School of Laboratory Medicine & Medical Sciences

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The development, optimisation and comparison of various virological assays and their uses in antiviral assessment of compounds wih potential anti-HIV activity.
(2009) Singh, Varish.; Parboosing, Raveen.
The development and optimization of anti-viral screening methods are essential to develop newer more effective, treatments against HIV. The XTT method is a widely described method for antiviral screening. Both continuous HIVinfected cells and experimentally infected T-cells have been used in the XTT assay. We compared these methods to screen several plant-derived extracts for cytotoxicity. Several considerations were taken into account when performing these tests (effect of media, solvents and plant enymes). Experiments were performed to investigate these effects. In addition, p24 and viral load quantification were compared as antiviral screening methods. The study showed that several modifications were necessary when performing the XTT assay on plant extracts, due to the effect of media, solvents and plant enymes. The XTT assays and p24 assays performed using experimentally infected cells are far more specific than those using chronically infected cells. The use of viral loads as an antiviral screening method consistently demonstrated the expected efficacy of AZT.
Gene expression analysis of squamous cell carcinoma of the oesophagus using a novel real time PCR probe system
(2010) Malik, Neelam.
Squamous cell carcinoma of the oesophagus (OSCC) is a common malignancy that occurs with high frequency in certain parts of the world, including South Africa. The aetiology of OSCC has remained unclear although many studies suggest that it is caused by a combination of variable risk factors. Recent reports implicate a variety of genetic factors in the carcinogenesis of OSCC but their involvement is yet to be defined.
A clinico-pathological and biochemical study of the toxicity of callilepis laureola (impila)
(1983) Bhoola, Keshavlal Daya Narotam.; Leary, W. P. P.
This study was undertaken as a result of the occurrence of a large number of deaths among the local Black population from the use of herbal medicines prepared from the rootstock of Callilepis laureola known to the Zulus as impila. The salient clinico-pathological features in these cases were hypoglycaemia, centrilobular zonal liver necrosis and acute renal tubular necrosis. The purpose of this study was to investigate fully the clinical, biochemical and pathological aspects of the toxicity produced by Callilepis laureola (impila). The first part of the investigation consisted of an assessment of all cases of death due to acute liver necrosis diagnosed by necropsy at King Edward VIII Hospital, Durban. A review of clinical and necropsy records of 21687 consecutive post-mortems performed on Black patients during a 20 year period showed that acute liver necrosis was the major contributing cause of death in 447 patients. In 263 cases the hepatic lesion was centri lobular zonal necrosis with associated acute tubular necrosis (Group A); while in 184 cases the I iver necrosis was of the massive or submassive type (Group B). A comparative assessment of these two groups as regards necropsy prevalence, age and sex distribution and the clinical, biochemical and pathological findings was undertaken. This study shows that the combination of hypoglycaemia, centri lobular zonal liver necrosis and acute renal tubular necrosis due to Callilepis laureola (impila) poisoning is a distinct clinico-pathological entity and differentiates this group from cases of acute massive and submassive liver necrosis resulting in most cases from fulminant viral hepatitis. In the search for the toxic components of the root of Callilepsis laureola several compounds were isolated. These were atractyloside, carboxyatractyloside, two thymol related oils and a carbohydrate. The thymol related oils as well as the carbohydrate were found to be non-toxic in laboratory rats. The crude methanol extract of the root of Callilepsis laureola, when injected intraperitoneally into laboratory rats, produced centrilobular zonal liver necrosis and acute renal tubular necrosis, the lesions identical to those seen in patients who had died after intake of impila prescribed by witchdoctors and other dispensers of herbal medicines. On the other hand intraperitoneal injections of the purified compound atractyloside caused acute renal tubular necrosis and hypoglycaemia in laboratory rats but failed to produce liver necrosis. Carboxyatractyloside also failed to cause liver necrosis. This indicated that there may be at least two toxins contained in the rootstock of Callilepsis laureola, one causing the liver lesion and the other (atractyloside) causing nephrotoxicity and hypoglycaemia. Repeated attempts at isolating the hepatotoxin have failed; the liver toxin or toxins being lost during the process of extraction and purification. Identification of the hepatotoxin awaits further investigation. It is possible that the liver necrosis may be caused by a metabolite or that it may be a synergistic effect of two or more compounds.
Cardiopulmonary exercise testing for high-risk South African surgical patients.
(2007) Biccard, Bruce McClure.
Aim: To determine the prognostic value of cardiopulmonary exercise testing (CPET) for major vascular surgery in South African patients. Methods: CPET has been used in Durban since October 2004 to predict cardiac risk for high-risk patients undergoing major vascular surgery. A submaximal 'anaerobic threshold' (AT) test was conducted on all high-risk patients. Patients were classified into two groups: 'low AT' where the oxygen consumption at the AT was <1 lml.kg^.min"1 for cycling or < 9ml.kg"1.mkf1 for arm cranking and 'high AT' when the patient surpassed these targets. Analysis of all in-hospital deaths following surgery was conducted by two independent assessors blinded to the CPET test result. Deaths classified as primarily 'cardiac in origin' have been used in this retrospective cohort analysis. Results: The AT measured during CPET was not a statistically significant pre-operative prognostic marker of cardiac mortality. However, the survivors of the patients with a 'low AT' may be identified by their response to increasing metabolic demand between 5 and 7 ml.kg^.min"1. Survivors were more dependent on increasing heart rate, while non-survivors were more dependent on oxygen extraction. When this information is added to the AT, CPET was the only test statistically associated with cardiac mortality, in comparison to Lee's Revised Cardiac Risk Index and the resting left ventricular ejection fraction which were not statistically associated with cardiac death. A hundred percent of patients with a positive test died of cardiac causes, while 11% of the patients with a negative test had cardiac deaths. The risk ratio associated with cardiac death following a positive test was 8.00 [95% CI 3.8-16.9]. The sensitivity was 0.25 [95% CI 0.04-0.64], the specificity was 1.00 [95% CI 0.90-1.00], the positive predictive value was 1.00 [95% CI 0.20-0.95] and the negative predictive value was 0.88 [95% CI 0.74-0.95]. Conclusions: CPET provides valuable prognostic information in our surgical population.
