TNF-beta Research Articles

Cellular sources of dysregulated cytokines in relapsing-remitting multiple sclerosis

BackgroundNumerous cytokines are implicated in the immunopathogenesis of multiple sclerosis (MS), but studies are often limited to whole blood (WB) or peripheral blood mononuclear cells (PBMCs), thereby omitting important information about the cellular origin of the cytokines. Knowledge about the relation between blood and cerebrospinal fluid (CSF) cell expression of cytokines and the cellular source of CSF cytokines is even more scarce.MethodsWe studied gene expression of a broad panel of cytokines in WB from relapsing-remitting multiple sclerosis (RRMS) patients in remission and healthy controls (HCs). Subsequently we determined the gene expression of the dysregulated cytokines in isolated PBMC subsets (CD4+, CD8+T-cells, NK-cells, B-cells, monocytes and dendritic cells) from RRMS patients and HCs and in CSF-cells from RRMS patients in clinical relapse and non-inflammatory neurological controls (NIND).ResultsRRMS patients had increased expression of IFN-gamma (IFNG), interleukin (IL) 1-beta (IL1B), IL7, IL10, IL12A, IL15, IL23, IL27, lymphotoxin-alpha (LTA) and lymphotoxin-beta (LTB) in WB. In PBMC subsets the main sources of pro-inflammatory cytokines were T- and B-cells, whereas monocytes were the most prominent source of immunoregulatory cytokines. In CSF-cells, RRMS patients had increased expression of IFNG and CD19 and decreased expression of IL10 and CD14 compared to NINDs. CD19 expression correlated with expression of IFNG, IL7, IL12A, IL15 and LTA whereas CD14 expression correlated with IL10 expression.ConclusionsUsing a systematic approach, we show that expression of pro-inflammatory cytokines in peripheral blood primarily originates from T- and B-cells, with an important exception of IFNG which is most strongly expressed by NK-cells. In CSF-cell studies, B-cells appear to be enriched in RRMS and associated with expression of pro-inflammatory cytokines; contrarily, monocytes are relatively scarce in CSF from RRMS patients and are associated with IL10 expression. Thus, our findings suggest a pathogenetic role of B-cells and an immunoregulatory role of monocytes in RRMS.

Circadian rhythmicity of inflammatory serum parameters: a neglected issue in the search of biomarkers in multiple sclerosis

Inflammatory serum parameters are intensely investigated in the search of biomarkers for disease activity and treatment response in multiple sclerosis (MS). A reason for contradictory results might be the timing of blood collection for analyzing serum concentrations of inflammatory parameters which are subject to diurnal changes. We included 34 untreated patients with relapsing-remitting MS and 34 age- and sex-matched healthy controls. 12 MS patients showed acute disease activity in corresponding MRI scans. Blood samples were obtained at 7.00, 11.00 am, 2.30, 6.00 and 9.30 pm within 1 day. We determined serum levels of cortisol and inflammatory markers including soluble tumor necrosis factor-beta (sTNF-β), soluble TNF-Receptor-1 (sTNF-R1) and -2 (sTNF-2), soluble vascular adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) by ELISA. We observed significantly higher serum levels of sTNF-R1 (p < 0.001) and sTNF-R2 (p < 0.001) in the morning and a significant decline of sICAM-1 (p < 0.005) and sVCAM-1 (p < 0.001) in the afternoon in both, MS patients and healthy controls. Comparison of diurnal serum levels between MS patients with active versus with non-active disease revealed significantly higher serum levels of sVCAM-1 (p < 0.05) around noon and in the early afternoon in MS patients with active disease. A significant decline of sICAM-1 (p < 0.05) in the afternoon was seen in MS patients with active and non-active disease. Our data indicate that increased awareness of potential diurnal serum concentration changes of biomarkers can eliminate one major cause of biased data as they occur in most of the investigated immunological parameters.

