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

<p>Abstract</p> <p>Background</p> <p>Rheumatoid 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.</p> <p>Methods</p> <p>We 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.</p> <p>Results</p> <p>The 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.</p> <p>Conclusion</p> <p>The 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.</p

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 (odds ratio (OR)=2.593; 95% confidence interval (CI) 1.353 - 4.971; p=0.0043) and GSTP1 A105/A105 OR=0.6011; 95% 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 relative risk (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.S Afr Med J 2012;102(7):627-630

Sirtuin 1 rs1467568 and rs7895833 in South African Indians with early-onset coronary artery disease

BACKGROUND : Sirtuin 1 (SIRT1), a class III histone deacetylase, has been identified as a candidate molecule affecting the epigenetic mechanisms of cardiovascular disease (CVD). Previous studies have shown that some SIRT1 single-nucleotide polymorphisms (SNPs) are associated with body mass index, diabetes, blood pressure, cholesterol metabolism and coronary artery calcification. We investigated two A>G SIRT1 SNPs, rs1467568 and rs7895833, in young South African (SA) Indians with coronary artery disease (CAD) and compared them to Indian and black controls. METHODS : For rs1467568, a total of 287 subjects were recruited into this study (104 CAD patients, 99 age-, gender- and race-matched controls, and 84 age- and gender-matched black controls). For rs7895833, a total of 281 subjects were recruited into this study (100 CAD patients, 99 age-, gender- and race-matched controls, and 82 age- and gender-matched black controls). All patients were male, of Indian ethnicity, stable CAD confirmed on angiography, mean age 37.5 years; range 24–45. All subjects were genotyped using TaqMan SNP genotyping assays. RESULTS : The variant allele for both SNPs was found at a higher frequency in the total Indian group compared to the total black population (rs1467568: 41 vs 18.5%, respectively, p < 0.0001, OR = 3.190, 95% CI: 2.058–40943; and rs7895833: 41 vs 22%, respectively, p < 0.0001, OR = 2.466, 95% CI: 1.620– 3.755). Indian controls presented with a higher frequency for both SNPs compared to black controls (rs1467568: 40 vs 18.5%, respectively, p < 0.0001, OR = 2.996, 95% CI: 1.850– 4.853; and rs7895833: 41 vs 22%, respectively, p < 0.0001, OR = 2.513, 95% CI: 1.578–4.004). No difference was seen in the distribution of both SNPs between CAD patients and either control group. We did not observe any association between the SNPs and clinical parameters in CAD patients and controls. CONCLUSION : Both SNP variant alleles occurred more frequently in SA Indians than in SA blacks. A larger study group and further analysis is required to assess whether these SIRT1 SNPs may serve as risk factors that contribute to Indians developing early-onset CAD.The National Research Foundation (NRF) for a scholarship and UKZN (College of Health Sciences).www.cvja.co.zaam2016Physiolog

TB/HIV pleurisy reduces Th17 lymphocyte proportion independent of the cytokine microenvironment

T-helper (Th) 17 cells are a pro-inflammatory subset of CD4+ effector T-cells critical in mucosal immunity. Imbalances in Th17 cell proportion have been implicated in the pathogenesis of several diseases; however, this has not been adequately explored in tuberculosis (TB) and human immunodeficiency virus (HIV) co-infection. Since Th17 cells are predominantly mucosally associated, we assessed Th17 proportion and associated microenvironment in pleural effusions from patients co-infected with TB/HIV. Our results show that TB+HIV+ pleurisy results in significantly reduced frequency of CD4+IL-17+RORC+STAT3+ Th17 cells compared to TB−HIV−ex vivo (p = 0.0054) and was confirmed in conditioned media studies in vitro (p = 0.0001). This was not associated with alterations in Th17 polarising cytokines IL-6, IL-21 and IL-23 or changes in Th17 signature cytokines IL-17A and F. However, the mRNA expression of Th17 signalling molecules, IL-6 (p = 0.0022), IL-6R (p = 0.0247), IL-1β (p = 0.0022) and signal transducer and activator (STAT) 3 (p = 0.0022) were significantly upregulated. Notably, TB+HIV+ pleural fluid contained significantly higher concentrations of IL-1β (p = 0.0008), IL-22 (p = 0.0115), IL-31 (p = 0.0210), TNF-α (p = 0.0251) and IFN-γ (p = 0.0026) than TB−HIV− pleural fluid ex vivo. Taken together, this suggests a reduced portion of Th17 lymphocytes in TB/HIV pleurisy is independent of locally mediated cytokine polarisation.The National Research Foundation, KwaZulu-Natal Research Institute for Tuberculosis and HIV and College of Health Sciences, University of KwaZulu-Natal.http://intl.elsevierhealth.com/journals/tube2017-07-31hb2016Physiolog

A comparative analysis of mycotoxin contamination of supermarket and premium brand pelleted dog food in Durban, South Africa

