Masters Degrees (Chemical Pathology)

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Experimental diabetes in the baboon.
(1979) Naidoo, Dayananthan.; Asmal, A. C.
The object of the present study was to determine simultaneously aspects of hepatic and peripheral glucose metabolism in the intact baboon. Isotopic techniques were used to study glucose turnover rates, glucose recycling, glucose pool and space, and the forearm technique to study peripheral exchange of glucose. The results obtained in the normal animals acted as reference values for each animal. Thereafter diabetes mellitus was produced with streptozotocin, a drug causing destruction of the beta cells of the pancreatic islets. Experiments were then repeated in the acutely diabetic baboon and the nature and extent of the abnormalities in glucose metabolism documented. Lactate metabolism and peripheral lipid metabolism were included in the study in order to establish any interrelationships with glucose metabolism and to determine the abnormalities resulting from the production of diabetes. In the normal animal the turnover rate of lactate was greater than glucose although the lactate pool was much smaller than the glucose pool. After producing diabetes glucose turnover rates increased threefold and correlated with the severity of hyperglycaemia. A significant increase in lactate turnover rates was noted but the increase was less than in the case of glucose turnover rates. The formation of glucose from lactate increased significantly but the fraction of the lactate turnover rate converted to glucose was unchanged. The glucose pool increased nearly threefold and correlated with the increase in glucose turnover rate. There was a significant but smaller increase in lactate pool which correlated with the increase in lactate turnover rate. Both glucose and lactate space decreased after diabetes but the decrease did not correlate with the severity of hyperglycaemia. In the majority of diabetic animals there was no glucose utilization in the forearm, and in fact glucose release was observed. Increased production of lactate occurred in the forearm of the diabetic baboon, despite decreased glucose utilization.Arterial levels of triglyceride and free fatty acid increased threefold after diabetes while the free glycerol level doubled. In the normal animal the general pattern of exchange in the forearm consisted of triglyceride and free fatty acid uptake and free glycerol release. In the diabetic animal triglyceride and free fatty acid release was observed, while the release of free glycerol was decreased. The pattern of forearm metabolism in the diabetic animals was variable and not as consistent as before the production of diabetes. Several interrelationships between glucose, lactate and lipid metabolism were noted. The baboons used in this study showed extreme sensitivity to the metabolic effects of Streptozotocin Diabetes. Hyperglycaemia increased in severity and ketoacidosis invariably developed in the second week. The animals were not treated with insulin and death from severe uncontrolled diabetes occurred in nearly all animals within two weeks. This study has demonstrated the severe abnormalities in hepatic and peripheral glucose metabolism in diabetes. The simultaneous pathogenesis of these abnormalities and their importance in the development of the acute diabetic syndrome have been defined. Associated abnormalities in lactate metabolism and lipid metabolism have also been documented.
Characteristics of insulin receptors in a human lymphoblastoid cell line (Raji).
(1988) Dunn, Rosanne Dorothy.
The notion that insulin binds to a specific site on the cell membrane was first proposed many years ago. However, experimental proof of a membrane bound insulin receptor did not come until the early 1970s when biologically active radiolabelled insulin was used in direct binding studies (Cuatrecasas, 1971). Recent advances in understanding the mechanism of insulin action are the result of studies on the structure and function of the insulin receptor. The membrane receptor would appear to have two functions: firstly, it must bind insulin and secondly, it must couple insulin binding to insulin action. Defects in either of these receptor functions will result in an impaired response to insulin, or insulin resistance (Taylor, 1985). Insulin resistance is a common disorder in a number of disease states in man. For example, non-insulin-dependent diabetes mellitus and obesity are associated with mild insulin resistance (Bar et aZ., 1976). There are also a number of relatively rare syndromes of extreme insulin resistance in which there is either impaired receptor function, or an immunological defect resulting in the development of auto-antibodies against the insulin receptor (Taylor et aZ., 1985).Studies on insulin receptor defects associated with these disease states have led to progress in understanding the molecular mechanisms of insulin action. Ideally when investigating these disease states one should study insulin action on classical target cells such as adipocytes, hepatocytes or muscle. However, it is now well established that the kinetics of insulin binding to its membrane receptor is similar in all human tissue whether or not it is a target for insulin action. This has led to a great deal of research on the more accessible human tissues such as monocytes, erythrocytes, cultured fibroblasts and Epstein-Barr virus (EBV) transformed B-Iymphocytes. The most convenient tissue to study is EBV transformed B-Iymphocytes, as these cells can be taken from individual patients and grown in culture in large quantities, which facilitates biochemical studies. Despite these advantages, it is important to establish that this virus-induced receptor is a true insulin receptor and not an artifact of viral transformation. Studies on B-Iymphocyte proliferation have shown that the insulin receptor appears on the cell membrane during the proliferative phase of B-cell activation. However , this is a transient event and once the cell reaches maturation the insulin receptor is no longer evident (Marchalonis & Galbraith, 1987). The insulin receptor has also been demonstrated in a number of cultured human lymphoblastoid cell lines (Gavin et aL, 1983; Maegawa et aL, 1983). It seems, therefore, that the insulin receptor is normally expressed by blast cells. The purpose of this study was to investigate insulin binding characteristics on a human lymphoblastoid cell line with B-cell characteristics which was originally derived from a patient with Burkitt's lymphoma. These cells, which are known as Raji cells, are unusual in that they carry multiple copies of the EBV genome in their DNA. For this reason they provide a useful model system for studying the insulin receptor in EBV transformed lymphocytes. In addition, studies on the mechanism of insulin action in these cells should give some insight into the function of the insulin receptor during B-cell proliferation. In this study four major characteristics of insulin binding to insulin receptors on Raji cells are described. Firstly, on the basis of kinetic studies a model for insulin-receptor interaction was established. Secondly, processing of insulin and the receptor was investigated to determine whether the receptor is functional. A third aspect was elucidation of the receptor structure and the insulin binding site. Finally, the cross-reaction between insulin and type I IGF receptors was studied, and the cellular response mediated by the insulin receptor and growth factor receptor was determined.