Peripheral blood T lymphocyte subsets and cytokines expression changes in cardia carcinoma

Objective To observing the immune status changing in cardia carcinoma post-operation patients.Methods Flow cytometry was used to detect T lymphocyte subsets (Th1,Th2,CD3 +,CD4 +,CD8 + ) in 97 cardia carcinoma patients,Enzyme linked immunosorbent assay (ELISA) assay was used to analyze the serum status of interleukin (IL)-2,interferon (IFN)-γ,IL-4,IL-10,soluble interleukin 2 receptor (sIL-2R) and tumor necrosis factor (TNF)-β.Results Peripheral blood Th1/Th2 ratio was significantly lower in pre-operation patients (0.43 ±0.13) than normal controls(0.58 ±0.12) ( P ＜0.05 ).cytokines status as IL-2 and IFN-γ expressed by Th1 was significantly lower than control group,while IL-4 and IL-10 expressed by Th2 was significantly higher (P ＜ 0.05).T cell mediated cell immunity has changed and disordered.The degree has related to TNM stage,The characteristics were:CD3 + and CD4 + were decreased and CD8 + increased( P ＜ 0.05 ) ; the Th1/Th2 ratio was significantly decreased ( P ＜ 0.05 ),sIL-2R and TNF-β were increased ( P ＜ 0.05 ).1 month post-operation,CD3 +,CD4 + were increased and CD8 + decreased,sIL-2R and TNF-β deaeased ( P ＜ 0.01 ).Conclusion Th1/Th2 cell ratio decreased in cardia carcinoma patients,cytokine expression imbalance in patients with suppressed immune function. Key words: Cardia carcinoma; Flow cytometry; Immunofunction

Abstract 273: P2Y2 Receptor-Mediated Lymphotoxin-α Secretion Regulates ICAM-1 Expression in Smooth Muscle Cells

Rationale: Single nucleotide polymorphism (SNP) in the LGASL2 galectin-2 (Gal-2) gene leads to altered secretion of lymphotoxin-α (LT-α) and is associated with coronary artery disease. Objective:Our aim was to determine whether factors other than genetic variations in LGASL2 regulate LT-α release and to define the role of this pro-inflammatory in vascular smooth muscle cells (SMC). Methods and results: The proinflammatory cytokine lymphotoxin-alpha (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in VSMC are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y 2 nucleotide receptor (P2Y 2 R) agonist UTP stimulates a strong and sustained release of LTA from wild-type but not P2Y 2 R -/- SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Re-introduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found UTP-stimulated LTA secretion is not sensitive to brefeldin A (BFA), which blocks the formation of vesicles involved in protein transport from the ER to the Golgi apparatus, suggesting that P2Y 2 R/filamin-mediated secretion of LTA is independent of the ER/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y 2 R deficient in LTA secretion. Conclusion: These data suggest that P2Y 2 R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on VSMC.

An Effector Phenotype of CD8+T Cells at the Junction Epithelium during Clinical Quiescence of Herpes Simplex Virus 2 Infection

Herpes simplex virus 2 infection is characterized by cycles of viral quiescence and reactivation. CD8(+) T cells persist at the site of viral reactivation, at the genital dermal-epidermal junction contiguous to neuronal endings of sensory neurons, for several months after herpes lesion resolution. To evaluate whether these resident CD8(+) T cells frequently encounter HSV antigen even during times of asymptomatic viral infection, we analyzed the transcriptional output of CD8(+) T cells captured by laser microdissection from human genital skin biopsy specimens during the clinically quiescent period of 8 weeks after lesion resolution. These CD8(+) T cells expressed a characteristic set of genes distinct from those of three separate control cell populations, and network and pathway analyses revealed that these T cells significantly upregulated antiviral genes such as GZMB, PRF1, INFG, IL-32, and LTA, carbohydrate and lipid metabolism-related genes such as GLUT-1, and chemotaxis and recruitment genes such as CCL5 and CCR1, suggesting a possible feedback mechanism for the recruitment of CD8(+) T cells to the site of infection. Many of these transcripts are known to have half-lives of <48 h, suggesting that cognate antigen is released frequently into the mucosa and that resident CD8(+) T cells act as functional effectors in controlling viral spread.

