Active tuberculosis is a frequent complication of Hodgkin's disease and it has been demonstrated recently that the circulating lymphocytes are abnormal in this disease (48). Furthermore, reactivation of tuberculosis following steroid therapy is a common complication (56) and it is now well established that steroids are toxic to lymphocytes (57). With this background evidence to suggest the importance of normal lymphocyte function in host resistance and the accidental finding of a poor lymphocyte response in a tuberculous patient, a study of the in vitro reaction of the lymphocytes in tuberculous patients was undertaken. In vitro cultures of peripheral white blood cells will undergo transformation and mitosis when in the presence of phytohemagglutinin (PHA), an extract of the red kidney bean Phaseolus vulgaris. The number of cells which transform or enter mitosis in the culture is representative of the individual's immune responsiveness. In the tuberculous patients studied, irrespective of the state of their disease, the mean mitotic index (the number of cells in mitosis per thousand cells) was found to be significantly lower than normal controls suggesting that their mean immunological capacity is below normal. During the course of the tuberculous study one of the individuals used as a normal control came down with the "flu" on the evening of the day her blood was taken. The PHA mitotic index was found to be severely depressed. To study the effect of such acute upper respiratory infections, presumably of viral origin, mitotic indices were determined on blood samples taken from laboratory volunteers who felt they were coming down with a cold. The indices were found to be depressed in all cases as compared to healthy normals. The reaction of delayed hypersensitivity and the relationship of this reaction to immunity and tuberculosis has been a matter of study for many years. It seems clear that the individuals in the population that later develop tuberculosis are drawn largely from those members who have a positive skin test while those with a negative skin test have been found unlikely to develop tuberculosis to the point of not being required to have X-rays in many instances. Tuberculin PPD (purified protein derivative) was one of the first antigens shown to be capable of transforming lymphocytes in vitro (60, 61) providing the individual had had prior sensitization. As all tuberculin negative student nurses entering training at the Vancouver General Hospital receive a bacillus Calmette Guerin vaccination (BCG), they were chosen for study in order to elucidate the relationship of in vivo and in vitro responses to the tubercle antigen, PPD. As the number of cells responding to PPD is much less than responding to PHA, the mitotic index is a very insensitive method to use in determination of in vitro responsiveness to antigen. Therefore, the response in vitro was determined by the incorporation of (3)H-thymidine into DNA of stimulated lymphocytes. It was found that in 12 to 18 months after vaccination with BCG nurses who had become skin test negative to PPD did not differ significantly in their in vitro response from nurses who had remained skin test positive. A correlation was found between the PHA and PPD in vitro responses which suggests that the response to the general stimulus, PHA, is an indicator of the reaction that can be mobilized to specific antigen. Varying the dose of BCG that was administered affected only in vivo responses. Finally, contrary to many reports, no quantitative correlation was found between in vivo and in vitro responses to PPD following BCG vaccination. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Tuberculosis has re-emerged as a global health problem due to co-infection with HIV and the emergence of drug resistant strains of Mycobacterium tuberculosis. There is a need for a reliable and fast serodiagnostic assay to reduce the time required for test results from weeks to hours, in order to better control the spread of the disease. Previous studies have shown that TB patients contain antibodies against M. tuberculosis mycolic acids. In standard immunoassays such as ELISA, an unacceptable number of false positive and negative test results were obtained. This study aimed at assessing the potential of detecting anti-mycolic acids antibodies in TB patient sera on a biosensor as surrogate marker for TB infection. Mycolic acid liposomes were immobilized reproducibly on a non-derivatized biosensor cuvette and blocked with saponin. A high dilution of serum in PBS/ AE was used to calibrate the signal of the two cells, followed