Development of a monoclonal antibody-based immunoradiometric assay for the measurement of the free alpha-subunit of human chorionic gonadotrophin.
(1990) Haneef, Raazia Be.; Pegoraro, Rosemary J.
Almost a century has elapsed since the antigen-antibody interaction was first recognised as the basis of an immune response (Ehrlich, 1897). However, it was only in the 1930s, with the development of improved technologies that this concept was better understood, and led to the discovery of the amazing diversity and specificity of antibody molecules (Landsteiner, 1933). Theoretically, it is possible to make antibodies to a variety of biological substances and other chemicals, and therefore they are ideally suited as specific recognition elements to be used for analytical, cytological, functional, therapeutic and biochemical purposes. The development of the radioimmunoassay (RIA) thirty five years ago, revolutionised research in many areas of clinical and scientific investigation. This technique evolved rapidly from the discovery made by Berson et al. in 1956 that antibodies to insulin could be detected in patients treated with this hormone, by measuring the binding of radiolabeled insulin to these antibodies. Although in the past RIAs have been the most important assay system employing antibody and labelled tracer, the limitation was that reliance had to be placed on the chance development of a good polyclonal antibody. These shortcomings stimulated the search for monospecific antibodies of reproducible quality and sufficient quantity. The development and introduction of monoclonal antibody technology brought about a revolution in immune serology (Kohler and Milstein, 1975). Establishment of immortal cell lines which contained the genetic elements of antibody-producing cells was achieved by fusion between a myeloma cell line and spleen cells from an immunised donor. The resulting hybrids had the essential properties of both parents, namely, permanent growth and a high capacity for the synthesis and secretion of immunoglobulins, normally characteristics of plasmacytomas, together with the genetic elements defining a specific antibody. Gestational trophoblastic disease (GTD) is a neoplastic condition of the trophoblast and occurs as molar pregnancy in a benign or invasive form, or as choriocarcinoma in a malignant form. Effective therapy has been developed for the treatment of both choriocarcinoma and molar pregnancy, but the key to successful management of these patients lies in their prompt diagnosis and careful monitoring of response to treatment (Green-Thompson, 1986). Fortuitously, these tumours elaborate the human chorionic gonadotrophin hormone (hCG) and its free alpha (a) and beta (B) subunits and hence a ready marker for the tumour exists. Human chorionic gonadotrophin is one of a group of glycoprotein hormones, which includes luteinising hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH). These hormones are composed of two dissimilar subunits designated a and B, which are bound non-covalently in the intact molecule. The B-subunit of each glycoprotein hormone is unique and is responsible for the respective biological and immunological properties of the glycoproteins. In contrast, all four hormones possess an identical a-subunit which is coded for by a single gene (Fiddes and Goodman, 1979). The measurement of hCG and its free B-subunit, as so-called BhCG, for the diagnosis and monitoring of therapy in patients with GTD is now routinely practised throughout the world (Vaitukaitis et al., 1972). However it has been demonstrated by Bagshawe (1975) that when serum BhCG can no longer be measured by current RIA methods, up to 10" tumour cells may remain undetected. In addition, there have been isolated reports of two patients with choriocarcinoma in whom BhCG was undetectable in the serum but who appeared to be secreting only the a-subunit (Dawood et al, 1977). Furthermore, it has been suggested that measurement of free a-subunit rather than intact hCG or the free B-subunit is a more effective means of detecting persistent trophoblastic disease as well as tumour recurrence following treatment (Quigley et al, 1980a and b). Radioimmunoassays which measure the free a-subunit of hCG have been developed, but in general lack the specificity and sensitivity required (Gaspard et al, 1980; Kohorn et al, 1981). These assays employ polyclonal antisera which also detect epitopes common to the pituitary gonadotrophins. Thus there is a need to