Devapregasan Moodley

Mycobacterium tuberculosis: Manipulator of Protective Immunity.

Mycobacterium tuberculosis (MTB) is one of the most successful pathogens in human history and remains a global health challenge. MTB has evolved a plethora of strategies to evade the immune response sufficiently to survive within the macrophage in a bacterial-immunological equilibrium, yet causes sufficient immunopathology to facilitate its transmission. This review highlights MTB as the driver of disease pathogenesis and presents evidence of the mechanisms by which MTB manipulates the protective immune response into a pathological productive infection.

Fumonisin B1 modulates expression of human cytochrome P450 1b1 in human hepatoma (Hepg2) cells by repressing Mir-27b

Fumonisin B₁ (FB₁), a common mycotoxin contaminant of maize, is known to inhibit sphingolipid biosynthesis and has been implicated in hepatocellular carcinoma promoting activity in humans and animals. MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression via translational repression. Human cytochrome P450 (CYP1B1) is highly expressed in oestrogen target tissues and catalyzes the metabolic activation of many procarcinogens. The aim of our study was to investigate the effect of FB₁ on miR-27b suppression and its effect on CYP1B1 modulation in a human hepatoma cell line (HepG2). MiR27b and CYP1B1 expressions were evaluated in HepG2 cells by quantitative PCR. In order to directly assess the effect of miR-27b on CYP1B1 mRNA levels, cells were transfected with the mimic to miR-27b. CYP1B1 protein expression was measured using Western blot. FB₁ significantly down-regulated (11-fold) expression of miR-27b in HepG2 cells; whilst CYP1B1 mRNA and protein expression was significantly upregulated by 1.8-fold and 2.6-fold, respectively. CYP1B1 is post-transcriptionally regulated by miR-27b after HepG2 exposure to FB₁. FB₁-induced modulation of miR-27b in hepatic cells may be an additional mode of hepatic neoplastic transformation.

Fumonisin B1 induces global DNA hypomethylation in HepG2 cells - An alternative mechanism of action

Fumonisin B1 (FB1), a common mycotoxin contaminant of maize, is known to inhibit sphingolipid biosynthesis and has been implicated in cancer promoting activity in animals and humans. FB1 disrupts DNA methylation and chromatin modifications in human hepatoma (HepG2) cells. We investigated the effect of FB1 on enzymes, DNA methyltransferases and demethylases, involved in chromatin maintenance and gross changes in structural integrity of DNA in HepG2 cells. We measured: (i) the expression of 84 key genes encoding enzymes known to modify genomic DNA and histones (superarray and qPCR); (ii) protein expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and the major demethylase (MBD2) (western blotting); (iii) degree of DNA methylation by FACS using anti-5-MeCyt and (iv) DNA migration (single cell gel electrophoresis). FB1 significantly decreased the methyltransferase activities of DNMT1, DNMT3A and DNMT3B, and significantly up regulated the demethylases (MBD2 expression and activity, and KDM5B and KDM5C expression). FACS data showed FB1 significantly increased DNA hypomethylation and resulted in gross changes in structural DNA as evidenced by the Comet assay. We conclude that FB1 induces global DNA hypomethylation and histone demethylation that causes chromatin instability and may lead to liver tumourigenesis.

Initiation but no execution - modulation of peripheral blood lymphocyte apoptosis in rheumatoid arthritis - a potential role for heat shock protein 70