Oxidative stress of tissue in hypertensive rats.
(2006) Govender, Melvin M.; Nadar, Anand.
Oxidative stress, resulting from an antioxidant/free radical imbalance, is considered to be an important etiologic factor in the patho-physiological changes associated with salt sensitive hypertension. An important unresolved issue in hypertension research is the mechanism for organ damage during the development of the syndrome. Reactive oxygen species (ROS) such as the superoxide radical (02) , hydrogen peroxide (H202), and the hydroxyl radical (OH), may playa critical role in the pathogenesis of hypertension by targeting the very tissue that is responsible for regulating blood pressure, during the hypertensive state. Thus, this study was undertaken to evaluate the antioxidant and free radical status in the DSS rat strain, which has been shown to be an excellent model of salt sensitive hypertension. The antioxidant status was evaluated on the basis of the vascular superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, and the free radical status was evaluated on the basis of the plasma H20 2 concentration. The levels of malonyldialdehyde (MDA), which is a bio-marker for lipid peroxidation was used to determine the level of oxidative stress in the kidney, liver and brain. The kidney and liver were also subjected to an induced free radical mediated lipid peroxidation, by exposing the tissue to increasing known concentrations of H202 (2.5mM - 15mM). The level of lipid peroxidation was used to assess the tissues antioxidant buffering capacity to an induced free radical "attack". The results have shown that the DSS strain may have a compensatory increase in vascular SOD levels, to counter an increase in 02-. SOD levels were significantly lower during salt loading. The GPx levels were significantly lower in the DSS strain, and showed a slight increase during salt loading. The results demonstrate that the DSS strain has a compromised antioxidant status compared to the DSR strain. The plasma H202concentration displayed non-significant changes in the DSS strain, however salt loading did result in a non-significant increase in the plasma H202 concentration in the DSS strain. The GPx : HZ02 ratio, demonstrated an inadequate increase in GPx levels during salt loading to neutralise this non-significant increase in HzOz concentration. The kidney showed an increased level of in vivo lipid peroxidation, which could implicate increased tissue damage, and thus confirm the kidney as being a target organ during the hypertensive state. The liver and brain showed non-significant differences in the level of in vivo lipid peroxidation and are therefore thought not to be target tissue in the hypertensive state. The kidney displayed a decreased antioxidant buffering capacity to the induced free radical "attack", thereby demonstrating the tissue's decreased ability to neutralise an increased free radical level. Although the liver displayed a "normal" level of in vivo lipid peroxidation, it also displayed a decreased antioxidant buffering capacity to an induced free radical "attack", showing that the liver is able to cope with in vivo free radical levels, but at higher free radical levels, its loses its ability to quench a free radical "attack" and thereby minimise lipid peroxidation. The in vivo lipid peroxidation levels of the kidney, liver and brain have shown that tissues have varying abilities to cope with tissue oxidative stress, and behave differently, in their free radical quenching abilities. These results have shown that a compromised free radical and antioxidant status results in oxidative damage to the tissue responsible for regulating blood pressure.
Evaluation of a measles immunisation campaign in Natal/KwaZulu.
(1990) Abdool Karim, Salim Safurdeen.
Routinely collected data on vaccines supplied and administered, measles notifications and hospital admissions for measles were used to evaluate the 1990 measles immunisation campaign in Natal/KwaZulu. comparisons of the monthly averages during the 12 month period before the campaign, 4 months of the campaign and 12 months after the campaign indicated that the 1990 measles campaign in Natal/KwaZulu demonstrated that the campaign was limited, not by design, to blacks only. The campaign galvanised a high degree of participation from almost all health services in this region and resulted in a rapid and marked plunge in the incidence of measles as reflected by declines in both measles notifications and measles hospital admissions. There was no deleterious shortterm residual effect of the measles campaign on routine measles immunisation services. The spillover effects of the measles campaign on routine immunisation services against polio, tuberculosis and tetanus was generally beneficial. While the campaign was a success in generating involvement of health services in Natal/KwaZulu and reducing the burden of measles in this region, this disease has not been eliminated. Vigilance and continued routine vaccination efforts are required to prevent further epidemics of measles in Natal/KwaZulu.
The kind of society required for human flourishing : a critical comparison of the formation of ethical character in Aristotelian and African ethics.
(2005) Oguamanam, Eugene Ezenwa.; Roberts, D.
One thing that ethics attempts to determine is the right way to live in order to attain human flourishing. Both Aristotelian and African ethics give us communitarian accounts of how flourishing is attained by individuals who are brought up to have the right sorts of character. I argue that there are significant similarities between the accounts of the formation of ethical character in Aristotelian and African ethics. I aim to show that through a critical comparison of these two accounts, an account of the kind of society required from human flourishing can be developed. This can then be used to critique a dominant view of human flourishing: that of contemporary individualism. First I set out the Aristotelian account showing how it depends on a certain conception of the nature of persons. Second, I explore the African account of ethics and ethical character and show how this account is based on a similar communitarian conception of the nature of persons. In both Aristotelian and African ethics, society and upbringing play a crucial role in the attainment of human flourishing. Thus, third, I examine in detail the kind of society required for the formation of ethical character according to Aristotelian and African ethics respectively. I argue that there are many fruitful structural similarities between the two accounts. Lastly, I use the work done in the third chapter, as well as the work of certain prominent communitarian theorists, to critique a contemporary individualist view of human flourishing.
The Use of an enzyme-linked immunosorbent assay (Elisa) for the determination and characterization of antiendotoxin antibodies.
(1994) Badsha, Nasima.; Gaffin, Stephen L.
