Made available in DSpace on 2015-12-10T14:22:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-26. Added 1 bitstream(s) on 2015-12-10T14:28:27Z : No. of bitstreams: 1 000849323.pdf: 2210676 bytes, checksum: 9a2b0916dc4afbd9f41047f5af8cb332 (MD5) / A escolha do regime antimicrobiano para o tratamento das peritonites em Diálise Peritoneal (DP) é fundamental para uma evolução clinica favorável. Não há consenso sobre a melhor terapia; poucos estudos prospectivos controlados foram publicados e a única revisão sistemática com meta-análise disponível não mostrou superioridade de alguma classe ou associação antimicrobiana. O objetivo desta revisão foi analisar os resultados do tratamento das peritonites em adultos em DP empregando duas metodologias, revisão sistemática com meta-análise de estudos clínicos randomizados (ECR) e a meta-análise proporcional de série de casos, uma nova estratégia que tem sido utilizada em outras condições clinicas. Método: Foi realizada uma revisão de literatura, sem restrição de idioma. Os estudos foram obtidos das bases CENTRAL, MEDLINE, EMBASE e LILACS. Os critérios de inclusão foram: (1) ECR e série de casos com número de pacientes em cada estudo maior que cinco (2) uso de algum antibiótico, ou esquema antibiótico para tratamento inicial de peritonite em DP (ex: cefazolina/gentamicina, vancomicina/gentamicina); para germes gram-positivos (ex:vancomicina/cefalosporina da primeira geração) e para gram-negativos (ex: gentamicina, ceftazidima e fluoquinonolonas) (3) estudos relatando a taxa de resolução. Para os ECR foram considerados os desfechos cura, resposta primária, remoção de cateter e recidiva epara metanálise proporcional de serie de casos foi considerado apenas o desfecho percentagem de cura. Resultados:Um total de 65 estudos (22 ECR e 43 série de casos) preencheram os critérios de inclusão. Nos RCT não foi observada superioridade de nenhuma classe ou associação antimicrobianautilizada para tratamento empírico ou para episódios causados por germes gram-positivos ou negativos. Entre as serie de casos, a associação ceftazidina/glicopeptídeo (proporção total= 87% [IC 95% = 0,83-0,91] ) apresentou... / The choice of antimicrobials for treatment of peritoneal dialysis (PD)-related peritonitis is crucial for a favorable outcome. There is no consensus about the best therapy; few prospective controlled studies have been published, and the only published systematic reviews did not report superiority of any class of antimicrobials. The objective of this review was to analyze the results of PD peritonitis treatment in adult patients by employing two methodologies, the systematic review with meta-analysis of randomized clinical trials (RCT) and the proportional meta-analysis of cases series, a new strategy that has been used in other clinical settings. Methods: A review of the literature was conducted. There was no language restriction. Studies were obtained from MEDLINE, EMBASE, and LILACS. The inclusion criteria were: (1) case series and RCTs with the number of reported patients in each study greater than five, (2) use of any antibiotic therapy for initial treatment of PD-related peritonitis (e.g., cefazolin plus gentamicin, vancomycin plus gentamicin), for gram-positive (e.g., vancomycin, first generation cephalosporin), or for gram-negative rods (e.g., gentamicin, ceftazidime, fluoroquinolone), and (3) studies specifying the rates of resolution. For RCT analyses the outcomes resolution, primary response, catheter removal, and relapse were considered, whilefor proportional meta-analysis only the outcome resolution rate was taken using a random-effects model, and the pooled resolution rates were calculated. Results: 64 studies (21 RCT and 43 case series) met all inclusion criteria. There were 21 comparisons between the antibiotics using in RCT. No superior antimicrobial class or associations were identified, when used for empirical treatment or for gram-positive or gram-negative episodes. Among the case series the pooled resolution rate of ceftazidime plus glycopeptide as initial treatment (pooled propor = ...

D.Tech. (Biomedical technology) / The majority of the South African rural black population remain is exposed to HIV/ AIDS and other chronic diseases, tuberculosis, malaria and cancer. The effect of single and combined mycotoxins on their health and particularly their immune system is unknown and remain of concern as these populations are on daily basis exposed more than one mycotoxin at once. The aim of this study was to evaluate the exposure of South African rural black populations to mycotoxins via animal products in comparison to urban populations and to assess the effect of the major mycotoxins (fumonisin B1 (FB1), aflatoxin B1 (AFB1) and ochratoxin A (OTA)) mostly present in their food on human and animals (pigs) mononuclear cells and by extrapolation, evaluate possibilities of these mycotoxins on the immune system. To achieve this, animal feed and animal products (milk, serum, and tissues) obtained from selected rural and commercial farms in selected areas of South Africa were analysed for fungal and mycotoxins contamination. It was found in this study that almost all of the samples from both areas were contaminated with the major mycotoxin producing fungal strains (Fusarium, Aspergillus and Penicillium spp.) with the most prominent among them being Aspergillus flavus (87%), A. parasiticus (43%), A. niger (69%), A. ochraceus (42%), A. candidus (23%), F. verticillioides (98%) F. graminearum (67%) and P. Verrucosum (48.9%) and in commercial samples A. flavus (98%), A. parasiticus (51%), A. ochraceus (65%), A. niger (31%), A. candidus (21%), F. verticillioides (F. moniliforme) (68%), F. graminearum (43%) and P. verrucosum (7%). While, the three main mycotoxins were also present and contaminated most samples with fumonisins (FBs) 0in rural and commercial samples at 90.6% and 93.3% respectively with respective means values of 10136.4 ppb and 1045.4 ppb. Aflatoxins (AFs) contamination was of 92.0% in rural samples and 96.2% in commercial samples with means concentrations of 168.8 ppb and 294.1 ppb respectively. While 85.4% and 83.7% of rural and commercial samples respectively were contaminated with ochratoxin A (OTA), with mean concentrations of 67.6 ppb and 89.4 ppb respectively. Zearalenone (ZEA) concentrations were of 43.6 ppb in rural samples and 62.7 ppb in commercial samples with respective contamination of 50.6% and 55.3%. In addition, a co-occurrence of fungi and mycotoxins contaminations was found in both rural and commercial samples. It was found that, 50.5% of rural and 53% of commercial samples were contaminated with all four analyzed mycotoxins. (FBs, AFs, OTA and ZEA), whereas, 81.2% and 79.5% of samples respectively from rural and commercial farms were contaminated with FBs, AFs and OTA mycotoxins simultaneously. The above-obtained results are of significance in this study as they confirm the hypothesis of fungal contamination and mycotoxin co-occurrence in South African feed and their possible combined effects on consumers.

