Thesis (M.A.)--University of North Carolina Wilmington, 2007. / Includes bibliographical references (leaves: 92-96)

Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Renal transplantation is undoubtedly the best treatment for patients with irreversible renal failure. As a prelude to establishing the nature of malignancies in renal transplant patients we sought to determine factors influencing the outcome of renal transplantation. The survival of renal allografts and of recipients is influenced by a number of demographic, clinical and therapeutic factors. Some of these factors have been better studied than others, and we sought to establish the influence of particular factors on our own patients and allografts. The total number and nature of malignancies developing in these patients subsequent to transplantation was also established. All patients transplanted in our unit between April 1, 1976 and March 31, 1999 were included in the study. In the study period, 542 patients received 623 renal allografts. Demographic details were analysed. Patient and graft outcomes were assessed using Kaplan-Meier survival analysis. The survival curves were compared using univariate analysis; results that were significant were subjected to multivariate analysis. The influence of a number of factors on graft and patient survival were assessed and compared. The impact of a variety of variables on the number and behaviour of malignancies was also established. Patient and graft survival were superior in recipients who were aged less than 40 years; cyclosporine improved graft survival but not patient survival. Early graft loss was associated with a high patient mortality rate. Contrary to the experience elsewhere, black and white patients had similar outcomes after renal transplantation. Of the 542 recipients 41(8.1%) developed malignancies with Kaposi's sarcoma occurring, in 21 patients and skin cancers in 13 patients. The relative risk for the Kaposi's sarcoma development was 235. Kaposi's sarcoma was the most common tumour in non-white patients (accounting for 79% of malignancies in this group) and occurred less than 2 years after transplantation. Kaposi's sarcoma was equally common in male and female recipients. Under cyclosporine the latent period to malignancies was reduced but the frequency remained unaffected. Kaposi's sarcoma skin lesions were present in all the affected patients, with the lower limbs the most common site of involvement. Kaposi's sarcoma responded to reduction of immunosuppression without the need for complete discontinuation, and with preservation of renal function. Extracutaneous involvement occurred in over one quarter of the patients and invariably proved fatal in all patients with visceral organ involvement. The histopathology of posttransplant Kaposi's sarcoma was the same as that described in the other epidemiological forms of the disease. White male recipients were at the greatest risk of developing skin cancers after renal transplantation. Squamous cell carcinomas were relatively more common and were found in sun-exposed areas. The lesions were treated only by local excision and none metastasized. Malignant lymphoma, breast cancer and lung cancer occurred in individual patients but the relative risk of all these lesions were close to unity. Patients with preexisting cancers did not develop recurrences following transplantation. SECTION 2 Both immunosuppression and immunostimulation are thought to play a role in the development of Kaposi's sarcoma after renal transplantation. We investigated the quantitative and qualitative aspects of the immune system of patients who had developed Kaposi's sarcoma. The lymphocyte phenotypes were established using flow cytometry while transformation studies were performed using mitogens. Pokeweed was used as the B-cell mitogen, and concanavalin A and phytahaemagglutinin were the T-cell mitogens. Cell mediated immunity was also tested using delayed type hypersensitivity skin tests and the serum immunoglobulin levels were estimated. Firstly, with regard