Recent clinical studies have highlighted the effectiveness of immunotherapy for Gram-negative bacteraemia in humans. Studies in America, undertaken on patients with Gram-negative bacteraemia, have shown that mortality was reduced by virtually 50% in patients who received specific antiendotoxin antiserum. In India, mortality from pseudomonas septicaemia was significantly reduced by the administration of small quantities of a anti-pseudomonas immunoglobulin. The antibodies in those studies were raised by vaccination of healthy volunteers with heat-killed Gram-negative bacteria or vaccines containing endotoxin. Adverse side effects in volunteers as well as logistic and legal problems make it difficult to produce antiserum on a large scale, in this manner. In Israel, S.L. Gaffin and coworkers found that approximately 7% of plasma units in a blood bank had antiendotoxin antibody concentrations of 40 ).1g/m1 or greater. This high titre human plasma significantly protected cats from lethal endotoxic shock secondary to haemorrhage. The immunoprecipitin technique used by them to measure antiendotoxin antibody concentrations was unsuitable for screening large numbers of blood samples. To overcome this problem we have devised an enzyme-linked imounosorbent assay (ELISA) for determining the level of antiendotoxin immunoglobulin G in human plasma. The assay, which is suitable for large scale use, was found to be specific for antiendotoxin antibodies. It was calibrated using a serum sample of specific antibody concentration as determined by an ilununoprecipitin assay. Serum samples found to be high in antiendotoxin titres (> 40_ug/m1) were tested for their specificity towards endotoxins from 12 bacterial iv strains and species. While each sample was found to have its own characteristic specificities, most were found to react strongly with Sh. flexneri, S. typhimurium and S. enteritidis. The Natal Blood Transfusion Service has found that in Natal, blood units containing high concentrations of specific antibodies occur with a frequency of 3,6% among all White donors and 10,35% among all African donors. They found that African females, in turn, had almost twice the frequency of high titre serum as African males. In this study, Indian female hospital patients did not have a statistically higher frequency of high-titre serum than Indian male patients. Blood units donated to the Natal Blood Transfusion Service are now routinely screened by ELISA for antiendotoxin antibodies and those units with high concentrations (> 40 ug/ml) of antibody were pooled and fractionated to obtain a gamma globulin, Lot LG-l. The binding capacity of the LG-1 antibodies towards 12 endotoxins was examined. Binding was found to be highest with endotoxin from Sh. flexneri, S. abortus equi and S. typhimurium and intermediate with S. enteritidis and E.coli 026:B6. Binding with the other endotoxins tested was relatively low. Differential absorption experiments showed that LG-1 was made up of a mixture of cross-reacting as well as specific antibodies For example, the antibodies binding Sh. flexneri endotoxin were mainly specific. Those binding E. coli 026:B6 endotoxin were specific and cross-reacting in almost equal proportions. Antibodies to the endotoxins from the salmonella strains tested were mainly cross-reacting. The specificities of the LG-1 antibodies towards endotoxins from the various Gram-negative bacteria did not in most cases reflect the incidence of these organisms in blood cultures taken from hospital patients. V The activity of LG-1 antibodies was compared to that of normal human immunoglobulin preparations obtained from the National Blood Fractionation Centre, Pinetown and to an anti-pseudomonas immunoglobulin prepared by Wellcome Laboratories, England. The binding capacity of the antibodies in the standard globulin preparations towards most of the endotoxins tested was less than 15% of that of the LG-1 antibodies. The anti-pseudomonas immunoglobulin was shown to bind poorly to most of the endotoxins tested in comparison with binding by LG-1 antibodies.
Molecular diagnosis and typing of HTLV-I in KwaZulu-Natal.
(1998) Tarin, Michelle Lucille.; York, Denis Francis.; Bhigjee, Ahmed Iqbal.
Two areas of the HTLV-I genome were targeted for an in-house molecular diagnostic test, namely the pol and env regions. The pol primers proved the most sensitive (100%)and specific (100%). Amplification using the env primer pair was not reproducible, and was not pursued further. The AmpliSensor assay (Acugen Systems, Lowell, MA) was also tested. The assay was very specific, but not as sensitive as our in-house PCR. To investigate the predominant HTLV-I subtype in the region, a 1535 by env gene was isolated from peripheral blood obtained from five local HTLV-I seropositive patients. Four of the patients presented with HAM/TSP, and the fifth presented with a skin disease. Nucleotide sequencing of the amplified products revealed the local strains to be very conserved, differing by 0.1% to 0.9% among themselves. No apparent difference was noted for the two clinical manifestations. Phylogenetic analysis was performed using repesentative strains from around the world. The local strains clearly fell within the cosmopolitan subtype. The local strains were most closely related to the North American strains suggesting an unexpected link between the two countries.
Microsatellite instability and cell cycle protein analysis in endometrial carcinoma.
(2006) Padayachy, Padmini.; Chetty, Runjun.
Mycotoxins in food with particular reference to fumonisin B1 : their health impact on a Kranskop rural community, KwaZulu Natal.
(1998) Chelule, Paul Kiprono.; Dutton, Michael Francis.; Gqaleni, Nceba.
