Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The immune system matures progressively from infancy to adulthood, thus children may differ from adults in their immune function. The immature immune system demonstrates a higher naive to memory T cell ratio, defective macrophage function and antigen presentation which, cumulatively, results in diminished production of cytokines such as IFN-y. This cytokine has been shown to play a pivotal role in protection against Mycobacterium tuberculosis (M. tuberculosis) disease. Other cytokines, such as IL-12 and TNF-a, are also involved in the defence against M. tuberculosis. Epidemiological evidence suggests an agerelated incidence of tuberculosis (TB) irrespective of prevalence in a given region. Reports in the literature also demonstrate depressed immune responses in TB patients, at diagnosis, (before TB therapy) with subsequent improvement after TB therapy. The aims of this study were to optimise a whole blood assay in order to characterise immune responses, as measured by proliferation and cytokine production, in TB patients (after TB therapy) and healthy controls of different ages. Immune responses of TB patients would also be compared, before, and after TB therapy. A total of 68 subjects were included in this study. These comprised 27 TB patients and 41 healthy Mantoux positive controls. All subjects were stratified into two age groups: <12 years and >12 years. Diluted whole blood was cultured and stimulated with the mitogen, phytohaemagglutinin (PHA) and the specific mycobacterial antigen, purified protein derivative (PPD) to measure proliferation and IFN-y, IL-2, TNF-a and IL-10 production in the supernatant of cultures. Age was a significant variable for the following PHA-stimulated cytokines: IFN-y, TNF-a and IL-10. Proliferation and IL-2 production after PHA stimulation did not demonstrate any relationship with age. None of the PPD-stimulated proliferative or cytokine responses demonstrated any correlation with age. Concentrations of PHA- and PPD-induced IFN-y for all subjects (patients and controls) were increased “after therapy”, compared to “before therapy”. This phenomenon could possibly be due to maturation in the capacity of the immune system to produce this cytokine. Patients >12yrs demonstrated improvement in all proliferative and cytokine responses (except for PPD-induced IL-2 and TNF-a) “after therapy”, compared to “before therapy”. This is probably a valid finding and is thus in accordance with the literature. The whole blood assay is a simple, non-laborious assay that, according to the literature, produces results that seem to correlate well with that of conventionally used PBMCs. Age appears to be an important variable in the quantitative assessment of cellular immune responses (when the mitogen, PHA is used as a stimulant) and immune responses of older TB patients appear to improve after TB therapy, compared to before TB therapy. / AFRIKAANSE OPSOMMING: Die immuunsisteem matureer stelselmatig van kind na volwassene. Dus sal kinders se immuniteit verskil van volwassenes s’n. Die immature immuunsisteem het ‘n hoer nai'witeit vir geheue T-sel verhouding, defektiewe makrofaag funksie en antigeen presentering wat gesamentlik lei tot verminderde produksie van sitokiene soos byvoorbeeld IFN-y. Daar is bewys dat hierdie sitokien ‘n deurslaggewende rol speel in die beskerming teen Mycobacterium tuberculosis (M. tuberculosis). Ander sitokiene, soos IL-12 en TNF-a speel ook ‘n rol in die beskerming teen M. tuberculosis. Epidemiologiese data dui aan dat daar ‘n ouderdomverwante insidensie van tuberkulose (TB) is sonder dat dit beinvloed word deur die voorkoms van TB in ‘n sekere area. Verslae in die literatuur wys ook op onderdrukte immuniteitrespons in TB-pasiente by diagnose (voor TB-behandeling) met uiteindelike verbetering na TB-behandeling. Die doel van hierdie studie was om ’n volbloed metode te optimaliseer in ’n poging om die immuunrespons te karakteriseer soos gemeet met behulp van proliferasie en sitokien produksie by TB-pasiente (na TB-behandeling) en gesonde kontrole persone van verskillende ouderdomme. Die immuunrespons van TB-pasiente word ook vergelyk voor en na TBbehandeling. ‘n Totaal van 68 gevalle is vir die studie gebruik. Dit sluit in 27 TB-pasiente en 41 gesonde Mantoux positiewe kontroles. A1 die gevalle is in twee ouderdomsgroepe verdeel: <12 jaar en >12 jaar. Kulture