Therese Dix-Peek

APOL1 Risk Variants Are Strongly Associated with HIV-Associated Nephropathy in Black South Africans

APOL1 variants are associated with HIV-associated nephropathy and FSGS in African Americans. The prevalence of these variants in African populations with CKD in HIV-1 infection has not been investigated. We determined the role of APOL1 variants in 120 patients with HIV-associated nephropathy and CKD and 108 controls from a South-African black population. Patients with CKD were selected on the basis of histology. Genotypes were successfully determined for APOL1 G1 and G2 variants and 42 single nucleotide polymorphisms, including 18 ancestry informative markers, for 116 patients with CKD (96.7%; 38 patients with HIV-associated nephropathy, 39 patients with HIV-positive CKD, and 39 patients with HIV-negative CKD), and 108 controls (100%). Overall, 79% of patients with HIV-associated nephropathy and 2% of population controls carried two risk alleles. In a recessive model, individuals carrying any combination of two APOL1 risk alleles had 89-fold higher odds (95% confidence interval, 18 to 912; P<0.001) of developing HIV-associated nephropathy compared with HIV-positive controls. Population allele frequencies were 7.3% for G1 and 11.1% for G2. APOL1 risk alleles were not significantly associated with other forms of CKD. These results indicate HIV-positive, antiretroviral therapy-naïve South-African blacks with two APOL1 risk alleles are at very high risk for developing HIV-associated nephropathy. Further studies are required to determine the effect of APOL1 risk variants on kidney diseases in other regions of sub-Saharan Africa.

The induction of bone formation by the recombinant human transforming growth factor-β3

Implantation of recombinant human transforming growth factor-β3 (hTGF-β3) with coral-derived calcium carbonate-based macroporous bioreactors with limited conversion to hydroxyapatite (7% HA/CC) in the rectus abdominis muscle of the non-human primate Chacma baboon Papio ursinus induces endochondral bone formation. The exact mechanisms by which hTGF-β3 signalling induces bone in heterotopic sites of P. ursinus are not known. Coral-derived 7% HA/CC bioreactors with and without 125 μg hTGF-β3 were implanted in triplicate in the rectus abdominis muscle of 6 adult baboons. 7% HA/CC bioreactors either with or without hTGF-β3 were loaded with 125 μg of recombinant human Noggin (hNoggin), a bone morphogenetic proteins (BMPs) antagonist. Tissues on day 15, 60 and 90 were analysed by histomorphometry and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Down-regulation of BMP-2 characterized 7% HA/CC constructs preloaded with 125 μg hNoggin with Noggin down-regulated on day 60 and 90 together with lack of TGF-β3 expression. Down-regulation of BMP-2 correlated with minimal bone formation by induction. hTGF-β3/hNoggin pre-treated bioreactors up-regulated BMP-2 but only on day 90 together with a significant down-regulation of Noggin on day 60 and 90, correlating with the induction of bone formation, albeit limited, on day 90 at the periphery of the macroporous bioreactors only. hTGF-β3 treated bioreactors significantly down-regulated BMP-2 on day 15 whilst up-regulating BMP-2 on day 60 and 90, together with down-regulation of Noggin on day 60 and 90 correlating with the prominent induction of bone formation. hTGF-β3 significantly up-regulated RUNX-2 and Osteocalcin expression on day 15 controlling the differentiation of progenitor stem cells into the osteoblastic lineage. The induction of bone as initiated by hTGF-β3 in the rectus abdominis muscle of P. ursinus is via the BMPs pathway with hTGF-β3 controlling the induction of bone formation by regulating the expression of BMPs via Noggin expression. These results unequivocally demonstrate that hTGF-β3 elicits bone induction by up-regulation of endogenous BMP-2 and is blocked by hNoggin.

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral-derived macroporous constructs.

Coral‐derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre‐loaded with either 500 μg of the calcium channel blocker, verapamil hydrochloride, or 240 μg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT‐PCR. On day 15, up‐regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate‐treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre‐loaded with the Ca++ channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down‐regulation of bone morphogenetic protein‐2 (BMP‐2) together with up‐regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre‐loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral‐derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca++ activating stem cell differentiation and the induction of bone formation.

Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer

Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell-cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines.

Volume overload and its risk factors in South African chronic kidney disease patients: an appraisal of bioimpedance spectroscopy and inferior vena cava measurements.