to humoral immunity, 2/3 of the patients had normal serum immunoglobulin levels, although the B-cell count was reduced in all the patients on immunosuppression. B-cell transformation tests with pokeweed mitogen revealed that B-cell function was not impaired in patients with Kaposi's sarcoma. The patients with decreased immunoglobulin levels also appeared to be malnourished as evidenced by low albumin levels. Secondly, CD3 and CD4, but not CD8, cell counts were reduced in patients with Kaposi's sarcoma. The transformation analyses revealed significant differences compared to controls, with reduced responses in patients with Kaposi's sarcoma. Thirdly, natural killer (NK) cell numbers were also reduced in patients with Kaposi's sarcoma. There were no significant differences in delayed type hypersensitivity skin reactions that could not be accounted for by racial differences. Cellular immunity is impaired in patients with Kaposi's sarcoma with a reduction in the number of NK cells. Both of these components of the immune system are important in protection against malignant transformation. SECTION 3 Kaposi's sarcoma is an important complication of renal transplantation. If the human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma, the virus should be present in all Kaposi's sarcoma lesions and be drastically reduced or cleared from involved tissue on remission of the Kaposi's sarcoma. Fourteen renal transplant patients with cutaneous Kaposi's sarcoma, including autopsy material from two cases, were investigated for the presence of HHV-8. A second skin biopsy was taken from 11 survivors, after remission of Kaposi's sarcoma, from normal skin in the same anatomical region as the first biopsy. Remission was induced by reduction or cessation of immunosuppression. A peripheral blood sample was collected simultaneously with the repeat biopsy. A nested polymerase chain reaction assay was used to detect HHV-8 DNA in the biopsy tissue and peripheral blood mononuclear cells followed by direct sequencing of polymerase chain reaction product to detect any nucleotide changes. HHV-8 DNA was detected in all the cutaneous Kaposi's sarcoma and all the visceral Kaposi's sarcoma samples, as well as a number of Kaposi's sarcoma-free organs including the thyroid, salivary gland, and myocardium that have not been described before. Mutations in the viral DNA could be demonstrated in all patients. The mutations found were related more to that seen in AIDS-Kaposi's sarcoma cases than that found in African endemic Kaposi's sarcoma cases. HHV-8 sequences could be detected in follow-up frozen skin biopsies of five patients but were negative in the equivalent formalin-fixed specimens. Viral DNA was also detected in 2 of 11 peripheral blood mononuclear cell samples collected at the time of the follow-up skin biopsies. Reduction or withdrawal of immunosuppression allows the immune system to recover sufficiently to reduce viral replication with subsequent viral persistence and low-grade viral replication that coincides with clinical remission of the Kaposi's sarcoma lesions. This provides further evidence for the important etiological role played by HHV-8 in the pathogenesis of posttransplant Kaposi's sarcoma. SECTION 4 The recently discovered HHV-8 is an important factor in the aetiopathogenesis of Kaposi’s sarcoma. The reason for the exceptionally high prevalence of Kaposi's sarcoma in our area, as well as that of other developing countries, remains unexplained. We investigated the seroprevalence of the virus in the different healthy subjects as well as organ donor-recipient pairs. All recipients were tested at the time of transplantation, as were the paired donors. Control subjects tested were healthy blood donors, Renal Unit staff, and household contacts of patients with Kaposi's sarcoma. An enzyme-linked immunoassay (ELISA) to the whole virus was used for screening and all positives were confirmed using ELISA to the latent ORF 73 antigen. The prevalence of HHV-8 was similar in all groups and averaged less than 6%. After