A functional polymorphism of lymphotoxin-alpha (LTA) gene rs909253 is associated with gastric cancer risk in an Asian population

BackgroundThe potentially functional polymorphism, rs909253 (+252 G>A), in the intron region of the LT-α (TNF-β) gene has been implicated in the risk of gastric cancer (GC) in some individually published studies, but others have shown inconsistent and inconclusive results. MethodsWe conducted a meta-analysis to assess the association between the lymphotoxin-α gene (LTA)+252 (G>A) polymorphism and gastric cancer susceptibility. ResultsWe demonstrate that there were no significant associations in single-locus analysis between the polymorphism of LTA and gastric cancer risk in all subjects; however, when studies were stratified by ethnicity, these polymorphisms of LTA were found to be associated with a significant cancer risk in different genetic models in an Asian population (heterozygote [GA genotype] comparison: odds ratio [OR]=1.29, 95% confidence interval [CI]: 1.01–1.65, P=0.038) in which the risk in the subjects was more than 70% (12 studies with 2074 cases and 3690 controls). Moreover, the susceptibility to gastric carcinogenesis has a substantial influence on the population-attributable risk by modulating the effects of environmental risk factors such as Helicobacter pylori infection (OR=1.77, 95%CI: 1.05–2.99, P=0.032). ConclusionsThe present meta-analysis results suggest that the LTA rs909253 GA genotype is a possible risk factor for developing gastric cancer in the Asian population, especially those with H. pylori infection.

Research of regulative factors on CD8(+)HLA-DR(+) effector T cells in severe aplastic anemia

To explore the regulative factors on CD8(+)HLA-DR(+) T cells in the patients with severe aplastic anemia (SAA) and examine the roles of these cells in the immunopathogenesis of SAA. CD8(+)HLA-DR(+) T cells were sorted from bone marrow mononuclear cells of 13 SAA patients from July 2011 to March 2012 by magnetic activated cell sorting system and were divided into 3 groups: interleukin 2 (IL-2) group (0, 0.1, 1, 10, 100 and 1000 U/ml), cyclosporine A (CsA) group (addition of 400 ng/ml CsA in each IL-2-containing well),receptor antagonist group (addition of IL-2 receptor antagonist 8 µg/ml in each IL-2-containing well). Then cell proliferation rate was evaluated by MTT assay after a 72-hour culturing. Bone marrow mononuclear cells of the SAA patients were divided into CsA group, IL-2 group and control group and cultured for 18 hours and another 4 hours following the dosing of phorbol ester. The expression of tumor necrosis factor β (TNF-β) in CD8(+)HLA-DR(+) T cells was analyzed by flow cytometry. The cell proliferations of IL-2 wells at the concentrations of 10, 100 and 1000 U/L (0.36 ± 0.12, 0.41 ± 0.12, 0.46 ± 0.14) were significantly higher than those of the control wells (0.23 ± 0.11), CsA group (0.18 ± 0.05, 0.19 ± 0.00, 0.20 ± 0.04) and receptor antagonist group (0.18 ± 0.05, 0.17 ± 0.04, 0.18 ± 0.03, all P < 0.05). No statistic difference existed between CsA and receptor antagonist groups (P > 0.05). The expressions of TNF-β of CD8(+)HLA-DR(+)T cells of the IL-2 group were higher than those of the control group (64% ± 25% vs 46% ± 22%) whereas the CsA group (27% ± 20%) were lower than those of the control group (both P < 0.05). IL-2 can significantly stimulate the proliferation of CD8(+)HLA-DR(+) T cells and accelerate the in vitro secretion of TNF-β in SAA patients. The proliferation may be inhibited by CsA and receptor antagonist. And the expression of TNF-β is suppressed significantly by CsA.