by binding of patient sera inhibited with either mycolic acid, cholesterol or placebo phosphatidylcholine liposomes at a lesser dilution. The inhibition was done to confirm the specificity of the binding response. There was no inhibition of binding when a sputum negative control serum (HIV-TB-) was pre-incubated with either cholesterol or mycolic acids on the biosensor coated with mycolic acid liposomes. The antibodies that are specific to mycolic acid were demonstrated in all TB positive patients on mycolic acids coated cuvette cell surfaces after pre-incubation of serum with mycolic acids. The patient sera that were false positive and false negative on ELISA tested negative and positive respectively on the biosensor. Only sera from two patients, both HIV positive, tested false positive on both ELISA and biosensor. The biosensor was able to detect anti-mycolic acids antibodies of even low affinity. In ELISA, these antibodies were washed away. No inhibition of antibody binding on cholesterol-coated cuvettes was found after pre-incubation of serum with mycolic acids or cholesterol liposomes. The cholesterol surface became unstable during pre-incubation of serum with mycolic acids. Mycolic acid appeared to be a stronger antigen than cholesterol. The anti-mycolic acids antibodies were specific and sensitive for diagnosis of TB on the biosensor. More sera should be analyzed on the biosensor to make a statistically accountable statement on whether the improved sensitivity and specificity is adequate for a simple, rapid, sensitive and accurate biosensor-based serodiagnostic assay. / Dissertation (MSc(Biochemistry))--University of Pretoria, 2006. / Biochemistry / unrestricted

DNA fingerprinting of Mycobacterium tuberculosis strain has been used in combination with conventional epidemiologic investigation, which has improved the understanding of tuberculosis transmission. Restriction Fragment Length Polymorphism (RFLP) based on IS6110 probe has become a standard method of fingerprinting of M tuberculosis. Since the technique is labour intensive and the discriminatory power of IS611 0 fingerprinting method for strains habouring only one to five copies is poor, other typing methods for typing M tuberculosis should be evaluated. In this regard, Amplified Fragment Length Polymorphism (AFLP) has the potential to overcome many of the RFLP problems. The first objective was to determine the suitability of the RFLP and AFLP techniques and to study the extent of transmission of tuberculosis in a referral hospital in South Africa. A total of 47 M tuberculosis isolates were differentiated using RFLP technique. The same samples were typed using the PCR- based AFLP technique and results were compared. The second objective was to determine the prevalence of isoniazid (INH) resistance and estimate the incidence of recent transmission of the disease in the Eastern-Cape (EC) and North-West province (NW) by using the best suited technique. RFLP grouped the 47 typed M. tuberculosis isolates into five families and four clusters. AFLP grouped the analyzed isolates (previously typed by RFLP) into two groups based on the banding patterns observed. As a result of the low degree of genotypic variation among the AFLP band pattern of M tuberculosis isolates, AFLP seemed less promising for individual strain differentiation of M tuberculosis. This technique can be used in future for differentiation of Mycobacterial species and The prevalence of INH resistance was found to be 6.7% in the EC and 8.4% in the NW province. The magnitude of recent transmission in the Eastern Cape studied by RFLP method, was found to be at 22% among the positive tuberculosis isolates identified. Transmission of TB in NW province was associated with reactivation rather than recent transmission due to lack of clustering of strains in that region. / Dissertation (MSc(Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Background: Tuberculosis (TB) is a major public health problem in Swaziland aggravated by the escalating HIV epidemic. Health services in five of the major industries in Swaziland represent the potential for the highest quality of TB care in the country, arising from increased supervision and better case holding. The guidelines of the national TB control programme (TBCP) are mostly adhered to, but there is a tendency to rely on clinical and radiological parameters for diagnosis due to problems with sputum microscopy. Aim and Objectives: The aim of this study was to evaluate current TB management protocols by describing case management and treatment outcomes in these five industries. Specific objectives included the determination of quantitative outcomes (cure and treatment completion, smear conversion, treatment interruption and failure, and mortality). Patient knowledge of TB and its treatment as well as health worker practices were also assessed. Methods: Descriptive questionnaire survey. Results: The majority of TB patients (79%) were young (mean age 38 yrs) males. 