The use of the multi-mycotoxin screen based on dialysis to analyze foods and feeds for mycotoxins, is well documented. This study investigated the possibility of incorporating FB I into the screen. Maize meal (25g) was spiked with AFB I , CPA, FB1, ST and ZEA and extraction was done using acetonitrile/4% potassium chloride (90:10 v/v). The recoveriesof the mycotoxins were 77.4, 61.5, 97.4, 79.8 and 98% respectively on analysis by HPLC. Fumonisin B1 could not be completely incorporated into the screen due to its reaction with sodium hydrogen carbonate, which is a component in the method. Thus, FB I was determined in a separate portion of the extract. The high cost of FBI standards which are often of inferior purity necessitated that FB I standards be locally produced in the laboratory using Fusarium moniliforme MRC 826, a good producer of FB 1 . In this study, production of FB I was carried out using a stirred jar fermenter and patty cultures. The yields were 160mg/1 and 6mg/g of FB I for the two methods respectively. Methyl esterification of tricaballylic acid moieties of FB I was done for effective clean-up. This was achieved by derivatizing FBI, with diazomethane. It was found that other functional groups besides the tricaballylic acid moieties of FB I were undesirably methylated as well, which made cleanup by this method difficult as shown by electrospray mass spectrometric analysis. Attempts to de-methylate FBI methyl esters with esterase was not successful. Analysis of human faecal samples was carried out with the view of developing a short term marker for assessing human exposure to FB I . Faeces from rural (20) and urban (23) volunteers were analyzed by high performance liquid chromatography. The results showed that 35% of the rural samples and 9% of the urban volunteers had detectable amounts of FB I ranging from 0.600 to 19.56 mg/kg. There was a significant difference (p = 0.04)between the two population groups. A study was carried out to assess the occurrence of FBI in a rural area of Tugela valley in Kranskop magisterial district of KwaZulu Natal. A questionnaire was administered to gather information on the family health and nutrition. Raw (stored) and processed foods and faeces, were collected for analysis of FB1. A similar control study was carried out in the urban area of Durban Metro. Homes were mapped out using the GIS for easy follow up. Oesphageal cancer (OC) incidence from the local hospital and weather data for the study area were collected from South African Weather Bureau, Johannesburg. The questionnaire results showed that the common diseases were mainly of respiratory origin (24% and 26%) from both rural and urban groups respectively. Food analysis (by HPLC) showed that the number of maize samples with FB I were higher in the rural area (31.9%) in comparison to the urban samples (6.1%). The level ranged from 0.092-22.225 mg/kg in food and 0.513-39 mg/kg in faeces. The mean concentration of FB i in the faeces and maize samples showed a similar significant difference of 0.014 between the two groups. However, these concentrations were much lower than those of high OC area in Transkei (117 mg/kg). There was no detection of FBI in fermented food products.
A hospital outbreak of multiresistant haemophilus influenzae type B.
(1996) Sattar, Kalawathie.; Hoosen, Anwar.
Following an outbreak of multi-resistant Haemophilus influenzae type b (Hib)infections in a tuberculosis hospital, this study was undertaken to determine carriage of Hib in 2 paediatric wards; to characterise all isolates of Hib, determine their antimicrobial susceptibility profile and the antibody response of the children to a conjugate vaccine. Prior to and one month after immunisation, oro- and nasopharyngeal swab specimens as well as venous blood were collected from each child. Isolates were tested for /3-lactamase and chloramphenicol acetyltransferase (CAT)production, their MIC's determined by the agar dilution method and characterisation of Hib isolates was performed by biotyping and analysis of outer membrane protein (OMP) profiles. An ELISA was also developed to determine serum antibody levels to polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hib. The study population comprised a total of 135 children who had been hospitalised for treatment for tuberculosis. The patients were aged 4 months to 14 years with a median of 37,5 months. During the study period, none of the children developed invasive Hib disease. The overall carriage rate of Hib increased from 38% (51/135) before immunisation to 62% (84/135) after immunisation (P 0,15 /ig/ml. After immunisation, 34%(45) of patients increased their antibody levels to > 1,0 /xg/ml. There was no statistical difference between the mean antibody concentrations of patients who were colonised by Hib and those who were not (p = 0,58). The vaccine did not reduce carriage of Hib in this study population of children being treated for tuberculosis and the immune response to the vaccine was not optimal. Production of /3-lactamase and the prevalence of rifampicin resistance has implications for treatment and chemoprophylaxis in this population. OMP analysis showed a diversity of types. Multi-resistant strains causing invasive disease had the same OMP type as some multiresistant strains which colonised the children.
The effects of nerve stimulation on pacemaking activities of biological tissues.
(1973) Bhagat, Chotoo Ichharam.; Reid, John Victor Oswald.
