ItemDeveloping and evaluating comprehensive multiplex peptide array for serological diagnostic and surveillance of infectious disease in Zimbabwe.(2021) Vengesai, Arthur.; Naicker, Thajasvarie.; Mduluza, Takafira.Introduction: Peptides that mimic B-cell linear epitopes may be used as biomarkers for the diagnosis and surveillance of diseases. Peptide microarray technology provide rapid and high-throughput immunoassay platforms, for the simultaneous of identification of B-cell linear epitopes. In this framework, a peptide microarray was designed for the integrated surveillance of infectious diseases endemic in Zimbabwe. The peptide microarray was evaluated against peptides derived from Ascaris lumbricoides, Necator americanus, Shistosoma haematobium, Schistosoma mansoni, Trichuris trichiura, Bacillus anthracis, Mycobacterium leprae, Wuchereria bancrofti, Rabies lyssavirus, Chlamydia trachomatis, Trypanosoma brucei and severe acute respiratory syndrome coronavirus (SARS-CoV-2). Methods: Published peptides were retrieved from the Tackling Infection to Benefit Africa infectious diseases epitope microarray. Novel peptides were predicted using ABCpred. The peptide microarrays were printed in a laser based approach. IgG and IgM reactivity against the derived peptides were evaluated using peptide microarray immunoassays. Positive response was defined as fluorescence intensity ≥ 500 relative fluorescence units. Immunodominant peptides were identified using heat maps and bar graphs reflecting the obtained fluorescence signal intensities. Receiver Operating Characteristic (ROC) analysis and Mann-Whitney-U test were performed to determine the diagnostic validity of the peptides. Results: Species-specific responses with at least one peptide derived from each NTD pathogen were observed. The reactive peptides included; for S. haematobium, XP_035588858.1-206-220 and XP_035588858.1-206-220 immunodominant for IgG and IgM respectively, for S.mansoni, P20287.1-58-72 immunodominant for both antibodies and for T. trichuria, CDW52482.1-326-340 immunodominant for IgG and CDW57769.1-2017-2031 and CDW57769.1-1518-1532 immunodominant for IgM. For SARS-CoV-2 derived peptides, 4 (QTH34388.1- 1-14, QRU89900.1-41-54, QTN64908.1-136-149 and QLL35955.1-22-35) showed reactivity against IgG. Four peptides (QRU89900.1-41-54, QSM17284.1-76-89, QTN64908.1-136-149 and QPK73947.1-8-21) also showed reactivity against IgM. The SARS-CoV-2 reactive peptide were derived from the membrane glycoprotein and nucleocapsid protein. Conclusion: Species-specific sero-reactivity was indicative of exposure to the different NTDs parasites antigens. Multiplex peptide microarrays are a valuable tool for integrated NTDs surveillance and for screening parasites exposure in endemic areas. In silico peptide prediction and peptide microarray technology may provide a powerful platform for the discovery of SARS-CoV-2 B-cell epitopes. Nhanganyaya: Peptides akafanana nema B-cell linear epitopes akakosha nekuti anogona kushandiswa kuongorora kuti munhu anechirwere here nekurakidza kutenderera kwezvirwere munharaunda . Peptide microarray inotibatsira kutsvaka ma B-cell linear epitopes nekukasika panguva imwe. Nekuda kweizvozvo, peptide microarray yakagadzirirwa kuti tiongorore zvirwere zvinotapukira munyika yeZimbabwe. Iyo peptide microarray yakaongororwa mashoja emuviri anorwisa zvirwere zvinosanganisira chirwere cheelephantiasis, bhiraziya, chirwere chemakonye emudumbu (intestinal worms), chirwere chemaziso chetrachoma, chirwere chemapere mbudzi, chirwere cherabis chirwere che COVID-19 nechirwere cheanthrax. Maitiro: Mamwe mapeptides akawanikwa kubva ku Tackling Infection to Benefit Africa infectious diseases epitope microarray. Mapeptides matsva akawandikwa pachishandiswa chirongwa cheABCpred. Ma peptide microarray akagadzirwa ku Germany nemhando ye laser printer tekinoroji. Masoja emumiviri anoti IgG ne IgM ezvirwere zvambotaurwa akarongororwa tichishandisa peptide microarray tekinoroji. Zvakabuda muwongororo: Takaona kuti vanhu vemuZimbabwe vane masoja emuviri anokwanisa kurwisa mapeptides anowanikwa pahosha dzinokonzeresa zvirwere zvinosanganisira chirwere cheelephantiasis, bhiraziya, chirwere chemakonye emudumbu (intestinal worms), chirwere chemaziso chetrachoma, chirwere chemapere mbudzi, chirwere cherabis chirwere che COVID-19 nechirwere cheanthrax. Takaona zvakare kuti peptide microarray tekinoroji inokwanisa kushandiswa kuongorora zvirwere zvakawanda panguva imwe. ItemGene polymorphisms of uric acid related proteins and the Angiotensin Receptor IV (AT4) in Pre-eclampsia.