Dry pelleted dog food in the South African market is available via supermarkets, pet stores (standard brands [SBs]) and veterinary channels (premium brands [PBs]). For the purpose of this study, the supermarket channel included the cheaper quality foods and PBs were sold via the veterinary channel (n = 20). These feeds were analysed for four main mycotoxins (aflatoxins [AF], fumonisin [FB], ochratoxin A [OTA] and zearalenone [ZEA]) using standard welldescribed extraction, characterisation and quantitation processes. Irrespective of the brand or marketing channel, all foods were contaminated with fungi (mainly Aspergillus flavus, Aspergillus fumigatus and Aspergillus parasiticus) and mycotoxins (most prevalent being aflatoxins and fumonisins). This was observed in all 20 samples irrespective of the marketing channel or perceived quality. Also, many samples within each marketing channel failed the 10 ppb limit for aflatoxin set by regulations in South Africa. Although fumonisin was detected in all samples, a single sample failed the Food and Drug Administration (FDA) limit of 100 ppb. Both OTA and ZEA were found at low concentrations and were absent in some samples. This study suggested that higher priced dog food does not ensure superior quality or that it is free from contamination with fungi or mycotoxins. However, analysis of the more expensive PBs did reveal contamination concentrations lower than those of the SBs

Fusaric acid decreases p53 expression by altering promoter methylation and m6A RNA methylation in human hepatocellular carcinoma (HepG2) cells

Fusaric acid (FA) is a food-borne mycotoxin that mediates toxicity with limited information on its epigenetic properties. p53 is a tumour suppressor protein that regulates cell cycle arrest and apoptotic cell death. The expression of p53 is regulated transcriptionally by promoter methylation and post-transcriptionally by N-6-methyladenosine (m6A) RNA methylation. We investigated the effect of FA on p53 expression and its epigenetic regulation via promoter methylation and m6A RNA methylation in human hepatocellular carcinoma (HepG2) cells. HepG2 cells were treated with FA [0, 25, 50, 104, and 150 µg/ml; 24 h] and thereafter, DNA, RNA, and protein was isolated. Promoter methylation and expression of p53 was measured using qPCR and Western blot. RNA immuno-precipitation was used to determine m6A-p53 levels. The expression of m6A methyltransferases (METTL3 and METTL14), demethylases (FTO and ALKBH5), and readers (YTHDF1-3 and YTHDC2) were measured using qPCR. FA induced p53 promoter hypermethylation (p < 0.0001) and decreased p53 expression (p < 0.0001). FA decreased m6A-p53 levels (p < 0.0001) by decreasing METTL3 (p < 0.0001) and METTL14 (p < 0.0001); and suppressed expression of YTHDF1 (p < 0.0001), YTHDF3 (p < 0.0001), and YTHDC2 (p < 0.0001) that ultimately reduced p53 translation (p < 0.0001). Taken together, the data shows that FA epigenetically decreased p53 expression by altering its promoter methylation and m6A RNA methylation in HepG2 cells. This study reveals a mechanism for p53 regulation by FA and provides insight into future therapeutic interventions

Fusaric acid induces hepatic global m6A RNA methylation and differential expression of m6A regulatory genes in vivo - a pilot study

N6-methyladenosine (m6A) is an abundant epitranscriptomic mark that regulates gene expression to execute cellular developmental programmes and environmental adaptation. Fusaric acid (FA) is a mycotoxin that contaminates agricultural foods and exerts toxicity in humans and animals; however, its epitranscriptomic effects are unclear. We investigated the effect of FA on global m6A RNA methylation and mRNA expression levels of key m6A regulatory genes in C57BL/6 mouse livers. C57BL/6 mice (n = 6/group) were orally administered 0.1 M phosphate-buffered saline (PBS) or 50 mg/kg FA. Mice were euthanized 24 h after oral administration, livers were harvested, and RNA was isolated. RNA samples were assayed for global m6A levels using an m6A RNA Methylation Quantification Kit. The mRNA expression of m6A regulators i.e. writers, erasers, and readers were measured by qRT-PCR. FA increased global m6A RNA methylation (p < 0.0001) in mouse livers. FA increased the expression of METTL3 (p = 0.0143) and METTL14 (p = 0.0281), and decreased the expression of FTO (p = 0.0036) and ALKBH5 (p = 0.0035). The expression of YTHDF2 (p = 0.0007), YTHDF3 (p = 0.0061), and YTHDC2 (p = 0.0258) were increased by FA in mouse livers. This study shows that the liver m6A epitranscriptome can be modified by FA exposure in an in vivo model and can be useful for identifying the molecular mechanisms whereby m6A RNA modifications influence the toxicological outcomes of FA exposure

A Critical Review of the Biochemical Mechanisms and Epigenetic Modifications in HIV- and Antiretroviral-Induced Metabolic Syndrome

Metabolic syndrome (MetS) is a non-communicable disease characterised by a cluster of metabolic irregularities. Alarmingly, the prevalence of MetS in people living with Human Immunodeficiency Virus (HIV) and antiretroviral (ARV) usage is increasing rapidly. This study aimed to look at biochemical mechanisms and epigenetic modifications associated with HIV, ARVs, and MetS. More specifically, emphasis was placed on mitochondrial dysfunction, insulin resistance, inflammation, lipodystrophy, and dyslipidaemia. We found that mitochondrial dysfunction was the most common mechanism that induced metabolic complications. Our findings suggest that protease inhibitors (PIs) are more commonly implicated in MetS-related effects than other classes of ARVs. Furthermore, we highlight epigenetic studies linking HIV and ARV usage to MetS and stress the need for more studies, as the current literature remains limited despite the advancement in and popularity of epigenetics