Importantly, IL-1β-primed MSCs possessed an enhanced ability to migrate to the inflammatory site of the gut via upregulation of chemokine receptor type 4 (CXCR4) expression. In summary, IL-1β-primed MSCs have improved efficacy in treating DSS-induced colitis, which at least partly depends on their increased immunosuppressive capacities and enhanced migration ability.     

Association of PPARγ2 (Pro12Ala) and neuropeptide Y (Leu7Pro) gene polymorphisms with obstructive sleep apnea in obese Asian Indians

Background: Obstructive sleep apnea (OSA) is prevalent in 7.5% in urban Asian Indians. Peroxisome proliferator activated receptor gamma2 (PPARγ2) has been implicated in adipocyte differentiation. Neuropeptide Y (NPY) is also considered as a candidate gene for excess body fat accumulation. The association of PPARγ2 (Pro12Ala) and NPY (Leu7Pro) gene polymorphisms with OSA has not been studied in Asian Indians. Objective: To study the distribution of PPARγ2 (Pro12Ala) and NPY (Leu7Pro) polymorphism in Asian Indians with and without OSA. Methods and results: This study was carried out in 252 obese subjects [(body mass index (BMI > 25 kg/m²)]; 142 with OSA and 110 without OSA. Measurements included anthropometric and biochemical parameters (fasting blood glucose, lipid profile, various circumferences and skin-fold thicknesses). PPARγ2 (Pro12Ala) and NPY (Leu7Pro) gene polymorphisms were studied in all subjects. The frequency of the variant allele (Ala12) of PPARγ2 gene was significantly higher in subjects with OSA (14.4%) when compared with subjects without OSA (5.5%; χ² = 9.7; p = 0.001). The distribution of the variant allele (Pro7) of NPY gene was comparable in subjects with OSA (3.5%) and without OSA (3.6%; χ ² = 0.001, p = 0.94). Conclusion: This study reveals a significantly higher frequency of PPARγ2 (Ala12) allele in obese Asian Indians with OSA when compared to obese Asian Indians without OSA.     

Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes-infected human macrophages

In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT-domain-, leucine-rich repeat- and PYD-containing protein 3 (NALP3) inflammasome complex in human monocyte-derived macrophages and the molecules and signalling pathways involved in GAS-induced inflammatory responses. We focused upon analysing the impact of dynamin-dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome–lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild-type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)-1β, IL-10, tumour necrosis factor (TNF)-α and chemokine (C-X-C motif) ligand (CXCL)-10 cytokines were produced after bacterial stimulation in a dose-dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin-dependent endocytosis in macrophages attenuates the induction of IL-1β, TNF-α, interferon (IFN)-β and CXCL-10 mRNAs. We also observed that pro-IL-1β protein was expressed and efficiently cleaved into mature-IL-1β via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS-stimulated inflammatory responses in human macrophages.     

Sequestosome-1 (SQSTM1) sequence variants in ALS cases in the UK: prevalence and coexistence of SQSTM1 mutations in ALS kindred with PDB

Mutations in the SQSTM1 gene have been reported to be associated with amyotrophic lateral sclerosis (ALS). We sought to determine the frequency of these mutations in a UK familial ALS (FALS) cohort. Sequences of all eight exons of the SQSTM1 gene were analysed in index cases from 61 different FALS kindred lacking known FALS mutations. Six exonic variants c.463G>A, p.(Glu155Lys), c.822G>C, p.(Glu274Asp), c.888G>T, p.(=), c.954C>T, p.(=), c.1038G>A, p.(=) and c.1175C>T, p.(Pro392Leu) were identified in five FALS index cases, three of which were non-synonymous and three were synonymous. One index case harboured three variants (c.822G>C, c.888G>T and c.954C>T), and a second index case harboured two variants (c.822G>C and c.954C>T). Only the p.(Pro392Leu) and p.(Glu155Lys) mutations were predicted to be pathogenic. In one p.(Pro392Leu) kindred, the carrier developed both ALS and Paget''s disease of bone (PDB), and, in the p.(Glu155Lys) kindred, the father of the proband developed PDB. All p.(Pro392Leu) carriers were heterozygous for a previously reported founder haplotype for PDB, where this mutation has an established causal effect. The frequency of the p.(Pro392Leu) mutation in this UK FALS cohort was 2.3% and 0.97% overall including three previously screened FALS cohorts. Our results confirm the presence of the p.(Pro392Leu) SQSTM1 mutation in FALS. This mutation is the most common SQSTM1 mutation found in ALS to date, and a likely pathogenicity is supported by having an established causal role in PDB. The occurrence of the same mutation in ALS and PDB is indicative of a common pathogenic pathway that converges on protein homeostasis.     

Retinol Binding Protein-4 Is Associated with TNF-α and Not Insulin Resistance in Subjects with Type 2 Diabetes Mellitus and Coronary Heart Disease

We studied the association between RBP4 and various markers related to insulin resistance and diabetic complications as well as inflammatory markers in Saudi population suffering from type 2 diabetes and coronary heart disease. Patients with type 2 diabetes were divided into 3 groups according to the type of treatment and involvement of coronary artery disease. Serum RBP4, TNF-α, insulin, CRP, resistin, leptin and adiponectin were analysed in all samples. RBP4 levels increased significantly in the group of diabetic subjects treated with oral hypoglycemic agents and diabetic patients with coronary heart disease (30.2 ± 11.8; 33.4 ± 13.6 respectively), while there was no significant change in the other group for diabetic subjects on low-carbohydrate diet (25.1 ± 10.9) compared to control group (22.6 ± 9.5). RPB4 levels were positively correlated with TNF-α in the group of diabetic subjects on oral hypoglycemic agents and diabetic patients with coronary heart disease (r = 0.52, P < 0.05; r = 0.58, P < 0.05 respectively). No correlations were found between RBP4 levels and insulin resistance in all studied groups. Our findings suggest that serum RBP4 levels is associated with pro-inflammatory cytokine (TNF-α) and is not associated with insulin resistance among patients with type 2 diabetes and coronary heart disease.