is gemaak van verdunde volbloed en gestimuleer met phytohaemaglutinin (PHA) en gesuiwerde proteien derivaat (purified protein derivative-PPD) om proliferasie en IFN-y, IL- 2, TNF-a en IL-10- produksie in die supernatant van die kulture te meet. Ouderdom was ‘n beduidende veranderlike vir die volgende PHA-gestimuleerde sitokiene: IFN-y, TNF- a en IL-10. Daar was geen korrelasie tussen proliferasie en IL-2-produksie na PHA-stimulasie aan die een kant en ouderdom aan die ander kant nie. Geen van die PPDgestimuleerde proliferasie response of sitokien response het enige korrelasie met ouderdom getoon nie. Konsentrasies van PHA- en PPD-geinduseerde IFN-y vir alle gevalle (pasiente en kontrole) was verhoog “na behandeling”, vergeleke met “voor behandeling”. Hierdie fenomeen kan moontlik toegeskryf word aan maturasie in die vermoe van die immuunsisteem om sitokiene te vervaardig. Pasiente >12 jaar het bewyse getoon van verbetering in alle proliferasie en sitokien response (behalwe vir PPD-gei'nduseerde IL-2 en TNF-a) “na behandeling”, vergeleke met “voor behandeling”. Dit is waarskynlik ‘n geldige bevinding en is dus in ooreenstemming met verslae in die literatuur. Die volbloed metode is ‘n eenvoudige metode wat nie baie arbeidsintensief is nie, wat volgens die literatuur, resultate lewer wat goed korreleer met die konvensionele gebruik van perifere bloed mononukliere selle (PBMC’s). Dit wil voorkom asof ouderdom ‘n belangrike veranderlike is in die kwantitatiewe beoordeling van sellulere immuunrespons (wanneer PHA gebruik word as ‘n stimulant), en of die immuunrespons van ouer TB-pasiente verbeter na TB-behandeling in vergeleke met die respons voor TB-behandeling.

Thesis (PhD)--Stellenbosch University, 2004 / ENGLISH ABSTRACT: It is estimated that about one third of the world's population is infected with Mycobacterium tuberculosis (M. tuberculosis). Of those infected, only 10 % will develop disease of which 3-5 % will relapse after completion of treatment. Susceptibility to M tuberculosis or relapse following treatment, may be due to environmental influences such as poverty-related factors including intestinal parasites (helminths), and/or genetic factors, all of which can influence the immune response to M. tuberculosis. In the current study, the epidemiology of mycobacterial infection and helminths was studied in two adjacent suburbs of Cape Town, South Africa. These communities had a tuberculosis notification rate of over 1 000/100 000 population with rampant infestations by helminths such as Ascaris lumbricoides and Trichuris trichiura. M. tuberculosis infection and Bacille Calmette Guerin (BCG) vaccination induce a Thl (type 1) immune response, while a Th2 (type 2) immune response is required for expulsion of intestinal parasites. Type 1 and type 2 responses negatively cross regulate each other in vitro and in experimental models. The interaction of these two immune responses in the study community, were investigated. It was hypothesised that susceptibility to M. tuberculosis and progression to disease may be increased in individuals mounting prominent type 2 immune responses, manifested by high serum IgE levels. Furthermore it is proposed that that poverty-related factors and intestinal parasites, specifically those trafficking through the lungs, could further augment the type 2 dominance in the study community. Results presented show that serum IgE concentrations, surrogate marker for type 2 activation, were high among healthy adults, confirming the dominance of type 2 responses. When characterised in census blocks or enumerator sub-districts (ESDs), IgE levels correlated with the tuberculosis notification rate per ESD. The notification rate of tuberculosis also correlated with the socio-economic status, female literacy and population density of the study population. Although these correlations do not necessarily imply a causal relationship, these factors are associated with susceptibility to M. tuberculosis. It was also shown that IgE concentrations decreased significantly after successful treatment of tuberculosis, showing that IgE concentrations in humans can be down-regulated under these circumstances, presumably due to enhancement of a type 1 response. Furthermore, as a reason for the high serum IgE concentrations in the study population, the helminth burden was subsequently measured in all primary school children in the study community. Results