Doctoral Degrees (General Internal Medicine)

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The association between tissue non-specific alkaline phosphatase expression and differentiation of mesenchymal stromal cells
(Stellenbosch : Stellenbosch University, 2017-03) Bartlett, Cara-Lesley; Ferris, William F.; Crowther, Nigel J.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. General Internal Medicine.
ENGLISH SUMMARY: Diseases resulting from the dysregulation of adipocyte and osteoblast differentiation include diabetes type II and osteoporosis. Both adipocytes and osteoblasts are derived from the same progenitor cell type, known as mesenchymal stromal cells (MSCs), which may also differentiate into cells of several mesenchymal lineages. A more detailed understanding of the mechanisms involved in the differentiation of MSCs would provide valuable insight into the underlying causes of, as well as facilitate the development of improved treatments for, diseases related to MSC differentiation dysregulation. Tissue-nonspecific alkaline phosphatase (TNAP) is highly expressed in several tissues including bone tissue, where it has a well-established role in skeletal mineralisation. In recent years TNAP expression has been reported in adipocytes, and has been identified as identical to the stem cell marker, mesenchymal stem cell antigen-1 (MSCA-1). The above findings indicate that TNAP has diverse roles, and may be one of the factors involved in determining the differentiation pathway of MSCs. Previous studies have found that inhibition of TNAP in the mouse preadipocyte cell line, 3T3-L1 resulted in a decrease in lipid accumulation during in vitro adipogenic differentiation, suggesting that TNAP is involved in adipogenesis. In the present study, rat-derived primary MSCs were isolated from bone marrow (bmMSCs) as well as subcutaneous (scADSCs) and peri-renal visceral adipose (pvADSCs) depots, and differentiated in vitro towards either an adipocytic or osteoblastic phenotype. The expression of TNAP was assessed in rat-derived MSCs undergoing both adipogenic and osteogenic differentiation. TNAP expression levels were highest in bmMSCs, followed by scADSCs and pvADSCs, with higher alkaline phosphatase (ALP) activity observed during adipogenesis compared to osteogenesis in all three MSC types. The addition of the reported TNAP inhibitor, levamisole during osteogenesis prevented mineralisation in all MSC types, but had no significant effect on lipid accumulation during adipogenesis. Other reported inhibitors were also examined; Histidine was not successful in reducing lipid accumulation or mineralisation, while Lhomoarginine was able to significantly reduce lipid accumulation in all MSC types. The inhibitor results were not conclusive due to possible off target effects within the cells. Attempts to inhibit adipogenic differentiation by knockdown of TNAP expression in scADSCs using shRNA were not successful, as indicated by the presence of lipid droplets in cells where TNAP-specific shRNA was present. This study also revealed that ALP activity was localised to the membrane of intracellular lipid droplets characteristic of adipocytes, and that the same TNAP mRNA transcript type which is preferentially expressed in bone tissue is also preferably expressed during adipogenic differentiation of bmMSCs and scADSCs, while expression in pvADSCs was below detectable levels. TNAP isoforms differ from one another due to differences in posttranslational glycosylation pattern. Glycosylation differences were observed between bmMSCs differentiated from a naïve state towards an adipogenic, compared to an osteogenic, phenotype. Differences were also observed between scADSCs and bmMSCs when differentiated towards adipocytes. This may indicate that a distinct isoform of TNAP exists in adipocytes. In conclusion, this study confirms earlier findings on the presence of TNAP in adipocytes. Differences in TNAP expression from MSCs isolated from different tissue depots were also discovered. This study provides a characterisation of the role of TNAP in adipogenic differentiation; however, the exact mechanisms remain to be elucidated.
A comparative study of the determinants of bone strength and the propensity to falls in black and white South African women
(Stellenbosch : Stellenbosch University, 2008-11-05) Conradie, Magda; Hough, F. S.; Stellenbosch University. Faculty of Health Sciences. Dept. of Medicine. Internal Medicine.
The comparative study presented in this dissertation specifically aimed to assess fracture risk in black (Xhosa) and white South African women by evaluating known determinants of bone strength as well as the propensity to falls. We thus compared the prevalence of clinical (historic) risk factors for osteoporosis, measured and compared vertebral and femoral bone mineral density (BMD) employing dual energy X-ray absorptiometry (DEXA), ultrasound variables using the Sahara sonometer, serum parathyroid hormone (PTH) and 25-OH Vitamin D, mineral homeostasis and modern biochemical markers of bone turnover, bone geometry and the propensity to falls. Finally, we determined the prevalence of vertebral fractures in these black and white South African females. 1. Significant ethnic differences were noted in the presence and frequency of historical clinical and lifestyle risk factors for osteoporosis. Blacks were heavier and shorter, they consumed less calcium, were more inactive, preferred depot-medroxyprogesterone acetate as contraceptive agent and were of higher parity. Whites smoked more, preferred oral oestrogen containing contraceptive tablets and were more likely to have a positive family history of osteoporosis. Hormone therapy was used almost exclusively by postmenopausal whites. Inter-ethnic differences in weight, physical activity and high parity was most marked in the older subjects. 2. We found that peak spinal BMD was lower, but peak femoral BMD similar or higher (depending on the specific proximal femoral site measured) in black South-African females compared with whites. The lower peak spinal BMD was mainly attributed to lower BMD’s in the subgroup of black females with normal to low body weight, indicating that obesity either protected black females against a low spinal BMD or enhanced optimal attainment of bone mineral. An apparent slower rate of decline in both spinal- and femoral BMD with ageing was noted in the black females compared with whites in this cross-sectional study – an observation which will require confirmation in longitudinal, follow-up studies. This resulted in similar spinal BMD values in postmenopausal blacks and whites, but significantly higher femoral BMD measurements in blacks. The volumetric calculation of bone mineral apparent density (BMAD) at the lumbar spine and femoral neck yielded similar results to that of BMD. Spinal BMAD was similar in blacks and whites and femoral neck BMAD was consistently higher in all the menopausal subgroups studied. Weight significantly correlated with peak- and postmenopausal BMD at all sites in the black and white female cohorts. Greater and better maintained body weight may be partially responsible for slower rates of bone loss observed in black postmenopausal females. Most of the observed ethnic difference in BMD was, in fact, explained by differences in body weight between the two cohorts and not by ethnicity per se. 3. A low body weight and advanced age was identified as by far the most informative individual clinical risk factors for osteopenia in our black and white females, whereas physical inactivity was also identified as an important individual risk factor in blacks only. Risk assessment tools, developed and validated in Asian and European populations, demonstrated poor sensitivity for identification of South African women at increased risk of osteopenia. The osteoporosis risk assessment instrument (ORAI) showed the best results, with sensitivities to identify osteopenic whites at most skeletal sites approaching 80% (78% - 81%). The risk assessment tool scores appear to be inappropriate for our larger sized study cohort, especially our black subjects, thus resulting in incorrect risk stratification and poor test sensitivity. General discriminant analysis identified certain risk factor subsets for combined prediction of osteopenia in blacks and whites. These risk factor subsets were more sensitive to identify osteopenia in blacks at all skeletal sites, compared with the risk assessment tools described in the literature. 4. Higher ultrasonographically measured broadband ultrasound attenuation (BUA) and speed of sound (SOS) values were documented in our elderly blacks compared with whites, even after correction for differences in DEXA determined BMD at the spine and proximal femoral sites. BUA and SOS showed no decline with ageing in blacks, in contrast to an apparent significant deterioration in both parameters in ageing whites. If these quantitative ultrasound (QUS) parameters do measure qualitative properties of bone in our black population, independent of BMD as has been suggested in previous work in Caucasian populations, the higher values documented in elderly blacks imply better preservation of bone quality in ageing blacks compared with whites. The correlation between QUS calcaneal BMD and DEXA measured BMD at the hip and spine was modest at best. QUS calcaneal BMD was therefore unable to predict DEXA measured BMD at clinically important fracture sites in our study population. 