Adequate maternal, intervillous and fetal blood flow are all necessary for fetal wellbeing. Compromise to any part of this exchange would be detrimental to pregnancy outcome. Preeclampsia is associated with reduced maternal spiral artery flow, resulting in reduced placental perfusion. This in turn creates an ischemic environment which may pre-dispose morphological changes in placental villi. This pilot study utilized morphometric image analysis to examine some features of the fetal component of the placenta in normotensive (NT) and pre-eclamptic (PE) groups. The features examined included: density of placental villi (expressed as percentage of field area occupied by placental tissue); stem vessel carrying capacity (expressed as percentage of stem villus area occupied by vessel lumina); the thickness of the stem arterial walls relative to artery size (expressed as percentage of artery area occupied by arterial wall) and the extent of fibrosis associated with villi (expressed as percentage of field area occupied by fibrosis). The results were as follows: density of placental villus arrangement NT:51.89±6.19, PE:64.78±6.93 (P<0.001); carrying capacity of stem villi NT:17.20±11.78, PE:8.67±8.51 (P<0.001); relative thickness of stem villi arterial walls NT:74.08±12.92, PE: 86.85±10.55 (P<0.001); and extent of fibrosis NT:0.727±0.310, PE:1.582±0.707 (P<0.001). These significant differences between normotensive and pre-eclamptic placentae suggest possible fetal maladaptations in response to the intervillous ischemia, compounding the existing maternal compromise to materno-fetal exchange. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

HIV and hypertensive disorders in pregnancy, in particular pre-eclampsia, are the main causes of maternal mortality in South Africa. In HIV associated pre-eclampsia, it is biologically plausible that the immune activation associated with pre-eclampsia may be neutralised by the immune suppression of HIV infection. The precise aetiology of pre-eclampsia is unknown, however leptin has been implicated in its development. Leptin is an adipocyte hormone, also produced by the placenta. It has a role in the development of inflammation. Adipose tissue is reduced in HIV infected individuals, resulting in lower leptin levels with consequent impaired immune function. This study aimed to compare serum and placental leptin levels in HIV infected and uninfected normotensive and pre-eclamptic pregnancies. Since insulin levels may affect the secretion of leptin, the study also compared insulin levels in these pregnancies. Following ethical clearance and hospital permission, 180 participants were recruited during their antenatal period. The groups were HIV- normotensive (n = 30), HIV+ normotensive (n = 60), HIV– pre-eclamptic (n = 30) and HIV+ pre-eclamptic (n = 60). Blood samples were collected ante-natally and placental samples post delivery. Serum leptin and insulin levels were determined by ELISA. Placental leptin levels were determined by ELISA and immunohistochemistry with morphometric image analysis. The placental production of leptin was determined by RT PCR. There was a non-significant increase in serum leptin levels in HIV- pre-eclampsia compared to HIV- normotensive pregnancies (p = 0.42). However leptin was decreased significantly in HIV+ pre-eclampsia compared to HIV- normotensive (p = 0.03). Based on HIV status leptin levels were decreased in HIV+ groups compared to HIV- groups in both pre-eclamptic (p < 0.01) and normotensive pregnancies (p < 0.01). Insulin levels of the HIV positive groups were lower than those of the HIV negative groups (p < 0.001). Insulin levels were also decreased in pre-eclampsia compared to normotensive pregnancies, irrespective of HIV status (p = 0.02). Immunohistochemistry demonstrated an increase in immuno-reactivity of leptin in the exchange villi of pre-eclamptic compared to normotensive placentae, irrespective of HIV status (p < 0.001). Supporting this finding, ELISA also demonstrated elevated leptin levels in the placenta of pre-eclamptic compared to normotensive pregnancies (p < 0.001). Placental leptin levels were similar in both HIV positive and negative pregnancies (p = 0.36). However, the placental leptin mRNA expression was up-regulated in HIV negative pre-eclampsia (p = 0.04) but not in HIV positive pre-eclampsia (p = 1.00). In conclusion, the elevated placental leptin in pre-eclampsia, irrespective of HIV status, is consistent with hypoxia. These elevated levels are not reflected in the maternal serum which raises the possibility of decreased leptin expression by adipose tissue especially in HIV infection where serum leptin levels are decreased. This would negate the increased placental leptin expression in pre-eclampsia. Furthermore, the elevated placental leptin levels are suggestive of an autocrine role of leptin in the placenta. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

Sub-Saharan Africa bears 70% of the global HIV burden with KwaZulu-Natal (KZN) identified as the epicenter of this pandemic. HIV related nephropathy (HIVRN) exceeds any other causes of kidney diseases responsible for end stage renal disease, and has been increasingly recognized as a significant cause of morbidity and mortality. There is nonetheless a general lack of surveillance and reporting for HIVRN exists in this geographical region. Consequentially, the aim of this study was to outline the histopathogical spectrum of HIVRN within KZN. Moreover, from a pathology standpoint, it is important to address whether HIVRN was a direct consequence of viral infection of the renal parenchyma or is it a secondary consequence of systemic infection. Additionally, an evaluation of the efficacy of Highly Active Anti-Retroviral Therapy (HAART) in combination with angiotensin converting enzyme inhibitors (ACE-I) was performed via a genetic appraisal of localized replication of HIV-1 in the kidney, ultrastructural review and immunocytochemical expression of a podocyte maturity and proliferation marker pre and post-HAART. Blood and renal biopsies were obtained from 30 children with HIV related nephropathy pre- HAART, followed-up clinically for a period of 1 year. This cohort formed the post-HAART group. Clinical and demographic data were collated and histopathology, RT-PCR, sequencing, immunocytochemistry and transmission electron microscopy was performed. The commonest histopathological form of HIVRN in children (n = 30) in KZN was classical focal segmental glomerular sclerosis (FSGS) presented in 13(43.33%); mesangial hypercellularity 10(30%); mesangial, HIV associated nephropathy 3(11%) and minimal change disease 2(6.67%). Post-HAART (n = 9) the predominant pathology was mesangial hypercellularity 5(55.56%); FSGS 3(33.33%) and sclerosing glomerulopathy 1(11.11%). This study also provides data on the efficacy of HAART combined with ACE-I. The immunostaining pattern of synaptopodin, Ki67 and p24 within the glomerulus expressed as a mean field area percentage was significantly downregulated in the pre-HAART compared to the post-HAART group respectively (1.14 vs. 4.47%, p = 0.0068; 1.01 vs.4.68, p < 0.001; 4.5% vs 1.4%, p = 0.0035). The ultrastructural assessment of all biopsies conformed to their pathological appraisal however, features consistent with viral insult were observed. Latent HIV reservoirs were observed within the podocyte cytoplasm but was absent in mesangial or endothelial cells. Real-Time polymerase chain reaction assays provided evidence of HIV-1 within the kidney. Sequence analysis of the C2-C5 region of HIV-1 env revealed viral diversity between renal tissue to blood. In contrast to a collapsing type of FSGS that occurs in adults, the spectrum of paediatric nephropathy in treatment-naive children within KwaZulu-Natal was FSGS with mesangial hypercellularity. Additionally, our study demonstrates podocyte phenotype dysregulation pre- HAART and reconstitution post therapy. Evidence of ultrastructural viral reservoirs within epithelial cells is supported by a genetic appraisal confirming the ubiquitous presence of HIV DNA in renal tissue. Moreover, sequence analysis showed viral evolution and compartmentalization between renal viral reservoirs to blood. Finally, the interplay of viral genes and host response, influenced by genetic background, may contribute to the variable manifestations of HIV-1 infection in the kidney in our paediatric population. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2012.