transplantation the seroprevalence increased to almost 20% but neither the transplanted kidney nor blood transfused perioperatively could account for the increase. Kaposi's sarcoma developed in 3 of the 116 patients transplanted. All patients with Kaposi's sarcoma were proven to be HHV-8 seropositive before the development of the disease. Two of the patients who developed Kaposi's sarcoma were seropositive before transplantation. No patient who received a graft from a seropositive donor developed Kaposi's sarcoma. We refute the notion that a high prevalence of HHV-8 in the general population is responsible for the high prevalence of Kaposi's sarcoma in our population or that the donor organ is a major source of infection in renal transplant recipients. Reactivation, rather than primary infection appears to be the source of the virus after renal transplantation. / AFRIKAANSE OPSOMMING: Nieroorplanting is ongetwyfeld die beste behandeling vir pasiente met onomkeerbare nierversaking. As ‘n aanloop om die aard van maligniteite in nierooorplantingspas'fente vas te stel het ons gepoog om die faktore wat die uitkoms van nieroorplantings bemvloed te bepaal. Die oorlewing van oorgeplante niere en van nierontvangers word deur ‘n aantal demografiese, kliniese en terapeutiese faktore bemvloed. Sommige van hierdie faktore is beter ondersoek as ander and ons het gepoog om die invloed van sekere faktore op ons eie pasiente en oorgeplante niere te bepaal. Die getal en aard van maligniteite wat ontwikkel het in hierdie pasiente na nieroorplanting is ook gedokumenteer. Alle pasiente in ons eenheid in wie ‘n nier tussen 1 April 1976 en 31 Maart 1999 oorgeplant was, is in die studie ingesluit. Tydens die studieperiode het 542 pasiente 623 niere ontvang. Demografiese detail is ontleed. Pasient- en nieroorplantings uitkomste is beraam deur gebruik te maak van Kaplan-Meier oorlewing analiese. Die oorlewingskurwes is vergelyk deur gebruik te maak van enkelveranderlike ontledings; noemenswardige resultate is onderwerp aan meerveranderlike ontledings. Die invloed van ‘n aantal faktore op oorgeplante nier- en pasientoorlewing is ondersoek en vergelyk. Die impak van ‘n verskeidenheid veranderlikes op die getal en gedrag van maligniteite is ook ondersoek. Pasient oorlewing asook oorlewing van oorgeplante niere was beter in ontvangers onder die ouderdom van veertig jaar. Vroee verlies van ‘n oorgeplante nier het verband gehou met ‘n hoe pasientmortaliteit. Siklosporien het die oorlewing van oorgeplante niere verbeter, maar nie die van pasiente nie. In teenstelling met die ervaring elders, het swart en wit pasiente soortgelyke uitkomste uitkoms gehad na ‘n nieroorplanting. Van die 542 ontvangers, het 41 (8.1%) maligniteite ontwikkel; Kaposi se sarkoom het in 21 pasiente voorgekom en velkanker in 13 pasiente. Die relatiewe risiko (“relative risk") vir die ontwikkeling van Kaposi se sarkoom was 235. Kaposi se sarkoom was die algemeenste tumor in swart en gekleurde pasiente (verantwoordelik vir 79% van maligniteite in die groep) en het binne twee jaar voorgekom. Kaposi se sarkoom was ewe algemeen in manlike en vroulike ontvangers. Met behandeling deur middel van siklosporien het die latente periode totdat maligniteite ontwikkel het verkort, maar die insidensie daarvan het onveranderd gebly. Velletsels geassosieer met Kaposi se sarkoom was teenwoordig in alle pasiente met die vel van die onderste ledemate die mees algemeen betrokke ligging. Die sarkoom het gereageer op vermindering van immuunonderdrukking, sonder die nodigheid vir volkome onttrekking, en met die bewaring van nierfunksie. Ekstrakutane betrokkenheid het in meer as ‘n kwart van die pasiente voorgekom en was altyd noodlottig in pasiente met viserale aantasting. Die histopatologie van postoorplanting Kaposi se sarkoom was dieselfde as die wat beskryf is vir die ander epidemiologiese vorms van die siekte. Wit mans het die hoogste risiko vir die ontwikkeling van velkankers na nieroorplanting gehad. Plaveiselsel karsinoom was betreklik meer algemeen en het in son-blootgestelde