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease, which causes synovial damage. Persistence of lymphocyte infiltrates in the rheumatoid synovium has been attributed to abnormal apoptosis. While not comprehensively investigated, perturbations in peripheral blood lymphocyte (PBL) apoptosis may also be involved in perpetuation of autoimmune processes in RA.MethodsWe investigated total, CD4+ and CD19+ PBL apoptosis in our study cohort by monitoring the translocation of phosphatidylserine using the Annexin-V assay. To examine the role of death receptor mediated apoptosis as well as activation-induced-cell-death (AICD), PBLs were labeled with CD95/Fas and CD69 markers and enumerated by flow cytometry. Proteolytic activity of initiator and executioner caspases was determined by luminometry. DNA fragmentation assays were used to examine whether apoptotic signals were transduced to the nucleus. Quantitative PCR arrays were used to investigate apoptotic pathways associated with RA-PBLs. Since heat-shock-protein-70 (HSP70) is an inducible protein which modulates apoptotic signals, we determined HSP70 levels by intra-cellular flow cytometry and western blots.ResultsThe RA-PBLs showed signs of elevated apoptosis whilst in circulation. These include increases in the loss of plasma membrane asymmetry, indicated by increased externalization of phosphatidylserine (especially in B-lymphocytes). RA-PBLs showed a bias to CD95/Fas mediated apoptotic pathways, but low levels of the CD69 marker suggested that this was not associated with immune activation. Although downstream markers of apoptosis such as caspase-3/7 activity, were increased, no DNA fragmentation was observed in RA-PBLs. Interestingly, elevated levels of apoptosis did not correlate with absolute lymphocyte counts in RA patients. Levels of HSP70 were highly elevated in RA-PBLs compared to controls.ConclusionThe results suggest that while apoptosis may be initiated in RA-PBLs, they may lack commitment to fully executing the apoptotic program. This may be related to inhibition on apoptotic transduction by HSP70. This study provides evidence that abnormalities in RA-PBLs apoptosis may occur whilst still in circulation and may contribute to pathogenesis of the disease.

GST polymorphisms and early-onset coronary artery disease in young South African Indians

BACKGROUND Glutathione S-transferases (GSTs) detoxify environmental agents which influence the onset and progression of disease. Dysfunctional detoxification enzymes are responsible for prolonged exposure to reactive molecules and can contribute to endothelial damage, an underlying factor in coronary artery disease (CAD). OBJECTIVES We aimed to assess 2 common polymorphic variant isoforms in GSTM1 and GSTP1 of GST in young CAD patients. METHODS All patients (N=102) were South Africans of Indian ancestry, a population associated with high CAD risk. A corresponding age-, sex- and race-matched control group (N=100) was also recruited. Frequency of the GSTM1 +/0 (v. +/0 and 0/0) and GSTP1 A105/G105 (v. wild-type A105/A105) genotypes was assessed by differential polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP), respectively. RESULTS The GSTM1 0/0 and GSTP1 A105/A105 genotypes occurred at higher frequencies in CAD patients compared with the control group (36% v. 18% and 65% v. 48%, respectively). A significant association with CAD was observed in GSTM1 0/0 (OR=2.593; 95% CI 1.353 - 4.971; p=0.0043) and GSTP1 A105/A105 (odds ratio (OR)=0.6011; 95% confidence interval (CI) 0.3803 - 0.9503; p=0.0377). We found a significant association between smoking and CAD; the presence of either of the respective genotypes together with smoking increased the CAD risk (GSTP1 A105 RR=1.382; 95% CI 0.958 - 1.994; p=0.0987 and GSTM1 null RR=1.725; 95% CI 1.044 - 2.851; p=0.0221). CONCLUSION Our findings support the association of genotypes GSTM1 0/0 and GSTP1 A105/A105 and smoking with CAD.

The interleukin-6 -147 g/c polymorphism is associated with increased risk of coronary artery disease in young South African Indian men.