Investigation of Host Candidate Malaria-Associated Risk/Protective SNPs in a Brazilian Amazonian Population

The Brazilian Amazon is a hypo-endemic malaria region with nearly 300,000 cases each year. A variety of genetic polymorphisms, particularly in erythrocyte receptors and immune response related genes, have been described to be associated with susceptibility and resistance to malaria. In order to identify polymorphisms that might be associated with malaria clinical outcomes in a Brazilian Amazonian population, sixty-four human single nucleotide polymorphisms in 37 genes were analyzed using a Sequenom massARRAY iPLEX platform. A total of 648 individuals from two malaria endemic areas were studied, including 535 malaria cases (113 individuals with clinical mild malaria, 122 individuals with asymptomatic infection and 300 individuals with history of previous mild malaria) and 113 health controls with no history of malaria. The data revealed significant associations (p<0.003) between one SNP in the IL10 gene (rs1800896) and one SNP in the TLR4 gene (rs4986790) with reduced risk for clinical malaria, one SNP in the IRF1 gene (rs2706384) with increased risk for clinical malaria, one SNP in the LTA gene (rs909253) with protection from clinical malaria and one SNP in the TNF gene (RS1800750) associated with susceptibility to clinical malaria. Also, a new association was found between a SNP in the CTL4 gene (rs2242665), located at the major histocompatibility complex III region, and reduced risk for clinical malaria. This study represents the first association study from an Amazonian population involving a large number of host genetic polymorphisms with susceptibility or resistance to Plasmodium infection and malaria outcomes. Further studies should include a larger number of individuals, refined parameters and a fine-scale map obtained through DNA sequencing to increase the knowledge of the Amazonian population genetic diversity.

Expression of lymphokines in CD8(+)HLA-DR(+) T lymphocytes of patients with severe aplastic anemia

To examine the expression of lymphokines damaging hematopoietic cells by CD8(+)HLA-DR(+) effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia (SAA) and explore further the heterogeneous immunopathogenesis of SAA. The CD8(+)HLA-DR(+) cells were sorted by immunomagnetic separation from the PB of 24 untreated SAA patients and 23 normal controls. The mRNA expressions of perforin, granzyme B, FasL and tumor necrosis factor β (TNF-β) of sorted cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels of perforin, granzyme B of CD8(+)HLA-DR(+)T cells in untreated group were higher than those of the controls (0.66 ± 0.25, 0.56 ± 0.26 vs 0.53 ± 0.14, 0.40 ± 0.13, P = 0.042, 0.012). The mRNA level of FasL in CD8(+)HLA-DR(+) T cells of untreated SAA patients was higher than that of the controls (0.77 ± 0.24 vs 0.61 ± 0.16, P = 0.011). The mRNA of TNF-β in CD8(+)HLA-DR(+) T cells of untreated SAA patients was also higher than that of the controls (0.58 ± 0.16 vs 0.46 ± 0.15, P = 0.011). CD8(+)HLA-DR(+) T cells may damage hematopoiesis through the actions of perforin, granzyme B, TNF-β and FasL. And it thus contributes to the immunopathogenesis of SAA.

Abstract 2617: Cytokine and cytokine receptors of adaptive immune response are differentially associated with breast cancer risk in women of African ancestry and European ancestry

