FGFR2 genetic variants in women with breast cancer.

Black African populations are more genetically diverse than others, but genetic variants have been studied primarily in European populations. The present study examined the association of four single nucleotide polymorphisms (SNPs) of the fibroblast growth factor receptor 2, associated with breast cancer in non­African populations, with breast cancer in Black, southern African women. Genomic DNA was extracted from whole blood samples of 1,001 patients with breast cancer and 1,006 controls (without breast cancer), and the rs2981582, rs35054928, rs2981578, and rs11200014 polymorphisms were analyzed using allele­specific Kompetitive allele­specific PCR™, and the χ2 or Fisher's exact tests were used to compare the genotype frequencies. There was no association between those SNPs and breast cancer in the studied cohort, although an association was identified between the C/C homozygote genotype for rs2981578 and invasive lobular carcinoma. These results show that genetic biomarkers of breast cancer risk in European populations are not necessarily associated with risk in sub­Saharan African populations. African populations are more heterogenous than other populations, and the information from this population can help focus genetic risks of cancer in this understudied population.

Altered microRNA expression in patients with sarcoidosis.

Background Sarcoidosis is a granulomatous multisystem disease of uncertain aetiology. The disease has major inflammatory and immune components; however, the immunopathogenesis is not well understood. Micro ribonucleic acids (microRNAs) are classes of miniature, single-stranded, non-coding RNAs. Their key recognised role includes mediating the silencing of target genes post-transcriptionally. Recently, the role of miRNAs has gained interest in numerous disorders, suggested as being involved in pathogenesis of those diseases and acting as disease markers. Very little is known about the role of miRNAs in sarcoidosis, with nothing known regarding miRNAs in South African patients. The main objective, therefore, was to investigate the serum expression of approximately 800 miRNAs in patients with sarcoidosis compared with race-, age- and gender-matched healthy controls. Methods A total of six patients and six matched controls participated in this study. Whole blood samples were collected in EDTA tubes, processed and the plasma retained. RNA was extracted from the stored plasma samples using the QIAGEN miRNeasy Mini Kit® and concentrated using a salt-ethanol precipitation. The extracted miRNA was profiled using an nCounter® miRNA human v3 expression assay and data analysed using the nSolver™ Analysis Software. Results After excluding one sample/control pair because of cellular RNA contamination, the remaining five patient and five matched control samples were analysed, and 145 miRNAs were found to be potentially differentially expressed. On applying a Bonferroni correction, the only miRNA that was significantly different was miRNA let-7a-5p, which was significantly overexpressed (141-fold change; p<0.0003) in patients compared with controls. Conclusion This is the first miRNA report of differentially expressed miRNAs in the serum of patients with sarcoidosis and matched healthy controls in South Africa. The results obtained suggest that miRNAs may play a role in sarcoidosis pathogenesis. Whether these molecules have diagnostic or prognostic implications, needs future studies recruiting larger patient cohorts.

PAM50 intrinsic subtypes, risk of recurrence score and breast cancer survival in HIV-positive and HIV-negative patients-a South African cohort study.

PURPOSE: Treatment decision making for patients with breast cancer increasingly depends on analysis of markers or systems for estimating risk of breast cancer recurrence. Breast cancer intrinsic subtypes and risk of recurrence (ROR) scores have been found to be valuable in predicting survival and determining optimal treatment for individual patients. We studied the association of breast cancer survival with the PAM50 gene expression assay in HIV-positive and HIV-negative patients. METHOD: RNA was extracted from formalin-fixed paraffin-embedded specimens of histologically confirmed invasive carcinoma and was purified using the AllPrep® DNA/RNA FFPE kit, Qiagen (Hilden, Germany). The NanoString RUO PAM50 algorithm was used to determine the molecular subtype and the risk of recurrence score of each sample. The overall and disease-free survival were determined with comparison made among HIV-positive and -negative patients. We then generated Kaplan-Meier survival curves, calculated p-values and estimated hazard ratios and their 95% confidence intervals using Cox regression models. RESULTS: Of the 384 RNA samples analysed, 98.4% met the required RNA quality standard and the specified QC threshold for the test. Luminal B was the most common PAM50 intrinsic subtype and 82.1% of patients were at high risk for disease recurrence based on ROR score. HIV infection, PAM50-based HER2-enriched and basal-like intrinsic subtypes, and high ROR were associated with poor overall and disease-free survival. HIV-positive patients with luminal A & B subtypes had significantly worse survival outcomes than HIV-negative luminal patents. CONCLUSION: Aggressive tumour biology was common in our cohort. HIV infection, PAM50 HER2-enriched,basal-like intrinsic subtypes and high ROR score were associated with poor overall and disease-free survival. HIV infection impacted survival in patients with luminal subtypes only.