areas voorgekom. Die letsels was uitsluitlik met lokale eksisie behandel en geen pasiente het metastases ontwikkel nie. Maligne limfoom, borskanker, en longkanker het in enkele pasiente voorgekom maar die relatiewe risiko van al die letsels was om en by een gewees. Nie een van die pasiente met vorige maligniteite het herhaling van die tumore na oorplanting ontwikkel nie. AFDELING 2 Die vermoede is dat beide immuunonderdrukking en immuunstimulasie ‘n rol speel in die ontwikkeling van Kaposi se sarkoom na ‘n nieroorplanting. Ons het die kwantitiewe en kwalitatiewe aspekte van die immuunsisteem van pasiente wat Kaposi se sarkoom ontwikkel het na ‘n nieroorplanting, ondersoek . Limfosiet fenotipes is met behulp van vloeisitometrie bepaal, terwyl transformasiestudies uitgevoer is deur gebruik te maak van mitogene. “Pokeweed” is gebruik as die B-sel mitogeen, en konkanavalien A en fitaheemagglutinien was die T-sel mitogene. Sel-gemedieerde immuniteit was ook getoets deur die gebruik van vertraagde tipe hipersensitiwiteit veltoetse. Die serum immunoglobulien vlakke was ook bepaal. Eerstens, met betrekking tot humorale immuniteit, het 2/3 van die pasiente normale serum immunoglobulienvlakke gehad, alhoewel die B-seltelling verminder was in al die pasiente op immuunonderdrukking. B-seltransformasie-toetse met “pokeweed” mitogeen het getoon dat B-sel funksie nie ingekort was in pasiente met Kaposi se sarkoom nie. Die pasiente met verminderde serum immunoglobulinvlakke het ook wangevoed voorgekom soos die verlaagde serum albumienvlakke uitgewys het. Tweedens was CD3 en CD4 seltellings, maar nie CD8 nie, verlaag in pasiente met Kaposi se sarkoom. Betekenisvolle verskille is ook aangetoon met T-sel transformasietoetse in vergelyking met kontroles, met verminderde response in Kaposi se sarkoom pasiente. Derdens was natuurlike dodersel (NK) getalle ook minder in pasiente met Kaposi se sarkoom. Daar was geen noemenswaardige verskille in vetraagde tipe hipersensitiwiteit velreaksies wat nie deur rasseverskille kon verklaar word nie. Sellulere immuniteit is ingekort in pasiente met Kaposi se sarkoom met ‘n verlaging in die aantal NK selle. Beide die komponente van die immuunstelsel is belangrik vir beskerming teen maligne transformasie. AFDELING 3 Kaposi se sarkoom is ‘n belangrike komplikasie van nieroorplanting. As die menslike herpesvirus-8 (HHV-8) Kaposi se sarkoom veroorsaak, behoort die virus teenwoordig te wees in alle letsels en as Kaposi se sarkoom remissie ondergaan behoort dit drasties te verminder of te verdwyn in weefsel waarin dit voorkom. Veertien nieroorplantingspasiente met Kaposi se sarkoom van die vel, insluitend outopsiemateriaal van twee gevalle, is ondersoek vir die teenwoordigheid van HHV- 8. ‘n Tweede velbiopsie van dieselfde anatomiese area as die eerste is uitgevoer op 11 oorlewende pasiente na remissie van die sarkoom. Remissie was deur die vermindering of onttrekking van immuunonderdrukking bewerkstellig. ‘n Perifere bloedmonster is by dieselfde geleentheid as die tweede biopsie geneem. ‘n Geneste polimerase kettingreaksietoets (“nested polymerase chain reaction”) is gebruik om die teenwoordigheid van HHV-8 DNA in die biopsieweefsel en perifere bloed mononukluere selle te bepaal, gevolg deur direkte volgordebepaling (“sequencing”) van die polimerase kettingreaksieproduk om enige nukleotiedveranderings te dokumenteer. HHV-8 DNA is waargeneem in al die kutane Kaposi se sarkoom en al die viserale Kaposi se sarkoom monsters, sowel as in weefsel waar die Kaposi se sarkoom nie voorgekom het nie en waar die teenwoordigheid van die virus nie tevore beskryf is nie, soos die skilklier, speekselklier, en hartspier. Mutasies in die virale DNA kon in alle pasiente aangetoon word. Die mutasies wat gevind is, was nader verwant aan die wat in VIGS-Kaposi se sarkoom beskryf is as die wat in endemiese Kaposi se sarkoom in Afrika gevind word. HHV-8 volgordes kon waargeneem word in bevrore opvolg-velbiopsies van vyf pasiente, maar was afwesig in die ekwivalente formaliengefikseerde monsters. Virus DNA is