No description available.

Pox viruses have been demonstrated in over 60 types of wild and exotic birds as well as domestic birds. Avipox viruses have been isolated and characterised from fowls, quails, canaries, parrots and lovebirds. This work describes the first isolation of a poxvirus from Jackass penguins (Spheniscus dermersus) and the characterisation of the virus as a separate species of penguinpox virus.

Bacteria and fungi have acquired the ability to interact and survive in many hostile environments both found in nature, as well as, the human body. Candida albicans, an opportunistic fungal pathogen, causes a variety of infections in immunocompromised or immunosuppressed individuals, but also asymptomatically colonizes 80% of the population within the intestinal tract, oral cavity, as well as, the female genitourinary system. A unique capability of C. albicans is the ability to change its morphology from benign circular yeast form, to oval pseudohyphal form, to cylindrical tissue-penetrating hyphal form. Our laboratory has previously identified a bacterium, Alcaligenes faecalis, which displays inhibitory characteristics towards C. albicans. A review of the literature shows that some bacteria have the ability to inhibit C. albicans, but only when in the hyphal form. We therefore wanted to explore if the morphological state of C. albicans dictated the degree of inhibition A. faecalis is able to exude, or simply “does morphology matter?” To determine this effect, a series of both solid and liquid media experiments were performed using a wild-type (able to convert between morphologies) strain of C. albicans, a mutant strain of C. albicans locked into the yeast morphological state, and a mutant strain of C. ablicans locked into the hyphal morphological state. For solid media experiments, the different strains of C. albicans were made into a lawn on agar plates, A. faecalis was spotted onto the lawns, and, after 24 hours, observed for signs of inhibition. For liquid media experiments, C. albicans strains were inoculated alone or co-cultured with A. faecalis for 24 hours and plated to enumerate colony forming units. Our results indicate that: (1) the morphological state of C. albicans is not a determining factor, which is a unique finding compared to other published reports; (2) Both A. faecalis and the closely related A. viscolactis both inhibit C. albicans showing that this is a shared ability among the Alcaligenes genus. (3) that the ability to inhibit C. albicans is thru some form of contact dependent mechanism, as the cell free supernatant of A. faecalis has no inhibitory action. Currently, the exact mechanism for this interaction is unknown, but could be one of the secretion systems bacteria use for interactions with other microbes. As there are very limited treatments for fungal infections and severe side-effects associated with current antifungals, exploiting these mechanisms are medically relevant to human health as they could potentially lead to novel treatments for problematic human fungal pathogens.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2005. / The aim of this study was to investigate the anti-inflammatory and immunosuppressive effects of the synthetic progestins, MPA and NET-A on human cells in vitro. These injectable contraceptives are used extensively throughout the world, including Africa. The potential of these two synthetic hormones to have certain immunosuppressive and GC properties have previously been shown. Therefore, it was of concern to us to investigate whether these two hormones could possibly demonstrate any of these GC-like properties at contraceptive doses. This was achieved by determining the effects of these two synthetic hormones in vitro on certain immunologic parameters. Chapter 1 is a literature review on MPA, NET and GCs. This chapter starts with a short introduction that sets the scene. The mode of action, effectiveness, sideeffects as well as previously reported relevant data on both MPA and NET-A is portrayed in this review. Research on the known GC, Dex, is also included in the section dealing with GCs, because this synthetic hormone was used as a comparative GC in all our experiments. This chapter soon makes the reader realize how much evidence exists that indicate the possible immunosuppressive effects these two contraceptive hormones, in particular MPA, could have. The possible anti-inflammatory or pro-inflammatory effects of MPA and NET-A are investigated in Chapter 2. This was done in vitro by measuring the effects of these two synthetic hormones on the inflammatory markers, IL-6 and TNFα, by means of ELISA. In this chapter we demonstrate that MPA, even at contraceptive doses, exhibits significant anti-inflammatory properties on both cytokines tested, while NETA displayed considerably less anti-inflammatory tendencies. In its true antiinflammatory manner, we found that Dex significantly inhibited the release of both inflammatory markers from human monocytes. In Chapter 3, we investigated the effects of MPA and NET-A on the activation of human lymphocytes. This was achieved by flow cytometric measurement of the expression of the activation membrane marker CD69 by CD4 and CD8 T cells. Here we discovered that MPA had a very significant inhibitory effect on the activation of both CD4+ and CD8+ T cells, while NET-A only significantly inhibited the activation of CD8+ T cells. In addition, we found that the inhibition of CD4+ and CD8+ T cell activation by MPA was more or less the same as the known GC, Dex, and in some cases even more potent. Chapter 4 consists of an investigation