Discordance between PAM50 intrinsic subtyping and immunohistochemistry in South African women with breast cancer.

PURPOSE: Breast cancer is a heterogeneous disease with different gene expression profiles, treatment options and outcomes. In South Africa, tumors are classified using immunohistochemistry. In high-income countries multiparameter genomic assays are being utilized with implications for tumor classification and treatment. METHODS: In a cohort of 378 breast cancer patients from the SABCHO study, we investigated the concordance between tumor samples classified by IHC and the PAM50 gene assay. RESULTS: IHC classified patients as ER-positive (77.5%), PR-positive (70.6%), and HER2-positive (32.3%). These results, together with Ki67, were used as surrogates for intrinsic subtyping, and showed 6.9% IHC-A-clinical, 72.7% IHC-B-clinical, 5.3% IHC-HER2-clinical and 15.1% triple negative cancer (TNC). Typing using the PAM50 gave 19.3% luminal-A, 32.5% luminal-B, 23.5% HER2-enriched and 24.6% basal-like. The basal-like and TNC had the highest concordance, while the luminal-A and IHC-A group had the lowest concordance. By altering the cutoff for Ki67, and realigning the HER2/ER/PR-positive patients to IHC-HER2, we improved concordance with the intrinsic subtypes. CONCLUSION: We suggest that the Ki67 be changed to a cutoff of 20-25% in our population to better reflect the luminal subtype classifications. This change would inform treatment options for breast cancer patients in settings where genomic assays are unaffordable.

High risk APOL1 genotypes and kidney disease among treatment naïve HIV patients at Kano, Nigeria.

INTRODUCTION: Racial disparities are known in the occurrence of kidney disease with excess risks found among people of African descent. Apolipoprotein L1 (APOL1) gene variants G1 and G2 are associated with kidney disease among HIV infected individuals of African descent in the USA as well as among black population in South Africa. We set out to investigate the prevalence of these high-risk variants and their effects on kidney disease among HIV infected patients in Northern Nigeria with hitherto limited information despite earlier reports of high population frequencies of these alleles from the Southern part of the country. METHODS: DNA samples obtained from the whole blood of 142 participants were genotyped for APOL1 G1 and G2 variants after initial baseline investigations including assessment of kidney function. Participants comprised 50 HIV positive patients with no evidence of kidney disease, 52 HIV negative individuals with no kidney disease and 40 HIV positive patients with chronic kidney disease (CKD) evidenced by persistent proteinuria and/or reduced eGFR, who also had a kidney biopsy. All the HIV positive patients were newly diagnosed and treatment naïve. RESULTS: The distribution of the APOL1 genotypes among the study participants revealed that 24.6% had a G1 risk allele and 19.0% a G2. The frequency of the High Risk Genotype (HRG) was 12.5% among those with CKD compared to 5.8% in the HIV negative group and zero in the HIV positive no CKD group. Having the HRG was associated with a higher odds for developing HIV Associated Nephropathy (HIVAN) (2 vs 0 risk alleles: OR 10.83, 95% CI 1.38-84.52; P = 0.023; 2 vs 0 or 1 risk alleles: OR 5.5, 95% CI 0.83-36.29; P = 0.07). The HRG was also associated with higher odds for Focal Segmental Glomerulosclerosis (FSGS) (2 vs 0 risk alleles: OR 13.0, 95% CI 2.06-81.91; P = 0.006 and 2 vs 0 or 1 risk alleles: OR 9.0, 95%CI 1.62-50.12; P = 0.01) when compared to the control group. CONCLUSION: This study showed a high population frequency of the individual risk alleles of the APOL1 gene with higher frequencies noted among HIV positive patients with kidney disease. There is high association with the presence of kidney disease and especially FSGS and HIVAN among treatment naive HIV patients carrying two copies of the HRG.

Demographic and clinical profile of black patients with chronic kidney disease attending a tertiary hospital in Johannesburg, South Africa.