produce monoclonal antibodies which recognise regions of the free a-subunit which are hidden in the intact gonadotrophins. Such antibodies would provide the required specificity for use in RIAs but are limited in their use by their inherent lack of high affinity for the antigen. Fortunately, this drawback may be overcome by using monoclonal antibodies as labelled reagents in an alternative assay system, the immunoradiometric assay (IRMA), described by Miles and Hales (1968). The IRMA, particularly the two-site sandwich version of the assay, has been shown to provide greater sensitivity in addition to allowing enhanced specificity. This is a consequence of the use of two antibodies in excess to detect the analyte, each directed at a different epitope on the target molecule. The first antibody, referred to as the capture antibody, is usually linked to a solid-phase to facilitate easy separation and is added in excess relative to the target hormone to enhance antibody-antigen interaction, thereby allowing increased sensitivity in the measurement of analyte. The second antibody, referred to as the detection antibody, is labelled with a radioactive isotope or an enzyme to detect antigen already bound to the capture antibody. The application of monoclonal antibodies specific for the free a-subunit to a highly sensitive IRMA format is an obvious need. Hence this study was undertaken firstly, to raise and characterise monoclonal antibodies to the free a-subunit, secondly to develop an IRMA using these antibodies and finally to establish whether measurement of free a-subunit has any clinical advantage.
Apolipoprotein E allele distribution in a South African Indian female population : effect on the lipid profile.
(1993) Gounden, Nirmala.; Pegoraro, Rosemary J.; Berger, G. Michael B.
Genetic polymorphism of apolipoprotein (apo) E has been shown to account for a significant amount of variance in plasma lipid and lipoprotein levels, thereby contributing to the incidence of cardiovascular disease across populations. In this cross-sectional study apo E genotypes were determined in a sample of 173 healthy, middle-aged Indian women using a restriction isotyping method, in which DNA was amplified by PCR and the Cfol restricted DNA fragments were separated on a polyacrylamide gel, allowing unambiguous typing of the common apo E genotypes. Considering the three common alleles, e2, e3 and e4, a reduced frequency of the e2 allele was observed in the study population in comparison to other populations around the world. This finding underlines the heterogeneity of apo E allele frequencies in different populations. This study also investigated possible effects of apo E genotype on lipoprotein changes in this sample of women spanning the menopause. Apo E polymorphism was associated with significant differences in plasma lipid levels. Notably, total and low density lipoprotein cholesterol and more especially plasma triglyceride concentrations were increased in carriers of the e3/4 genotype. Two-way analysis of variance of the effect of apo E genotype and menopausal status on the lipid profile showed no significant interaction effect, indicating that the effects of apo E genotype on the lipid profile do not differ significantly between premenopausal and postmenopausal women. Age and to a lesser extent the waist hip ratio also correlated with lipid concentrations, but menopausal status had no apparent effect in this sample. This study confirms the potentially deleterious effect of the e4 allele, in a vulnerable population. The reduced occurrence of the E2 isoform, which is considered to offer a measure of protection against cardiovascular disease, may contribute to the relatively high incidence of coronary heart disease in the South African Indian population. However, the relatively low incidence of the e2 allele may protect this population against the occurrence of type III hyperlipoproteinaemia precipitated by diabetes and obesity in e2/2 homozygotes.
The relative contribution of the p53 tumour suppressor gene and the human papilloma virus to cancer of the cervix in Black women.
(1995) Arteta, Ana Maria Aguirre.; Pegoraro, Rosemary J.
Abstract available in PDF.
Microsatellite instability and cell cycle protein analysis in endometrial carcinoma.
(2006) Padayachy, Padmini.; Chetty, Runjun.
Red cell membrane abnormalities in hereditary spherocytosis patients of KwaZulu-Natal.
(2006) Bridgemohan, Roshini.; Jogessar, Vinod B.; Coetzer, Theresa Helen Taillefer.