5. Bone turnover, as assessed biochemically, was similar in the total pre- and postmenopausal black and white cohorts, but bone turnover rates appeared to differ with ageing between the two racial groups. A lower bone turnover rate was noted in blacks at the time of the menopausal transition and is consistent with the finding of a lower percentage bone loss at femoral sites at this time in blacks compared with whites. Bone turnover only increased in ageing postmenopausal blacks, and this could be ascribed, at least in part, to the observed negative calcium balance and the more pronounced secondary hyperparathyroidism noted in blacks. Deleterious effects of secondary hyperparathyroidism on bone mineral density at the proximal femoral sites were demonstrated in our postmenopausal blacks and contest the idea of an absolute skeletal resistance to the action of PTH in blacks. The increase in bone turnover and the presence of secondary hyperparathyroidism due to a negative calcium balance may thus potentially aggravate bone loss in ageing blacks, especially at proximal femoral sites. 6. Shorter, adult black women have a significantly shorter hip axis length (HAL) than whites. This geometric feature has been documented to protect against hip fracture. The approximately one standard deviation (SD) difference in HAL between our blacks and whites may therefore significantly contribute to the lower hip fracture rate previously reported in South African black females compared with whites. Average vertebral size was, however, smaller in black females and fail to explain the apparent lower vertebral fracture risk previously reported in this population. Racial differences in vertebral dimensions (height, width) and/or other qualitative bone properties as suggested by our QUS data may, however, account for different vertebral fracture rates in white and black women – that is, if such a difference in fact exists. 7. The number of women with a history of falls was similar in our black and white cohorts, and in both ethnic groups the risk of falling increased with age. There is a suggestion that the nature of falls in our black and white postmenopausal females may differ, but this will have to be confirmed in a larger study. Fallers in our postmenopausal study population were more likely to have osteoporosis than non-fallers. Postmenopausal blacks in our study demonstrated poorer outcomes regarding neuromuscular function, Vitamin D status and visual contrast testing and were shown to be more inactive with ageing compared with whites. An increased fall tendency amongst the black females could not however be documented in this small study. Quadriceps weakness and slower reaction time indicated an increased fall risk amongst whites, but were unable to distinguish black female fallers from non-fallers. 8. Vertebral fractures occurred in a similar percentage of postmenopausal blacks (11.5%) and whites (8.1%) in our study. Proximal femoral BMD best identified black and white vertebral fracture cases in this study. Quite a number of other risk factors i.e. physical inactivity, alcohol-intake, poorer physical performance test results and a longer HAL were more frequent in the white fracture cases and could therefore serve as markers of increased fracture risk, although not necessarily implicated in the pathophysiology of OP or falls. However, in blacks, only femoral BMD served as risk factor. Similar risk factors for blacks and whites cannot therefore be assumed and is deserving of further study. White fracture cases did not fall more despite lower 25-OH-Vitamin D, poorer physical performance and lower activity levels than non-fracture cases. Calcaneal ultrasonography and biochemical parameters of bone turnover were similar in fracture and non-fracture cases in both ethnic groups. Our study data on vertebral fractures in this cohort of urbanized blacks thus cautions against the belief that blacks are not at risk of sustaining vertebral compression fractures and emphasize the need for further studies to better define fracture prevalence in the different ethnic populations of South Africa. 9. In our study, hormone therapy in postmenopausal white women improved bone strength parameters and reduced fall risk. In hormone treated whites compared with non-hormone users, a higher BMD at the spine and proximal femur as determined by DEXA were documented and all QUS measurements were also significantly higher. The biochemically determined bone turnover rate, as reflected by serum osteocalcin levels, was lower in hormone users. Fall frequency was lower in the older hormone treated women (≥ 60yrs) and greater quadriceps strength and reduced lateral sway was noted. Only one patient amongst the hormone users (2%) had radiological evidence of vertebral fractures compared with four patients (6%) amongst the never-users. As hormone therapy was used almost exclusively by whites in this study population, the impact of hormone therapy on postmenopausal black study subjects could not be assessed.
Development and usability evaluation of a multimedia e-learning resource for electrolyte and acid-base disorders
(Stellenbosch : Stellenbosch University, 2015-03) Davids, Mogamat Razeen; Halperin, Mitchell L.; Chikte, Usuf M. E.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. General Internal Medicine.
ENGLISH ABSTRACT: We have developed an innovative multimedia e-learning resource, the Electrolyte Workshop, to provide students and clinicians with instruction and the opportunity for simulated practice in managing electrolyte and acid-base disorders. Our teaching approach is built around relevant physiology and makes use of real cases and storytelling to engage the learner. We have documented the challenges encountered during the development process and have made recommendations for the managing of similar projects. While there are many factors that must be in place to ensure successful e-learning, this dissertation focuses on an important but under-appreciated factor, namely the usability of the computer interface. Usability describes how easy technology interfaces are to use and is routinely evaluated and optimized in the software development industry. This is not yet the case with e-learning, especially in the area of medical education. Poor usability limits the potential benefit of educational resources, as this means that learners will struggle with the interface as well as with the challenges of the content presented. A comprehensive usability evaluation of our Electrolyte Workshop was completed. This included testing with typical end-users, where data were collected via standardized questionnaires and by observing and analysing their interactions with our application. We employed heuristic evaluation as an additional approach and assembled a panel of experts to evaluate our application against a set of heuristics, or principles of good interface design. Many serious usability problems were identified, thus severely limiting the potential educational impact of our Electrolyte Workshop. There was a striking disconnect between the objective measures of usability and self-reported questionnaire data. Our user-testing data make a useful contribution to the debate on how many users are required to find most of the usability problems in an interface. Heuristic evaluation proved to be a very efficient approach. However, both user testing and heuristic evaluation detected serious problems which were missed with the other method. These evaluations informed a comprehensive revision of our application and we could then compare the original with an optimized version in a randomized trial. We found large improvements in objective usability measures, which are likely to increase the satisfaction and motivation of learners. There were similar scores on measures of learning. This was not surprising as our participants were all relatively high-knowledge learners and not novices as regards the subject matter. Our study clearly indicates that the usability evaluation of e-learning resources is critical, and provides an example of how clinician-teachers can improve the usability of the resources they develop. Usability should be evaluated as a routine part of the development and implementation of e-learning materials, modules and programmes. This should start with the earliest versions of the resource, when making changes is easier and less costly. We have demonstrated that a combination of methods should be employed and have highlighted the utility of heuristic evaluation. An iterative approach should be followed, with several cycles of testing and re-design. User testing should always include the study of objective usability measures and not rely only on self-reported measures of user satisfaction.
The effect of highly active antiretroviral therapy on Human Papilloma Virus Infection and Cervical Dysplasia in women living with HIV
(Stellenbosch : Stellenbosch University, 2014-04) Zeier, Michele D.; Nachega, Jean; Botha, Matthys Hendrik; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. General Internal Medicine.