81 % of patients were treated for TB for the first time. The HIV status of a third of patients was known, and 82.7% of these were positive. There were significant differences between the perceptions of health workers and patients on the delivery of TB care and the time lapse between diagnosis and treatment. Chest X-ray was the main diagnostic tool used. In more than 97% of cases the TBCP prescribed treatment regimen was used. Directly observed treatment was provided to 77.4% of patients. The majority of patients had some knowledge of TB and its spread. 73.4% of patients knew about available TB treatment, and 75% about treatment duration. Coughing was identified as an important symptom by 84.5% of patients. There was a significant difference between calculated and estimated adherence to treatment. In 55.6% of patients no sputum smear was done at two months. Treatment outcome was favourable in 83.7 % of patients, compared to only 62.1 % of TBCP patients in 2001. Outcome analysis showed that the participating industries had a highly successful TB control programme compared to the national TBCP, with outcome indicators meeting international standards. A serious deficiency detected was the lack of smear microscopy for diagnosis and treatment monitoring. Limitations: The possibility exists that patients presenting to the Health Centres were not registered sequentially. The usual limitations relating to questionnaires are applicable. Recommendations: Directly observed treatment coverage and supervision can be improved in industry as the patient group is well-defined and captive. Sputum microscopy should become the mainstay of diagnosis and monitoring. Health care providers should be primed to detect co-existing lung disease and HIV, and TB drug side effects. Accurate recording and reporting systems should be introduced without delay. Communication between the TBCP and the non-governmental health institutions in Swaziland needs improvement. / Dissertation (MMed)--University of Pretoria, 2005. / School of Health Systems and Public Health (SHSPH) / Unrestricted

The need for new anti- TB drugs is increasingly rising because of the resistance of M. tuberculosis to existing drugs. The mycobacterial cell wall serves as an impermeable protective barrier for the bacilli from toxins and chemotherapeutic agents, mainly due to the mycolic acids waxy outer layer. The mycolic acids play an important role in the architecture and physical properties of the mycobacterial cell wall. This study was based on the observed mimicry and association between the host cholesterol and the mycolic acids. This may present yet another way in which the TB bacilli survives by manipulating its host and using some of its components for its survival. The research focused on whether the cholesterol-like molecules on the mycobacterial cell surface can be targeted for effective delivery of anti-mycobacterial agents. In order to exploit the ability of M tuberculosis to accumulate cholesterol or interact with it, a cholesterol¬binding molecule was used for targeting an anti- TB drug to the mycobacterial cell wall or to the cell membrane of infected macrophages. It was observed that the drug does possess anti-mycobacterial activities even though higher concentrations of the compound were required. This supports the idea that the ability of cholesterol to interact with the mycobacterial mycolic acids can be exploited for designing of anti- TB agents. It was also demonstrated in this study that cholesterol has a negative effect on the activity of INH. Thus cholesterol, which is required for entry and survival of M tuberculosis in the host cells, has yet another protective effect on this pathogen. The possible ability of cholesterol to target the same enzyme(s) as INH is another small piece of knowledge to complete the puzzle to understanding the mode of virulence and pathogenesis of this pathogen and develop of new ways to fight the old enemy. / Dissertation (MSc(Biochemistry))--University of Pretoria, 2006. / Biochemistry / unrestricted