BACKGROUND Interleukin-6 (IL-6) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases such as coronary artery disease (CAD). The -174 IL-6 G/C promoter polymorphism influences mRNA levels and protein expression and is implicated in CAD. The Indian population in South Africa, unlike the black community, has a high prevalence of premature CAD. This polymorphism has not been fully explored in this population. The present study assessed the -174 IL-6 G/C polymorphism in young Indian patients with angiographically documented CAD and compared them with age- and gender-matched Indian and black control subjects. METHODS Polymorphic variants were assessed by polymerase chain reaction-restriction fragment length polymorphism, and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS The -174 IL-6 C allele was found with a higher frequency (23%) in the total Indian group compared to 2% in the black participants [P<0.0001, odds ratio (OR)=0.05, 95% confidence interval (CI) 0.018-0.14). The difference in frequency was more pronounced when Indian controls were compared to black controls (29% vs. 2%, respectively) (P<0.0001, OR=0.05, 95% CI 0.02-0.17). A significant association between the -174 IL-6 G allele and CAD was found in Indian patients compared to Indian controls (84% in cases vs. 71% in Indian controls; P=0.043, OR=0.47 95% CI 0.23-0.95). Levels of IL-6 in circulation were higher in black controls (6.62±0.63 pg/mL) compared to Indian controls (2.51±0.57 pg/mL) and CAD patients (1.46±0.36 pg/mL) (P<0.0001). Levels of IL-6 were higher in all groups with homozygous -174 IL-6 C alleles, but only significant in the healthy Indian control group (GG 3.73±0.94 pg/mL vs. GC/CC 0.89±0.5 pg/mL, P=0.0001). CONCLUSION The presence of the IL-6 -174 G allele influences levels of IL-6 and increases the risk of CAD in South African Indians.

Functional analysis of the p53 codon 72 polymorphism in black South Africans with rheumatoid arthritis—a pilot study

The p53 tumor-suppressor protein plays an integral role in apoptosis. Perturbations in peripheral lymphocyte (PL) apoptosis may be associated with rheumatoid arthritis (RA). Polymorphisms at codon 72 of p53 (arginine (Arg72) to proline transition) confers differences in mitochondrial translocation and apoptosis inducing capabilities of p53 in vitro. We examined associations of this polymorphism with PL apoptosis, mitochondrial depolarization, and clinical markers of disease activity in a cohort of black South African RA patients. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. PL apoptosis was measured using the annexin-V assay and mitochondrial membrane potential with the JC-1 assay. Clinical and laboratory parameters were recorded for all patients. Statistical differences in these parameters were investigated according to genotype. Genotype distribution did not differ significantly between RA patients and controls (Arg/Arg, Arg/Pro, Pro/Pro: 12%, 46%, and 42% versus 3%, 34%, and 63%), despite significantly higher frequency of the Arg72 allele in patients (p = 0.0406). There was no significant difference in PL apoptosis and mitochondrial depolarization based on p53 codon 72 genotype. In addition, clinical markers of disease activity were not significantly different between genotypes. We conclude that p53 codon 72 genotype does not influence PL apoptosis or mitochondrial depolarization and is not associated with clinical markers of disease in RA.

Atorvastatin increases miR-124a expression: a mechanism of Gamt modulation in liver cells.

Atorvastatin is used to control cholesterol and lipid levels in hyperlipidaemic and hypercholesterolaemic patients. Myopathy and hepatotoxicity, however, have been reported as side effects in a small percentage of statin users. This study aimed to investigate the cytotoxicity and the effect of atorvastatin on microRNA expression in HepG2 cells. The methylthiazol tetrazolium assay was used to assess hepatocyte viability and at 20 μM atorvastatin (24 h) treatment were 82 ± 1.5% viable (P = 0.0002). Levels of intracellular ATP in cells treated with 20 μM atorvastatin were reduced by 1.25‐fold, P = 0.002. Cytotoxicity, measured by the release of intracellular lactate dehydrogenase, was increased from 0.95 ± 0.29 units in control cells to 1.12 ± 0.02 units (P = 0.002) in atorvastatin treated cells. A panel of 84‐miRNA species was used to evaluate the effect of atorvastatin on miRNA expression. MiR‐124a was significantly up‐regulated by atorvastatin (12.94‐fold). A significant decrease in GAMT expression (3.54‐fold) was observed in atorvastatin treated cells following quantitative PCR analysis. In addition, western blotting data showed GAMT protein levels were significantly lower than the controls (3.02‐fold) and analysis of creatine levels in treated cells showed a significant decrease in the atorvastatin treated culture supernatant compared to control culture supernatant (32.33 ± 3.51 μM/l vs. 59.67 ± 1.52μM/l, P = 0.0056). This is the first study to show that atorvastatin up‐regulates miR‐124a levels and consequently modulates GAMT expression in hepatocytes. J. Cell. Biochem. 116: 2620–2627, 2015.