of the effects of MPA and NET-A on the cytokines belonging to TH1 and TH2 subsets of CD4 T cells. This was achieved by determining whether MPA and/or NET-A targeted specific subsets of T helper cells by measuring the distinct regulatory cytokines, IFNγ and IL-4. The mechanism and role of the T helper subsets are discussed in the introduction of this chapter. Our results were portrayed as a ratio of TH2: TH1 on which the statistical analysis was done. In addition to the analysis done on the ratio, we analyzed the helper subsets separately in order to determine which subset(s) were influenced. The results of this chapter showed that neither MPA nor NET-A significantly affected either one of the helper subsets, while Dex significantly decreased this ratio. After our observed effects of MPA and NET-A on CD8 T cells, it became of interest in Chapter 5 to investigate the effects of these two synthetic hormones on the CD8 T cell-specific chemokine, RANTES. This was achieved by measuring the effects MPA and NET-A had on RANTES production in vitro by means of ELISA. Surprisingly, we discovered in this chapter that MPA and NET-A enhanced RANTES production before and after activation of CD8 T cells. We also found that Dex had the same effect on RANTES production, but to a lesser degree. Finally, a general conclusion depicting the significance and implications of our results as well as possible future research that is required is presented in Chapter 6. It was of great importance to discuss and interpret the magnitude of data generated out of all our experiments to the utmost of our capabilities. We found that MPA, even at contraceptive doses, displayed significant immunosuppressive as well as anti-inflammatory properties. NET-A, on the other hand, demonstrated weaker immunosuppressive properties in our research and no significant anti-inflammatory properties. These findings could have clinical implications in females being treated with these synthetic contraceptives. We also demonstrated significant variation found amongst genders in response to MPA, NET-A and Dex.

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2005. / There has recently been a significant transformation in the medical world, in particular regarding the relation between the mind/health and mind/illnesses. The changes are briefly a revolution whereby the new approach sees the development of an illness as an interaction between the psychological, biochemical and physiological factors. Music, which is used as a clinical intervention, is perceived first through the brain, affirms this interaction between the body systems, as well as having the capacity to modify the mind and thus the biochemistry of the body. The aim of this study was essentially to supply empirical data by measuring selective parameters while the patients were receiving music intervention during the physiotherapeutic treatment for pneumonia and bronchitis. Forty adult patients who were divided into an experimental and control group, according to a random scale, participated in the research. The dependant variables that had shown significant changes amongst the experimental group after three days of physiotherapeutic treatment were as follows: the cortisol, the cortisol: DHEA ratio plasma levels, the POMS scale (that measures different moods), the peak flow measurements of the lung functions and the immune parameters, namely, CD4+ : CD8+ ratio and B-cells. The results showed that the experimental group that was exposed to the acoustic stimuli of the Magnificat in D, BWV 243 of JS Bach, experienced a more positive mood and lower cortisol levels, while the immune markers as well as the peak flow of the lungs had improved. The results of the control group showed significant implications, in that its cortisol levels increased and the POMS subscale of anger and depression showed no significant change, while the tension decreased significantly. This research provided sufficient scientific evidence to confirm the concept of a bidirectional communication between the brain and the immune system. It also showed clearly that music had the capacity to modify emotional conditions, which again influenced the endocrine and autonomic nervous system and modulated the immune systems.

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Due to modern high-throughput technologies, large numbers of compounds are produced by parallel synthesis and combinatorial chemistry. The pharmaceutical industry therefore requires rapid and accurate methods to screen new drugs leads for membrane permeability potential in the early stages of drug discovery. Around 50 % of all investigational new drugs fail in pre-clinical and clinical phases of development due to inadequate absorption/permeation, distribution, metabolism, excretion and/or unacceptable toxicity. This may be decreased by applying in vitro screening methods early in the discovery process. Reliable in vitro models can be applied to determine permeation of the test compounds, which will help avoid the wasting of valuable resources for the development of drugs that are destined to fail in preclinical and clinical phases due to insufficient permeability properties. It is important to decide as early as possible on the most promising compound and physical formulation for the intended route of administration. With awareness of the increasing importance of in vitro models in the investigations of the permeability properties of drug compounds, this research project was specifically devoted to determine the suitability of our in vitro model to evaluate and predict drug permeability. A