BACKGROUND: The prevalence of chronic kidney disease (CKD) is increasing worldwide; black patients have an increased risk of developing CKD and end stage kidney disease (ESKD) at significantly higher rates than other races. METHODS: A cross sectional study was carried out on black patients with CKD attending the kidney outpatient clinic at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) in South Africa, between September 2019 to March 2020. Demographic and clinical data were extracted from the ongoing kidney outpatient clinic records and interviews, and were filled in a questionnaire. Patients provided blood and urine for laboratory investigations as standard of care, and data were descriptively and inferentially entered into REDcap and analysed using STATA version 17. Multivariable logistic regression analysis was used to identify demographic and clinical variables associated with advanced CKD. RESULTS: A total of 312 black patients with CKD were enrolled in the study with a median age of 58 (IQR 46-67) years; 58% patients had advanced CKD, 31.5% of whom had grossly increased proteinuria, 96.7% had hypertension, 38.7% had diabetes mellitus and 38.1% had both hypertension and diabetes mellitus. In patients with advanced CKD, the median age was 61 (IQR 51-69) years, eGFR 33 (30-39) mL/min/1.73 m2, serum bicarbonate 22 (IQR 20-24), haemoglobin 12.9 (IQR 11.5-14.0) g/dl and serum uric acid 0.43 (IQR 0.37-0.53). The prevalence of metabolic acidosis was 62.4%, anemia 46.4% and gout 30.9% among those with advanced CKD, while the prevalence of metabolic acidosis and anaemia was 46.6% and 25.9% respectively in those with early CKD. Variables with higher odds for advanced CKD after multivariable logistic regression analysis were hypertension (OR 3.3, 95% CI 1.2-9.2, P = 0.020), diabetes mellitus (OR 1.8, 95% CI 1.1-3.3, P = 0.024), severe proteinuria (OR 3.5, 95% CI 1.9-6.5, P = 0.001), angina (OR 2.5, 95% CI 1.2-5.1, P = 0.008), anaemia (OR 2.9, 95% CI 1.7-4.9, P = 0.001), hyperuricemia (OR 2.4, 95% CI 1.4-4.1, P = 0.001), and metabolic acidosis (OR 2.0, 95% CI 1.2-3.1, P = 0.005). Other associations with advanced CKD were loss of spouse (widow/widower) (OR 3.2, 95% CI 1.4-7.4, P = 0.006), low transferrin (OR 2.4, 95% CI 1.1-5.1, P = 0.028), hyperkalemia (OR 5.4, 95% CI 1.2-24.1, P = 0.029), use of allopurinol (OR 2.4, 95% CI 1.4-4.3, P = 0.005) and doxazosin (OR 1.9, 95% CI 1.2-3.1, P = 0.006). CONCLUSION: Hypertension and diabetes mellitus were strongly associated with advanced CKD, suggesting a need for primary and secondary population-based prevention measures. Metabolic acidosis, anemia with low transferrin levels, hyperuricemia and hyperkalemia were highly prevalent in our patients, including those with early CKD, and they were strongly associated with advanced CKD, requiring clinicians and dietitians to be proactive in supporting the needs of CKD patients in meeting their daily dietary requirements towards preventing and slowing the progression of CKD.

Profiling Biomarkers in HIV Glomerular Disease - Potential for the Non-Invasive Diagnosis of HIVAN?

BACKGROUND: There is a wide spectrum of kidney pathology in human immunodeficiency virus (HIV) infection, affecting all structures of the kidney. The histology of HIV chronic kidney disease (CKD) is diverse, ranging from HIV-associated nephropathy (HIVAN) to focal glomerulosclerosis (FSGS), HIV-immune complex disease (HIV-ICD), other glomerulopathies and tubulo-interstitial nephritis. Definitive diagnosis is by kidney biopsy, an invasive procedure. However, serum and urinary biomarkers may be useful in predicting the histological diagnosis of HIVAN. PURPOSE: We wished to determine the utility of serum and urinary biomarkers in predicting the histological diagnosis of HIVAN. PATIENTS AND METHODS: We measured neutrophil gelatinase-associated lipocalin (NGAL), cystatin C, transforming growth factor (TGF)-ß isoforms and bone morphogenetic protein (BMP)-7 in the serum and urine in patients with different histological forms of HIV glomerular disease. RESULTS: In HIVAN, we demonstrated increased levels of serum cystatin C and increased levels of serum and urinary NGAL. Urinary TGF-ß1 and TGF-ß2 levels were elevated in HIV-positive patients with CKD but were not significantly different in the different HIV histologies, while urinary BMP-7 levels were elevated in minimal change disease. CONCLUSION: This study confirmed the presence of increased serum and urinary biomarkers of tubular injury in patients with HIVAN, and increased urinary biomarkers of fibrosis in HIV CKD, and may indicate their value as a non-invasive diagnostic tool for the diagnosis of HIVAN.

Survival of south african women with breast cancer receiving anti-retroviral therapy for HIV.