ENGLISH ABSTRACT: Title The Effect of Highly Active Antiretroviral Therapy on Human Papilloma Virus Infection and Cervical Cytological Abnormalities in Women Living With HIV Background Human Papillomavirus (HPV) infection causes cervical cancer. The prevalence of HPV-related dysplastic lesions is significantly higher in patients co-infected with the HI virus and thought to be linked to possible more persistent HPV infection. There is, however, conflicting evidence as to whether treatment of Human Immunodeficiency Virus (HIV) infection with antiretroviral agents may influence cervical HPV infection and the behaviour of Squamous Intraepithelial Lesions (SIL). Aims To examine the effect of the initiation of combination antiretroviral therapy (cART) on: 1) the persistence of cervical Low-grade SIL (LSIL); 2) The progression of cervical LSIL to High-Grade SIL (HSIL); 3) The effectiveness of excision treatment of HSIL 4) HPV genotypes detected, in HIVinfected and uninfected women at the Infectious Diseases Clinic and the Colposcopy Clinic, Tygerberg Teaching Hospital, Cape Town, South Africa. Design and Methods We conducted a retrospective cohort analysis of 1720 women with LSIL of the survival of progression-free-time or time-to-clearance. Time to progression or persistence was compared according to HIV status, antiretroviral treatment and CD4 count. In another retrospective cohort analysis, we investigated the effectiveness of excision treatment in 1848 women who underwent LLETZ or CKC biopsy was used. Logistic regression and survival analysis were used to compare excision treatment failure and recurrence-free time between groups according to HIV status, antiretroviral therapy and CD4 count. To investigate the effect of antiretroviral therapy on the cervical HPV infection, 300 HIV-infected women were prospectively enrolled and followed at 6-monthly interval. Cytological testing and cervical HPV sampling were done at each visit. Biopsy of suspicious lesions and excision treatment were done at colposcopy clinic according to standard a protocol. The Roche Linear array HPV genotyping test was used for HPV detection. Generalized Estimating Equation (GEE) multivariate analysis was applied to investigate the effect of cART on the detection of HPV infection, while adjusting for time-dependent covariates such as CD4 count, sexual activity and excision treatment. The effect on each HPV type was then also compared to the effect on HPV16. Results Overall, we found that there was no difference between the progression of LSIL to HSIL by HIV status. However, among HIV-infected patients, those who started ART before first LSIL had a significantly lower risk for progression (HR 0.66, 95% CI 0.54-0.81). CD4 count did not have an impact on the risk for progression. We also found lower persistence of SIL in the HIV uninfected group (HR 0.69, 95% CI 0.57-0.85) and that cART was independently associated with decreased persistence of LSIL. On the other hand, a higher CD4 count at the time of first LSIL was not associated with lower persistence of the lesion. HIV infected women with HSIL experienced much higher excision treatment failure than uninfected women (53.8% vs. 26.9%, p<0.001). Factors that improved outcome were higher CD4 count and complete excision. cART reduced the risk of detection of any HPV type by 47% (OR 0.53, 95% 0.49-0.58, p<001). When adjusted for covariates, time of exposure to cART and CD4 had a stronger effect. Every month of cART exposure reduced the risk detection of any HPV type with 7%. The effect was also significant on HPV16 alone (OR 0.93, 95% CI 0.90-0.95). All non-oncogenic subtypes were influenced similarly or more strongly than HPV16, as well as oncogenic HPV52. Only one oncogenic subtype HPV subtype, HPV39, was influenced marginally less (ratio of OR 0.95, CI 0.90-0.99, p=0.04). There was an increased risk for any HPV detection at CD4 count<200 (OR 1.63, 95% CI:1.50-1.77), but when adjusted, the time of cART exposure again remained the strongest predictor of risk (OR 0.94, 95% CI:0.93-0.95). Conclusion cART impact the outcome of cervical HPV infection by increasing clearance, decreasing progression of LSIL and recurrence after excision treatment. This effect is time dependent and also associated with CD4 count. Specifically, HPV16 detection risk is also reduced by cART, and all HPV types are influenced at least as much as HPV16, except possibly HPV39. It seems that increased cervical HIVproviral load is associated with HPV detection risk, and both are lowered by cART time.
The effect of statins on bone and mineral metabolism
(Stellenbosch : University of Stellenbosch, 2003-04) Maritz, Frans Jacobus; Hough, F. S.; Hulley, P. A.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Internal Medicine.
ENGLISH ABSTRACT: The Effect of Statins on Bone and Mineral Metabolism Both statins and amino-bisphosphonates reduce the prenylation of proteins which are involved in cytoskeletal organization and activation of polarized and motile cells. Consequently statins have been postulated to affect bone metabolism. We investigated the effects of different doses of simvastatin (1,5,10 and 20mg/Kg/day), administered orally over 12 weeks to intact female Sprague-Dawley rats, and the effect of simvastatin 20mg/Kg/day in sham and ovariectomised rats, on femoral bone mineral density (BMD) and quantitative bone histomorphometry (QBH), compared to controls. Similarly, the affect of atorvastatin (2,5mg/Kg/day) and pravastatin (10mg/Kg/day) on BMD was investigated and compared to controls. BMD was decreased by simvastatin 1mg/Kg/day (p = 0.042), atorvastatin (p = 0,0002) and pravastatin (p = 0.002). The effect on QBH parameters differed with different doses of simvastatin (ANOVA; p = 0.00012). QBH parameters of both bone formation and resorption were equivalently and markedly increased by simvastatin 20mg/Kg/day in two independent groups of intact rats, and reflected by a relatively unchanged BMD. At lower doses, simvastatin 1mg/Kg/day decreased bone formation while increasing bone resorption as reflected by a marked decrease in BMD. Ovariectomised animals receiving simvastatin 20mg/Kg/day showed no change in BMD relative to the untreated ovariectomised controls, their increase in bone formation was smaller than in sham-operated rats receiving simvastatin and there was no change in bone resorption. The dose response curves of simvastatin for bone formation and resorption differed from each other. From these studies it is concluded that:- a) low-dose simvastatin (1mg/Kg/day), atorvastatin 2.5mg/Kg/day) and pravastatin 10mg/Kg/day) decrease BMD in rodents;b) 1mg/Kg/day simvastatin decreases bone formation and increases bone resorption and is reflected by a reduced BMD; c) 20mg/Kg/day simvastatin increases bone formation and resorption and results in an unchanged BMD; d) the effects of simvastatin on QBH differ at different dosages; e) the dose-response curves for QBH parameters of bone resorption and bone formation differ from each other; f) the effects of simvastatin seen in intact rats are not observed in ovariectomised rats; g) simvastatin is unable to prevent the bone loss caused by ovariectomy.
Familial adult myoclonus epilepsy : a clinical, neurophysiological and genetic study of a familial form of myoclonic epilepsy
(Stellenbosch : University of Stellenbosch, 2009-12) Carr, Jonathan; Brink, P. A.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Medicine. Internal Medicine.
ENGLISH ABSTRACT: Progressive Myoclonic Epilepsies (PME) are characterized by progressive neurological impairment with myoclonus, seizures and dementia. In contradistinction, Familial Adult Myoclonic Epilepsy (FAME) is characterized by a benign course with rare seizures and cortical tremor. Both conditions have neurophysiological features suggestive of a cortical origin for their myoclonus. This dissertation reports on a novel form of PME. Many of those who were affected had no or minimal progression of their illness, low seizure frequency and were cognitively intact, suggestive of non-progressive disorders linked to the FAME loci. The majority of patients had features of cortical myoclonus, with generalized spike and wave discharges on electroencephalography, enlarged evoked potentials, enhanced C reflexes, and evidence of cortical excitability with magnetic stimulation. However, there was evidence of cerebellar dysfunction both pathologically and on imaging. With regard to similar conditions, dentatorubral pallidoluysian atrophy and Unverricht-Lundborg syndrome were excluded by linkage analysis. Similarly, linkage was not present for either the FAME 1 or FAME 2 loci. This syndrome is both clinically and genetically novel, and has a nosology which is difficult to characterize, in which the condition appears to lie on the spectrum between FAME and PME. The dissociation between the pathological and radiological findings which suggest subcortical dysfunction, and the neurophysiological findings of cortical myoclonus is striking. Review of the literature associated with the neurophysiology of related conditions associated with PME and FAME suggests that: 1. The assumption that generalized forms of myoclonic disorders represent multifocal forms of focal cortical discharges is an oversimplification. 2. The dissociation between initial and later components of the evoked potential is less robust than is generally supposed, and that subcortical inputs may affect later components of the evoked potential. 3. In a high proportion of cases the latency from cortical spike discharge to myoclonic jerk obtained with jerk locked averaging is incompatible with a cortical origin for the spike discharge. 4. The proposal that myoclonus is a form of long latency reflex and that myoclonus represents a reflex arising from subclinical sensory input, is unproven.
Hepatitis-B-associated glomerular disease : a clinicopathological study of Hepatitis B virus associated Membranous Glomerulonephritis in Namibian and South African children 1974 - 2005 and a comparison with Hepatitis B associated Membranous Glomerulonephritis as well as Idiopathic Membranous Glomerulonephritis in adults
(Stellenbosch : Stellenbosch University, 2011-12) Bates, William D.; Moosa, Mohammed Rafique; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. General Internal medicine.