continuous flow-through diffusion system was employed to evaluate the permeability of nine different compounds/drugs with different chemical properties, across three biological membranes. The biological membranes chosen for the present study were human vaginal mucosa, human skin tissue and human small intestine mucosa. The continuous flow-through diffusion system was furthermore utilised to investigate the effects of de-epithelialisation of mucosal surfaces, chemical enhancers, temperature, permeant concentration and formulation on the permeability of the test compounds/drugs. The in vitro permeability information and data from the flow-through diffusion model were compared to in vitro and in vivo literature studies and drug profile. An in vitro model that is able to reliably predict in vivo data will shorten the drug development period, economise resources and may potentially lead to improved product quality. In this thesis research results are reported on the permeability of the mentioned biological membranes to the various chemical markers, including anti-HIV (human immunodeficiency virus) drugs. The permeability studies will be discussed in three sections: vaginal mucosa, skin tissue, small intestine mucosa. The results of the vaginal permeability studies showed that the three peptides (MEA- 5, MDY-19 and PCI) readily penetrated the vaginal mucosa. MDY-19 had a higher flux rate than MEA-5, commensurate with its smaller molecular size (weight). The surfactant enhanced the flux rate of MDY-19 approximately 1.3 times and decreased the lag time of the peptide. Removal of the vaginal epithelium increased the flux rates of the peptides across the mucosa and may have implications for a more rapid uptake of these and other microbicides in vivo. The permeability of 1 mM MDY-19 and PCI at 37 °C were significantly (p<0.05) higher than at 20 °C. At 37 °C the AUCs of the overall mean flux values of MDY-19 and PCI increased with concentration according to well-established diffusion theory. The experiments on the permeability of different terbinafine hydrochloride formulations through human skin demonstrated that the terbinafine hydrochloride formulations used in this study, readily diffused into the skin tissue. However, no flux values for any of the terbinafine hydrochloride formulations through the skin into the acceptor fluid were found. The mean terbinafine concentrations in the skin after 24 h exposure to the three commercial, terbinafine hydrochloride formulations were 3.589, 1.590 and 4.219 μg/ml respectively. The mean terbinafine concentration in the skin exposed to the 10 mg/ml PBS/Methanol solution was higher than those from the three commercial formulations. The results of the temperature study demonstrated that an increase of 5 ºC caused a significant increase in flux values of tritiated water across skin. The flux values for tritiated water across skin at 37 ºC were on average double those at a temperature of 32 ºC. The permeability of excised human small intestine mucosa to different oral dosage drugs was investigated over a 24 h period. The four drugs selected were zidovudine, propranolol hydrochloride, didanosine and enalapril maleate. They were selected as representative model compounds of drug classes 1 (high solubility, high permeability) and 3 (high solubility, low permeability) according to the Biopharmaceutics Classification System. The flux rates of the four chosen test drugs were influenced by the length of the experiment. Between the time periods 2-4 h and 4-6 h, zidovudine’s mean flux values across small intestine tissue were respectively 1.8 and 2.0 times higher than didanosine and 2.3 and 2.2 times higher than enalapril. Propranolol’s mean flux values were respectively 1.2 and 1.4 times higher than didanosine and 1.6 higher than enalapril during both the 2-4 and 4-6 h time periods. Between both the time periods 2-4 and 4-6 h AZT’s mean flux values were 1.4 times higher than propranolol and didanosine’s mean flux values were respectively 1.3 and 1.1 times higher than enalapril during the mentioned time periods. Class 1 drugs showed a significantly higher flux rate across the jejunal mucosa compared to the class 3 drugs and these results are in line with their Biopharmaceutics Classification System classification. The in vitro model has proved to be reliable to predict permeability of class 1 and 3 drugs and also showed correlation with human in vivo data. It seems that the in vitro flow-through diffusion model used in the present study have the potential to overcome some of the problems and limitations demonstrated by other in vitro techniques and may potentially serve as a future tool for pharmaceutical companies to predict the diffusion characteristics of new drugs and different formulations, across different biological membranes. Furthermore, it may serve as a prospective method for assessing the bioequivalence of alternative (generic) vehicles or formulations containing the same drug/compound. / AFRIKAANSE OPSOMMING: As gevolg van moderne hoë spoed tegnologie kan groot hoeveelhede middels vervaardig word deur ooreenkomende sintese en kombinasieleer chemie. Die farmaseutiese industrie benodig dus vinnige en akkurate metodes om nuwe geneesmiddels te evalueer t.o.v. membraan deurlaatbaarheid. Hierdie evaluasie moet verkieslik so vroeg moontlik in die geneesmiddel se ontwikkelingsproses geskied. Ongeveer 50 % van alle potensiële geneesmiddels misluk in pre-kliniese en kliniese fases van geneesmiddelontwikkeling. Die mislukte pogings kan toegskryf word aan onvoldoende absorbsie/deurlaatbaarheid, distribusie, metabolisme, ekskresie