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Setting Study conducted in Tygerberg, Cape Town in South Africa. Hypothesis Host biomarkers associated with the antimycobacterial immune response during active infection with M. tuberculosis and during anti-tuberculosis chemotherapy are indicative of bacterial killing in the host and can be used in models to predict eventual treatment outcome. Objectives 1. To investigate immune parameters that were selected in a biological context as biomarkers of the extent of disease and early response to anti-tuberculosis treatment. 2. To use selected immune parameters to characterise fast and slow responders to anti-tuberculosis therapy. Findings Evaluation of cytokine multiplex fluorescent bead-based immunoassays as a screening tool in the search for biomarkers The data showed that cytokine multiplex fluorescent bead-based immunoassays achieved acceptable recoveries to detect antigen-specific IFN- responses in whole blood supernatant making it attractive for biomarker screening. However, proper optimisation needs to be done and proper controls included when using these kits. Markers of extent of disease High levels of CRP at diagnosis were found to be associated with the presence of multiple cavities on chest X-rays. A high level of suPAR and sICAM-1 at diagnosis were associated with the extent of alveolar disease. Also significant were the associations between the level of granzyme B, LAG-3 at diagnosis and the size of the cavities. No significant associations were observed between sTNFRs or DR5 with the chest X-ray grading of tuberculosis disease. Early classification of fast and slow responders to anti-tuberculosis treatment After cross-validation classification, discriminant analysis (DA) and support vector machine (SVM) analysis of selected immune parameters (sICAM-1 CRP, granzyme B, suPAR, sTNFRs, LAG-3 and CD3dim/CD56+ (% of CD45+) resulted in a 75% to 100% correct classification of the fast responders and a 82% to 100% correct classification of the slow responders when using DA. For SVM, the correct classification of the fast responders ranged from 88% to 100%, and that for the slow responders ranged from 95% to 100%. Differential gene expression in fast and slow responders to treatment Direct comparison of fast and slow responders showed that IL-4 transcripts were significantly higher in the fast responders at week one after initiation of treatment when compared to slow responders. IL-42 was also differentially expressed. Although IL- was significantly up-regulated in both fast and slow responders after one week of treatment compared to diagnosis, IL- expression was more than two folds higher in slow responders than in fast responders. No significant differences between the fast and slow responders were observed in the expression of TGF-, TGF-RII, Foxp3 and GATA-3. Conclusion Predictive models for differential anti-tuberculous treatment responses combining host proteins are promising and should be included in larger prospective studies to find the optimal markers for inclusion into clinical trials of new drugs and for implementation into clinical practice. / AFRIKAANSE OPSOMMING: Ligging Studie onderneem in Tygerberg, Kaapstad, Suid-Afrika. Hipotese Gasheerbiomerkers wat verband hou met die antimikobakteriële immuunrespons tydens aktiewe infeksie deur M. tuberculosis en tydens teentuberkulose chemoterapie dui op bakteriële doding in die gasheer en kan in modelle gebruik word om die uiteindelike uitkoms van die behandeling te voorspel. Doelwitte 1. Om gekose immuunparameters in ’n biologiese konteks as biomerkers van die omvang van siekte en vroeë reaksie op behandeling te ondersoek. 2. Om gekose immuunparameters te gebruik om vinnige en stadige reageerders op teentuberkulosebehandeling te karakteriseer. Bevindings Evaluering van die sitokien veelvuldige fluoresseer-pêrelbaseerde immuuntoets (cytokine multiplex fluorescent bead-based immunoassays) as ’n siftingsinstrument in die soeke na biomerkers Die data het getoon dat die sitokien veelvuldige fluoresseer-pêrelgebaseerde immuuntoets in staat was om antigeenspesifieke IFN--respons te meet wat dit aanloklik maak vir biomerkersifting. Sorgvuldige optimering moet egter gedoen word en behoorlike beheer moet ingesluit word wanneer hierdie stelle gebruik word. Merkers van omvang van siekte Hoë vlakke van CRP by diagnose is getoon om verband te hou met die teenwoordigheid van veelvoudige holtes op die pasiënte se borskas x-strale. Hoë vlakke van suPAR en sICAM-1 by diagnose was assosieer met die omvang van alveolêre siekte. Die assosiasie tussen die vlakke van granzyme B, LAG-3 by diagnose en die grootte van die holtes was ook betekenisvol. Daar was geen betekenisvolle assosiasies toe sTNFRs of DR5 en die borskas x-straalgradering