PURPOSE: Breast cancer outcomes in sub-Saharan Africa is reported to be poor, with an estimated five-year survival of 50% when compared to almost 90% in high-income countries. Although several studies have looked at the effect of HIV in breast cancer survival, the effect of ARTs has not been well elucidated. METHODS: All females newly diagnosed with invasive breast cancer from May 2015-September 2017 at Charlotte Maxeke Johannesburg Academic and Chris Hani Baragwanath Academic Hospital were enrolled. We analysed overall survival and disease-free survival, comparing HIV positive and negative patients. Kaplan-Meier survival curves were generated with p-values calculated using a log-rank test of equality while hazard ratios and their 95% confidence intervals (CIs) were estimated using Cox regression models. RESULTS: Of 1019 patients enrolled, 22% were HIV positive. The overall survival (95% CI) was 53.5% (50.1-56.7%) with a disease-free survival of 55.8% (52.1-59.3) after 4 years of follow up. HIV infection was associated with worse overall survival (HR (95% CI): 1.50 (1.22-1.85), p < 0.001) and disease-free survival (OR (95% CI):2.63 (1.71-4.03), p < 0.001), especially among those not on ART at the time of breast cancer diagnosis. Advanced stage of the disease and hormone-receptor negative breast cancer subtypes were also associated with poor survival. CONCLUSION: HIV infection was associated with worse overall and disease-free survival. HIV patients on ARTs had favourable overall and disease-free survival and with ARTs now being made accessible to all the outcome of women with HIV and breast cancer is expected to improve.

Hepcidin and GDF-15 are potential biomarkers of iron deficiency anaemia in chronic kidney disease patients in South Africa.

BACKGROUND: Anaemia is a common presenting feature among patients with chronic kidney disease (CKD) and it is associated with poor clinical outcomes and quality of life. It is not clear if growth differentiation factor-15 (GDF-15) or hepcidin are useful as early markers of iron deficiency anaemia (IDA) among non-dialysis CKD patients. We therefore evaluated the diagnostic validity of GDF-15 and hepcidin as biomarkers of IDA among non-dialysis CKD patients in Johannesburg, South Africa. METHOD: An analytic cross-sectional study was conducted among non-dialysis CKD patients (n = 312) and apparently healthy controls (n = 184) from June to December 2016 at an Academic Hospital, in Johannesburg, South Africa. An interviewer administered proforma was used to obtain the socio-biological and clinical characteristics of the participants. Serum levels of GDF-15 and hepcidin were determined. Predictive logistic regression models were built and post estimation receiver operator characteristics were determined to evaluate diagnostic validity of hepcidin and GDF-15 for absolute and functional iron deficiency anaemia. RESULTS: About half (50.6%) of the participants were female while the participants' mean age was 49.7 ± 15.8 years. The predictive value of diagnosing absolute IDA among CKD patients using GDF-15 was 74.02% (95% CI: 67.62-80.42%) while the predictive value of diagnosing functional IDA among CKD patients using hepcidin was 70.1% (95% CI: 62.79-77.49%).There was a weak negative correlation between hepcidin levels and GFR (r = - 0.19, p = 0.04) in anaemic CKD patients, and between serum GDF-15 and haemoglobin (r = - 0.34, p = 0.001). Serum ferritin (ß = 0.00389, P-value< 0.001), was a predictor of log hepcidin. MCHC (ß = - 0.0220, P-value 0.005) and CKD stage (ß = 0.4761, P-value < 0.001), race (ß = 0.3429, P-value = 0.018) were predictors of log GDF-15. Both GDF-15 (adj OR: 1.0003, 95%CI: 1.0001-1.0005, P = 0.017) and hepcidin (adj OR: 1.003, 95%CI: 1.0004-1.0055, P = 0.023) were associated with iron deficiency anaemia after multiple linear regression modelling. CONCLUSION: Serum GDF-15 is a potential biomarker of absolute IDA, while hepcidin levels can predict functional IDA among CKD patients.

Association of chronic inflammation and accelerated atherosclerosis among an indigenous black population with chronic kidney disease.

INTRODUCTION: Inflammation plays a major role in the development of atherosclerosis and cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients. Toll-like receptor-4 (TLR4) is a major receptor for lipopolysaccharides (endotoxin) and other ligands involved in the pathogenesis of inflammation. We determined whether endotoxin levels and the presence of TLR4 polymorphisms are associated with markers of inflammation and atherosclerosis among South African CKD patients. MATERIALS AND METHODS: Endotoxin, lipopolysaccharide binding protein (LBP), serum CD14 (sCD14), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and carotid intima media thickness (CIMT) were measured in 160 participants (120 CKD patients and 40 controls). Associations between endotoxins and CIMT in the presence of sCD14, IL-8 and MCP-1, were assessed using odds ratios. Participants were screened for the presence of Asp299Gly and Thr399Ile TLR4 polymorphisms, and CIMT and inflammatory markers were compared between subjects with and without TLR4 polymorphisms. RESULTS: Endotoxin levels correlated with sCD14 (r = 0.441, p<0.001) and MCP-1 (r = 0.388, p<0.001) levels while increased CIMT was associated with MCP-1 (r = 0.448, p<0.001), sCD14 levels (r = 0.476, p<0.001), LBP (r = 0.340, p<0.001), and IL-8 (r = 0.395, p<0.001). Atherosclerosis was associated with endotoxin levels (odds ratio: 4.95; 95% confidence interval: 2.52-9.73; p<0.001), and was predicted by higher serum levels of inflammatory markers. Analysis of patients with TLR4 polymorphisms showed reduced serum levels of inflammatory markers and CIMT values compared with the patients carrying the wild type TLR4 alleles. CONCLUSION: The study demonstrated associations between circulating endotoxaemia, systemic inflammation and accelerated atherosclerosis among South African CKD patients, and showed that the atherogenic predictive power of endotoxaemia was significantly increased by the presence of elevated levels of inflammatory markers. Additional findings, which must be confirmed, suggest that TLR4 polymorphisms are associated with low levels of inflammatory markers and CIMT values.