The effects on the cardiac cycle length of stimulating the vagus nerves with single supramaximal electrical shocks depended upon when they were stimulated during the cycle. A maximum prolongation of the cardiac cycle was obtained when the vagi were stimulated 167 msec (SD±64) after the peak of an electrocardiogram P wave. The interval between a P wave and the subsequent vagal stimulation was called Pl-St interval. Pl-St(max) was the Pl-St interval at which maximum prolongation of the cardiac cycle occurred. Pl-St(max) increased significantly (p (0.001) with longer cardiac cycles. When the Pl-St intervals were shorter or longer than 167 msec (SD±64) the effects of vagal stimulation were less. The latent period for the effects of vagal stimulation was 195 msec (SD±32) The latent period also increased significantly (p(O.Ol) with longer cardiac cycles. The rise time of the vagal effect, obtained by subtracting (Pl-St(max)+ latent period) from the control cardiac cycle length, was 124 msec (SD+31) and occurred between Pl-St intervals of 167 msec (SD±64) and 291 msec (SD±70). The rise time did not vary with cardiac cycle length (p) 0.1), but the magnitude of the maximum response to vagal stimulation was inversely proportional to rise time (p <. 0.02). The peak response to vagal stimulation must have occurred when the vagal effects pegan somewhere in the middle of diastolic depolarization of the pacemaker cells in the S-A node. The reasons for this were discussed. The half-decay time for the effects of vagal stimulation was 210 msec (SD±102). The slope of the curve relating the prolongation of the cardiac cycle length to Pl-St is positive at Pl-St intervals less than 167 msec (SD±64) and negative at Pl-St intervals between 167 msec (SD±64) and 291 msec (SD±90). The positive slope ranged from 0.13 to 0.48 with a mean of 0.23. The paradoxical responses of the S-A node to vagal inhibitory input obtained by Reid (1969), Levy et al (1969)and Dong and Reitz (1970) would be explained by the dependence of the cardiac cycle length upon the time of arrival of vagal stimulus in relation to the previous P wave and upon the slope of the curve relating the prolongation of the cardiac cycle length to Pl-St interval being positive and between zero and two at Pl-St intervals less than 167 msec (SD±64. The effects of single shock stimulation of the vagus nerves persisted for 3.890 sec (SD+l.255)7 the number of cardiac cycles involved varied between 5 and 11. The duration of the effects of vagal stimulation did not depend upon when during the cardiac cycle the vagi were stimulated. A "dip" in the response to vagal stimulation was present in all the experiments. The possibility of the "dip" phenomenon being due to simultaneous stimulation of the sympathetic fibres in the vago-sympathetic trunk was ruled out. It is suggested that the "dip" phenomenon may be due to transient accumulation of K+ in the interstitial fluid surrounding the pacemaker cells in the S-A node.There was no paradoxical response of the smooth muscle in the distal colon of the adult rabbit when the frequency of sympathetic inhibitory input was continuously increased. A paradoxical response in the frequency but not in the size of the contraction of the smooth muscle was obtained when the sympathetic nerves were stimulated with bursts of stimuli, each burst consisting of 5-40 impulses, 10 msec apart. One may conclude from this that the delay of the next spontaneous contraction but not the inhibition of the size of smooth muscle contraction is dependent upon the arrival time of a burst of stimuli during a contraction cycle. This was confirmed in an experiment when the sympathetic nerves were stimulated with single bursts of stimuli applied at different times during the contraction cycle. It is unlikely that such a paradoxical response would occur under physiological conditions as this would require the natural sympathetic efferent discharges to the smooth muscle to occur in regular bursts, each burst consisting of impulses at a high frequency. Stimulation of the sympathetic nerves at 3, 5, 10 and 25 PPS caused an inhibition of the size and frequency of smooth muscle contraction in the distal colon of the newborn rabbit. Assuming that the cholinergic fibres are excitatory there is therefore no evidence for the sympathetic fibres to the distal colon being cholinergic in the newborn rabbit. This is contrary to Burn's (1968) report of the sympathetic fibres being motor and cholinergic to the small intestinal smooth muscle in the newborn rabbit.The heart rate increased rapidly at the onset of exercise and then more gradually over the rest of the exercise period. The initial increase in the heart rate during exercise was not affected by adrenergic blockade but the subsequent increase in heart rate was significantly reduced by adrenergic blockade. Hence the increase in heart rate at the onset of exercise is due primarily to a decrease in the cardiac vagal efferent discharge, whereas the subsequent increase in heart rate is due to both a further decrease ln vagal discharge and an increase in sympathetic discharge to the S-A node. In almost all the sub jects there was initially a rapid decline in the heart rate in the post-exercise period, but subsequently the heart rate returned to resting levels in a variety of ways. These were classified into 5 types. Of particular interest to the present study was the Type V pattern of heart rate change. This was characterised by an increase in heart rate of 6 beats or more per minute during the post-exercise period, with or without superimposed arrhythmia. The Type V pattern may be the equivalent of the paradoxical responses to inhibitory input demonstrated in animal experiments i.e. an increase in the heart rate with increasing vagal stimulation frequency. Type V pattern occurred more frequently at mild exercise levels (4 out of 14) than at moderate exercise level (lout of 14) and also more frequently in adrenergic blocked individuals (11 out of 28) than in control subjects (5 out of 28) It is suggested that the sympathetic effects on the P-R interval and arterial baroreceptor modulation of vagal efferent discharge protect again st the occurrence of paradoxical responses to vagal inhibitory input. They may do so by confining the vagal discharge to the rise time of vagal effect during the cardiac cycle. On the other hand the Type V pattern in p-adrenergic blocked individuals may be due to a decrease in the vagal discharge, in which case Type V pattern would not be a paradoxical response. The changes in minute ventilation in the post-exercise period were also variable. Besides a gradual decline in minute ventilation there were also gradual increases and sudden increases and decreases in minute ventilation. These may represent a form of paradoxical response to increasing inhibitory input and decreasing excitatory input to the respiratory neurones in man. However, all the changes in minute ventilation could also be explained by fluctuating excitatory and inhibitory neural input to the respiratory neurones.
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.
A histopathological and immunohistochemical evaluation of scar basal cell carcinomas.
(2006) Sydney, Clive.; Ramdial, Pratistadevi K.; Madaree, Anil.