van tuberkulosesiekte nie. Vroeë klassifikasie van vinnige en stadige reageerders op teentuberkulosebehandeling Ná klassifikasie op grond van kruisstawing het diskriminant-analise (DA) en ondersteuningsvektormasjiene (SVM) van geselekteerde immuunparameters (sICAM-1 CRP, gransiem B, suPAR, sTNFRs, LAG-3 en CD3dim/CD56+ (% van CD45+)) gelei tot ’n 75% tot 100% korrekte klassifikasie van die vinnige reageerders met DA en ’n 82% tot 100% korrekte klassifikasie van stadige reageerders. Vir SVM het die korrekte klassifikasie van vinnige reageerders gewissel van 88% tot 100%, en vir stadige reageerders het dit gewissel van 95% tot 100%. Differensiële geenuitdrukking in vinnige en stadige reageerders op behandeling In vergelyking met die vlak by diagnose is die uitdrukkingsvlak van IL-4 in die vinnige reageerders betekenisvol opgereguleer met ’n faktor van 9.2 teen die eerste week ná die aanvang van behandeling, in kontras met die stadige reageerders. Daar was geen verskille tussen die vinnige en die stadige reageerders met betrekking tot die uitdrukking van TGF-, TGF-RII, Foxp3 en GATA-3 nie. Gevolgtrekking Voorspellende modelle vir differensiële tuberkulose behandelingsresponse wat gasheerproteïene kombineer, hou belofte in en behoort in groter prospektiewe studies ingesluit te word om die mees geskikte merkers te vind vir insluiting in kliniese proewe van nuwe middels en vir implementasie in kliniese praktyk.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Sequence analysis showed that the essential glnA1 gene of Mycobacterium tuberculosis might be closely related to an actinomycetes progenitor sequence and that this sequence may have undergone duplication into other non-essential GS encoding genes in some Actinobateria, notably the mycobacteria. Also, the M. tuberculosis glnA1 sequence remains conserved throughout the evolution of M. tuberculosis. It was also shown that glnA1 is widely expressed in M. tuberculosis infected human pulmonary tissue, where M. tuberculosis might be present in altered phenotypes. These results imply that glnA1 is under selective pressure against evolutionary change. At transcriptional level it was shown that M. tuberculosis glnA1 might be expressed from two alternate promoter sites, but that these promoter sites may not be controlled by environmental nitrogen (in the form of ammonium) variation. We also showed that M. tuberculosis GS is effectively exported by M. smegmatis to the cell wall, but that GS secretion into the exogenous environment does not occur. Also, evidence has been presented which suggested that M. tuberculosis GS might be modified at the C-terminus, probably as part of a mechanism that retains GS in the cytosol. This hypothesis was further substantiated where it was demonstrated that two GS isoforms of different size (short isoform in cytosol, longer isoform in cell wall) are present in M. bovis BCG. It is unknown what causes this modification, since it couldn’t be observed in M. smegmatis, but it was suggested that it might be through the action of a cis- or trans-acting element present in proximity of the M. tuberculosis glnA1 gene. It was also shown that a cluster of genes found immediately downstream of the M. tuberculosis glnA1 sequence might be regulated in an operonic fashion under conditions of elevated environmental nitrogen concentrations. Two of the genes (glnE and glnA2) in this operon arrangement have been previously shown to be involved in nitrogen and glutamine metabolism. The function of the other gene, Rv2223c, is unknown. It was shown that Rv2223c homologs are mostly found in the mycobacteria and that it may encode an exported protease. It was hypothesised that this sequence and its adjacently located progenitor sequence, Rv2224c, might be involved in M. tuberculosis GS mediated metabolism. It was showed that over-expression of Rv2223c and Rv2224c may be toxic to E. coli and mycobacterial hosts, such as M. smegmatis, but that inhibition of transcription of these genes may be fatal to M. bovis BCG and M. tuberculosis H37Rv. It was also shown that Rv2223c is widely expressed in M. tuberculosis infected human tissue, which was comparable to that of glnA1. The results presented in this study shed more light on the distribution and transcriptional regulation of GS in mycobacteria and has identified new genes that might be involved in GS regulation. These results may present new approaches to tuberculosis control and thereby contribute to alleviate the burden of the disease. / AFRIKAANSE OPSOMMING: Genetiese en proteien volgorde analise het aangedui dat die glnA1 (kodeer vir glutamien