APOL1 Genetic Variants Are Associated with Serum-Oxidized Low-Density Lipoprotein Levels and Subclinical Atherosclerosis in South African CKD Patients.

INTRODUCTION: Apolipoprotein L1 (APOL1) plays an important role in cholesterol metabolism and attenuation of low-density lipoprotein (LDL) oxidation. While protecting against Trypanosoma brucei rhodesiense infection, APOL1 risk alleles confer greater risk for CKD and cardiovascular disease among patients of African descent. OBJECTIVES: We investigated whether APOL1 risk variants are associated with atherosclerosis and oxidized LDL (OxLDL) levels among black South African CKD patients. METHODS: A cross-sectional study of 120 adult CKD patients and 40 controls was undertaken. DNA samples of participants were genotyped for APOL1 G1 and G2 variants. High-sensitivity C-reactive protein, serum lipids, and OxLDL levels were measured, and carotid doppler ultrasonography was performed on all participants. RESULTS: APOL1 alleles rs73885319, rs60910145, and rs71785313 had minor allele frequencies of 9.2, 8.8, and 17.5%, respectively, in the patients, and 8.8, 8.8, and 13.8%, respectively, in the controls. Of the 9 patients with 2 APOL1 risk alleles, 77.8% were compound G1/G2 heterozygotes and 22.2% were G2 homozygotes. Carriers of at least 1 APOL1 risk allele had a 3-fold increased risk of subclinical atherosclerosis (odds ratio 3.19; 95% confidence interval: 1.64-6.19; p = 0.01) compared to individuals with no risk alleles. Patients with 1 or 2 APOL1 risk alleles showed a significant increase in OxLDL levels when compared with those without the APOL1 risk allele. CONCLUSION: These findings suggest an increased risk for atherosclerosis in carriers of a single APOL1 risk variant, and the presence of APOL1 risk variants was associated with increased serum OxLDL levels in black South African CKD patients.

Biomarkers for Diagnosis and Prediction of Outcomes in Contrast-Induced Nephropathy.

BACKGROUND: Serum creatinine is suboptimal as a biomarker in the early diagnosis of contrast-induced nephropathy (CIN). In this study, we investigated a panel of novel biomarkers in the early diagnosis of CIN and in assessing patient outcomes. METHODS: This single-centre, nested, prospective case-controlled study included 30 patients with CIN and 60 matched controls. Serum and urine samples were collected before contrast administration and at 24 hours, 48 hours, and ≥5 days after contrast administration. Concentrations of NGAL, cystatin C, ß 2M, IL18, IL10, KIM1, and TNFα were determined using Luminex and ELISA assays. Outcomes were biomarker diagnostic discrimination performance for CIN and mortality after generation of area under receiver operating characteristic curves (AUROCs). RESULTS: Median serum levels for 24 h cystatin C (p < 0.01) and 48 h ß 2M levels (p < 0.001) and baseline urine NGAL (p=0.02) were higher in CIN patients compared to controls with AUROCs of 0.75, 0.78, and 0.74, respectively, for the early diagnosis of CIN. Serum ß 2M levels were higher in CIN patients at all time points. Elevated baseline serum concentrations of IL18 (p < 0.001), ß 2M (p=0.04), TNFα (p < 0.001), and baseline urine KIM (p=0.01) and 24 h urine NGAL (p=0.02) were significantly associated with mortality. Baseline serum concentrations of IL18, ß 2M, and TNFα showed the best discrimination performance for mortality with AUROCs, all >0.80. Baseline NGAL was superior for excluding patients at risk for CIN, with positive and negative predictive ranges of 0.50-0.55 and 0.81-0.88, respectively. Cystatin C (p=0.003) and ß 2M (p=0.03) at 24 h independently predicted CIN risk. ß 2M predicted increased mortality of 40% at baseline and 50% at 24 hours. CONCLUSION: Serum cystatin C at 24 h was the best biomarker for CIN diagnosis, while baseline levels of serum IL18, ß 2M, and TNFα were best for predicting prognosis.