ook waargeneem in 2 van 11 perifere bloed mononukluere selmonsters wat versamel is tydens die opvolg velbiopsies. Vermindering of onttrekking van immuunonderdrukking laat die immuunsisteem toe om genoegsaam te herstel om virale replikasie te verminder met daaropvolgende teenwoordigheid en laegraadse virale replikasie wat ooreenstem met kliniese remissie van letsels van Kaposi se sarkoom. Dit verskaf verdere bewyse van die belangrike oorsaaklike rol wat deur HHV-8 gespeel word in die patogenese van postoorplanting Kaposi se sarkoom. AFDELING 4 Die onlangs-ontdekte HHV-8 is ‘n belangrike faktor in die etiopatogenese van Kaposi se sarkoom. Die rede vir die buitengewone hoe prevalensie in ons gebied, sowel as die van ander ontwikkelende lande, is nie voor die hand liggend nie. Ons het die seroprevalensie van die virus in verskillende gesonde persone, sowel as orgaan donor-ontvanger pare ondersoek. Alle nierontvangers en hul gepaarde donors is getoets ten tye van die nieroorplanting. Getoetste kontrole persone was gesonde bloedskenkers, niereenheid personeel en huishoudlike kontakte van pasiente met vorige Kaposi se sarkoom. ‘n Ensiemgekoppelde immuuntoets (enzyme-linked immnoassay; ELISA) vir die volledige virus was gebruik vir sifting en bevestiging is verkry vir alle positiewe toetse deur gebruik te maak van van ‘n ELISA vir die latente ORF 73 antigeen. Die prevalensie van HHV-8 was vergelykbaar in alle groepe en was gemiddeld minder as 6%. Na oorplanting het die prevalensie gestyg tot byna 20%, maar nog die oorgeplante nier nog perioperatiewe bloedoortappings kom die styging verklaar. Kaposi se sarkoom het in 3 van 116 van die pasiente wat ‘n oorplanting ondergaan het, ontwikkel. Al die pasiente met Kaposi se sarkoom was HHV-8 seropositief voor die ontwikkeling van die Kaposi se sarkoom. Twee van die pasiente wat Kaposi se sarkoom ontwikkel het was seropositief voor die oorplanting. Geen pasient wat ‘n nier van ‘n seropositiewe donor ontvang het, het Kaposi se sarkoom ontwikkel nie. Ons weerle die stelling dat ‘n hoe prevalensie van HHV-8 in die algemene bevolking verantwoordelik is vir die hoe prevalensie van Kaposi se sarkoom onder ons nieroorplantingspasiente of dat die donornier ’n belangrike bron van infeksie is. Dit wil voorkom of heraktivering, eerder as primere infeksie, ‘n bron van die virus is na nieroorplanting.

Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Please see fulltext for abstract. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) affekteer die hele wêreld, maar dit is veral in ontwikkelende lande 'n groot probleem. In Suid-Afrika, soos in baie ander lande, veroorsaak die immuun-paraliserende uitwerking van HIV -koïnfeksie dat die TB-epidemie voortwoeker. Daar is bewyse dat genetiese faktore in die gasheer die uitkoms van die siekte bepaal, aangesien slegs 10% van die individue wat deur Mycobacterium tuberculosis (M tuberculosis) geïnfekteer word, uiteindelik die aktiewe siekte ontwikkel. Die aangebore immuunsisteem is die liggaam se eerste verdedigingslinie, waarna die verworwe immuunreaksie geïnisieer word. Die bakterium se lotgevalle word moontlik bepaal in hierdie vroeë stadium pas nadat dit ingeasem is. Die kollektien-molekule is veral in die long 'n belangrike deel van die aangebore immuunrespons en sluit die mannose-bindende lektien en die surfaktantproteïene A (SP-A) en D (SP-D) in. Dit is al aangetoon dat hierdie drie kollektien-molekule in die gasheer 'n rol speel in die verdediging teen M tuberculosis. In hierdie ondersoek is veral klem gelê op die surfaktantproteïene, wat voorkom asof dit belangrike en kenmerkende rolle speel in die reaksie teen die ingeasemde bakterieë. SP-A versterk die aanhegting van M tuberculosis aan die alveolêre makrofage en verhoog fagositose, terwyl SP-D die bakterieë agglutineer en so verhoed dat dit deur die makrofage gefagositeer word. Gekontroleerde assosiasiestudies in pasiënte is gedoen deur polimorfismes in hierdie gene, wat geassosieer is met TB in ander bevolkings as ons eie, te bestudeer. 