sintetase (GS), ‘n essentiele protein) geen van Mycobacterium tuberculosis die naaste verwant is aan ‘n actinomycetes voorloper volgorde wat duplikasie ondergaan het om die ander nieessensiele GS koderende gene in sommige Actinobakterieë te vorm, veral in die mikobakterieë. Voords het die glnA1 geen geneties gekonserveerd gebly gedurende die evolusie van M. tuberculosis. Dit is ook aangetoon dat volop transkribasie van die glnA1 geen voorkom in die M. tuberculosis geïnfekteerde pulmonêre weefsel waar M. tuberculosis moontlik mag voorkom. Op transkriptionele vlak is dit aangetoon dat die M. tuberculosis glnA1 geen vanaf twee onderskeie promotors uitgedruk mag word, maar dat hierdie twee promotors nie deur variasies in die konsentrasie van stikstof (in die vorm van ammonium) in die omgewing beïnvloed word nie. Daar is ook aangedui dat M. tuberculosis GS effektief deur M. smegmatis oor die selmembraan na die selwand vervoer word, maar dat daar nie GS sekresie na die ekstrasellulêre omgewing geskied nie. Ook is bewyse gevind dat M. tuberculosis GS modifikasie aan die C-terminus mag ondergaan wat waarskynlik dien om GS beweging uit die sitosol te verhoed. Hierdie hipotese is versterk deurdat twee isoforms van verskillende grootte (klein in sitosol en groter in die selwand) gevind is in M. bovis BCG. Dit modifikasie meganisme is onbekend, maar vind moontlik nie plaas in nie-patogeniese mikobakterieë soos M. smegmatis nie en mag moontlik deur cis- of trans-werkende elemente gefasiliteer word. Daar is aangedui dat ‘n groepering van vier gene lanksaan die glnA1 lokus in ‘n operoniese meganisme gereguleer mag word onder variërende konsentrasies van stikstof in die omgewing. Dit is bekend dat twee van die gene in die operon (glnE en glnA2) betrokke in stikstof en gultamien metabolisme is. Die funksie van die ander twee gene (Rv2223c en Rv2224c) is onbekend. Daar is aangetoon dat volgordes soortgelyk aan Rv2223c beperk is tot die mikobakterieë en dat die geen ‘n protease, wat moontlik gesekreteer word vanuit die sitosol, kodeer. Daar is aangetoon dat die oor-produksie van die Rv2223c en Rv2224c proteine toksies is vir E. coli en mikobakterieë, soos M. smegmatis, maar dat transkripsie inhibisie hierdie gene dodelik is vir M. tuberculosis H37Rv en M. bovis BCG. Daar is ook angedui dat die ekspresie van hierdie gene volop verspreid is in M. tuberculosis geïnfekteerde menslike weefsel, soortgelyk aan diè van glnA1. Die resultate vervat in hierdie studie werp meer lig op die verspreiding en transkiptionele regulasie van GS in mikobakteriee en nuwe gene is ontdek wat betrokke by GS regulasie mag wees. Hierdie resultate mag bydra tot nuwe maniere om tuberkulose te bekamp en daardeur die voorkoms van die siekte te beperk.

Background: A post-exposure regrowth model utilizing the well-tried Bactec radiometric system, which would simulate in vivo situations at the site of invasive disease, was developed to measure drug activity against multiplying Mycobacterium tuberculosis. Aims: The aims of this dissertation were to (a) construct a radiometric model simulating drug efficacy relating to the combined bactericidal activity and delays in regrowth due to the action of anti¬tuberculosis (TB) agents, (b) compare the killing kinetics of drugs singly and in combinations by the time-kill curve method, with the radiometrically-determined regrowth model, and (c) assess whether the Bactec radiometric regrowth model could predict likely bactericidal activities of drugs. Design and methods: Drug concentrations in the time-kill curve method were in a range of achievable drug concentrations at the site of infection and in multiples of the minimal inhibitory concentration (MIC), (1x, 2x or 3x, and 8x). Exposure times of 6h, 24h, 48h and 72h were used and these were based on pharmacokinetic data reflecting likely periods of in vivo exposure in TB lesions. Standardized inocula of approximately 106CFU/mi of actively multiplying M. tuberculosis strains were used. The same concentrations, exposure times and bacterial concentrations were used for the assessment of radiometrically-determined post-exposure regrowth times of M. tuberculosis. Growth times were recorded as the number of days required to reach a predetermined growth index (GI) level in the Bactec system, and were expressed as T400 readings in days. Simple linear regression and a mathematical logistic model were used to assess whether the radiometric post-exposure regrowth model could predict the bactericidal activity of the drugs. For drug combination studies, 1MIC of isoniazid (INH) and rifampicin (RMP) were used singly and