show that more than 50 % of the children recruited were infected with A. lumbricoides and/or T. trichiura. Schools situated in the poorest areas with the highest tuberculosis notification rates, presented with the highest prevalences of helminths. All the children, irrespective of their helminth status or their participation in the study, subsequently received ant-helminthic treatment. The BCG vaccination scar status and Mantoux skin test responses were available on a sub-sample of the above-mentioned school children. Although it is assumed that most children receive BCG vaccination in the neonatal period, only two thirds of the children had evidence of a BCG scar. The results show that the prevalence of BCG scar positivity, while independent of age, was lower in children around 11 years of age. In contrast to the broad constancy of BCG scar expression, the percentage of children showing Mantoux reactivity increased with age, from 13 % at 6 years to 65 % at mid teenage. The time course of Mantoux conversion with age indicated that any tuberculin sensitivity, induced by the BCG, waned within the first few years of life and that PPD responsiveness thereafter was induced by environmental exposure to M. tuberculosis. Contrary to the ThllTh2 paradigm, the prevalence of helminth infection in children with a BCG scar was marginally lower than in those without one. A relatively weak positive correlation was found between tuberculin responsiveness and helminth infection and this correlation was most marked in children without a BCG scar. In this subgroup, children who were infected with helminths were more likely to be PPD responsive than those who were not infected. The data showed that conversion to PPD sensitivity predisposed to helminth infection. The results suggest that the effect of helminth infection on the development of clinical tuberculosis is such that those with large worm burdens and who make good PPD responses are likely to be resistant whereas those who deal very effectively with these parasites and who make weaker PPD responses are more likely to be susceptible. The data also indicate that the BCG vaccine used in this study does not give rise to a latent infection whereas the pathogenic M. tuberculosis does so and repeatedly stimulates an immune response to it. In a separate study, it was demonstrated how the host response to M. tuberculosis differs in patients at risk for developing tuberculosis after successful completion of treatment, compared to those who have protective immunity. Individuals participating in the study were also interviewed to understand their social and economic background and how it relates to the disease. Purified protein derivative (PPD) and M. tuberculosis-induced cytokine responses were determined in the study groups. The results show that single immunological marker of susceptibility could not be distinguished, but rather immunological patterns of susceptibility were observéd. Individuals who have had tuberculosis once before and who had been cured, presented with an immuno-suppressive profile, which included high concentrations of IL-lO, TGF-13 as well as high IgE levels. This type of profile suggests that although these individuals have had tuberculosis once before, they have not acquired protective immunity and would be susceptible to reinfection and progression to disease. Furthermore, the interviews conducted showed that most of the people included in this study were poor, unemployed, undernourished and lived in overcrowded conditions. It seems inevitable that those individuals with the immuno-suppressed profile living in poverty would present with a second episode of tuberculosis in the near future. We conclude that in the study community, which has a typical third world setting, poverty-related factors including helminths, could contribute to a dominant type 2 immune response which in tum, would down-regulate the protective type I response, resulting in an enhanced susceptibility to M. tuberculosis and progression to disease. / AFRIKAANSE OPSOMMING: Dit word beraam dat ongeveer een derde van die wêreld se populasie geïnfekteer is met Mycobacterium tuberculosis (M. tuberculosis). Van diegene wat wel geïnfekteer is, sal slegs 10 % siekte ontwikkel met 3-5 % wat 'n relaps episode sal ervaar na voltooiing van behandeling. Vatbaarheid vir M. tuberculosis of 'n relaps episode gevolg na behandeling, mag