(2019) Khaliq, Olive Pearl.; Naicker, Thajasvarie.; Moodley, Jagidesa.Background: Hypertensive disorders of pregnancy remain one of the major contributions to maternal and fetal morbidity and mortality around the globe. Pre-eclampsia is a hypertensive disorder of pregnancy which complicates 3-10% of pregnancies worldwide. It is a multi-organ disorder affecting the maternal system, thereby creating a major setback in terms of targeting the aetiology. One of the main organs disrupted is the kidneys and a dysregulation of uric acid levels and the renin angiotensin system have been implicated in pre-eclampsia. Therefore, the aim of this study is to investigate the gene polymorphisms of uric acid, aminopeptidase-N, the angiotensin receptor IV, and plasma levels of the receptor in pre-eclampsia compared to normotensive pregnancies. Materials and Methods: This was a retrospective study consisting of 637 blood samples of which 280 were normotensives and 357 pre-eclamptic. Pre-eclampsia was subdivided into early (n=187) and late onset pre-eclampsia (n=170). DNA was isolated from blood samples using the Thermo Scientific GeneJet whole blood Genomic DNA purification mini Kit. Single nucleotide polymorphisms of uric acid (rs505802, rs1014290, rs12129861, rs2231142), the angiotensin receptor IV (rs18059) and aminopeptidase-N (rs6496603) were amplified using the TaqMan genotyping assay. Plasma levels of angiotensin receptor IV were also measured using the ELISA in pre-eclampsia and compared to normotensives. Results: We found that rs505802 was higher in late onset pre-eclampsia compared to early onset pre-eclampsia and the normotensive group. We also observed a significant elevation of rs1014290 in early onset pre-eclampsia compared to late onset pre-eclampsia and the normotensive group. Gene polymorphisms of the angiotensin IV receptor (rs18059) and aminopeptidase-N (rs6496603) showed no significant association with pre-eclampsia. However, plasma levels of angiotensin IV receptor were significantly lower in pre-eclampsia than in normotensives. Furthermore, we found that the levels decreased with the severity of pre-eclampsia. Conclusion: The single nucleotide polymorphisms of uric acid (rs505802, rs1014290) are associated with the pathogenesis of pre-eclampsia. Furthermore, plasma levels of angiotensin IV are decreased in pre-eclampsia, indicating a dysregulation of the renin angiotensin system in pre-eclampsia. ABSTRACT-ISIZULU Amathuluzi asetshenzisiwe: Lolucwaningo lubizwa nge-retrospective lusebenzise amagazi awu 637 aphuma kwabanomfutho osesimweni sempilo (normotensives) abangu 280 Kanye nabanomfutho ophakeme wegazi kubakhulelwe (pre-eclamptic) abangu 357. Abanomfutho ophakeme wegazi baphinde bahlukaniswa izigaba ezimbili, ezibizwa nge early (n=187) ne late onset (n=170). Ufuzo egazini lutholakale ngokusebenzisa I Thermo Scientific GeneJet whole blood Genomic DNA purification mini Kit. Ama Single nucleotide polymorphisms e uric acid (rs505802, rs1014290, rs12129861, rs2231142), angiotensin receptor IV (rs18059) nawe aminopeptidase-N (rs6496603) acubulungwe ngokusebenzisa i- TaqMan genotyping assay. Izinga le-angiotensin receptor IV egazini likalwe ngokusebenzisa i-ELISA kwabanomfutho wamandla ophakeme kwabakhulelwe (pre-eclamptics) makuqhathaniswa nabanomfutho osesimweni sempilo (normotensive). Imiphumela: Sithole ukuthi amazinga e rs505802 abephezulu ku late onset pre-eclampsia makuqhataniswa ne early onset pre-eclampsia ne normotensive. Siphinde sabona ukukhuphuka kwenani le rs1014290 ku-early onset pre-eclampsia makuqhathaniswa ne late onset pre-eclampsia Kanye ne normotensive group. Ama gene polymorphisms we angiotensin IV receptor (rs18059) Kanye ne aminopeptidase-N (rs6496603) awakhombisanga mahluko ekuhlobaneni ne pre-eclampsia. Kodwa, inani le angiotensin IV receptor egazini abehlile kulabo abanomfutho wamadla ophakeme egazini (pre-eclampsia) makuqhathaniswa nalabo abanomfutho osesimweni sempilo (normotensive). Siphinde sathola ukuthi lokwehla kuhambisana nokubhebhetheka kwesifo somfutho wamadla ophakeme egazini. Ukuvala: I single nucleotide polymorphisms ye uric acid (rs505802, rs1014290) ihlobene nokuqala kwesifo somfutho wamadla ophakeme egazini kwabakhulelwe (pre-eclampsia). Futhi, izinga le angiotensin IV egazini lehlile kulabo abanomfutho wamadla ophakeme egazini kwabakhululwe (pre-eclampsia), okukhombisa ukungalawuleki kwe renin angiotensin system kulabo abanomfutho wamadla ophakeme egazini (pre-eclampsia). ItemThe role of neuroinflammatory markers in a preeclamptic rat model.