Abstract Disparities in breast cancer biology are evident between women of African ancestry (AA) and women of European ancestry (EA), and may be due, in part, to differences in inflammatory pathway genes. Studies on the relationships between cytokines and breast cancer risk have focused on a small number of genes and have not included AA women. In a large case-control study of 1008 AA (n=547 cases) and 763 EA (n=381 cases) women, we systematically tested the association between risk of breast cancer and single-nucleotide polymorphisms (SNPs) in 22 cytokine and cytokine receptor genes involved in adaptive immune response pathways. Logistic regression was used to estimate odds ratios and 95% confidence intervals in a genotypic (co-dominant) model adjusting for major breast cancer risk factors including age, education, body mass index (BMI), family history of breast cancer and proportion of European ancestry. All analyses were stratified on race. SNPs rs17810546 in IL12A (interleukin 12A), rs1041981 in LTA (lymphotoxin alpha), and rs3135932 in IL10RA, were associated with breast cancer risk in AA but not EA women. When stratifying by menopause and ER status, SNPs rs2070874 in IL4, rs1041981 in LTA and rs2430561 in IFNG (interferon gamma) were associated with breast cancer risk in pre-menopausal AA women, and SNPs rs2243248 in IL4 and rs746868 in LTA were associated with ER-negative breast cancer in EA women. Common variants in inflammatory pathway genes may help to define populations at high risk of breast cancer. Notable differences in distributions of SNPs and associations by ancestry may be related to the differential epidemiology of breast cancer in AA and EA women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2617. doi:1538-7445.AM2012-2617

Tumor necrosis factor (TNF) and lymphotoxin-alpha (LTA) single nucleotide polymorphisms: Importance in ARDS in septic pediatric critically ill patients

Accumulating evidence indicates that genetic background influences the outcome of sepsis, which despite medical advances continues to be a major cause of morbidity and mortality. This study aimed to evaluate the influence of SNPs LTA +252A>G, TNF-863C>A and TNF-308G>A on susceptibility to sepsis, acute respiratory distress syndrome (ARDS), septic shock and sepsis mortality. A prospective case-control study was carried out in a Brazilian pediatric intensive care unit and included 490 septic pediatric patients submitted to mechanical ventilation and 610 healthy children. No SNP association was found with respect to sepsis susceptibility. Nevertheless, a haplotype was identified that was protective against sepsis (+252A/-863A/-308G; OR=0.65; p=0.03). We further observed protection against ARDS in TNF-308 GA genotype carriers (OR=0.29; p=0.0006) and -308A allele carriers (OR=0.40; p=0.003). In addition, increased risk for ARDS was detectable with the TNF-863 CA genotype (OR=1.83; p=0.01) and the -863A carrier status (OR=1.82; p=0.01). After stratification according to age, this outcome remained significantly associated with the -308GA genotype in infants. Finally, protection against sepsis-associated mortality was found for the TNF-308 GA genotype (OR=0.22; p=0.04). Overall, our findings document a protective effect of the TNF-308 GA genotype for the ARDS and sepsis mortality outcomes, further providing evidence for an increased risk of ARDS associated with the TNF-863 CA genotype. Trial registration (www.clinicaltrials.gov): NCT00792883.

SNPs in PTGS2 and LTA predict pain and quality of life in long term lung cancer survivors

PurposeLung cancer survivors report the lowest quality of life relative to other cancer survivors. Pain is one of the most devastating, persistent, and incapacitating symptoms for lung cancer survivors. Prevalence rates vary with 80–100% of survivors experiencing cancer pain and healthcare costs are five times higher in cancer survivors with uncontrolled pain. Cancer pain often has a considerable impact on quality of life among cancer patients and cancer survivors. Therefore, early identification, and treatment is important. Although recent studies have suggested a relationship between single nucleotide polymorphisms (SNPs) in several cytokine and inflammation genes with cancer prognosis, associations with cancer pain are not clear. Therefore, the primary aim of this study was to identify SNPs related to pain in lung cancer survivors. Patients and methodsParticipants were enrolled in the Mayo Clinic Lung Cancer Cohort upon diagnosis of their lung cancer. 1149 Caucasian lung cancer survivors (440 surviving <3 years; 354 surviving 3–5 years; and 355 surviving >5 years) completed study questionnaires and had blood DNA samples available. Ten SNPS from PTGS2 and LTA genes were selected based on the serum-based studies in the literature. Outcomes included pain, and quality of life as measured by the SF-8. ResultsOf the 10 SNPs evaluated in LTA and PTGS2 genes, 3 were associated with pain severity (rs5277; rs1799964), social function (rs5277) and mental health (rs5275). These results suggested both specificity and consistency of these inflammatory gene SNPs in predicting pain severity in lung cancer survivors. ConclusionThese results provide support for genetic predisposition to pain severity and may aid in identification of lung cancer survivors at high risk for morbidity and poor QOL.