toegeken word aan armoede-verwante faktore wat intestinale parasiete (helminte) asook genetiese faktore, insluit. Hierdie faktore het die vermoë om die immuun respons teen M. tuberculosis te beïnvloed. In die huidige studie, is die epidemiologie van die mikobakteriele infeksie en helminte bestudeer in twee aangrensende voorstede van Kaapstad, Suid Afrika. Hierdie gemeenskappe het 'n tuberkulose aanmeldings koers van 1 000/1 00 000 populasie met verpreide infestasies met helminte soos Ascaris lumbricoides and Trichuris trichiura. Infeksie met M tuberculosis en vaksinasie met Bacille Calmette Guerin (BeG), induseer 'n Th1 (tipe 1) immuun respons, terwyl 'n Th2 immuun respons benodig word vir die eliminasie van intestinale parasiete. Die interaksie tussen die twee immuun response was in die huidige studie populasie bestudeer. Dit word gepostuleer dat persone met 'n sterk tipe 2 immuun respons, gemanifesteer deur hoë serum IgE vlakke, vatbaar is vir infeksie met M. tuberculosis en progressie tot siekte. Verder was dit voorgestel dat armoede-verwante faktore en intestinal parasiete, veral parasiete wat deur die longe beweeg, 'n dominante tipe 2 respons verder kan versterk. Die resultate voorgestel, wys daarop dat serum IgE konsentrasies, 'n surrogaat merker vir tipe 2 aktivering, hoog was in gesonde volwassenenes. Dit het die siening van 'n dominante tipe 2 respons bevestig. IgE vlakke was bereken vir elke sensus blok of enumerator sub-distrik (ESD) en het gekorreleer met die tuberkulose annmeldings koers per ESD. Die aanmeldings koers het ook gekorreleer met die sosio-ekonomiese status, vroulike geletterdheid en populasie digtheid. Alhoewel hierdie korrelasies nie noodwending dui op 'n oorsaak en gevolg verhouding nie, is dit duidelik dat hierdie faktore kan bydra tot vatbaarheid vir M. tuberculosis. Dit was ook getoon dat IgE konsentrasies beduidend afneem na suksesvolle behandeling van tuberkulose. Dit wys daarop dat IgE konsentrasies in mense afgeruleer kan an waarskynlik dui op 'n verhoogde tipe 1 respons. Verder, as 'n rede vir die hoë IgE konsentrasies in die studie populasie, is die helmint ladings gevolglik in alle prim ere skool kinders in die studie populasie, gemeet. Die resultate dui daarop dat meer as 50 % van die kinders ingesluit, geïnfekteer was met A. lumbricoides en/of T. trichiura. Skole in areas met die hoogste armoede syfer en tuberkulose annmeldings koers, het ook die hoogste prevalensie van helminte gehad. Alle kinders, ongeag hulle helmint status of hulle deelname in die studie, het gevolglik anti-helmintiese behandeling ontvang. BeG vaksinasie littekens en Mantoux vel toets response was beskikbaar op 'n subpopulasie van die bogenoemde skool kinders. Alhoewel dit aanvaar word dat die meerderheid van kinders BeG vaksinasie in die neonatale periode ontvang het, het slegs twee derdes van die kinders 'n BeG litteken getoon. Die resulatate dui daarop dat die prevalensie van BeG litteken positiwiteit, onafhanklik van ouderdom, laer was in kinders rondom die ouderdom van 11 jaar. In kontras met die konstante uitdrukking van BeG littekens, het die persentasie van Mantoux reaktiwiteit verhoog met ouderdom vanaf 13 % by 6 jaar tot 65 % teen 15 jarige ouderdom. Die tyd koers van Mantoux omskakeling met ouderdom dui daarop dat tuberkulin sensitiwiteit, geïnduseer deur BeG, afneem binne die eerste paar jaar van lewe en dat PPD responsiwiteit daarna deinduseer word deur omgewings blootstelling aan M. tuberculosis. In kontras met die ThllTh2 paradigma, was die prevalensie van helmint infeksies in kinders met 'n BeG litteken marginaal laer teenoor hulle sonder 'n litteken. 'n Relatiewe swak posititiewe korrelasie was gevind tussen tuberkulin responsiwiteit en helmint infeksie. Hierdie korrelasie was meer beduidend in kinders sonder 'n litteken. In hierdie sub-groep, was die helmintgeïnfekteerde kinders meer geneig om PPD responsief te wees teenoor hulle wat nie geïnfekteer was nie. Die data wys daarop dat omskakeling na PPD sensiwiteit kan lei tot infeksie met helminte. Die resultate stel voor dat die effek van helmint infeksie op die ontwikkeling van kliniese tuberkulose van so 'n aard is dat diegene met groot wurm ladings en wat goeie PPD response toon, meer geneig sal wees om weerstandig te wees. Diegene egter wat die parasiete beter kan beheer and wat goeie PPD response toon, sal meer geneig wees om vatbaar te wees vir tuberkulose. Die data