in combination while 2MIC of ethambutol (EMB), streptomycin (SM), ofloxacin (OFL) and amikacin (AMK) were used in combinations studies. Colony counts at Oh and following 24h exposures were performed and regrowth patterns were determined using the T400 method. M. tuberculosis H37Rv was tested and subsequently resistant strains. Results: INH and RMP were highly bactericidal while EMS showed moderate activity in the time-kill curve method. The three drugs produced the best curves, showing longer regrowth times and markedly depressed rates of regrowth in the Sactec post-exposure regrowth model. Using simple linear regression, a linear relationship between bacterial survivors and the radiometric regrowth times, T400, was achieved for all drugs tested. Even better agreement was found when control-related regrowth times, (T-C) 400, were used in the analysis. Conditions compromising the linear relationsbip in the radiometric regrowth model, for OFL and less markedly EMS and AMK, were likely postantibiotic effects (PAEs) brought on by the short exposure time (6h), and drug carry-over effects due to concentrations ≥ 8 MIC for INH, RMP and 8M (10x and 20x MICs). The mathematical logistic model showed good correlation between bactericidal activity and regrowth for INH and RMP but not for EMB, SM, OFL and AMK. Drug combination effects in the two techniques depended on the criteria used to describe synergy. Generally, it was found in drug combination experiments that the drugs did not influence each other to a meaningful extent. Discussion and conclusions: For prediction of bactericidal activity, interpretation of the radiometrically-determined regrowth model needs to accommodate PAEs and the effect of subinhibitory concentrations. The validity of the mathematical logistic model is not clear. Technical aspects of future studies such as better organism dispersal, need to be improved to achieve a more reliable evaluation based on the logistic model. For drug combination studies, the radiometric regrowth model yielded findings that were difficult to interpret in relation to published data, reinforcing the need for the use of internationally standardized techniques which would give statistically reliable data. The radiometrically-determined regrowth model showed good discrimination between the standard activities of anti- TB agents, correlating with clinical efficacy. It is simple to perform and could prove to be useful for the screening of candidate anti- TB drugs. Improved technical stringency and the evaluation of poorly active control drugs, are however needed before proof of validity of the model can be established. / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2007. / Medical Microbiology / unrestricted

TB is still one of the world's biggest killers. Immunosuppresion induced by AIDS caused a rise in the incidence of TB during the past decade. The search for new drugs to effectively treat TB remains one of the big challenges facing the scientific community. Drugs from plants have been used for centuries to treat various human diseases with varying degrees of success. South Africa with its big resource of plants and ethnobotanical knowledge is an ideal place to screen for anti- TB compounds. The Zulu tribe of South Africa used the root bark of Euclea nata/ensis A.DC. to treat TB related symptoms. Naphthoquinones isolated from E. nata/ensis proved to have good activity against TB. Nine compounds were isolated from the chloroform extract of E. nata/ensis root material. Three of these compounds were newly isolated from this species (mamegakinone, neodiospyrin and 5-hydroxy-4-methoxy-2-napthaldehyde). The structures of the isolated compounds were confirmed using NMR methods and where possible the HPLC and TLC results were compared to authentic standards. Most of the compounds were tested for anti- TB activity with only mamegakinone, lupeol and betulin not showing any activity (5-hydroxy-4-methoxy-2-napthaldehyde still needs to be tested). The activity of the naphthoquinones, especially 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin, show promise that these compounds could develop into an affordable medicine to treat TB. The activity of the crude extract against the resistant DP48 110 1 TB strain showed that there are probably unknown active compounds remaining in the extract. The most active compound, 7 -methyljuglone, was synthesised and an improved synthetic pathway was developed. The synthesis of naphthoquinones remains important in order to produce the compounds on a larger scale. This will make further studies into the mode of