Infiltrative morphological mimicry at sites of biopsy-proven nodular basal cell carcinoma has been described. The immunoprofile of scar BCCs (scar BCCs,SBCCs) has not been documented. The aim of this study was to assess the histopathological spectrum, stromal (fibronectin, laminin, actin, desmin and vimentin) response and proliferation (bcl-2, MIB1 and p53) status of SBCCs. Twenty nine BCCs occurring in scars, unrelated to previous malignancy (de novo scar BCCS, DN-SBCCs), 27 BCCs that were incompletely excised and regrew at the same site (regrowth scar BCCs, RG-SBCCs) and 25 BCCs that were completely excised with tumour free margins, but recurred at the same site (recurrent scar BCCs, R-SBCCs) were accessed from the files of the Department of Pathology and Plastic and Reconstructive Surgery of the Faculty of Medicine, University of KwaZulu Natal, and formed the basis of this study. The morphological features of DN-SBCCs was pure (3%), predominantly nodular (79%), micronodular (7%) and infiltrative (11 %). RG-SBCCs were predominantly nodular (82%), micronodular (7%) and infiltrative (11%). RSBCCs were predominantly nodular (80%), micronodular (4%) and infiltrative (16%). The majority of DN-SBCCs, RG-SBCCs and R-SBCCs showed intact basement membrane laminin staining, while two (7%) DN-SBCCs showed 1 + and 2+ loss of basement membrane laminin staining. Three (11 %) and two (8%) RG-SBCCs and R-SBCCs,respectively, showed 2+ or 3+ basement membrane laminin discontinuity. The majority of DN-SBCCs (83%), RGSBCCs (75%) and R-SBCCs (88%) were actin negative. No desmin immunopositivity was demonstrated in the epithelial or stromal components of DN-SBCCs, RG-SBCCs and R-SBCCs. All BCC groups showed high 3+ or 4+ vimentin immunopositivity. The majority (>50%) of the SBCCs showed low (2+) bcl-2 immunopositivity. There was no significant difference in p53 immunopositivity in all SBCCs. SBCCs demonstrate phenotypic and immunophenotypic heterogeneity. That DN-SBCCs with the infiltrative and micronodular patterns have not recurred implies that the histomorphology is a pseudo-aggressive pattern. A similar view could pertain to RG-SBCCs, but because the scar did not cicatrise the incompletely excised BCC implies that the histomorphology of RG-BCC may be a potentially more aggressive phenotype. The recurrence of a completely excised basal cell carcinoma may be viewed as a feature of an aggressive tumour, especially when the recurrent BCC contains micronodular and infiltrative components. However, as most R-SBCCs occurred at head and neck sites that are exposed to ultraviolet light, it is also possible that these are simply new BCCs occurring within scars in head and neck sites prone to BCCs. Furthermore, these R-SBCCs were not destructive tumours. CONCLUSION: None of the infiltrative foci of DN-SBCCs demonstrated laminin loss. Three of 5 with intra-epithelial actin immunopositivity also demonstrated low bcl-2 and high p53 staining, immunoprofiling these with an aggressive infiltrative component. Of 11 RG-SBCCs with high p53 staining, 4 had high p53 staining in the infiltrative component, but only one had a low bcl-2 composite score and low bcl-2 score in the infiltrative focus. In addition, these infiltrative foci demonstrated intraepithelial MSA positivity and a "VA" immunophenotype of the stromal cells, indicating one RG-SBCC with an established, aggressive immunophenotype. Those positive with one or more, but not all, aggressive immunostains, are hypothesised to be RG-SBCCs evolving/developing an aggressive immunophenotype. Only one R-SBCC, with a predominantly infiltrative pattern, had a "full-house" of aggressive immunostaining in the infiltrative foci: low bcl-2, high p53, 2+ laminin discontinuity and intra-epithelial and stromal MSA positivity. Of significance is that 7 with a predominant nodular pattern had a high p53 score. Of these, 5 had high bcl-2 scores. Hence, while high p53 may be a feature of aggressive growth, it is important that this staining be complemented with that of bcl-2, laminin and MSA.
Optimisation of an analytical method for the analysis of folic acid derivatives in biological materials.
(2007) Khanyi, Purity Duduzile.
Folic acid is a water-soluble, B-complex vitamin influencing a number of biological processes in humans and particularly important in the prevention of neural tube defects (associated with spinal bifida) in unborn children. Reliable analytical methods are therefore needed for quantisation of the amount of total folic acid (FA) in biological materials of quality assurance and regulatory purposes. What is particularly needed are rapid and reliable methods for ensuring that the correct amount of FA is consumed and the degradation rates of these compound is kept at minimum during the extraction process. Analytical methods for determination of folic acid in biological materials have been around for decades and the most common procedures include microbiological assay; biomolecular interaction analysis (BIA); immunoassay; conventional chromatographic procedure such as thin-layer column chromatography (TLC) and high performance liquid chromatography (HPLC). These procedures were replaced by HPLC, which is more rapid and in many instances yields a better resolution. Current HPLC methods uses C-18 column and reverse phase conditions in combination with ion-pair or ion suppression techniques; fluorescence or electrochemical detector, unfortunately, excitation and emission of folic acid is found not sufficiently to allow physiological levels of the form of the vitamin to be detected. In addition, ion-pair reagent nullifies the mobile phase and interferes with the absorption! fluoresce spectrum resulting in poor separation. Therefore this study was carried out to address and improve the problems that are in the existing HPLC methods. Currently scarce information is available on the determination of folic acid in biological materials by HPLC with UV detection. Serum samples were spiked with folic acid standard to check the efficiency of the method. Other wavelengths from 200 nm to 300 nm were attempted for detection of folic acid, in which the wavelength 250 nm was found to have better absorbance compared to other wavelengths. Folic acid was detected at 250 nm wavelength under isocratic elution using a mobile phase consisting of citrate phosphate buffer: acetic acid: methanol. Folic acid in maize meal was detected at 290 nm using mobile phase containing potassium phosphate containing ascorbic acid/sodium ascorbate mixture and 2-mercaptoethanol under gradient elution. The mobile phase used for gradient and isocratic elution was suitable for separation of folic acid from other compounds with flow rate of 3 ml/min modified to Iml/mim to avoid overloading of the column under isocratic elution. For good separation of folic acid under gradient elution the flow rate was set at 0.8ml/min with pH of mobile phase modified from pH 2.2 to pH 2.5. The recovery of folic acid added to human serum was 91% -100% and recovery of folic acid added in unfermented maize meal and fermented maize meal ranged from 55% - 73%. Folic acid level from unfermented maize meal and fermented maize meal ranged between 1.29 - 1.3 [!g/g and 1 - 2.1 [!g/g respectively. In conclusion the optimised method in this study gives better analytical results when compared with earlier HPLC method in terms of efficiency, reproducibility and sensitivity for folic acid in human serum and maize meal. However, there is a need to minimise the loss of folic acid during the sample treatment. The outcome of this work indicated that more work has to be done to improve extraction procedure for specific foods with minimum time preparation to sample analysis.
A microsatellite evaluation of the genetic status of the p27Kip1 and p21Cip1/WAF1 genes in oesophageal cancer.