dui ook daarop dat die BeG vaksien wat in die studie gebruik was, nie lei tot latente infeksie nie, terwyl patogene M tuberculosis dit wel doen en herhaardelik die immuun respons sal stimuleer. In 'n aparte studie, was dit gedemonstreer dat die gasheer-respons teen M. tuberculosis in pasiënte wat die gevaar loop om na suksesvolle voltooiing van behandeling, weer tuberkulose te ontwikkel, verskil van diegene wat beskermende immuniteit het. Onderhoude was ook gevoer met indiwidue wat deelgeneem het aan die studie, om ten einde hul sosiale en ekonomiese agtergrond te verstaan en hoe dit gekoppel is aan die siekte. Purified protein derivative (PPD) en M. tuberculosis-geinduseerde sitokien response was in die studie groepe bepaal. Die resultate wys daarop dat alhoewel 'n enkele immunologiese merker nie geidentifiseer kon word nie, was immunologiese patrone vir vatbaarheid welopgemerk. Indiwidue wat reeds een episode van tuberkulose gehad het en suksesvolle behandeling ontvang het, het 'n onderdrukte immuun profiel getoon. Dit het ingesluit hoë vlakke van die sitokiene, IL-lO en TGF-J3 asook hoë vlakke van serum IgE. Hierdie tipe profiel stel voor dat ten spyte van die vorige tuberkulose episode, hierdie persone nie beskermende immunitiet ontwikkel nie en dus vatbaar is vir herinfeksie en progressie tot siekte. Die onderhoude het getoon dat die meerderheid van mense in die studie populasie onder armoedige oorbevolkte omstandighede lewe, wat werkloosheid en ondervoeding insluit. Die studie het verder getoon dat hierdie indiwidue met die onderdrukte immuun profiel en wat in armoede lewe, in die nabye toekoms vatbaar is vir 'n tweede episode van tuberkulose. In die studie gemeenskap, met 'n tipiese derde wêreld opset, is daar gewys dat armoedeverwante faktore en helminte, mag bydra tot 'n dominante tipe 2 immuun respons wat op sy beurt, die beskermende tipe 1 response sal af-reguleer. Dit sal uiteindelik lei tot verhoogde vatbaarheid vir M. tuberculosis en uiteindelik progressie tot siekte (tuberkulose).

published_or_final_version / Surgery / Master / Master of Philosophy

Ticks are obligate haematophagous arthropods that represent a major economic drain upon the world's livestock as well being a significant medical and veterinary risk through the transmission of tick-borne pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. The tick-host relationship is a function of both ecological and physiological factors. Successful feeding requires the effective acquisition and digestion of a bloodmeal by the tick. Acquisition relies upon the ability of the tick to counteract host immune responses induced by the extended feeding periods of ixodid ticks (up to 2 weeks). The host response to tick infestation and the consequent countermeasures employed by the tick, constitute the tick-host interface. The immune response of hosts to Ixodes ricinus infestations was examined through antigenic profiling. The antigens exposed to the host were shown to vary throughout the feeding period and differed between the different development stages of I. ricinus. It was also shown that different host species infested with I. ricinus recognised different antigens. This was true of both natural and non-natural hosts, and even closely related species. Anti-complement activity was investigated in the salivary glands of Ixodes ticks. This activity was shown to inhibit some host species but not others. The pattern of inhibitory activity varied between the tick species tested in a way that was consistent with known tick host-preferences. The mechanisms of anti-complement activity in I. ricinus salivary glands were explored. The alternative but not the classical pathway of complement was inhibited. Activity was present in unfed ticks and throughout the feeding period. Three targets of the complement system were identified as being modulated by the tick. Digestion of the bloodmeal was explored and a haemolytic activity was associated with the salivary glands of I. ricinus ticks. The activity was demonstrated to be Mg²⁺- dependent. In addition, a subtractive cDNA library enriched for saliva-associated transcripts was successfully produced. Random sampling identified putative differentially expressed genes. The results of this thesis illustrate the complexity of tick-host interactions at the molecular level. It is apparent that the research described poses many more questions than answers.