Hereditary Spherocytosis (HS) is a common inherited haemolytic anaemia with variable clinical expression. Fifty subjects with HS from KwaZulu-Natal were studied with the aim of providing further information on the protein abnormalities of the red blood cell (RBC) membrane and their relationship with clinical presentations. Haematological and biochemical tests were performed by routine procedures. Mean Corpuscular Haemoglobin Concentration ( MCHC) in the HS group was 35.1g /dl. This was significantly higher than in normal control subjects (33.6g /dl) (p value < 0.001); indicating its usefulness for the screening of HS. Mean Red Cell Distribution Width (RDW) was also significantly higher in subjects with HS (p<0.001); thus providing an additional screening tool. Erythrocyte membrane proteins from 21 subjects were analysed by SDS - polyacrylamide gel electrophoresis (SDS-PAGE) using the Laemmli and Fairbanks methods. The most common abnormality was a deficiency of band 3 (10 subjects), followed by a combined spectrin and ankyrin deficiency in five subjects. One subject had increased band 6 and in five cases no abnormality was detected. A decreased ratio of protein 4.1a / 4.1b on the Laemmli SDS PAGE correlated with an increased reticulocyte count. The degree of haemolysis and clinical findings did not correlate with the type of red cell membrane protein defect. In this study red cell membrane analysis did not contribute further to the initial laboratory diagnosis. In addition it did not influence clinical management. The presence of red cell membrane abnormalities, either single or multiple, did not correlate with disease severity. Red cell membrane analysis, however, will play an important role for future management such as gene therapy. Red cell membrane analysis is also useful as a research tool to determine the underlying molecular defect and to assess racial or ethnic differences. It is also of value as a differential diagnostic tool in cases where the clinical and laboratory findings are not conclusive for HS.
Evaluation of the utilisation of semi-quantitative procalcitonin versus C-reactive protein for the diagnosis of infection in a Hospital paediatric population,with particular reference to utilisation in suspected bacterial meningitis.
(2019) Ngxamngxa, Unathi.; Gounden, Verena.
Background: The high mortality and morbidity due to bacterial infections such as meningitis, alongside the long turnaround times for gold standard microscopy and culture testing, warrants alternative laboratory tests such as C-reactive protein (CRP) and procalcitonin(PCT), which demonstrate adequate test sensitivity and specificity. In addition, there is a need for more studies a in Africa, which will show the diagnostic value of CRP and PCT in bacterial infections. The aims of this study were a) to describe the utilisation of semiquantitative serum PCT in aiding with the diagnosis of bacterial infections in a South African paediatric population and b) to compare the utility of PCT in a subset of patients with suspected bacterial meningitis. Methods A retrospective observational study with charts review was done. It included data for all paediatric patients admitted to the King Edward Hospital (Durban, South Africa) for which semi quantitative PCT testing was performed for the period April 2013- April 2016. Descriptive statistical methods were employed for the analysis of data. Results The semi-quantitative PCT results correlated well with the serum CRP levels and showed significant correlation with extent of rise in CRP. A statistically significant (p=0.035) difference of median CRP levels across different categories of PCT levels, with higher values associated with PCT categories associated with greater degrees of infection. Kappa statistic analysis performed to determine agreement between positive and negative culture results with positive and negative semi quantitative PCT results, showed poor observed agreement of 53.13% (acceptable >75%). Conclusions Findings of the current study do not demonstrate added benefit of use of semi-quantitative PCT over traditional CRP. Further studies will need to be done to examine utility of the semiquantitative PCT test for early diagnosis specific bacterial infections including meningitis and pneumonia in this specific population.
A comparison of 24-hour urine versus random urine samples for determination and quantification of Bence Jones protein in a South African population.
(2019) Reddy, Ashandree.; Gounden, Verena.
Objectives: The International Myeloma Working Group (IMWG) and College of American Pathologists recommend a 24-hour collection for Bence Jones proteins(BJP). Although a 24-hour urine collection is a definitive means to determine BJP excretion, it has several issues related to sample collection and is prone to inaccuracy. Protein to creatinine ratios have demonstrated good correlation with 24-hour urine. The aim of this study was to compare measured 24-hour urine to random urine collections for the quantitation of BJP in a South African population. Method: Known patients with multiple myeloma(MM) collected 24-hour urine as part of their routine clinical assessment for BJP, random urine samples were submitted following completion of the 24-hour collection. The measured 24-hour urine BJP was then compared to 2 estimated 24-hour BJP excretions which were calculated as follows; Estimation 1 (E1): Estimated 24-hour BJP (mg/24hour) = Urine BJP/Creatinine ratio (mg/mmol) ´ 10, Estimation 2 (E2): Estimated 24- hour BJP (mg/24hour) = Urine BJP/Creatinine ratio (mg/mmol) ´ 15mg/kg for women or ´20mg/kg for men. All the 24-hour BJP results were classified according to IMWG treatment response criteria. Results: When using the Wilcoxon paired test analysis, the measured 24-hour urine BJP was significantly different to both the E1 (p=0.049) and E2 (p=0.049) equations. But analysis following categorization of each patient per IMWG BJP response criteria, indicated no significant difference in classification of treatment response using either the E1 or E2 estimation equations (P=0.69). Conclusion: 24-hour urine collections are cumbersome. Random urine BJP estimates are simple, rapid and inexpensive. This study demonstrates that both the estimates of 24-hour BJP can be used to monitor response in patients with MM. This can be added to the body of evidence that random samples can be used to monitor patients’ treatment response in MM.

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