ENGLISH ABSTRACT: Background and Objective: The most common cause of severe proteinuria/nephrotic syndrome (NS) in children worldwide is minimal change disease (MCD). This is also the pattern observed in white and Indian children in South Africa (SA). By contrast, black and mixed race/coloured children of Southern Africa in the 1960s to 1990s were shown to have a different pattern of NS. One of the main differences was the frequency of hepatitis B virus (HBV) associated glomerulonephritis, usually membranous glomerulonephritis (MGN). The objective of this project was a clinicopathological study of this subgroup of nephrotic children to document the disease further and in particular to seek correlations between pathological and clinical features including prognosis. A central focus was to document the detailed ultrastructural examination of the renal biopsies of these children and to correlate the spectrum of pathological features with demographic, clinical, laboratory and prognostic features. The hypothesis was that the clinicopathological features of HBV MGN in children differed substantially from idiopathic MGN in general (children and adults) and also from HBV MGN in adults and that HBV MGN in children should be viewed as a distinct disease. Patients and methods: The childhood (12 years and younger) patient cohort was 309 children with severe proteinuria/nephrotic syndrome who presented at Tygerberg Hospital (TBH) over a 21 year period from 1974-1995, including 67 children from Namibia. The study group was 71 children with HBV MGN who were followed up to 2005. The comparative adult group was 45 adults with MGN of whom 12 had HBV MGN and 33 idiopathic MGN. (A comparison could not be made with idiopathic MGN in childhood as this centre only had 2 such patients during the study period.) Demographic, clinical, laboratory and renal pathology data were collected, compared and correlated. Results: HBV associated MGN was the most frequent cause of NS in the Namibian subgroup, 25/67 (37%) and the third most frequent, 71/309 (23%) in the childhood cohort as a whole. The MGN group was 86% (71/83) of the total HBV childhood nephrotic cohort, by far the dominant subgroup. The average age of the 71 children with HBV MGN was 6.0 years (range 2-12 years) at presentation and boys comprised 80% of the group. Hepatitis B envelope antigen (HBeAg) was identified in the serum of 87% of children tested. Laboratory features different from idiopathic MGN included more prominent haematuria, mildly raised serum transaminases and more frequently lowered serum C3 and C4 levels. Light microscopic examination of renal biopsies showed mesangial proliferation in all patients but with minimal glomerular sclerosis and interstitial disease. On ultrastructural examination mesangial and subendothelial deposits were common and prominent as was mesangial interposition. The MGN of HBV in children therefore frequently showed mesangiocapillary glomerulonephritis (MCGN) features in addition to the subepithelial deposits of MGN. The subgroup of 23 whose renal biopsies displayed severe mesangial interposition in addition to the subepithelial deposits of MGN were termed the mixed HBV MGN-mesangiocapillary GN group. Virus like bodies and tubuloreticular inclusion bodies were both found in more than 80% of biopsies of childhood HBV MGN. HBeAg was identified in the subepithelial deposits in the glomeruli. This was the first time this feature was demonstrated in Africa. The 46 South African children with HBV MGN showed a cumulative remission rate of 25% at 2 years and 52% at 4 years. Seven of the children (10%) of the total cohort developed chronic renal failure (CRF). Age of 6 years and above at presentation and severe mesangial deposits on biopsy correlated with fewer remissions and poorer outcome. In 3 patients the interval between the diagnosis of HBV MGN and the onset of CRF was more than 19 years with the longest being 23 years. The 358 cases of childhood HBV MGN from Southern Africa constitute 37% of the reported childhood patients. Comparative data A comparison was made between the 71 children with HBV MGN, 12 adults with HBV MGN and 33 adults with idiopathic MGN. The main differences were that both HBV MGN groups included only coloured and black patients and were more predominantly male while the idiopathic MGN group included all races. In the HBV patients, haematuria was more frequent and severe, liver enzymes were frequently raised and C3 more frequently reduced than in the idiopathic cohort. Both groups of adult MGN patients had normal C4 levels while the childhood HBV MGN group had reduced C4 levels. The immune complex pattern in both of the HBV MGN adult and childhood groups on biopsy was similar with more mesangial and subendothelial deposits as well as mesangial interposition than the idiopathic group. Despite this similarity between the two HBV groups, both adult groups showed more glomerular sclerosis and interstitial disease than the childhood group. The clinical outcome of the children’s cohort was better than the other 2 groups with remission (52%) more frequent at 4 years (p<0.01) and better renal and patient survival. Including the 83 cases from this series, at least 1243 renal biopsy proven cases of HBV MGN have been reported in the English literature; children (80%) and adults (20%). The male gender predominance in both age groups for HBV MGN is similar (children 79%; adults 84%) and significantly greater than for idiopathic MGN. Conclusions: The findings confirm that HBV MGN in children is a distinct form of GN which broadens the classical morphologic description of MGN by often including a number of mesangiocapillary GN features. The subgroup of renal biopsies with the most severe mesangiocapillary GN features was classified as the mixed HBV MGNmesangiocapillary GN group. The MGN spectrum as a whole comprised 86% of the HBV positive childhood group. HBV MGN was the most frequent association with NS/severe proteinuria in the Namibian subgroup (37%) and the third largest group (19%) in the SA children. It showed a relatively high spontaneous remission rate but at least 10% of the children developed renal failure. Age of 6 years and above at presentation and severe mesangial deposits on biopsy correlated with fewer remissions and poorer outcome. Extended follow up (more than 15 years) was required to demonstrate renal failure in some patients in the poor outcome group. Urbanisation, associated with lower HBV carrier rates, and HBV vaccination (initiated routinely in 1995 in SA), have already lead to a sharply decreasing incidence of this disease in SA. HBV MGN has been a valuable and possibly unique model of human GN and MGN in particular in that the HBeAg has been identified in both the serum and glomeruli enabling confirmation of the aetiological role of HBeAg. het ’n kumulatiewe remissie koers van 25% teen 2 jaar en van 52% teen 4 jaar getoon. Sewe van die kinders (10%) van die hele kohort het kroniese nierversaking (KNV) ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale neerslae in ‘n biopsie het met minder remissies en ’n swakker uitkoms gekorreleer. Drie pasiënte het meer as 19 jaar na aanvanklike voordoening ooglopende KNV ontwikkel, waarvan 23 jaar die langste interval was. Die 358 gevalle van kinderjare HBV MGN van Suidelike-Afrika maak 37% uit van die gerapporteerde kinder pasiënte. Vergelykende data ’n Vergelyking is getref tussen die 71 kinders met HBV MGN, 12 volwassenes met HBV MGN en 33 volwassenes met idiopatiese MGN. Die hoof verskille was dat beide HBV groepe net kleurling en swart pasiënte ingesluit het en meer oorwegend manlik was, terwyl die idiopatiese groep alle rasse ingesluit het. In die HBV pasiënte was hematurie meer algemeen en erg, lewer ensieme meer dikwels verhoog en C3 meer dikwels verlaag as in die idiopatiese kohort. Beide groepe van volwasse MGN pasiënte het normale C4 vlakke getoon terwyl die kindergroep met HBV MGN verlaagde C4 vlakke bewys het. Die immuunkompleks patroon in biopsies van die HBV MGN volwasse en kindergroepe was soortgelyk met meer mesangiale en subendoteliële neerslae asook meer mesangiale interposisie as in die idiopatiese groep. Ten spyte van hierdie ooreenkoms tussen die twee HBV groepe, het die twee volwasse groepe meer glomerulêre sklerose en interstisiële siekte as die kindergroep vertoon. Die kliniese uitkoms van die kinderkohort was beter as die ander twee groepe met remissie (52%) wat meer algemeen was teen 4 jaar (p< 0.01) en met beter nier- en pasïent oorlewing. Ingeslote die 83 gevalle van hierdie reeks, is ten minste 1243 nierbiopsie bewysde gevalle van HBV MGN in kinders (80%) en volwassenes (20%) in die Engelse literatuur gerapporteer. Die manlike oorheersing in beide ouderdomsgroepe van HBV MGN is soortgelyk (kinders 79%; volwassenes 84%) en betekenisvol meer as vir idiopatiese MGN. Gevolgtrekkings: Die bevindinge bevestig dat HBV MGN in kinders ’n afsonderlike vorm van GN is wat die klassieke beskrywing van MGN verbreed deur die algemene insluiting van ’n aantal mesangiokapillêre GN kenmerke. Die ondergroep van nier biopsies met erge mesangiokapillêre GN kenmerke is as die gemengde HBV MGNmesangiokapillêre GN groep geklassifiseer. Die MGN spektrum in geheel het 86% van die HBV positiewe kindergroep behels. HBV MGN was die mees algemene assosiasie met NS/erge proteïenurie in die Namibiese subgroep (37%) en die derde grootse groep (19%) onder die SA kinders. Die siekte het ’n relatiewe hoë spontane remissiekoers getoon, maar ten minste 10% van die kinders het nierversaking ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale neerslae in ‘n nierbiopsie het met minder remissies en ’n slegter uitkoms gekorreleer. Uitgebreide opvolg (meer as 15 jaar) was nodig om nierversaking in sommige van die swak uitkomsgroep aan te toon. Verstedeliking is geassosieerd met laer HBV draersyfers en hierdie faktor saam met algemene HBV inenting in die kinderjare (wat in 1995 in SA begin was), het ’n skerp daling in die voorkoms van hierdie siekte in SA teweeg gebring. HBV MGN is ’n waardevolle en moontlik unieke model van menslike GN en MGN, veral omdat die HBeAg in beide die serum en glomeruli identifiseer kon word om die etiologiese rol van HBeAg te bevestig.