Endothelial dysfunction in HIV-positive patients with acute coronary syndromes.

AIM: This study investigated endothelial function in HIV-positive patients with acute coronary syndrome (ACS). Flow-mediated dilatation, pulse-wave velocity, carotid intima-media thickness and endothelial biomarkers were used to non-invasively investigate endothelial dysfunction. METHODS: Twenty HIV-positive patients with ACS (HIV+/ACS) were compared to 20 HIV-negative patients with ACS (HIV-/ACS) and 20 HIV-positive patients without ACS (HIV+/no ACS). RESULTS: Endothelial function measured by flow-mediated dilatation (FMD) was similar in both the HIV+/ACS (5.2; IQR 1.4-13.4%) and HIV-/ACS groups (3.7; IQR 2.3-4.4%) (p = 0.78). Arterial stiffness, measured by pulse-wave velocity (PWV) was low in all three cohorts. Carotid intima-media thickness (CIMT) was also low in all three cohorts. The vascular cellular adhesion molecule-1 (VCAM-1) levels in HIV-positive patients with and without ACS were significantly higher than in the HIV-/ACS cohort (p = 0.033 and 0.024, respectively). CONCLUSIONS: Non-invasive investigations such as FMD, CIMT and PWV did not identify patients with HIV who were at high risk of ACS. Endothelial biomarkers may be more useful markers to identify HIV-positive patients who have endothelial dysfunction and increased risk of ACS.

Interleukin-6 gene polymorhisms and interleukin-6 levels are associated with atherosclerosis in CKD patients.

Inflammation is a major risk factor for atherosclerosis. Genetic polymorphisms in the inflammatory cytokine genes have been associated with atherosclerosis. Because levels of inflammatory cytokines are markedly elevated in patients with chronic kidney disease (CKD), we hypothesized that genotypic variations in the interleukin-6 (IL-6) gene are a cause of systemic inflammatory states and atherosclerosis in South African CKD patients. 120 CKD patients and 40 healthy controls were included. Serum IL-6 and high-sensitivity C-reactive protein (hs-CRP) levels were measured. Functional polymorphisms in the IL-6 genes were genotyped using polymerase chain reaction-sequence specific primer (PCR-SSP) methods. Carotid intima-media thickness (CIMT) and the presence of plaque were assessed by B-mode ultrasonography. Serum IL-6 and hs-CRP levels were increased in patients with CKD compared with healthy controls (p < 0.001). In CKD patients, serum IL-6 above the median value was associated with carotid plaque (OR: 2.11; 95% CI: 1.74 - 2.57, p = 0.004), with excess risk confined to the group with high IL-6 levels. Significant associations were found between the IL-6 gene and atherosclerosis in the CKD group (for G/G genotype: OR = 1.21, 95% CI = 1.05 - 1.39, p = 0.012; for GG+GC vs. CC: OR = 1.14, 95% CI = 1.02 - 1.28, p = 0.035). Patients with GG+GC genotype of the IL-6 gene polymorphism had higher levels of IL-6 than those with CC genotype (p = 0.029). In South African CKD patients, the IL-6 gene promoter polymorphism is associated with high serum IL-6 levels and atherosclerosis. The relationship between atherosclerosis and -174G/C polymorphism in the IL-6 gene suggests that IL-6 may be a potential pro-inflammatory mediator of atherosclerosis in CKD patients.

Biofilm formation and induction of stress response genes is a common response of several serotypes of the pneumococcus to cigarette smoke condensate.

OBJECTIVES: Exposure to cigarette smoke impacts on the virulence of Streptococcus pneumoniae (pneumococcus) by mechanisms including induction of biofilm formation. Most studies, however, have focused on individual strains of the pneumococcus. Accordingly, the current study has investigated the commonality of the pneumococcal stress response to cigarette smoke condensate (CSC), using five different strains of the pathogen. METHODS: Following exposure to CSC at final concentrations of 80 and 160 µg mL-1 during a 16 h incubation period, biofilm formation was measured using a crystal violet-based spectrophotometric procedure. Expression of stress genes seemingly linked to biofilm formation viz. hk11 and rr11 [histidine kinase and response regulator of the two-component regulatory system 11 (TCS11) respectively], cat eff (cation efflux system protein), abc (ATP-binding component of an ATP-binding cassette transporter) and 2005-hyp (hypothetical gene) was measured by sequential extraction of RNA, cDNA synthesis and real-time qPCR. RESULTS: Exposure of all five strains of the pneumococcus to CSC, resulted in significant biofilm formation, as well as induction of all five test stress genes. CONCLUSIONS: Augmentation of induction of selective stress genes and biofilm formation are common, possibly linked, responses of various serotypes of the pneumococcus to CSC, favouring both persistence of the pathogen and decreased efficacy of antibiotics.

