Doctoral Degrees (General Internal Medicine)

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    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.
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    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.
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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.
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    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.
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    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.