(2008) Gaffoor, Zakir.; Naidoo, Richard.
p21 C/P 1/"El and p 2 7K/P 1 are cyclin-dependant kinase inhibitors that fonn an integral part of the cell cycle process. These proteins function as cell-cycle inhibitors, and are able to induce cell cycle arrest by binding to cyclin complexes at key stages. p21 and p27 have been found to be down-regulated in various cancers. This study investigated aberrations at microsatellite markers linked to the p21 and p27 cell cycle genes, in a large cohort of oesophageal squamous cell carcinomas in South Africa. Fluorescent-based PCR were performed on markers linked to both the p21 and p27. The products were run with a 50-500hp marker on 6% denaturing polyacrylamide gels, on the ALFexpresstm' DNA sequencer. The detection and analysis of PCR products was achieved using the AL F e xp res sT M and Fragment M an a aerTm software programmes. Our findings indicate that markers linked to p27 display infrequent aberrations, with loss of heterozygosity ranging from 19% to 37%, and microsatellite instability at 3% to 7%. However, significant relationships between decreased survival time, and aberrations in markers DI2S391 and Dl2S364, were found to exist. Marker D6S1575 linked to p21 displayed frequent allelic loss at 47%, and was comparable to similar studies on the 6p region Further, LOH-Al in this marker was found to be significantly associated with poorly differentiated tumours. The findings from our study indicate that microsatellite aberrations occur infrequently at the p21 and p27 loci in oesophageal cancer. with the exception of marker D6S1575. In addition,this study clearly demonstrates the accuracy and sensitivity of the technology employed. This is the first microsatellite-based investigation of the p21/p27 gene loci in oesophageal cancer in South Africa, using a fluorescent-based PCR assay.
A comparative assessment of local, commercial and homemade amahewu with respect to nutritional value, hygiene, and other health benefits to the community.
(2003) Mbongwa, Hlengiwe Prosperity.; Gqaleni, Nceba.
Fermentation is a process by which primary food products are modified biochemically by the action of microorganisms and/or their enzymes. Several societies have, over the years, intentionally carried it out to enhance the taste, aroma, shelf-life, texture, nutritional value and other properties of food. It is used in many parts (lithe world. However, there are regional differences in use and these depend on the availability of raw materials, consumption habits. and other socio-cultural factors. This study was aimed at (comparatively) assessing, local commercial and homemade amahewu with respect to nutritional value, hygiene and other health benefits to the commirn ity. Methods employed were Thin Layer Chromatography (TLC) (mycotoxins), High Perliffmance Liquid Chromatography (HPLC) (mycotoxins, sugars and amino acids), Dumas (proteins), SOxhlet (lipids) and intubation technique (metabolisable energy) to analyse maize meal and amahewu samples from various regions. The regions sampled included mal3heleni (South Coast) and kwaNgcolosi (North Coast) villages. Commercial amahewu was analysed with kind permission from Clover SA. Species from the following genera were isolated and identified from amahewu samples: Lactobacillus, Saccharonivccs, Lcuconostoc, Lactococcus, Panioca, Entcrobacter and kleb•iella. Saccharotnyces was detected in commercial samples only. Gram-negative strains were identified in most of manheleni village samples. No traceable amounts of aflatoxin BI (AFB1), fumonisin B 1 (FBI) and zearalenone (ZEA) were found in Clover SA samples. AFB I was detected in 40% of both maize meal and amahewu samples from maBheleni (range 0.55 — 0.84ng/g and 8.3x10 5 — 9.1x10-5ng/g respectively). From the same village, 100% of the maize meal and 80% of the amahewu samples were contaminated with FBI (range 4.1 47.2ng/g and 1.4 ---- 6.9ng/g respectively). ZEA was detected in all maize meal samples (range 0.9 — 4.3ng/g). None of the amahewu samples contained detectable levels of ZEA. All maize meal and amahewu samples from kwaNgcolosi were contaminated with AF13 1 (range 8.3 — 30.I ng/g and 0.04 - 0.102ng/g respectively). FB I was detected in 75% of both maize meal and amahewu samples from the same village (range 0.5 — 4.1ng/g and 0.04 0.56ng/g respectively). ZEA was also found in all maize meal samples and 75% of amahewu samples (range 3.7 — 16.4ng/g and 0.03 -- 0.06ng/g respectively). MaBheleni, Clover SA and kwaNgcolosi maize meal and amahewu samples contained vitamins B1, 13 2 and B6 with a range of 0.31+0.21 - 4.48±0.81 B 1 ; 0.15±0.14 - 1.67±0.33 B2 and 0.05±0.07 - 0.77±1.45 lig/g B6. Fat levels ranged from 0.28±0.40 to 4.54±0.05 percentage by weight. The levels of proteins varied from 4.02±0.02 to 8.40±0.04 percentage by weight. Starch concentrations ranged from 31.51.5.28 to 75.911.92g/100g. Maize meal samples contained glucose and maltose, while glucose, fructose, sucrose, maltose, M-triose, DP 4 and 5 and DP >15 were detected in amahewu. Apparent and true metabolisable energy for homemade and commercial Freeze-dried amahewu was 13.194 and 13.696MJ/kg (AME N ); and 13.605 and 14.106M.Ekv ( 1 MEN ), respectively. This study has shown that lactic acid maize fermentation reduce' the levels of AF13 1 , FB I and ZEA toxins in maize meal, inhibits the growth of most Gram-negative bacteria, and in some instances, fermentation did improve the nutritional value. Metabolisable energy analysis represents an important tool to assess whether or not compounds ingested are converted to sources of energy in the body and utilised. Amahewu fermentation yielded beneficial products (probiotics: reduced mycotoxins levels and reduced starch). In conclusion, natural lactic acid maize fermentation to produce amahewu will do more good than harm to the consumer, therefore, people need to be advised on how to safely store their maize and also to be encouraged to consume their stored maize in fermented form.
The fate of mycotoxins in non-alcoholic lactic acid maize meal fermentation.