Secretory products of multiple sclerosis B cells are cytotoxic to oligodendroglia in vitro

B cells are important in the pathogenesis of multiple sclerosis (MS) and some of the effects are not dependent on maturation of B cells into immunoglobulin (Ig) producing plasmablasts and plasma cells. B cells present antigen, activate T cells, and are involved in immunoregulation and cytokine secretion. To determine if B cells from MS patients secrete products that have deleterious effects on glial cells not mediated by Ig, and to compare effects with secretory products of normal controls (NC), we isolated B cells from 7 patients with relapsing remitting MS (RRMS) and 4 NC. B cells were cultured alone or after stimulation with CD40 ligand (CD40L), CD40L+cross-linking of the B cell antigen receptor (xBCR) and CD40L+xBCR+stimulation of toll like receptor 9 (TLR9). Supernatants were harvested and incubated with mixed central nervous system (CNS) neonatal rat glial cells. Supernatants from unstimulated NC B cells induced on average death of 7% (range 0–24%) of differentiated oligodendrocytes (OL); in contrast, supernatants from unstimulated B cells from RRMS patients induced death of 57% (range 35–74%) of OL. Supernatants of stimulated B cells from NC did not increase the minimal OL death whereas stimulation of B cells from RRMS had variable results compared to unstimulated B cells. Supernatants from both NC and RRMS induced microglial enlargement and loss of normal resting bipolar morphology. OL death did not correlate with levels of tumor necrosis alpha (TNF-α), lymphotoxin alpha (LT-α), interleukin 6 (IL-6), IL-10, transforming growth factor beta 1 (TGF-β1) or any combination or ratio of these cytokines. Analysis of 26 supernatants from NC and RRMS patients failed to detect IgM. There were very low levels of IgG in 8 of the 26 supernatants, and no correlation between of OL death and presence or absence of IgG. Sera used in both the B cell and glial cell cultures were heated, which inactivates complement. The effects of B cell supernatants on OL could be direct and/or indirect involving either microglia and/or astrocytes. The identity of the toxic factor(s) is as yet unknown. Thus we have demonstrated that B cells from patients with RRMS but not NC secrete one or more factors toxic to OL. It is possible that such factors produced by peripheral blood B cells when within the CNS could contribute to demyelination in MS patients.

P2Y2 Receptor-mediated Lymphotoxin-α Secretion Regulates Intercellular Cell Adhesion Molecule-1 Expression in Vascular Smooth Muscle Cells

The proinflammatory cytokine lymphotoxin-α (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in vascular smooth muscle cells (VSMC) are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y(2) nucleotide receptor (P2Y(2)R) agonist UTP stimulates a strong and sustained release of LTA from WT but not P2Y(2)R(-/-) SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Reintroduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found that UTP-stimulated LTA secretion is not sensitive to brefeldin A, which blocks the formation of vesicles involved in protein transport from the endoplasmic reticulum to the Golgi apparatus, suggesting that P2Y(2)R/filamin-mediated secretion of LTA is independent of the endoplasmic reticulum/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y(2)R deficient in LTA secretion. These data suggest that P2Y(2)R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on vascular smooth muscle cells.

