Biochemical, apoptotic and cellular stress studies in rheumatoid arthritis.

Ph. D. University of KwaZulu-Natal, Westville 2009.Abstract available in PDF file

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

Association of -308 TNF-alpha promoter polymorphism with viral load and CD4 T-helper cell apoptosis in HIV-1 infected black South Africans

Objective. To determine whether the -308 TNF-α promoter polymorphism is associated with markers of HIV progression in the South African population. Methods. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the -308 TNF-α polymorphism in 75 patients and 76 healthy controls. Serum TNF-α concentrations were measured using ELISA in each cohort. CD4+ T cell apoptosis and HIV-1 RNA viral load were determined using Annexin-V-FITC assay and Nuclisens Easy Q HIV-1 assay respectively. CD4 + T cell counts were measured flow cytometrically. Results. The frequency of -308 G allele was similar in the HIV-1 and control cohorts. The -308GG genotype was associated with lower TNF-α concentrations and markers of increased HIV progression indicated by higher TH lymphocyte apoptosis, lower TH lymphocyte count and higher plasma viral load, irrespective of treatment. Conclusion. The presence of the TNF-α -308 G allele in HIV-1 patients may be associated with increased risk of HIV-1 progression. Further research is required to investigate the nature of this association. S Afr J HIV Med 2012;13(2):72-77

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

Apoptosis-promoting effects of Sutherlandia frutescens extracts on normal human lymphocytes in vitro

Sutherlandia frutescens (SF), an indigenous medicinal plant to South Africa, is traditionally used to treat a diverse range of illnesses. More specifically, the immune-enhancing potential of SF has been recognised to the extent that SF extracts have been recommended as an adjuvant in HIV/AIDS treatment by the South African Ministry of Health, despite a lack of knowledge of its mechanism of action or potential immune toxicity. As yet, unsubstantiated data support the notion of immunostimulatory effects of SF extracts in HIV-infected patients. This was suggested by post-treatment recovery of CD4+ cells brought about by the reduction of the impact of virus-induced apoptosis. This study investigated the apoptotic effects of SF extracts on normal human lymphocytes in vitro. Initially, an acute cytotoxic profile of SF extract was formulated, from which an IC50 of 7.5 mg/mL was calculated and administered for 3 h, 6 h and 12 h to cell populations. At 12 h, SF caused a significant increase in apoptosis in the total lymphocyte population and CD4+ cells as evidenced by increased phosphatidylserine (PS) translocation, caspase-3/7 activity, and decreased ATP content. After 12 h, the SF extract initiated lymphocyte activation in both total lymphocyte and CD4+ subpopulations, indicated by a doubling of the number of cells expressing the CD69 activation marker. The apoptosis observed may thus be the result of activation-induced lymphocyte cell death (AICD). Our results are in conflict with preliminary clinical evidence which has suggested SF extracts are possibly beneficial in the treatment of HIV infection. More extensive evaluations of the effects of SF extracts on the immune system in such subjects are urgently needed

The effects of <i>Sutherlandia frutescens</i> extracts in cultured renal proximal and distal tubule epithelial cells

Sutherlandia frutescens (SF), a medicinal plant indigenous to South Africa, is traditionally used to treat a diverse range of illnesses, including cancer and viral infections. The biologically active compounds of SF are polar, thus renal elimination increases susceptibility to toxicity in that organ. This study investigated the antioxidant potential, lipid peroxidation, mitochondrial membrane potential and apoptotic induction by SF extracts on proximal and distal tubule epithelial cells. Cell viability was determined using the MTT assay. Mitochondrial membrane potential was determined using a flow cytometric JC-1 Mitoscreen assay. Cellular glutathione and apoptosis were measured using the GSH-Glo™ Glutathione assay and Caspase-Glo® 3/7 assay, respectively. The IC50 values from the cell viability results for LLC-PK1 and MDBK were 15 mg/mL and 7 mg/mL, respectively. SF extracts significantly decreased intracellular glutathione in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells, while lipid peroxidation increased in treated LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. JC-1 analysis showed that SF extracts promoted mitochondrial membrane depolarization in both LLC-PK1 and MDBK cells by up to 80% (p < 0.0001). The activity of caspase 3/7 increased in both LLC-PK1 (11.9-fold; p < 0.0001) and MDBK (2.2-fold; p < 0.0001) cells. SF extracts at high concentrations appear to increase oxidative stress, to alter mitochondrial membrane integrity, and to promote apoptosis in renal tubule epithelia

The effects of Sutherlandia frutescens extracts in cultured renal proximal and distal tubule epithelial cells

Sutherlandia frutescens (SF), a medicinal plant indigenous to South Africa, is traditionally used to treat a diverse range of illnesses, including cancer and viral infections. The biologically active compounds of SF are polar, thus renal elimination increases susceptibility to toxicity in that organ. This study investigated the antioxidant potential, lipid peroxidation, mitochondrial membrane potential and apoptotic induction by SF extracts on proximal and distal tubule epithelial cells. Cell viability was determined using the MTT assay. Mitochondrial membrane potential was determined using a flow cytometric JC-1 Mitoscreen assay. Cellular glutathione and apoptosis were measured using the GSH-Glo™ Glutathione assay and Caspase-Glo® 3/7 assay, respectively. The IC50 values from the cell viability results for LLC-PK1 and MDBK were 15 mg/mL and 7 mg/mL, respectively. SF extracts significantly decreased intracellular glutathione in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells, while lipid peroxidation increased in treated LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. JC-1 analysis showed that SF extracts promoted mitochondrial membrane depolarization in both LLC-PK1 and MDBK cells by up to 80% (p < 0.0001). The activity of caspase 3/7 increased in both LLC-PK1 (11.9-fold; p < 0.0001) and MDBK (2.2-fold; p < 0.0001) cells. SF extracts at high concentrations appear to increase oxidative stress, to alter mitochondrial membrane integrity, and to promote apoptosis in renal tubule epithelia