Cigarette smoke exposure induces expression of the pneumococcal erm(B) macrolide resistance gene.

INTRODUCTION: Cigarette smoking is a well-recognized risk factor for development of severe, invasive pneumococcal disease. However, little is known about the direct effects of exposure to cigarette smoke on the virulence mechanisms of the pathogen, particularly in respect of resistance to macrolide antibiotics, which are widely used in the treatment of pneumococcal infection. This study aimed to investigate the effects of exposure to cigarette smoke condensate (CSC, 80 and 160 mg/L) and clarithromycin (2 and 8 mg/L), alone and in combination in vitro, on expression of the erm(B) and mef(A) macrolide resistance genes of strains 2507 and 521 (both serotype 23F), respectively, of the pneumococcus. METHODS: Following exposure to CSC or clarithromycin, individually and in combination, erm(B) and mef(A) gene expression were measured by sequential extraction of RNA, conversion to and amplification of cDNA, and detection by qRT-PCR. RESULTS: As expected, exposure of both test strains of the pneumococcus to clarithromycin resulted in substantial upregulation of both macrolide resistance genes, which was significantly (p<0.001) augmented by prior exposure to CSC in the case of erm(B), but not mef(A). Somewhat unexpectedly, exposure of strain 2507 to CSC (160 mg/L) alone (in the absence of clarithromycin) also resulted in significant (p<0.05) expression of the erm(B) gene. CONCLUSION: Although the possible clinical significance remains to be established, these findings suggest that smoking may impede the efficacy of macrolide-based antimicrobial therapy by accelerating the onset and magnitude of erm(B)-mediated resistance, representing a novel pro-infective mechanism of smoking.

Dynamic Changes in the Molecular Signature of Adverse Left Ventricular Remodeling in Patients With Compensated and Decompensated Chronic Primary Mitral Regurgitation.

BACKGROUND: There is no proven medical therapy that attenuates adverse left ventricular remodeling in patients with chronic primary mitral regurgitation (CPMR). Identification of molecular pathways important in the progression of left ventricular remodeling in patients with CPMR may lead to development of new therapeutic strategies. METHODS AND RESULTS: We performed baseline echocardiographic, cardiac catheterization, and serum NT-pro-BNP analysis in patients with severe CPMR awaiting mitral valve surgery and stratified the study population into compensated or decompensated CPMR. We obtained left ventricular endomyocardial biopsies (n=12) for mRNA expression analysis, and compared baseline transcript levels of 109 genes important in volume-overload left ventricular remodeling with levels in normal hearts (n=5) and between patients with compensated (n=6) versus decompensated (n=6) CPMR. Patients were then randomized to treatment with and without carvedilol and followed until the time of surgery (mean follow-up 8.3 months) when repeat endomyocardial biopsies were obtained to correlate transcriptional dynamics with indices of adverse remodeling. CPMR was associated with increased NPPA expression levels (21.6-fold, P=0.004), decreased transcripts of genes important in cell survival, and enrichment of extracellular matrix genes. Decompensated CPMR was associated with downregulation of SERCA2 (0.77-fold, P=0.009) and mitochondrial gene expression levels and upregulation of genes related to inflammation, the extracellular matrix, and apoptosis, which were refractory to carvedilol therapy. CONCLUSIONS: Transition to decompensated CPMR is associated with calcium dysregulation, increased expression of inflammatory, extracellular matrix and apoptotic genes, and downregulation of genes important in bioenergetics. These changes are not attenuated by carvedilol therapy and highlight the need for development of specific combinatorial therapies, targeting myocardial inflammation and apoptosis, together with urgent surgical or percutaneous valve interventions.

JC Virus and APOL1 Risk Alleles in Black South Africans With Hypertension-Attributed CKD.

INTRODUCTION: The polyomaviruses, John Cunningham (JC) and BK, infect humans, with primary infection occurring in childhood. First-degree relatives of African American individuals with nondiabetic chronic kidney disease (CKD) who had 2 apolipoprotein L1 (APOL1) risk variants had a lower prevalence of kidney disease in the presence of JC viruria. This study determined the prevalence of polyomavirus infections and their effects, in the presence APOL1 risk alleles, on CKD. METHODS: Sixty-four black South African individuals with hypertension-attributed CKD with an estimated glomerular filtration rate (eGFR) ≤60 ml/min per 1.73 m2, 44 first-degree relatives, and 56 unrelated controls were included. Viral DNA was extracted from urine and genomic DNA from blood using the Maxwell automated platform. Viral-load quantification was determined using Genesig polyomavirus kits. Genotyping of the APOL1 G1 and G2 variants was by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The prevalence of JC viruria was significantly higher in controls (36%) and first-degree relatives (20%) than in patients with CKD (3%, P < 0.001). Although patients with CKD and their first-degree relatives had similar socioeconomic status scores, we found a lower prevalence of JC viruria in patients with CKD compared with their first-degree relatives, who had normal kidney function. The absence of John Cunningham virus (JCV), DNA was a strong predictor of CKD (odds ratio [OR] 43.43; 95% confidence interval [CI] 7.39-255.20; P < 0.001). CONCLUSION: There was a strong association between the absence of JC viruria and CKD. Studies with a larger sample are essential to determine the renoprotective effects of JCV and its interactions with APOL1.