en/of onaanvaarbare middel toksisiteit. Dit is daarom belangrik om so vroeg moontlik in die geneesmiddelontwikkelingsproses te besluit op die mees belowende middel, asook die geskikte formulasie vir die spesifieke roete van toediening van die middel. Die farmaseutiese industrie benodig tans in vitro modelle met die potensiaal om die deurlaatbaarheid van geneesmiddels te bepaal en te voorspel. Betroubare in vitro modelle kan aangewend word om die deurlaatbaarheid van potensiële geneesmiddels te toets. Sodoende sal die onnodige uitgawes op die ontwikkkeling van geneesmiddels wat in elk geval later gaan faal in pre-kliniese en kliniese fases van geneesmiddelproewe a.g.v. deurlaatbaarheidseienskappe, vermy word. Hierdie navorsingsprojek was dus spesifiek onderneem om die waarde en toepaslikheid van ‘n in vitro deurlopende-vloei perfusie model te ondersoek. Die model se potensiaal om geneesmiddels se deurlaatbaarheid en absorpsie te voorspel was geëvalueer. Die deurlopende-vloei perfusie apparaat was gebruik om die deurlaatbaarheidsvloede van drie verskillende biologiese membrane t.o.v. nege chemiese stowwe (MEA-5, MDY-19, PCI, terbinafien hidrochloried, getritieerde water, zidovudien, propranolol, hidrochloried, didanosien, enalapril maleaat) te bepaal. Die drie biologiese membrane wat gebruik was, was vaginale weefsel, vel en klein intestinale weefsel. Al drie weefsel tipes was van menslike oorsprong. Die deurlopende-vloei perfusie apparaat was ook gebruik om die effek wat verwydering van die mukosa se epiteellaag op deurlaatbaarheidsvloede het, te ondersoek. Verder was navorsing gedoen op die effek van temperatuur en die konsentrasie en formulasie van die toetsmiddels op hulle diffusie vloedwaardes. Daar was ook gekyk na die invloed van ander chemiese stowwe op die toetsmiddels se diffusie vloedwaardes. Die in vitro deurlaatbaarheidsinformasie en -gegewens was vergelyk met ander in vitro en in vivo literatuurstudies en geneesmiddel databasisse. ‘n In vitro model wat in staat is om in vivo resultate betroubaar te voorspel, het die potensiaal om die tyd wat dit neem om geneesmiddels te ontwikkel, te verkort, finansiële uitgawes te besnoei en om geneesmiddelkwaliteit te verseker. In die tesis word dan die resultate gerapporteer van die deurlaatbaarheidsvloede van die verskillende tipes weefsel ten op sigte van verskeie chemiese stowwe, insluitende anti-MIV (menslike immuniteitsgebreksvirus) middels. Die deurlaatbaarheidstudies word bespreek in drie afdelings: vaginale mukosa, vel en klein intestinale mukosa. Die resultate van die deurlaatbaarheidstudies op die vaginale weefsel dui daarop dat die drie peptiede (MEA-5, MDY-19 and PCI) die vaginale mukosa goed penetreer. Soos verwag, het MDY-19 hoër diffusie vloedwaardes as MEA-5 gehad. Dit kan toegeskryf word aan MDY-19 se kleiner molekulere grootte (gewig). Surfaktant het die diffusie vloedwaardes van MDY-19 1.3 keer vergroot en het ook die tyd na vaste vlak verminder. Die verwydering van die vaginale epiteel het die diffusie vloedwaardes van die peptiede verhoog en mag dus dui op die vinniger opname van peptiede en moontlike ander mikrobisiede in vivo, wanneer die belyning van die epiteel onderbreek. Die deurlaatbaarheid van 1 mM MDY-19 en PCI by 37 °C was satisties beduidend (p<0.05) hoer as teem 20 °C. Die area onder die kurwe (AOK) van die gemiddelde vloedwaardes van MDY-19 en PCI by 37 °C, het toegeneem met ‘n toename in die konsentrasie van hierdie peptiede. Die toename vloedwaardes ondersteun dus die alombekende diffusie teorie. Die transdermale diffusie eksperimente van verskillende terbinafien formulasies het getoon dat terbinafien geredelik vrygestel word vanuit hierdie formulasies na die vel. Geen terbinafien vloedwaardes, van enige van die formulasies, was egter gevind in die ontvangselle van die deurlopende-vloei perfusie apparaat nie. Die gemiddelde terbinafien konsentrasies in die vel na 24 h se blootstelling aan drie kommersiële terbinafien hidrochloried formulasies was onderskeidelik 3.589, 1.590 en 4.219 μg/ml. Die gemiddelde terbinafien konsentrasie in die vel wat aan 10 mg/ml PBS/metanol blootgestel was, was hoër as die konsentrasies in die vel wat aan die drie kommersiële formulasies blootgestel was. Die resultate van die temperatuurstudie op vel het aangetoon dat ‘n temperatuur toename van 5 ºC ‘n statisties beduidende toename in vloedwaardes van getritieerde water oor vel veroorsaak. Die vloedwaardes van die getritieerde water oor vel teen ‘n temperatuur van 37 ºC was gemiddeld dubbeld so veel as teen 32 ºC. Die deurlaatbaarheidsvloede van klein intestinale mukosa ten opsigte van verskillende geneesmiddels (wat oraal toegedien word) was ondersoek gedurende ‘n 24 h eksperiment. Die vier geneesmiddels wat gebruik was, was zidovudine, propranolol hidrochloried, didanosien en enalapril maleaat. Hierdie geneesmiddels is verteenwoordigers van die Biofarmaseutiese Klassifikasie Sisteem se klas 1 (hoë oplosbaarheid, hoë deurlaatbaarheid) en klas 3 (hoë oplosbaarheid, lae deurlaatbaarheid) geneesmiddels. Die vloedwaardes van die vier geneesmiddels het gewissel na aanleiding van die tydsverloop in die eksperiment. Zidovudien se gemiddelde vloedwaardes tussen 2-4 en 4-6 h was onderskeidelik 1.8 en 2.0 keer hoër as didanosien se gemiddelde vloedwaardes vir hierdie tyd periodes en onderskeidelik 2.3 en 2.2 keer hoër as enalapril se gemiddelde vloedwaardes. Tydens hierdie selfde periodes was propranolol se gemiddelde vloedwaardes 1.2 en 1.4 keer hoër as didanosien en vir beide periods 1.6 keer hoër as enalapril se gemiddelde vloedwaardes. Gedurende