Introduction and aims. South Africa is the epicenter of the HIV/AIDS pandemic. Hypertensive disorders of pregnancy (15.7%) are the second cause of maternal deaths of which pre-eclampsia represents 83%. Normal pregnancy requires a balance between pro- and anti-angiogenic factors to necessitate effective vasculogenesis, angiogenesis and placental development, however, pre-eclampsia is characterised by an excess anti-angiogenic state. The hypoxic placenta releases excess anti-angiogenic factors into the maternal circulation causing endothelial dysfunction. However, there is no data to verify if HIV infection affects pre-eclampsia, or if the angiogenic imbalance is affected. Contradictory data exists on the association between HIV infection and pre-eclampsia. In an attempt to assess the role of HIV infection in pre-eclampsia, this study examined the immunolocalisation of sFlt-1, sEng, PlGF and VEGF in placentae of HIV negative and positive normotensive and pre-eclamptic pregnancies at term using immunohistochemistry (IHC) and immunoelectron microscopy (IEM). Additionally, we estimated the placental expression of sFlt-1, sEng, PlGF and VEGF to verify if the HIV negative differed from the HIV positive cohorts. We further evaluated the maternal serum to determine if variations existed in the circulating levels of these factors in HIV negative and positive normotensive and pre-eclamptic pregnancies. Methods. Following institutional ethical approval and informed consent, placental biopsies and maternal serum were collected post-delivery. For IHC and IEM, 130 and 25 placentae were evaluated, respectively. Following conventional immunohistochemical processing, 5μm sections were immunostained & immunoexpression of the various antibodies were evaluated with the Zeiss Axioscope A1 interfaced with an AxioVision Image analysis software package (version 4.8.3) in combination with the auto-measurement module (Carl Zeiss, Germany). Post-conventional immunoelectron processing, ultra-thin sections were immunolabelled. Sections were post-fixed, contrast enhanced with uranyl acetate and Reynolds lead citrate and viewed on a Jeol 1011 Transmission Electron Microscope. Additionally, the placental expressions of these factors were assessed using RT-PCR. In an attempt to confirm if maternal circulating levels of these factors differed, we quantitatively evaluated these factors in serum from HIV negative normotensives, HIV negative pre-eclamptics, HIV positive normotensives, and HIV positive pre-eclamptics using ELISA techniques. Results and Discussion. The expression of sFlt-1, sEng, PlGF and VEGF was confirmed using immunohistochemistry, RT-PCR and ELISAs. Irrespective of the HIV status, sFlt-1 and sEng was elevated with the concomitant reduction in PlGF in pre-eclamptic compared to normotensive pregnancies. The levels of VEGF were however undetectable across all study groups. It is plausible that this lack of effect of HIV status on the factors under study may be attributed to the treatment regimen as HAART is known to restore the immune response of HIV positive preeclamptic women. However, a concise anti-retroviral treatment history in our study was unavailable. Additionally, this study is novel in that it ultrastructurally immunolocalises sFlt-1, sEng, PlGF and VEGF within the placenta. This immunoelectron localisation data corresponds to our immunohistochemical data. Our study further demonstrates strong immunoreactivity of both placental sFlt-1 and sEng in pre-eclampsia with concurrent elevations in the maternal circulation. A qualitative increase in the occurrence of syncytial knots in the pre-eclamptics compared to the normotensive pregnancies was noted. These observations support the detachment of antixxx angiogenic rich microparticles from syncytial knots in the pre-eclamptics compared to the normotensive pregnancies was noted. These observations support the detachment of antiangiogenic rich microparticles from syncytial knots and their subsequent deportation and elevation in the maternal circulation. Moreover, their consequent antagonistic effects on VEGF, PlGF and TGF-β, disrupts the vascular endothelial maintenance. The strong immunoreactivity of sFlt-1, sEng, PlGF and VEGF was observed in villous endothelial cells. Moreover, a strong sFlt-1 and sEng but a weak PlGF and VEGF immunoreactivity was noted in syncytio- and cytotrophoblasts. This immunoexpression within trophoblasts is suggestive of their autocrine mode of action on normal trophoblast functions including invasion, differentiation and production. It is plausible that the angiogenic imbalance observed in our study, will impact on placental function, by modifying trophoblast activity thereby contributing to abnormal placentation. Conclusion. Our study supports the hypothesis that pre-eclampsia is characterized by an imbalance between pro- and anti-angiogenic factors. Whether the pregnancy is complicated by immune insufficiencies or not, does not affect the role of the anti-angiogenic factors in pre-eclampsia development. Nevertheless, the neutralising effect of HIV infection on the immune system may be insufficient in the development of pre-eclampsia. To our knowledge, the quantification of serum pro-/anti-angiogenic factors in HIV-associated pre-eclampsia is novel. In conclusion, our data reinforces the hypothesis that increased concentrations of sFlt-1 and sEng are involved in the pathogenesis of pre-eclampsia and indicates their possible use as discriminatory factors between diseases. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.