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Doctor of Philosophy / Profound perturbation of epithelial structure is a characteristic feature of the immunopatholoical response to bacterial antigens considered to be central in the pathogenesis of the destructive lesion of periodontitis. The pathological basis for the disturbance of epithelial structure is not understood. It was demonstrated that the structural integrity and functional differentiation of the lining epithelium is compromised in relation to inflammatory changes associated with destructive periodontitis. In the pathological lining epithelium of the periodontal pocket there was a marked reduction of epithelial cadherin important in intercellular adhesion, of involucrin, a marker of terminal differentiation, and of the gap junction connexions that form intercellular communication channels. These changes were associated with alterations of filamentous actin expression, collectively indicating profound perturbation of epithelial structure. The data reported support the concept that the ability of the pathological lining epithelium to function as an effective barrier against the ingress of microbial products into the tissues is severely compromised (Ye et al., 2000). In addition, a recent study (Ye et al., 2003) by Western analysis of serum IgG from all 22 patients with chronic periodontitis tested indicated recognition of multiple epithelial components in individual patterns. In contrast, subjects with a healthy periodontium displayed only trace recognition of epithelial antigens. Levels of epithelial-reactive antibodies were significantly correlated with attachment loss as an indication of disease activity. To investigate a possible relationship between the bacterial flora adjacent to the diseased sites and the presence of epithelial-reactive antibodies, subgingival plague samples were taken from deep periodontal pockets and cultured anaerobically. Gram positive bacteria containing antigens potentially cross-reactive with epithelial cells were reproducibly isolated by probing membrane colony lifts with affinity-isolated (epitheial-specific) antibodies. The bacteria were identified as streptococci (S. mitis, S. constellatus and two S. intermedius strains) and Actinomyces (A. georgiae, and A. sp. oral clone) by 16S rDNA sequence homology. Recognition by affinity-isolated antibodies of antigens from the captured organisms was confirmed by Western analysis. Conversely, absorption of affinity-isolated antibodies with bacterial species specifically reduced subsequent recognition of epithelial antigens. To identify the auto-antigens, a human keratinocyte cDNA expression library in Lambda phage was probed using a pooled sera. Groups of responders were detected for CD24 (a recently described adhesion molecule also known as P-selectin ligand), antioxidant protein 2 (a newly recognised member of the thiol-dependment anti-oxidant proteins), lavtate dehydrogenase A, the transcription factor NFAT5, and for three genes encoding novel proteins. Six identified bacteria, especially S intermedius were demonstrated to absorb antibodies reaching with identified auto-antigens in patterns varying between individuals. This evidence indicated that during the course of periodontits, subjects develop increased levels of antibodies to common oral bacteria amongst which are included tissue cross-reactive antigens. Periodontitis could therefore present a risk for the subsequent initiation or exacerbation of a broad spectrum of disease processes including autoimmune, inflammatory, proliferative and degenerative disorders.

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Bibliography: leaves 113-144. Investigates the functions of the maternal, protein secretions of the endoparasitoid wasps Venturia canescens Gravenhorst regarding their role in providing protection against the host's immune system. Also investigates mucinous protein secretions on the egg surface and coagulation reactions and their role in protecting eggs against host cellular attacks.