A historical perspective of allogeneic and autologous immunohaematopoietic stem cell transplantation in South Africa and a study of the non-haematologic consequences
(Stellenbosch : Stellenbosch University, 2015-03) Wood, Lucille; Jacobs, Peter; Irusen, Elvis; Abayomi, Akin; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. General Internal Medicine.
ENGLISH ABSTRACT: HISTORICAL PERSPECTIVE Stem cell therapy was commenced after using rabbits as research models. Once this process was successful, the first human transplant was done in 1974. Certain prerequisites were necessary and these were achieved - a protected environment, an apheresis unit, protocols and accreditation with International Registries. Initially, unmanipulated bone marrow and peripheral blood stem cells were used together with immunosuppressive drugs followed by the use of Cyclosporin A then the addition of ex vivo Campath®. AUDIT OF ACUTE ASSOCIATIONS (468 subjects in initial cohort) NEPHROLOGY Creatinine was used as an indication of renal function. Of the 76 available for analysis, 47% had acute kidney injury. Dialysis had a poor outcome as reported in the literature. Renal complications occurred frequently mostly due to infection. CARDIOLOGY A total of 119 individuals were available for analysis. Echocardiograms and electrocardiograms were part of pre-transplant assessment. Left ventricular systolic dysfunction predicted for increasing post transplant problems. Cardiac complications occurred at a lower frequency than other post-transplants side-effects consistent with the published data. DERMATOLOGY Cases were evaluated on a daily basis and referred to a dermatologist when necessary. To confirm Graft-Versus-Host Disease (GVHD), a skin biopsy was done to differentiate it from drug hypersensitivity or viral infections. The exposure to ex vivo Campath® significantly improved outcome by reducing the incidence and severity of GVHD. Quality of life was enhanced with substantial cost saving. GASTROENTEROLOGY Foregut symptoms occurred in 90% of patients. Nutritional problems were encountered. Altered liver functions were relatively common attributable to drugs, sepsis and conditioning regimens. Liver biopsies were not performed in this series and endoscopy performed only when necessary. A STUDY ON LATE COMPLICATIONS (55 subjects) RESPIRATORY Spirometry and diffusing capacity were done in this cohort. All the lung function studies were within the predicted normal range apart from some marginal reduction in diffusing capacity. In none of these patients did late consequences such as Bronchiolitis Obliterans Organising Pneumonia and Late Onset Non-Infectious Pulmonary complications occur. Cytomegalovirus reactivation was common but early intervention prevented serious complications. IMMUNOLOGY An in vitro functional study was done. Both the innate and adaptive systems were evaluated. Taken into consideration were the type of transplant, age from transplant, diagnosis and conditioning. The granulocyte Burst-test was done for the innate profile. Reduced activity was shown in all the subgroups. It appears as if the innate response of the granulocytic cells never recovered due to reduced granulocytic function in vitro. The adaptive responses were evaluated in vitro and only the autografts showed better CD4+ and CD8+ cytokine production. No major differences were seen in other groups. Normal cytokine production by CD4+ and CD8+ T cells were present when these were activated in vitro to produce regulatory cytokines, implying that their lymphoid component was intact post-transplant. BONE DISEASE Here both the Dual energy X-ray Absorptiometry (DXA) and Quantitative Computed Tomography (QCT) were used to evaluate bone mineral density. There was a discrepancy present between the two modalities. DXA showed no osteoporosis but QCT 22%. Biomarkers were normal in all. There was no history of fracture and no objective evidence of vertebral fractures using vertebral fracture assessment. Although QCT was used for the study, DXA remains the gold standard in South Africa. CONCLUSION This doctoral provided information on the non-haematological consequences in South Africa with the use of Campath® ex vivo.
The immunopathogenesis and treatment of tuberculous pericardial effusions in a population with a high prevalence of infection with the human immunodeficiency virus
(Stellenbosch : University of Stellenbosch, 2005-12) Reuter, Helmuth; Doubell, Anton F.; Burgess, Lesley J.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Medicine. Internal Medicine.
Mycobacterium tuberculosis (M. tuberculosis) accounts for more adult deaths than any other infectious agents. The present study included 162 patients with tuberculous pericarditis; 50% of the tuberculous pericarditis patients studied were human immunodeficiency virus (HIV) positive, compared to only 4.2% of patients who presented with non-tuberculous pericardial effusions. A steady year-to-year rise in HIV prevalence was observed in this 6-year study. Although the prognosis of pericardial tuberculosis (TB) is excellent with appropriate medical treatment, untreated pericardial TB has a mortality of 80-85%. It is thus important to diagnose tuberculous pericarditis efficiently. Traditionally, the diagnosis of pericardial TB is established by positive mycobacterial culture and/or histological evidence of necrotising granulomatous inflammation of the pericardium. Our study confirmed the insensitivity of pericardial fluid culture and pericardial biopsy in the diagnosis of pericardial TB, and at the time of clinical decision-making, results were usually not available. To overcome these difficulties, we explored various alternative strategies and this resulted in two diagnostic tools, namely a diagnostic rule and a diagnostic algorithm or classification tree. By means of classification and regression tree analysis, we allocated a weighted diagnostic index to each of five independently predictive features (fever, night sweats, weight loss, serum globulin >40 g/L and peripheral blood leukocyte count <10x109/L). A total diagnostic index of 6 or more corresponded to 82-86% sensitivity and 76-87% specificity for a diagnosis of tuberculous pericarditis. When possible, pericardial fluid should be aspirated to determine adenosine deaminase (ADA) levels and pericardial differential leukocyte counts. Fluid should also be sent for Gram stain and culture. The proposed diagnostic classification tree utilises the independently predictive attributes of pericardial adenosine deaminase levels, pericardial fluid lymphocyte/neutrophil ratios, peripheral leukocyte counts and the HIV status. Applying this prediction model to our entire data set of 233 patients resulted in 96% sensitivity and 97% specificity for the correct diagnosis of tuberculous pericarditis. Generally, patients were critically ill at the time of enrolment; 90% of tuberculous pericarditis presented with echocardiographic features of cardiac tamponade. Echoguided percutaneous pericardiocentesis with an indwelling catheter and intermittent daily aspiration was highly effective and safe. It is likely that the combination of this drainage technique and the early initiation of anti-tuberculous chemotherapy contributed to the almost complete absence of constriction in the patients studied, and our data do not support the routine use of adjunctive corticosteroids in patients with tuberculous pericarditis. Tuberculous exudates result from a Th1 mediated immune response characterised by lymphocyte dominance, significantly elevated levels of gamma-interferon (IFN-γ) and undetectable levels of interleukin-4 (IL-4). IFN-γ levels were not influenced by HIV status in spite of the severely diminished pericardial CD4+ lymphocyte counts observed in this study. It is thus likely that in HIV positive patients IFN-γ production is partly maintained by activated CD8+ T cells, which were significantly elevated in HIV positive patients compared to HIV negative tuberculous pericarditis patients. This finding underlines the importance of IFN-γ in the human immune response against M. tuberculosis. We also demonstrated that the presence of ADA in pericardial fluids reflects the activity of the cellular immune response. Both IFN-γ and ADA can be utilised as sensitive and specific diagnostic tools for pericardial TB.
An investigation of the clinical profile and extent of Long QT Syndrome (LQTS) associated with the KCNQ1-A341V mutation in South Africa and with the KCNH2-A1116V mutation in an Italian family and the role that autonomic nervous system (ANS) activity and genetics play in clinical variability
(Stellenbosch : University of Stellenbosch, 2007-12) Crotti, Lia; Brink, Paul A.; Schwartz, Peter J.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Medicine. Internal Medicine.