beide genoemde tyd periodes was zidovudien se gemiddelde vloedwaardes 1.4 keer hoer as propranolol en didanosien se gemiddelde vloedwaardes was onderskeidelik 1.3 en 1.1 keer hoër as enalapril tydens 2-4 en 4-6 h. Die klas 1 geneesmiddels het statisties beduidende hoër vloedwaardes gehad as die klas 3 geneesmiddels. Hierdie resultate stem ooreen met die geneesmiddels se Biofarmaseutiese Klassifikasie Sisteem klassifikasie. Dit wil dus voorkom asof die in vitro model wat gebruik was in die studie, gebruik kan word om die deurlaatbaarheidsvloede van klas 1 en 3 te voorspel. Die resultate van hierdie studie stem ooreen met ander in vivo studies. Dit wil voorkom asof die in vitro deurlopende-vloei perfusie apparaat die potensiaal het om sommige van die probleme en tekortkominge van ander in vitro modelle te oorkom en dat dit moontlik die potensiaal het om die diffusie-eienskappe van nuwe geneesmiddels en verskillende formulasies oor verskillende biologiese membrane te voorspel. Die model kan verder moontlik dien as ‘n potensiële toestel om biogelykbaarheid van alternatiewe (generiese) formulasies, wat dieselfde geneesmiddel/chemiese stof bevat, te bepaal.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: In South Africa alone, 30% of women of child-bearing age are infected with HIV. With the increasing focus and success of prevention of mother-to-child transmission (PMTCT) programmes, an estimated 300 000 infants are born exposed to HIV every year. The underlying impact of in utero HIV exposure on infant immune health has not been extensively characterised. Clinical follow-up of these HIV-exposed uninfected (HEU) infants reveals increased infectious morbidity and mortality compared to their unexposed (UE) counterparts. Objectives: (i) To evaluate and characterise adaptive immune properties by measuring vaccine-specific antibody levels in children from 2 weeks to 2 years of age in the presence and absence of maternal HIV infection. (ii) To investigate specific cellular markers of immune activation, immune regulation, apoptosis and B cell memory on T and B cell populations in HEU and UE children measured at 18 and 24 months of age. Methods: This sub-investigation formed part of a collaborative pilot study between the universities of British Columbia (Vancouver, Canada) and Stellenbosch. A total of 95 HIV-positive and HIV-negative mothers were recruited after delivery at Tygerberg Hospital, and signed informed consent for their infants to be included in the study. Of these infants, only 27 HEU and 30 UE infants were eventually enrolled and followed up at various time points, starting at two weeks of age. Four of these infants were confirmed to be HIV-positive at 2 weeks and clinically followed up according to the protocol, but were excluded from statistical data analyses. Blood was collected at 2, 6 and 12 weeks and again at 6, 12, 18 and 24 months of age. Quantitative IgG-specific antibodies to Haemophilus influenzae B (Hib), Bordetella pertussis, tetanus and pneumococcus were measured at each time point, using commercially available ELISA (Enzyme-Linked ImmunoSorbent) kits. Cellular markers of immune activation, immune regulation, apoptosis and memory were measured in various populations of T and B cells at 18 and 24 months only, by using four-colour flow cytometry and validated whole-blood staining methods. In addition, a functional assay was developed to evaluate cell susceptibility to apoptosis (spontaneously) by measuring the expression of Annexin V on both CD4+ T and CD20+ B cells after 16 and 24-hour incubation periods. The statistical analysis of the antibody data was conducted by repeated-measures ANOVA (i.e. analysis of variance), using a mixed-model approach. Differences in the expression of the two groups’ cellular markers were compared by employing one-way ANOVA. An F test p value (which assumes normality) was reported, while the non-parametric Mann-Whitney U test served as confirmatory tool. Repeated-measures ANOVA was used for the evaluation of the functional spontaneous apoptosis assay at three time points (ex vivo, 16 and 24 hours) on the 18-month samples, while one-way ANOVA was used for the 24-month samples. Results: The HEU group (n = 23) displayed significantly lower levels of antibodies to pertussis (20.80 vs 28.01 Food and Drug Administration [FDA] U/ml; p = 0.0237), tetanus (0.08 vs 0.53 IU/ml; p < 0.001) and pneumococcus (31.67 vs 80.77 mg/l; p = 0.003) than the UE group (n = 23) at 2 weeks of age. No statistical differences were noted for Hib antibody levels between the two groups at this time point. At 6 weeks of age, HEU infants displayed lower mean levels of all antibodies measured; however, these differences did not reach statistical significance. Following vaccination, compared to UE controls, the HEU group presented with statistically significantly higher antibody levels to pertussis at 6 months (155.49 vs 63.729 FDA U/ml; p = 0.0013), 12 months (26.54 vs 8.50 FDA U/ml; p < 0.001) and 18 months of age (1658.94 vs 793.03 FDA U/ml; p = 0.0362). A significant difference in tetanus antibody levels between the two groups was only evident at 24 months, with the HEU group displaying higher levels (3.28 vs 1.70 IU/ml; p = 0.018) than the UE group. No differences were observed between the two groups following vaccination for Hib. At 18 and 24 months, the HEU group showed increased expression of cellular markers of immune activation (CD69 and CD40L) on CD4+ T cells compared to UE controls. The two groups showed similar expression of the cellular marker of activation CD38 on CD8+ T cells. The HEU group displayed