Polymorphisms of LTA, LGALS2, and PSMA6 genes and coronary atherosclerosis: A pathological study of 1503 consecutive autopsy cases

ObjectiveRecent genome-wide association studies have identified polymorphisms of lymphotoxin-α (LTA), galectin-2 (LGALS2), and proteasome subunit a type 6 (PSMA6) genes as genetic risk factors for myocardial infarction (MI). However, their effects on coronary atherosclerosis, an intermediate phenotype of MI, remain largely unknown. MethodsWe investigated the correlation between polymorphisms of the LTA, LGALS2, and PSMA6 genes and the severity of pathological coronary stenosis index (CSI) and MI in 1503 consecutive autopsy cases of Japanese elderly patients. ResultsThe polymorphisms LTA rs1041981 and LGALS2 rs7291467 were associated with CSI with odds ratios of 1.54 (95% CI, 1.17–2.01; AA+CA over CC) and 1.62 (95% CI, 1.11–2.37; TT over CC+CT), respectively. PSMA6 rs1048990 was not associated with CSI. None of the SNPs was associated with MI in our sample. ConclusionOur findings indicate that the LTA and LGALS2 polymorphisms affect the subclinical phenotype of the coronary artery, which predisposes to the incidence of MI.

Dysfunctional B-cell activation in cirrhosis resulting from hepatitis C infection associated with disappearance of CD27-Positive B-cell population

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis and hepatocellular carcinoma (HCC). Both advanced solid tumors and HCV have previously been associated with memory B-cell dysfunction. In this study, we sought to dissect the effect of viral infection, cirrhosis, and liver cancer on memory B-cell frequency and function in the spectrum of HCV disease. Peripheral blood from healthy donors, HCV-infected patients with F1-F2 liver fibrosis, HCV-infected patients with cirrhosis, patients with HCV-related HCC, and non-HCV-infected cirrhotics were assessed for B-cell phenotype by flow cytometry. Isolated B cells were stimulated with anti-cluster of differentiation (CD)40 antibodies and Toll-like receptor (TLR)9 agonist for assessment of costimulation marker expression, cytokine production, immunoglobulin (Ig) production, and CD4(+) T-cell allostimulatory capacity. CD27(+) memory B cells and, more specifically, CD27(+) IgM(+) B cells were markedly less frequent in cirrhotic patients independent of HCV infection. Circulating B cells in cirrhotics were hyporesponsive to CD40/TLR9 activation, as characterized by CD70 up-regulation, tumor necrosis factor beta secretion, IgG production, and T-cell allostimulation. Last, blockade of TLR4 and TLR9 signaling abrogated the activation of healthy donor B cells by cirrhotic plasma, suggesting a role for bacterial translocation in driving B-cell changes in cirrhosis. Profound abnormalities in B-cell phenotype and function occur in cirrhosis independent of HCV infection. These B-cell defects may explain, in part, the vaccine hyporesponsiveness and susceptibility to bacterial infection in this population.

A Simple PCR–RFLP Method for Genetic Phase Determination in Compound Heterozygotes

When susceptibility to diseases is caused by cis-effects of multiple alleles at adjacent polymorphic sites, it may be difficult to assess with confidence the genetic phase and identify individuals carrying the risk haplotype. Experimental assessment of genetic phase is still challenging and most population studies use statistical approaches to infer haplotypes given the observed genotypes. While these statistical approaches are powerful and have been proven very useful in large scale genetic population studies, they may be prone to errors in studies with small sample size, especially in the presence of compound heterozygotes. Here, we describe a simple and novel approach using the popular PCR–RFLP based strategy to assess the genetic phase in compound heterozygotes. We apply this method to two extensively studied SNPs in two clustered immune-related genes: The −308 (G > A) and the +252 (A > G) SNPs of the tumor necrosis factor (TNF) alpha and the lymphotoxin alpha (LTA) genes, respectively. Using this method, we successfully determined the genetic phase of these two SNPs in known compound heterozygous individuals and in every sample tested. We show that the A allele of TNF −308 is carried on the same chromosome as the LTA +252(G) allele.