Background Although great progress has been made in defining genes conferring the majority of genetic risk in Long QT Syndrome (LQTS) patients, there remains a substantial challenge to explain the widely observed variability in disease expression and phenotype severity, even among family members, sharing the same mutation. Identifying clinical and genetic variables capable of influencing/predicting the clinical phenotype of LQTS patients would allow a more accurate risk stratification, important for determining prognosis, selecting patients for the most appropriate therapy, and counseling asymptomatic mutation carriers (MCs). To address these questions an Italian LQT2 family and a South African Founder LQT1 population have been used. Methods and Results Italian LQT2 family. The proband, a 44-yr-old white woman, presented with ventricular fibrillation and cardiac arrest. Intermittent QT prolongation was subsequently observed and LQT2 was diagnosed following the identification of a missense KCNH2 mutation (A1116V). The proband also carried the common KCNH2 polymorphism K897T on the non-mutant allele. Relatives who carried A1116V without K897T were asymptomatic but some exhibited transient mild QTc prolongation suggesting latent disease. Expression studies in Chinese Hamster Ovary (CHO) cells, demonstrated that the presence of KCNH2-K897T is predicted to exaggerate the IKr reduction caused by the A1116V mutation. These data explain why symptomatic LQTS occurred only in the proband carrying both alleles. South African LQT1 population. The study population involved 320 subjects, 166 MCs and 154 non mutation carriers (NMCs). Off ß-blocker therapy, MCs had a wide range of QTc values (406-676 ms) and a QTc>500 ms was associated with increased risk for cardiac events (OR=4.22; 95%CI 1.12-15.80; p=0.033). We also found that MCs with a heart rate <73 bpm were at significantly lower risk (OR=0.23; 95%CI 0.06-0.86; p=0.035). In a subgroup of patients Baroreflex Sensitivity (BRS) was determined both in presence and absence of ß-blocker therapy. BRS, analyzed in subjects in the 2nd and 3rd age quartiles (age 26-47) to avoid the influence of age, was lower among asymptomatic than symptomatic MCs (11.8±3.5 vs 20.1±10.9 ms/mmHg, p<0.05). A BRS in the lower tertile carried a lower risk of cardiac events (OR 0.13, 95%CI 0.02-0.96; p<0.05). This study also unexpectedly determined that KCNQ1-A341V was associated with greater risk than that reported for large databases of LQT1 patients: A341V MCs were more symptomatic by age 40 (79% vs 30%) and became symptomatic earlier (7±4 vs 13±9 years), both p<0.001. Accordingly, functional studies of KCNQ1-A341V in CHO cells with KCNE1, identified a dominant negative effect of the mutation on wild-type channels. Conclusion Our findings indicate that risk stratification for LQTS patients must be more individually tailored and may have to take into account the specific mutation and probably additional clinical and genetic variables capable of influencing/predicting the clinical phenotype of LQTS patients. As a matter of fact, we have provided evidence that a common KCNH2 polymorphism may modify the clinical expression of a latent LQT2 mutation and the availability of an extended kindred with a common mutation allowed us to highlight that KCNQ1-A341V is associated with an unusually severe clinical phenotype and to identify two autonomic markers, HR and BRS, as novel risk factors.
A longitudinal study of the stability of the dentition following orthodontic treatment
(Stellenbosch : Stellenbosch University, 1992) Rossouw, Paul Emile; Preston, C. B.; Nortje, C. J.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Dentistry.
ENGLISH ABSTRACT: The maintenance of dental alignment following orthodontic treatment has been, and continues to be, a challenge for the orthodontist (McReynolds and Little, 1991). Orthodontists should endeavour to establish normal occlusions and function to the end that physiologic balance and retentive stability may be achieved (Goldstein, 1953). Many philosophies and theories have been formulated in response to this challenge, but few have successfully withstood the test of rigorous post-orthodontic evaluation. The present study comprises longitudinal assessments of dentofacial changes which occurred in South African Caucasian subjects during their orthodontic treatment as well as a mean of 7 years following active treatnent. The sample consists of 88 Caucasian subjects; 33 males and 55 female sUbjects who have undergone conventional edgewise orthodontic treatment (Lindquist; 1985). The treatment includes extraction (56%) and nonextraction (44%) therapy. Due to the intricate structure of the craniofacial complex, it is deemed important to discuss the major components of this complex separately and then to compare the variables describing the area with post-orthodontic lower incisor crowding. Lower incisor crowding or irregularity, most often referred to as relapse when occurring in the post-orthodontic dentition, is a phenom~non that is clinically visible and easily assessed using the Little Irregularity Index (Little, 1975). A variety of orthodontic study cast and cephalometric variables represent the changes which occur at the three time intervals selected for this study, namely pre-treatment (T1), post-treatment (T2) and following active treatment (T3). statistical analysis of th~ data was undertaken by the Institute for Biostatistics of the Medical Research Council, Tygerberg, RSA utilising the SAS (1985). The significance level of the results of this study is set at p = 0.05. x No previous study has documented the evaluated and described the various craniofacial skeleton in this format. literature parts or has of the The thesis is divided into thirteen chapters.
Management of lymphoma in a centre with high HIV and tuberculosis prevalence
(Stellenbosch : Stellenbosch University, 2021-12) Bassa, Fatima Cassim; Irusen, Elvis; Warwick, James; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: General Internal Medicine.
Background The staging and management of patients with lymphoma (PWL) can be challenging and is particularly so in environments of high HIV and TB prevalence such as South Africa, for which there is little guidance. The additional contribution of positron emission tomography/computerised tomography (PET/CT) for assessing bone marrow involvement (BMI) in this setting is also uncertain. Methods This cross-sectional analytic study evaluated the clinical profiles, therapeutic interventions, including PET/CT scans, and outcomes of PWL at the Haematology unit, Tygerberg Hospital. We evaluated the potential utility of differential uptake of 18F- flouro deoxyglucose (FDG) on PET/CT in identifying second pathologies such as HIV and TB, in conjunction with clinical information and the use of additional testing, such a biopsy of discrepant uptake. The relative frequencies and causes of discrepancies of intensity of visual uptake of FDG, which the study termed the two-tone sign (2TS), was evaluated in both HIV- positive and HIV-negative patients. Results Two hundred and eighty patients, 101 HIV-positive and 178 HIV-negative, were recruited from a pool of 516 PWL, referred to the unit from March 2015 to March 2020. The median age of the patients was 42 years, with HIV-positive patients significantly younger than the HIV-negative cohort (p= 0.02). Significantly more HIV- positive patients had a history of TB or were on therapy for TB at presentation (p=<0.01). However, HIV-negative patients had significantly more other co- morbidities (p=<0.01). The 2 main subtypes of lymphoma were diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL), with DLBCL being commoner in HIV-positive and HL in HIV- negative patients. Seventy-six percent of patients presented with advanced disease, similar in HIV-positive and HIV-negative patients (75.2 vs.76.4%). Complete remission rate was significantly better in HIV-negative patients with DLBCL while, with HL, there was no significant difference in outcomes between HIV- positive and HIV-negative patients. The 2TS was found with a higher frequency in the HIV-positive group than HIV- negative group (39.5% vs 25.3%), with a trend towards statistical significance (p=0.056). Causes of a 2TS were HIV, TB, lymphoma, synchronous malignancies or reactive uptake. In most patients, the cause of the discrepancy was identifiable and disease burden and response to therapy could be assessed. With respect to bone marrow involvement with lymphoma, congruence was demonstrated between the bone marrow biopsy (BMB) and PET/CT in 63.2% of patients with no significant difference between HIV positive and negative patients. The overall sensitivity of PET/CT was 87% (CI: 77.4-93.6%) and specificity 75.2% (CI: 66.7-82.5%). There was no impact of HIV on the BMB patterns on PET/CT, despite there being HIV-associated changes on the BMB in some patients. None of the patients evaluated, had TB on the BMB. Incongruence between BMB and PET/CT was found in 17 HIV negative patients with HL, with diffuse bone marrow uptake on PET/CT and a negative BMB. Conclusions This study demonstrated the complexities of patients with lymphoma managed at the unit, both HIV-positive and HIV-negative. Despite the aggressive rollout of antiretroviral therapy, many HIV-positive patients were not virologically controlled and presented with advanced, aggressive subtypes of lymphoma. The study found that it is possible to stage HIV-positive patients and those with TB using PET/CT, provided this is done with all available clinical information. We propose that in patients with HL and in most patients with DLBCL in our setting, both HIV-positive and HIV-negative, BMB is not required for assessment of BMI.
miRNA signature of healthy vs impaired mesenchymal stem cells as a biomarker for autologous stem cell therapy
(Stellenbosch : Stellenbosch University, 2021-12) Seboko, Ascentia Mathapelo; Van de Vyver, Mari; Scholefield, Janine; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine.