Urinary Uromodulin Levels and UMOD Variants in Black South Africans with Hypertension-Attributed Chronic Kidney Disease.

Uromodulin, the most abundant protein in urine, is synthesized in the thick ascending loop of Henle and distal convoluted tubules. Patients with chronic kidney disease (CKD) have reduced urinary uromodulin levels secondary to tubular damage. Genome wide association studies identified significant single nucleotide polymorphism (SNP) associations with CKD at the uromodulin (UMOD) locus. We examined the association of urinary uromodulin concentrations with CKD and with SNP rs1333226 in the UMOD gene. The study included 71 black South Africans with hypertension-attributed CKD with an eGFR ≤ 60ml/min/1.73m2, 52 first-degree relatives, and 58 unrelated controls. Urinary uromodulin concentration was measured using Luminex® multiplex kits. After DNA extraction from blood using the Maxwell® automated platform, genotyping of rs13333226 was performed using real-time PCR using TaqMan® genotyping assays. Urinary uromodulin levels were significantly lower in CKD cases compared to both controls and first-degree relatives and correlated negatively with age, serum uric acid, serum creatinine, and systolic BP and positively with CKD-EPI eGFR. For each 1-standard deviation increase in uromodulin level, the multivariable-adjusted odds ratio for CKD was 0.6 (95% CI [0.48 to 0.81]; p <0.01). There were no significant differences in the minor allele frequency between CKD cases and controls (p = 0.59) nor between first-degree relatives and controls (p = 0.98). There were no significant associations between genotype at rs13333226 and urine uromodulin levels (p = 0.43). Higher levels of urinary uromodulin are associated with lower odds of hypertension-attributed CKD. We did not detect associations of genotype at rs13333226 with urinary uromodulin levels in our sample population. Larger sample size studies from ethnically disparate populations are essential to further categorize this association.

Associations of plasma fibroblast growth factor 23 and other markers of chronic kidney disease-Mineral and bone disorder with all-cause mortality in South African patients on maintenance dialysis: A 3-year prospective cohort study.

INTRODUCTION: Few studies have linked high levels of plasma C-terminal fibroblast growth factor 23 (FGF23) with poor clinical outcomes in patients on maintenance haemodialysis (MHD), while the association between intact FGF23 and mortality in this group of patients remains inconclusive. Therefore, the aim of this study was to evaluate the association between plasma levels of intact FGF23 and mortality in dialysis patients. METHODS: A prospective multicenter study involving patients undergoing dialysis at three dialysis centers in Johannesburg was undertaken between 1st October 2014 and 31st December 2017. RESULTS: The study comprised 165 chronic dialysis patients (111 blacks, 54 whites) with a mean age of 46.6 ±14.2 years. During a three year follow up period, there were 46 deaths (1.03 per 100 person-years). The median plasma FGF 23 level was 382 pg/ml (interquartile range [IQR], 145-2977). In adjusted multivariable analyses, there was a non-statistically significant increase in the risk of mortality with higher quartiles of FGF 23 levels: the adjusted hazard ratios (HR) for the second, third and fourth quantiles were HR 3.20 (95% CI, 0.99-10.35; P = 0.052), HR 2.43(95% CI,0.65-9.09; P = 0.19), and HR 2.09 (95% CI, 0.66-7.32; P = 0.25),respectively. Corrected serum calcium 2.38-2.5 mmol/l [HR 2.98 (95% CI, 1.07-8.29; P = 0.04] and > 2.50 mmol/l [HR 5.50 (95% CI, 1.84-16.48; P = 0.002] were independently associated with increased risk of death. Likewise, patients with intact parathyroid hormone > 600 pg/ml had a 3.46-fold higher risk of death (HR 3.46, 95% CI, 1.22-9.82 P = 0.019). These findings persisted in time -dependent analyses. CONCLUSION: Higher levels of intact FGF 23 appear not to be independently associated with all-cause mortality in our dialysis patients, while hypercalcaemia and severe hyperparathyroidism were found to be independent predictors of mortality in this cohort of patients.