action, biosynthesis, bioactivity etc. of these compounds possible. Attempts were made to synthesise diospyrin with 7 -methyljuglone as the starting material. These experiments failed up to now. By altering the reaction parameters such as pH and temperature it should be possible to synthesise diospyrin in future attempts. Neodiospyrin were synthesised from reduced 7 -methyljuglone. This synthesis will yield information on the naphthoquinone chemistry and on how to synthesise diospyrin and isodiospyrin. The enzymatic synthesis of naphthoquinones was also investigated with the use of a cell-free extract. These experiments indicated that it might be possible to enzymatically synthesise diospyrin and the other dimers. / Dissertation (MSc (Plant Physiology))--University of Pretoria, 2005. / Plant Science / unrestricted

The intra- and extracellular activities of 2 novel tetramethylpiperidine (TMP)-substituted phenazines, B4121 and B4128 against Mycobacterium tuberculosis H37R (ATCC 27294) were determined and compared with those of clofazimine (B663). Clofazimine, together with B4121 and B4128, were also tested for their activities against drug-resistant strains of M.tuberculosis. Both B4121 and B4128 were significantly more active than clofazimine against M.tuberculosis, including multidrug-resistant clinical strains of this microbial pathogen, demonstrating a lack of cross resistance between the riminophenazines and standard anti-tuberculous drugs. Using M.tuberculosis-infected monocyte-derived macrophages both B4121 and B4128 were found to possess intracellular activity, which was superior to that of both clofazimine and rifampicin. The relationship between anti mycobacterial action of the TMP-subsitituted phenazines and clofazimine and the effects of these agents on microbial PLA2 activity, cation (K+, Ca2+) fluxes and energy metabolism (ATP) was also investigated. PLA2 and cation fluxes were measured by radiometric procedures, while microbial ATP was assayed using a luciferin/luciferase chemiluminescence method. All 3 riminophenazines, particularly B4128 caused dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+-influx and enhancement of uptake of Ca2+. The results of kinetics studies demonstrated that riminophenazine-mediated enhancement of PLA2 activity and inhibition of K+ uptake in mycobacteria are rapidly-occurring and probably related events that precede, by several minutes, any detectable effects on microbial ATP concentrations and uptake of Ca2+. Inclusion of the extracellular and intracellular Ca2+-chelating agents EGTA and BAPTA, respectively, individually or in combination, did not prevent the effects of the riminophenazines on mycobacterial PLA2 (enhancement) or K+ transport (inhibition), whereas α-tocopherol, which neutralizes PLA2 primary hydrolysis products, antagonized the inhibitory effects of the riminophenazines on microbial K+ uptake. These results demonstrated that the riminophenazine-mediated enhancement of PLA2 is a Ca2+-independent event. The involvement of PLA2 in the antimicrobial activity of the riminophenazines was supported by the observation that added, exogenous Iysophosphotidylcholine (a primary hydrolysis product of PLA2 action on membrane phospholipids) also inhibited K+ transport and growth of mycobacteria. Enhancement of endogenous PLA2 as a mechanism of riminophenazine-mediated disruption of cation transport and antimycobacterial activity was further investigated using the conventional calcium-mobilizing stimuli, calcium ionophore A23187 and thapsigargin. Both agents, but A23187 in particular caused in dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+ influx and growth. Influx of Ca2+ into A23187- and thapsigargin-treated mycobacteria was observed using both radiometric and FURA-2-based spectrofluorimetric procedures. Exposure of the mycobacteria to these agents resulted in an immediate increase in uptake of Ca2+, which implies that enhancement of PLA2 activity in calcium-mobilizing stimuli-treated mycobacteria is Ca2+ dependent. In conclusion, the TMP-substituted phenazines possess anti mycobacterial properties which are superior to those of clofazimine, particularly against intraphagocytic M.tuberculosis. The superior anti mycobacterial properties of these agents is paralleled by their potentiating effects on microbial PLA2 and consequent inhibitory action on uptake of K+, particularly in the case of B4128. Mycobacterial PLA2 and K+ transporters may therefore represent novel targets for antimicrobial chemotherapy. / Thesis (DPhil (Medical Immunology))--University of Pretoria, 2007. / Immunology / unrestricted