(2020) Ijomone, Olayemi Kafilat.; Naicker, Thajasvarie.; Shallie, Philemon Dauda.Introduction: Preeclampsia (PE) is a clinical complication of pregnancy characterised with the new onset of hypertension and proteinuria, and/or organ dysfunction. Globally, it is associated with maternal and perinatal morbidity and is a major cause for concern. If undiagnosed, preeclampsia can lead to eclampsia, which is characterised by an onset of seizures (convulsions). Thus, neurological consequences have been reported in both PE mothers and their offspring. Materials and Methods: This study investigated the role of neuroinflammation and oxidative stress in the brain of an Nꭃ-nitro-L-arginine methyl (L-NAME) induced Preeclamptic rat model through birth to late postnatal days in the mother and the offspring. Pregnant rats were divided into control, early onset and late onset PE groups. Blood pressure, urine volume and proteinuria were measured on gestational day (GD) 0, 12 and 17 to establish PE. At GD 19, postnatal day (PND) 1 and 60, rats and their pups were sacrificed and brain excised. Prior to sacrifice at PND 60, the offspring were subjected to neurobehavioural studies to test for memory performance and locomotor activity. Ionized calcium binding adaptor molecule 1 (IBA1) and Excitatory amino acid transporter 1 (EAAT1), and oligodendrocyte transcription factor 2 (OLIG2) expression in the cortex and cerebellum were analysed by immunohistochemistry. Additionally, cortical and cerebellar tissues were homogenised for further biochemical analysis of oxidative stress such as Nitric oxide (NO), lipid peroxidase superoxide dismutase (SOD), glutathione (GSH), lipid peroxidase (LPO) and purinergic enzyme activities at PND 60. Results: We found an increase in blood pressure accompanied by proteinuria and a low foetal count in the L-NAME treated groups. Neuroinflammation was evident in the treated group at birth and PND 60 as shown by an increase in the number of IBA1 expressing activated microglia with a simultaneous reduction in the immunoexpression of EAAT1. PE- induced axonal damage was noted as shown by a reduced number of oligodendrocytes. At PND 60, PE groups show alteration in oxidative stress markers, increased acetylcholinesterase activity, and decreased purinergic enzymes activities such as adenosine triphosphatase (ATPase) and ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase). The offspring at PND 60 also display reduced memory performance and locomotor activity, which was accompanied by an increased number of activated microglia, down-regulated the immunoexpression of EAAT1 and reduced number of oligodendrocyte cells. Discussion and conclusion: This study demonstrates neuroinflammation and axonal damage in PE at delivery which also persists into later life. This finding might be attributed to systemic inflammation and vulnerability of blood-brain barrier associated with PE which can cause crossing over of substances from the systemic environment thereby causing insult to the brain. Alteration in oxidative stress markers and an increase in acetylcholinesterase in the brain usually pinpoint to neurovascular/ neurodegenerative disease, this might be an indication of PE been predisposing to neurodegenerative disease later in life. We, therefore, conclude that a history of PE predisposes mothers and their offspring to a higher risk of neurological complications later in life. ItemThe role of primigravidae, lymphatic vessel endothelial receptor-1 and podoplanin in HIV associated early and late onset pre-eclampsia.(2017) Onyangunga, Onankoy Atshakala.; Naicker, Thajasvarie.; Moodley, Jagidesa.Abstract available in PDF file. ItemThe role of peripheral natural killer cells in immunocompromised pre-eclamptic and normotensive pregnant black South Africans.(2015) Ajith, Anushka.; Naicker, Thajasvarie.; Moodley, Jagidesa.Abstract available in PDF file. ItemThe spectrum of HIV related nephropathy in KwaZulu-Natal : a pathogenetic appraisal and impact of HAART.(2012) Ramsuran, Duran.; Naicker, Thajasvarie.; Bhimma, Rajendra.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. ItemThe role of leptin in HIV associated pre-eclampsia.(2013) Haffejee, Firoza.; Naicker, Thajasvarie.; Singh, Moganavelli.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. ItemThe role of soluble FMS-like tyrosine-kinase-1, vascular endothelial growth factor and placental growth factor in HIV associated pre-eclamptic pregnancies : a South African perspective.(2013) Govender, Nalini.; Naicker, Thajasvarie.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.