ENGLISH ABSTRACT: Background: Globally, approximately 463 million adults are living with diabetes. This growing prevalence of diabetes places an increased burden on the health systems of low-to-middle income countries such as South Africa. The pathological microenvironment associated with diabetes leads to severe co-morbidities that include chronic foot ulcers. Non-healing ulcers affect approximately 15% of diabetic patients. Novel approaches in the treatment of chronic ulcers make use of mesenchymal stem cell (MSC) therapies. However, autologous MSCs from diabetic patients are less successful than allogeneic donor MSCs. This is due to the impairment of MSCs as result of the pathological diabetic environment. MicroRNAs (miRNAs) have gained immense popularity as biomarkers of disease and have been associated with the development of metabolic diseases. This study hypothesised that MSCs with compromised therapeutic potential, have a unique miRNA signature that could potentially be used as a biomarker to predict if a specific patient would be a good candidate for autologous MSC therapy. Methods: Three models were utilised: 1) Healthy control and impaired diabetic bone marrow MSCs (BMSC) isolated from wildtype (C57BL6/J) and obese diabetic B6.Cg-Lepob/J (ob/ob) mice. BMSC function was assessed by comparing the a) ex vivo growth rate, b) secreted and intracellular IL6 (ELISA), and c) osteogenic (Alizarin Red S staining) and adipogenic (Oil Red O staining) differentiation capacity. Broad miRNA profiling (> 600 miRNAs) was performed using the NanoString mouse v1.5 miRNA Expression Assay (BMSCs) and RT-qPCR (whole blood). 2) Synthetic miRNA mimics were transfected into C3H10T1/2 cells with subsequent assessment of cellular proliferation and migration capacity. 3) The expression of miRNAs of interest was assessed in the serum and PBMCs of overweight/obese (OW/OB) (n=14) and diabetic (n=16) patients who were subdivided into: a) predicted good healers and b) predicted poor healers based on serum MMP9 levels. Health, lifestyle, and dietary questionnaires were completed by participants and blood samples collected to determine their metabolic (blood glucose, HbA1c) profile. Results: The miRNA profile of BMSCs was affected by both sex (male vs female) and metabolic phenotype (healthy vs impaired). MSC impairment was prominent in male ob/ob mice and coincided with the upregulation of 19 miRs and downregulation of 3 miRs compared to their healthy control counterparts (p < 0.05). Five miRNAs of interest (miR-142-5p, miR-200b, miR-202-5p, miR-384-3p and miR-466g) emerged as having a potential role in the functional capacity of BMSCs. C3H10T1/2 cells transfected with miR-202-5p had an increased proliferation rate (p < 0.01) compared to non-transfected cells. All 5 miRNAs of interest reduced the rate of wound closure in vitro following transfection (p < 0.05) compared to non-transfected cells. This was due to more random migration patterns and a reduction in directionality. No association could be observed between the metabolic profile, predicted healing capacity and miRNA expression in the PBMCs of participants. Conclusion: The data confirmed that a unique miRNA signature exists between healthy vs impaired BMSCs. Numerous confounding factors, including sex-specific differences, make the use of these miRNAs as biomarkers unlikely. This study did, however, confirm a role of miRNAs in BMSCs function.
Refinements and innovations in biopsy and analysis techniques for pleural and lung disease
(Stellenbosch : University of Stellenbosch, 2007-12) Diacon, Andreas Henri; Bolliger, Chris T.; Wright, Colleen A.; Walzl, Gerhard; University of Stellenbosch. Faculty of Health Sciences. Dept. of Medicine. Internal Medicine.
1.1. Background Tumors arising from the lung, pleura, or chest wall are a frequent problem in clinical pulmonary medicine. Most lesions are either infectious, neoplastic or granulomatous in nature, but a variety of other differential diagnoses must be considered. An accurate diagnosis is important because the available treatments differ substantially, and because any delay will impair the prognosis in potentially curable patients with lung carcinoma. The investigations involve the disciplines of radiology, pulmonology, surgery, microbiology, and anatomical pathology and consume a respectable amount of resources. The aim of the work covered in this thesis was to optimize the available diagnostic methods for the routine use in a health care setting with limited resources. 1.2. Methods The general idea of this work was to identify conventional sampling methods that could be developed further to become more useful for the diagnosis of chest tumors in a low resource health care setting. The key method was research performed: a) to revise and expand the indication for a sampling method, b) to technically improve the sampling process, and c) to optimize sample transport, preparation and analysis in collaboration with the analytical laboratory. 1.3. Results A list of invasive diagnostic procedures, imaging methods and analytical processes were developed, evaluated and integrated into clinical practice. A) transbronchial needle aspiration, B) transthoracic cutting needle biopsy, C) transthoracic fine needle aspiration, D) transthoracic ultrasound, and E) rapid on-site evaluation of needle aspirates by a cytopathologist. Five studies pertaining to this thesis were published in international peerreviewed journals: â ¢ Safety and yield of ultrasound-assisted transthoracic biopsy performed by pulmonologists (Respiration 2004;71:519-22) This paper established that ultrasound-assisted transthoracic biopsy performed by pulmonologists is feasible, safe, practical, low-cost and has a high yield. â ¢ Utility of rapid on-site evaluation of transbronchial needle aspirates (Respiration 2005;72:182-8) This paper demonstrated the economical advantages of on-site evaluation of transbronchial specimens in a low-resource setting. â ¢ Transbronchial needle aspirates: comparison of two preparation methods (Chest 2005;127:2015-8) This paper demonstrated that preparing smears on-site has a far better yield than pooling samples into a vial. This means that the yield is improved over the current standard at no additional cost. â ¢ Transbronchial needle aspirates: how many passes per target site? (European Respiratory Journal 2007;29:112-6) This paper investigated the most economical and effective approach to serial sampling with transbronchial needle aspiration during flexible bronchoscopy. â ¢ Ultrasound assisted transthoracic biopsy: fine needle aspiration or cutting needle biopsy? (European Respiratory Journal 2007;29:357-62) This paper compared two common methods of sampling and demonstrates that the less expensive method is sufficient in the majority of cases. 1.4. Conclusion This work has impacted on current practice in multiple ways. Conventional methods have been optimized by improving technical factors and with the integration of interdisciplinary collaboration. The initiated research is ongoing with the aim to achieve continued technical and economical improvements in the diagnosis of chest tumors.
The role of phosphatase activity and expression in glucocorticoid modulation of preosteoblasts
(Stellenbosch : University of Stellenbosch, 2011-12) Sanderson, Micheline; Ferris, W. F.; Moolman-Smook, J. C.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Medicine. Internal Medicine.