The Arg72 variant of the p53 functional polymorphism (rs1042522) is associated with coronary artery disease in young South Africans of Indian ancestry.

BACKGROUND AND AIM Tumor protein p53 (p53), classically referred to as a tumor suppressor gene, is involved in cell cycle regulation and may be related to atherosclerosis by affecting smooth muscle cell proliferation, a feature of atherogenesis. A polymorphism at codon 72 (rs1042522) results in functional variability and hence plays a role in the pathophysiology of coronary artery disease (CAD). This polymorphism has been well established for its role in cancer and has only recently been investigated in CAD. Limited data is available on South Africans (SA) of Indian ancestry. We examined associations of this polymorphism and clinical markers in a cohort of young SA Indian CAD patients. METHODS A total of 284 subjects were recruited into this study which included 100 CAD patients (diagnosed on angiography, mean age 37.5, range 24-45years), 100 age- and sex-matched Indian controls and 84 age- and sex-matched Black controls. Polymorphic variants were assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Data for clinical markers were obtained from pathology reports. RESULTS Genotype distribution differed significantly between CAD patients and Indian controls (Pro/Pro, Pro/Arg, Arg/Arg: 24%, 48%, 28% vs. 30%, 61%, 9% respectively, p=0.0025). There was a significant genotype distribution between Indian and Black controls (Pro/Pro, Pro/Arg, Arg/Arg: 30%, 61%, 9% vs. 45.2% 40.5%, 14.3% respectively, p=0.0212). A significantly higher frequency of the p53 Arg72 allele was found in CAD patients compared to controls (52% vs. 39.5% respectively, p=0.0159). The variant allele was slightly higher in Indian controls (39.5%) compared to Black controls (34.5%), but this did not reach statistical significance (p=0.3324). The levels of total cholesterol, LDL, HDL, triglycerides, fasting glucose, fasting insulin and %HbA1c were not significantly influenced by the p53 genotypic variants. CONCLUSION Although the p53 codon 72 SNP is not associated with clinical markers of disease in CAD, the higher frequency of the variant allele in SA Indians may be a contributing factor for this population having an increased risk of developing premature CAD.

Fumonisin B1 inhibits apoptosis in HepG2 cells by inducing Birc-8/ILP-2

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium sp., a common contaminant of maize. FB1 inhibits sphingolipid biosynthesis, alters sphingosine/sphinganine ratios and modifies cell survival and cell death processes at varying propensities at both species- and tissue-specific level. We investigated the effect of FB1 on the apoptotic pathway in human hepatoma (HepG2) cells. We measured: (i) the level of cell proliferation and cell death mechanism of HepG2 cells (MTT assay, annexin V and propidium iodide staining, JC-1 assay, γH2AX and cleaved PARP and Hoechst staining); (ii) initiator and executioner caspase activity (luminometric enzyme activity assays); (iii) regulation of mRNA expression of pro- and anti- apoptotic molecules using an apoptosis array (qPCR) and (iv) levels of significantly altered apoptosis-related proteins (Western blotting) following a 24 h incubation. FB1 caused a dose-dependent decrease in cell viability with an inhibitory concentration for 50% of cell growth at 200 μM. FACS data showed FB1 induced a 2.5-fold increase in annexin V staining, however, caspase activity and mitochondrial depolarization was not significantly influenced. Cleaved PARP and γH2AX were significantly lower in treated cells with minimal DNA condensation and fragmentation observed with the Hoechst stain. BIRC-8/ILP-2 was most significantly up-regulated (8-fold; apoptosis array). ILP2 protein levels were elevated (2.3-fold) with a corresponding decrease in Smac/DIABLO protein levels (1.7-fold). Further analysis showed a dose-dependent increase in BIRC-8/ILP-2 mRNA and protein expression in HepG2 cells. We conclude that FB1 modulates apoptosis in a complex dose-dependent regulation of pro- and anti-apoptotic molecules.