(2003) Mokoena, Mduduzi Paulos.; Gqaleni, Nceba.
This study was aimed at investigating the potential of lactic acid fermentation in reducing myco toxin concentration in maize meal products. Maize meal was spiked separately with aflatoxin Bi, fumonism Bi, and zearalenone, and fermented for four days. During this period the concentration of each toxin and the pH of the fermented maize meal were monitored. There was a significant (p= 0.000) decrease in the concentration of all the mycotoxins, with a percentage reduction of 55-69 by the third day and 68-75 by the fourth day, respectively. Commercial amahewu samples were also screened for the presence of these three mycotoxins, and the results indicated that the samples were not contaminated with detectable levels of these toxins. An attempt was made to characterise the metabolic derivatives (by-products) of each mycotoxin following lactic acid maize meal fermentation. To achieve this maize meal samples were separately spiked with each of mycotoxin, fermented for four days and screened for specific mycotoxin derivatives (by-products) using GC/MS, HPLC and relevant standards (i.e. partially hydrolysed fumonisin Bi, aflatoxin B2a, a- and Pzearalenol). None of the targeted derivatives could be detected in the fermented maize meal samples. The potential cytotoxicity of the mycotoxin-spiked fermented samples was investigated using an SNO cell line. The fermented toxin-spiked maize meal samples with a starter culture were comparatively less toxic (29 - 36%) to SNO oesophageal cells than samples spiked with toxin without a starter culture (24 - 30%). However, this observed difference was not statistically significant (p = 0.295 - 0.681). Furthermore, cells that were only inoculated with the cell culture medium had significantly (p = 0.000) high percentage cell viability. This study indicates that it is possible to significantly reduce the concentration of mycotoxins using lactic acid maize fermentation to trace levels. However, such a reduction will not significantly alter the possible chronic toxic effects of such toxins in the diet, particularly a maize based diet containing poor quality protein. The trace amounts of these toxins in fermented and unfermented maize meal should continue to be a cause for concern.
Non-insulin-dependent diabetes in young Indians : a clinical and biochemical study.
(1982) Jialal, Ishwarlal.
One of the earliest recorded references to polyuria is found in the Papyrus Ebers (1500 BC) and much later the occurrence of "honey urine" was noted by an ancient Hindu physician, Sushrutha, in old Indian Sanskrit (400 BC). However, the first good clinical description of the disease is ascribed to Celsus, although the name "diabetes" was introduced by Aretaeus of Cappadocia. The body of knowledge which has accumulated since these early recordings to the present state of the art reflects a most impressive sojourn, punctuated by many milestones, each adding impetus to future attempts in a relentless endeavour to unravel the aetiopathogenesis of this common malady. However, this "sweet evil" (diabetes) remains an enigma in many ways. There is little doubt today that there are 2 major types of diabetes: juvenile onset diabetes, presently known as insulin-dependent diabetes mellitus (IDDM) and maturity onset diabetes, referred to as non-insulin dependent diabetes mellitus (NIDDM). In NIDDM aggregation of HLA types, evidence of cell mediated immunity and the presence of circulating islet cell antibodies, which are characteristically associated with IDDM, are not found. There is also a vast difference in concordance of diabetes in the co-twins between the two types of diabetes suggesting that a different mixture of genetic and environmental factors is operative in the pathogenesis of these two types of diabetes. In I960, Fajans and Conn drew attention to the existence of a form of diabetes with an onset before the age of 35 years. Their patients showed a substantial improvement in glucose tolerance when treated with an oral hypoglycaemic agent, tolbutamide. Subsequent to this report numerous studies from various parts of the world confirmed this entity of non-insulin dependent diabetes in the young (NIDDY) in White Caucasians. There are, however, several different syndromes presenting as mild carbohydrate intolerance in the first two to three decades of life. The classical form of NIDDY is a mild non-insulin requiring form of diabetes in which the disorder is inherited as a dominant trait; there is little progression of glucose intolerance, if any, with time, and the diabetes is rarely accompanied by vascular complications. This subtype of diabetes is referred to as MODY (maturity onset diabetes in the young) and thus constitutes a subset under the broad umbrella of NIDDY. However, recently compelling evidence for heterogeneity within MODY has been presented. This evidence is based on the prevalence of certain HLA antigens, insulin responses to oral glucose, occurrence of vascular complications, progression of hyperglycaemia to the stage of insulin requirement and failure to demonstrate autosomal dominant inheritance in some families studied. In the South African Indian population which has a high prevalence of diabetes, Campbell was the first to draw attention to NIDDY in Indians more than two decades ago. Since this initial report, nobody has really studied NIDDY in any depth in South Africa and certainly not in the Indian population. NIDDY in the local Indian population is of particular interest for the obvious reason that diagnostic and management problems arise daily in a population with a high prevalence of non-insulin dependent diabetes. It is vital that the clinical features, endocrine and associated biochemical aberrations be known in detail if this condition is to be managed appropriately and adequately. A study of these aspects therefore became the primary task of this thesis. To pre-empt any challenge that patients were not really diabetic, the strict criteria of the W.H.O. for the diagnosis of diabetes were chosen. It should therefore be borne in mind throughout this study that a group of rather severe diabetics were selected by design. The patients studied represent the rather extreme end of the spectrum. But, in the event, this selection proved advantageous in that it covered an unstudied part of the spectrum and some light could be shed on the natural history of the disorder. In the long term the purpose was to prepare the ground for what must become the thrust of future studies, namely the biochemical pathogenesis of NIDDM. If it is true that some forms of NIDDY are inherited dominantly, existing techniques should make it possible to identify a gene(s) locus and if this is done the biochemical basis of this disorder must be identifiable. In the present study direct examination of these aspects were not undertaken, but an attempt was certainly made to pinpoint those biochemical abnormalities which are perhaps primary or central to the whole disorder.

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