ENGLISH ABSTRACT: The increase in the prescription and use of glucocorticoids (GCs) to treat various diseases and resulting decrease in bone density and development of osteoporosis is of growing concern. Glucocorticoid-induced osteoporosis (GCIO) is a relatively under-researched disease with the mechanism by which GCs affect bone metabolism not yet fully delineated. This holds especially true for the early events in bone development. The negative effects of GCs are predominantly seen in osteoblasts, the cells responsible for bone formation, in that GCs diminish both the numbers and function of osteoblastic cells. Osteoblast precursor cell proliferation is crucial to ensure the existence of a healthy pool of osteoblastic cells needed to form new bone after bone resorption by osteoclasts. Previously, it was shown that GCs reduce the proliferation of immortalised osteoblastic cell lines. In addition, early immortalised preosteoblasts were more sensitive to GCs than their mature counterparts. However, these cells have corrupted cell cycles; therefore, primitive primary mesenchymal stromal cells (MSCs) were used in this study to examine the effect of GCs on the mitogen-induced proliferation of early osteoblast precursor cells (naïve MSCs and preosteoblasts) using the synthetic GC, dexamethasone (Dex). Mitogenic conditions established for naïve rat mesenchymal stromal cells (rMSCs) indicated that mild (5% FBS) stimulation is sufficient to induce proliferation, whereas a higher FBS concentration (20% FBS) was mitogenic in primary preosteoblasts. It was also found that pharmacological doses of Dex drastically decreased the mitogen-induced proliferation of both naïve rat MSCs (rMSCs) and preosteoblasts. Mitogen-activated protein kinase (MAPK) signalling pathways, such as ERK1/2, govern cell proliferation. GCs have been shown to decrease the activity of ERK1/2, which is associated with decreased proliferation in osteoblastic cells. In the present study, western blot analysis showed that Dex reduced the proliferation-associated shoulder of the ERK1/2 activity profile in both naïve rMSCs and preosteoblasts. Moreover, the ERK1/2 signalling pathway was shown to be essential for mitogen-stimulated growth of naïve rMSCs and preosteoblasts as the MEK1/2 inhibitor, U0126, inhibited mitogen-induced proliferation. Using western blot analysis, it was shown that, after mitogen administration, ERK1/2 activity exhibited a typical proliferation profile, which was blocked by U0126. Protein tyrosine phosphatases (PTPs) dephosphorylate and inactivate ERK1/2. Utilising sodium vanadate, an inhibitor of PTPs, in vitro phosphatase assays revealed that PTP activity was the predominant phosphatase activity present in naïve rMSCs and preosteoblast lysates after concomitant mitogen and Dex stimulation. The mRNA of the dual specificity phosphatase, MKP-1, was rapidly (within 30 minutes) upregulated after mitogen and Dex administration in both naïve rMSCs and preosteoblasts. However, the protein expression pattern of MKP-1 did not correspond to the mRNA induction, suggesting that the MKP-1 protein could be subjected to rapid degradation. These findings suggest that MKP-1 could possibly be involved in the GC regulation of mitogen-induced proliferation of early osteoblast precursor cells, but closer investigation is needed to fully elucidate this role. In addition, the involvement of other PTPs should not be excluded and warrants further investigation. During the course of the present study, it was found that strong mitogenic stimulation with 20% FBS led to oncogene-induced senescence (OIS). Flow cytometry analysis revealed the presence of two populations in naïve rMSCs preparations and DNA content analysis was consistent with that of cells undergoing OIS. These results indicated that the more primitive osteoblast precursor cells (naïve rMSCs) are more responsive to mitogens than their mature counterparts (preosteoblasts). In addition, it was found that the magnitude of ERK1/2 activation was increased in naïve rMSC after strong mitogenic stimulation, indicating that naïve rMSCs are still highly sensitive to stimulation with strong mitogens. In summary, these findings show that Dex decreased the proliferation of naïve rMSCs and preosteoblasts concomitantly with a decrease in ERK1/2 activity. In addition, Dex upregulated MKP-1 mRNA, but the same effect was not seen on the MKP-1 protein levels. Therefore, this suggests that PTP/s other than MKP-1 could be responsible for the inactivation of ERK1/2 by Dex, leading to decreased proliferation in naïve rMSCs and preosteoblasts. Further identification of PTPs that regulate osteoblast precursor cell numbers and function could lead to the elucidation of the mechanism through which GCs act to negatively influence bone density. This will improve our insights into the pathogenesis of GCIO and aid in the identification of therapeutic targets which can be exploited to develop new agents to treat osteoporosis.
A study of non-alcoholic fatty liver disease (NAFLD) in South African patients and analysis of candidate genes in insulin resistance and fatty acid oxidation.
(Stellenbosch : Stellenbosch University, 2008-12) Kruger, F. C.; Kotze, Maritha J.; Van Rensburg, C. J.; Stellenbosch University. Faculty of Health Sciences. Dept. of Medicine. Internal Medicine.
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in Western countries, extending from steatosis (FLD) to steatohepatitis (NASH). Differentiation between NASH and nonprogressive NAFLD is difficult on clinical grounds therefore a need exists to identify reliable biomarkers of disease progression. The aims of the study were 1) to describe the disease profile of NAFLD/NASH in South African patients of the Western Cape, 2) to investigate the metabolic derangements associated with this condition, including insulin resistance, lipid abnormalities and liver fibrogenesis, and 3) to assess the possible involvement of candidate genes in relation to the disease phenotype in the patient cohort. A total of 233 patients (73% female) were enrolled in this study, consisting of 69% Cape Coloured, 25% Caucasian, 5% Black and 1% Asian individuals. All subjects were obese or overweight based on the assessment of body mass index (BMI). Screening for NAFLD identified 182 patients (87%) with ultrasonographical evidence of fatty infiltration and/or hepatomegaly. Liver biopsies were performed on patients with persistently abnormal liver functions and/or hepatomegaly. NAFLD was confirmed histologically in 111 patients of whom 36% had NASH and 17% advanced liver fibrosis. None of the Black patients had advanced fibrosis.
Ultrasound-assisted transthoracic diagnostic techniques
(Stellenbosch : University of Stellenbosch, 2011-12) Koegelenberg, Coenraad Frederik Nicolaas; Bolliger, Chris T.; Diacon, Andreas H.; Wright, Colleen A.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Medicine. Internal Medicine.
ENGLISH ABSTRACT: Although transthoracic ultrasonography is a well established modality, it is still underutilised by chest physicians. The aim of this research project was to investigate the feasibility, diagnostic yield and safety of ultrasound(US)-assisted transthoracic biopsies performed by clinicians in various settings relevant to daily practice of respiratory medicine. We conducted four clinical trials which are summarised below: 1. In a prospective study on the feasibility of US-assisted transthoracic fine needle aspiration (TTFNA) of drowned lung secondary to a proximal mass lesion, a novel indication for US-assisted TTFNA was described. TTFNA passes >20mm from the visceral pleura had a sensitivity of 74.2% and were also more likely to contain malignant cells than more superficial passes. The surprisingly high yield and the fact that no serious complications were observed validated this approach, which may be an alternative to bronchoscopy. 2. In the largest single-centre study on US-assisted TTFNA with rapid on-site evaluation (ROSE) and cutting needle biopsy (CNB) in the setting of superior vena cava (SVC) syndrome ever reported, we were able to accurately diagnose 96% of all patients who presented with an associated mass lesion that abutted or infiltrated the chest wall. No pneumothoraces or major haemorrhage was caused. We also validated the single-session approach, and were able to conclude that US-assisted TTFNA (with ROSE) is the initial investigation of choice in suspected bronchogenic carcinoma, whereas both TTFNA and CNB need to be performed in all other cases. 3. We continued to validate the novel single-session sequential approach in a study on anterosuperior mediastinal masses. US-assisted TTFNA with ROSE was performed on 45 consecutive patients, immediately followed by CNB where a provisional diagnosis of epithelial carcinoma or probable tuberculosis (TB) could not be established. An accurate cytological diagnosis was made in 73.3%, and was more likely to be diagnostic in epithelial carcinoma and TB than all other pathology (p<0.001). CNB yielded a diagnosis in 88.2%. Overall 93.3% of patients were diagnosed by the single-session approach. No pneumothorax or major haemorrhage was observed. 4. In a prospective study, we compared US-assisted Abrams and Tru-Cut needle biopsies with regard to their yield for pleural TB. Pleural biopsy specimens obtained with Abrams needles contained pleural tissue in 91.0% of cases and were diagnostic in 81.8%, whereas Tru-Cut needle biopsy specimens only contained pleural tissue in 78.7% (p=0.015) and were diagnostic in 65.2% (p=0.022). In conclusion, we investigated the feasibility of US-assisted biopsies performed by respiratory physicians in various settings, and consistently found acceptable to very high diagnostic yields with minimal complications. Furthermore, we were able to validate a novel indication for US-assisted TTFNA (US-assisted TTFNA of drowned lung), validate the use of a single-session sequential approach (USassisted TTFNA with ROSE followed by CNB where indicated) in at least two clinical settings (SVC syndrome and anterosuperior mediastinal masses) and we were able to show that US-assisted Abrams needle biopsy is superior to Tru-Cut needles biopsy when histological confirmation of TB pleuritis is required.

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