significantly higher levels of CD127, the interleukin (IL) 7 receptor, on CD4+ T cells compared to UE controls at 18 months of age. The HEU group also showed increased expression of cellular markers of apoptosis on both CD4+ T and CD8+ T cells. No statistical significance was noted for the expression of Fas on CD4+ T cells at 18 and 24 months of age. However, at 24 months, the HEU group showed significantly increased expression of FasL on both CD4+ T and CD8+ T cells. During cell culture experiments, the HEU group displayed increased susceptibility to spontaneous apoptosis shown by increased Annexin V expression on CD4+ T cells after a 16-hour incubation period at both 18 and 24 months. At 18 and 24 months, no difference was noted in the two groups’ immune regulation as measured by the expression of CTLA-4. The HEU group displayed increased levels of the cellular markers of immune activation CD80 on CD20+ B cells at 18 and 24 months of age. The HEU group also showed significantly increased levels of CD69 on CD19+ B cells at 24 months. No statistical significance was reached for the expression of CD62L and CD10 at either 18 or 24 months. Although the HEU group displayed increased levels of apoptosis (Fas) on CD20+ B cells, no statistical significance was reached at 18 or 24 months of age. In addition, the HEU group showed no difference in the expression of programmed death 1 (PD-1) at 18 and 24 months. HEU and UE groups showed similar expression of Annexin V after 16 hours of incubation in the 18 and 24-month samples. The expression of the biomarker of B cell memory CD27 on CD20+ B and CD19+ B cells was comparable between the two groups at both time points. Conclusion: At 2 and 6 weeks, lower mean antibody responses in HEU infants suggest poor placental transfer due to maternal HIV infection, while increased responses to specific antibodies may reflect an exaggerated immune response to immunisation. These robust responses may be due to the lack of competition with maternal antibodies, or may be ascribed to indirect stimulation of B cells via the activation of T cells. A hyper-inflammatory state is an imminent danger, with increased expression of cellular markers of immune activation and apoptosis that may be consistent with early HIV exposure that persists following infancy. These observations may serve as contributing factors to the extensively documented increased susceptibility to infections in the HEU population. Although these findings are consistent with a primed immune system, larger studies are required to confirm these observations in relation to clinical outcomes and to assess further whether these differences persist in later years. / AFRIKAANSE OPSMOMMING: Agtergrond: In Suid-Afrika alleen het 30% van vroue van ʼn vrugbare leeftyd MIV. Met die toenemende fokus en sukses van programme vir die voorkoming van moeder-na-kind-oordrag (sogenaamde PMTCT-programme) word ongeveer 300 000 babas jaarliks aan MIV blootgestel. Die onderliggende impak van intra-uteriene MIV-blootstelling op ʼn baba se immuunstelsel is nog nie omvattend beskryf nie. Kliniese opvolgondersoeke van hierdie MIV-blootgestelde dog onbesmette babas (sogenaamde HEU’s) dui op ʼn hoër siekte- en sterftesyfer weens infeksies as hul nieblootgestelde eweknieë (sogenaamde UE’s). Doelstellings: (i) Om kinders met MIV-positiewe en MIV-negatiewe moeders se aangepaste (verworwe) immuuneienskappe te beoordeel en te beskryf deur hulle vaksienspesifieke teenliggaamvlakke vanaf die ouderdom van twee weke tot twee jaar te meet. (ii) Om ondersoek in te stel na bepaalde sellulêre merkers van immuunaktivering, immuunregulering, apoptose en B-selgeheue by die T- en B-selgroepe van sowel HEU’s as UE’s op die ouderdom van 18 en 24 maande. Metodes: Hierdie subondersoek het deel uitgemaak van ʼn samewerkende loodsondersoek tussen die universiteite van Brits-Columbië (Vancouver, Kanada) en Stellenbosch. Altesaam 95 MIV-positiewe en MIV-negatiewe moeders is gewerf nadat hulle by Tygerberghospitaal geboorte geskenk het, en het ingeligte toestemming verleen dat hul babas by die studie ingesluit kon word. Van dié babas is slegs 27 HEU’s en 30 UE’s uiteindelik in die studie opgeneem en in verskillende stadia vanaf die ouderdom van twee weke opgevolg. Vier van die babas is op twee weke as MIV-positief bevestig en volgens die protokol klinies opgevolg, maar is van die statistiese dataontleding uitgesluit. Bloedmonsters is op twee, ses en 12 weke en weer op ses, 12, 18 en 24 maande geneem. Kwantitatiewe IgG-spesifieke teenliggame teen Haemophilus influenzae B (Hib), Bordetella pertussis, tetanus en pneumokokkus is telkens met behulp van kommersieel verkrygbare ELISA- (“Enzyme-Linked ImmunoSorbent”-)stelle bepaal. Sellulêre merkers van immuunaktivering, immuunregulering, apoptose en geheue is op slegs 18 en 24 maande by verskillende populasies T- en B-selle deur middel van ʼn vierkleurvloeisitometrie en geldig verklaarde volbloedkleuringsmetodes bepaal. Voorts is ʼn funksionele toets ontwikkel om selvatbaarheid vir apoptose te bepaal deur die ekspressie van Annexin V op sowel CD4+ T- as CD20+ B-selle ná 16 en 24 uur van inkubasie te meet. Die statistiese ontleding van die teenliggaamdata is met behulp van herhaaldemetings-ANOVA (d.w.s. afwykingsontleding) volgens ʼn gemengdemodel-benadering gedoen. Verskille in die twee groepe se sellulêre merkervlakke is deur middel van eenrigting-ANOVA vergelyk. ʼn F-toets-p-waarde (wat normaliteit veronderstel) is bereken, terwyl die nieparametriese Mann-Whitney-U-toets as bevestigende instrument gedien het. Vir die 18 maande-monsters is herhaaldemetings-ANOVA