BACKGROUND Fluid retention occurs early in chronic kidney disease (CKD) resulting in increased cardiovascular morbidity and mortality. This study aimed to assess volume and nutritional status among South African CKD participants and determine the relationship between malnutrition, inflammation, atherosclerosis, and volume overload using a body composition monitor (BCM). We also evaluated the usefulness of BCM measurement in assessing volume overload. METHODS 160 participants comprising hemodialysis, peritoneal dialysis, stage 3 CKD patients, and healthy controls (40 in each group) were studied. A BCM was used to assess fluid and nutritional status. Cardiac dimension measurements, and inferior vena cava diameter (IVCD) and carotid intima media thickness were assessed by echocardiography and ultrasonography, respectively. Serum interleukin-6 (IL-6) and C-reactive protein (CRP) levels were measured as markers of inflammation. RESULTS Fluid overload and malnutrition were present in 68% and 63% of studied patients, respectively. Using physical examination findings as the reference measurements for volume overload, the area under the concentration curves for BCM and IVCD measurements were 0.866 (sensitivity 82%, specificity 74%, p < 0.001) and 0.727 (sensitivity 57%, specificity 70%, p < 0.001), respectively. Lean tissue index, inflammation, and atherosclerosis were associated with volume overload. CONCLUSIONS Volume overload and malnutrition were common across the spectrum of South African CKD cohorts; volume overload was associated with malnutrition, inflammation, and atherosclerosis. Bioimpedance spectroscopy (BIS) is a useful and sensitive tool for the assessment of fluid status in clinically euvolumic nondialytic CKD patients.

Atherosclerotic vascular disease and its correlates in stable black South African kidney transplant recipients

Background Despite remarkable improvement in renal function attributable to kidney transplantation, the burden of cardiovascular disease (CVD) among kidney transplant recipients (KTRs) remains high in the post-transplant period. Aggressive use of statins in KTRs may make lipoprotein ratios correlate better with atherosclerotic vascular disease (AsVD) when compared with traditional lipid profile parameters. We therefore evaluated the clinical and echocardiographic correlates of AsVD among non-diabetic, stable, black KTRs in South Africa. Methods This was a cross-sectional study of 41 adult (18–65 years), non-diabetic, stable KTRs and 41 age- and sex-matched healthy controls. An interviewer-administered questionnaire was used to obtain information on participants’ sociodemographic and cardiovascular risk factors. Anthropometric parameters were measured. Urine and blood samples were obtained and analyzed. Echocardiography was performed and carotid intima media thickness (CIMT) was assessed in both right and left carotid arteries. Spearman’s rank correlation and binary logistic regression were performed to determine the relationship between CVD risk factors and AsVD. Results AsVD was present in 46.3% of KTRs compared to 17.1% of healthy controls (p = 0.004). Left ventricular hypertrophy was present in 92.7% of the KTRs. There were statistically significant differences in waist–hip ratio, systolic blood pressure, mean arterial pressure, urine albumin–creatinine ratio, serum fibrinogen, serum creatinine, estimated glomerular filtration rate, left atrial diameter, left ventricular mass (LVM), and left ventricular mass index (LVMI) between KTRs and controls. A positive relationship was seen between CIMT and certain risk factors for CVD including LVM, LVMI, and mitral valve deceleration time, (p < 0.001). Castelli index 2 and lipoprotein combine index (LCI) showed positive correlation with CIMT. On multivariate analysis, increasing age and kidney transplant status were independent predictors of AsVD after controlling for other risk factors. Conclusion AsVD was common among KTRs. Older age and kidney transplant status independently predicted AsVD. Castelli index 2 and LCI correlated with AsVD better than serum lipid parameters.

Transforming Growth Factor-β Protects against Inflammation-Related Atherosclerosis in South African CKD Patients

Background Transforming growth factor-β (TGF-β) may inhibit the development of atherosclerosis. We evaluated serum levels of TGF-β isoforms concurrently with serum levels of endotoxin and various inflammatory markers. In addition, we determined if any association exists between polymorphisms in the TGF-β1 gene and atherosclerosis in South African CKD patients. Methods We studied 120 CKD patients and 40 healthy controls. Serum TGF-β1, TGF-β2, TGF-β3, endotoxin, and inflammatory markers were measured. Functional polymorphisms in the TGF-β1 genes were genotyped using a polymerase chain reaction-sequence specific primer method and carotid intima media thickness (CIMT) was assessed by B-mode ultrasonography. Results TGF-β isoforms levels were significantly lower in the patients with atherosclerosis compared to patients without atherosclerosis (p<0.001). Overall, TGF-β isoforms had inverse relationships with CIMT. TGF-β1 and TGF-β2 levels were significantly lower in patients with carotid plaque compared to those without carotid plaque [TGF-β1: 31.9 (17.2 – 42.2) versus 45.9 (35.4 – 58.1) ng/ml, p=0.016; and TGF-β2: 1.46 (1.30 – 1.57) versus 1.70 (1.50 – 1.87) ng/ml, p=0.013]. In multiple logistic regression, age, TGF-β2, and TGF-β3 were the only independent predictors of subclinical atherosclerosis in CKD patients [age: odds ratio (OR), 1.054; 95% confidence interval (CI): 1.003 – 1.109, p=0.039; TGF-β2: OR, 0.996; 95% CI: 0.994–0.999, p=0.018; TGF-β3: OR, 0.992; 95% CI: 0.985–0.999, p=0.029). TGF-β1 genotypes did not influence serum levels of TGF-β1 and no association was found between the TGF-β1 gene polymorphisms and atherosclerosis risk. Conclusion TGF-β isoforms seem to offer protection against the development of atherosclerosis among South African CKD patients.