Effect of buckminsterfullerenes on cells of the innate and adaptive immune system: an in vitro study with human peripheral blood mononuclear cells

C60 nanoparticles, the so-called buckminsterfullerenes, have attracted great attention for medical applications as carriers, enzyme inhibitors or radical scavengers. However, publications evaluating their immunological mechanisms are still rather limited. Therefore, we aimed to analyze systematically the in vitro influence of polyhydroxy-C60 (poly-C60) and N-ethyl-polyamino-C60 (nepo-C60) on peripheral blood mononuclear cells (PBMC) from healthy individuals, angling their effect on proliferation, expression of surface markers, and cytokine production. We isolated PBMC from 20 healthy subjects and incubated them in a first step only with poly-C60 or nepo-C60, and in a second step together with recall antigens (purified protein derivative, tetanus toxoid, bacillus Calmette-Guérin). Proliferation was determined by 3H-thymidine incorporation, activation of PBMC-subpopulations by flow cytometry by measurement of the activation marker CD69, and secretion of T helper cell type 1 (TH1)- (interferon-gamma [IFN-γ], tumor necrosis factor beta [TNF-β]), TH2- (interleukin-5 [IL-5], −13, −10) and macrophage/monocyte-related cytokines (IL-1, IL-6, TNF-α) into the supernatants by enzyme-linked immunosorbent assay. Both fullerenes did not influence T cell reactivity, with no enhanced expression of CD69 and production of T cell cytokines observed, the CD4/CD8 ratio remaining unaffected. In contrast, they significantly enhanced the release of IL-6 and CD69-expression by CD56 positive natural killer cells. PBMC, which had been cultured together with the three recall antigens were not affected by both fullerenes at all. These data indicate that fullerenes do not interact with T cell reactivity but may activate cells of the innate immune system. Furthermore, they seem to act only on ‘naïve’ cells, which have not been prestimulated with recall antigens, there are however, large inter individual differences.

Cytokines in cerebrospinal fluid of children with West syndrome

In this study we aimed to determine the levels of tumor necrosis factor beta (TNF-b), interleukin (IL) 1-beta (IL-1b), IL-5, IL-10, and interferon gamma (IFN-g ) in CSF from children during the onset of West syndrome (WS). We observed elevated levels of IL-1b and IFN-g correlated to clinical, EEG, therapeutic response, and follow-up suggesting the involvement of immune response in WS. These results suggest that inflammatory and immunologic mediators may play a role in the pathophysiologic mechanisms of infantile spasms. Our findings may explain the perfusion and cognitive disfunctions and actions of adrenocorticotropic hormone (ACTH), corticosteroids, and intravenous immunoglobulin (IVIg) observed in WS. In conclusion, WS results from association of neurophysiological mechanisms and structural abnormalities with participation of cytokines mainly in symptomatic group.

Association between TNF-β NcoI (A252G) gene polymorphism and type I diabetes mellitus in Turkish population

Objective: Diabetes mellitus is a common metabolic disorder characterized by hyperglycemia. Cytokines induce signal transduction and secondary messenger pathways after binding their specific ligands. In this way, these proteins control and also modulate activity of the target cells. Tumor necrosis factor plays an important role in diabetes and is a member of the family of cytokines. In this study, we aimed to investigate the effect of A252G (TNFβ NcoI, rs909253) polymorphism in the tumor necrosis factor beta gene on the Type I Diabetes Mellitus in the Turkish population. Materials and Method: Distribution of A252G polymorphism was determined in 96 patients with Type I diabetes and 101 healty individuals by PCR-RFLP. Results: When obtained data was evaluated, the A allele of A252G polymorphism were observed more frequently than G allele in patients and controls. Moreover, G alleles were observed more frequently among patients than in controls. This high frequency of G allele found in patients demonstrates a 1.6 fold increased risk of susceptibility to type I diabetes in Turkish population (P=0.032). However, there was no significant difference between patients and controls in terms of the distribution of genotypes (P=0.132). Conclusion: As a result, although presence of a G allele demonstrated an increased risk of susceptibilty to type I diabetes, TNF-β A252G polymorphism’s genotype distrubution was not associated with type I diabetes in Turkish population. Polymorphisms involved in the development of Type I Diabetes Mellitus results in better understanding of the development and the progression of disease.