gebruik om die funksionele toets vir spontane apoptose in drie stadia (ex vivo, op 16 uur en op 24 uur) te beoordeel. Vir die 24 maande-monsters is eenrigting-ANOVA gebruik. Resultate: Op die ouderdom van twee weke het die groep HEU’s (n = 23) aansienlik laer teenliggaamvlakke teen kinkhoes (20.80 vs 28.01 Food and Drug Administration [FDA] U/ml; p = 0.0237), tetanus (0.08 vs 0.53 U/ml; p < 0.001) en pneumokokkus (31.67 vs 80.77 mg/l, p = 0.003) as die UE-groep (n = 23) getoon. In dié stadium is geen statistiese verskille in Hib-teenliggaamvlakke tussen die twee groepe opgemerk nie. Op ses weke het die groep HEU’s laer gemiddelde vlakke van ál die betrokke teenliggame getoon, hoewel hierdie verskille nie statisties beduidend was nie. In vergelyking met die UE-kontrolegroep het die groep HEU’s ná inenting statisties beduidend hoër teenliggaamvlakke teen kinkhoes getoon op ses maande (155.49 vs 63.729 FDA U/ml; p = 0.0013), 12 maande (26.54 vs 8.50 FDA U/ml; p < 0.001) én 18 maande (1658.94 vs 793.03 FDA U/ml; p = 0.0362). ʼn Beduidende verskil in die twee groepe se tetanus-teenliggaamvlakke het eers op 24 maande geblyk, met die groep HEU’s s’n hoër (3.28 vs 1.70 IE/ml; p = 0.018) as die UE’s s’n. Ná inenting teen Hib is geen verskille tussen die twee groepe waargeneem nie. Op 18 en 24 maande het die HEU’s verhoogde ekspressie van sellulêre merkers van immuunaktivering (CD69 en CD40L) op CD4+ T-selle getoon in vergelyking met die UE-kontrolegroep. Soortgelyke vlakke van die sellulêre merker van aktivering CD38 is ook op die CD8+ T-selle van die twee groepe opgemerk. Op 18 maande het die HEU-groep ʼn beduidend verhoogde ekspressie van CD127, die IL-7-reseptor, op CD4+ T-selle getoon in vergelyking met die UE-kontrolegroep. Die HEU groep het ook verhoogde ekspressie van sellulêre merkers van apoptose op sowel CD4+ T- as CD8+ T-selle getoon. FAS-ekspressie op CD4+ T-selle op 18 en 24 maande was nie statisties beduidend nie, hoewel die HEU-groep op 24 maande beduidend verhoogde ekspressie van FasL op CD4+ T- sowel as CD8+ T-selle getoon het. In selkwekingseksperimente het die HEU-groep ʼn verhoogde vatbaarheid vir apoptose getoon na aanleiding van die ekspressie van Annexin V op CD4+ T-selle ná 16 uur van inkubasie op sowel 18 as 24 maande. Op 18 en 24 maande was immuunregulering, aan die hand van die ekspressie van CTLA-4, bykans dieselfde by albei groepe. Op sowel 18 as 24 maande toon die HEU’s verhoogde ekspressie van die sellulêre merker van immuunaktivering CD80 op CD20+ B-selle. Op 24 maande het die HEU’s ook aansienlik hoër vlakke van CD69 by CD19+ B selle getoon. Op nóg 18 nóg 24 maande was die ekspressie van CD62L en CD10 statisties beduidend. Hoewel verhoogde vlakke van apoptose (Fas) by CD20+ B-selle by die HEU-groep opgemerk is, was dit nie statisties beduidend op 18 óf 24 maande nie. Daarbenewens was daar ook geen verskil in die ekspressie van geprogrammeerde seldood 1 (PD-1) op 18 en 24 maande nie. Op 18 en 24 maande het die HEU’s en UE’s ʼn soortgelyke ekspressie van Annexin V ná 16 uur van inkubasie getoon. Op sowel 18 as 24 maande was die twee groepe se ekspressie van die biomerker van B-selgeheue CD27 op CD20+ B- en CD19+ B-selle vergelykbaar. Gevolgtrekking: Op twee en ses weke dui laer gemiddelde teenliggaamreaksies by HEU’s op swak plasentale oordrag weens die moeder se MIV-infeksie, terwyl verhoogde reaksies op bepaalde teenliggame weer op oordrewe immuunreaksie op inenting dui. Hierdie robuuste reaksie kan toegeskryf word aan die gebrek aan mededinging met die moeder se teenliggame, of kan deur indirekte stimulasie van die B-selle via die aktivering van die T-selle veroorsaak word. ʼn Hiperinflammatoriese toestand is ʼn dreigende gevaar, met verhoogde ekspressie van sellulêre merkers van immuunaktivering en apoptose wat met vroeë MIV-blootstelling met ʼn latere nawerking verbind kan word. Hierdie waarnemings kan bydraende faktore wees tot HEU’s se goed gedokumenteerde verhoogde vatbaarheid vir infeksies. Hoewel hierdie bevindings met ʼn geaktiveerde immuunstelsel strook, moet groter studies dit aan die hand van kliniese uitkomste bevestig en ook bepaal of hierdie verskille in later jare voortduur. / The Harry Crossley Foundation, Poliomyelitis Research Foundation (PRF) / NHLS Research Grant Trust

Thesis (MMed (Medical Microbiology))--University of Stellenbosch, 2006. / One of the major routes of transmission of human immunodeficiency virus (HIV) in the developing world is vertical transmission from mother to infant – pre-, intra-, or post-partum. In the Western Cape, HIV-1 subtype C is the predominant subtype in the heterosexual population, and this trend was expected to be seen amongst cases of mother-to-child transmission of HIV. The aim of this study was to perform genetic characterisation and phylogenetic analysis of the HIV-1 genome in positive serum/plasma samples obtained from children (age 0 to 18 months) from 2000-2002, and temporally related specimens from their mothers. We obtained 27 suitable pairs of samples taken within 6 months of delivery. From this pool, we obtained 21 infant DNA sequences and 17 maternal sequences, resulting in 16 mother-infant pairs. All patient sequences were identified as HIV-1 subtype C, and, as expected, mother and infant viral sequences clustered together. In some cases where a mother was suspected to have two dominant quasispecies based on the electropherogram, only one sequence was detectable in the infant. Single or multiple amino acid deletions were consistent between mothers and infants, and some pairs showed the same amino acid deletions seen in other pairs.