'n Polimorfisme in die amino-terminaal area van die SP-D-geen is positief geassosieer met vatbaarheid vir TB. 'n Familie-gebaseerde studie is ook gedoen om die resultate van die gekontroleerde assosiasiestudie te repliseer. Verskillende resultate is verkry en word moontlik bepaal deur die familiestruktuur wat gebruik is. Die aantal families wat bestudeer is, was relatief min en daarom kan daar nie afgelei word dat die assosiasie wat voorheen waargeneem is vals is nie. 'n Groter studie sal gedoen moet word. Die impak van hierdie polimorfisme is verder ondersoek om te bepaal of dit die totale struktuur van die proteïen beïnvloed. Die effekte van hierdie polimorfisme op die konsentrasie van SP-D in die serum van aktiewe TB-pasiënte is ondersoek en vergelyk met die van kontroles. Ons kon nie vasstel watter rol, indien enige, die polimorfisme in die totale struktuur van die SP-D-molekule speel nie, maar ons het bewys dat die serumkonsentrasie van SP-D beduidend verhoog was in aktiewe TB-pasiënte in vergelyking met kontroles (p < 0.0001). Verder het ons ook gedemonstreer dat die konsentrasie van SP-D beduidend verhoog was in die IT-genotipe van die aktiewe TB pasiënte vergeleke met die kontroles (p < 0.0001). Die IT-genotipe is al voorheen positief geassosieer met vatbaarheid vir TB (T. Roos, ongepubliseerde inligting). Verskeie alleliese variante is geïdentifiseer in die SP-A-gene (SP-A1 en SP-A2) wat saam die volle funksionele SP-A-proteïen vorm. Polimorfismes wat onlangs in die kollageen-agtige area van SP-A 1 en SP-A2 gevind is (Madan et al., 2002) en een nuwe polimorfisme wat in hierdie studie geïdentifiseer is, is ondersoek in 'n Suid-Afrikaanse bevolking. Ons het beduidende positiewe assosiasie tussen 'n polimorfisme in die kollageen-agtige area van die SP-A2 geen en vatbaarheid vir TB (p = 0.007) aangetoon. Ons bevindinge bewys die belangrikheid van die bestudering van mensgenetika, wat die immuunkompetensie rig, om vatbaarheid vir infektiewe siektes te verstaan.

Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The solute carrier family 11 member 1 gene (SLC11A 1) is a divalent metal ion transporter with various pleiotropic effects on macrophage function. This gene that regulates iron, and is also regulated by cellular iron levels, has previously been linked to many infectious and autoimmune diseases. In this analysis, in vitro studies using the luciferase reporter system as well as case-control association studies were applied to investigate the significance of SLC11 A1 allelic variation in patients with diverse disease phenotypes. For in vitro studies, five different SLC11A 1 promoter constructs were generated, followed by transfection into U937 and THP-1 cells. The inserted fragments included two previously described alleles (alleles 2 and 3), two novel alleles identified in this study (alleles 8 and 9) and a C to T point mutation at nucleotide position -237 in the presence of allele 3. The most striking finding was the opposite effect observed for allele 3 in the presence of the -237C~ T polymorphism, similar to that of allele 2. Although the SLC11A 1 gene has previously been implicated in iron transport, we have demonstrated, for the first time, that the various alleles investigated cause differential expression of the gene upon iron loading. Association studies were performed by investigating diseases including oesophageal cancer (DC), inflammatory bowel disease (lBO) and hereditary haemochromatosis (HH) (or primary iron overload). Significant associations (P<O.05) were observed with allele 3 for all three conditions investigated only after stratification according to the presence of the -237C~ T polymorphism. Re-assessment of the promoter alleles according to expression profiles determined by the in vitro studies, showed statistically significant associations for allele 3 with DC and primary iron overload, compared with the respective population-matched control groups. Additionally, several novel variants were identified in exon 2 (112G~A, 148deIGACCAGCCC, 157insGACCAGCCCAG) and intron 1 (IVS1-28C~T), with variant IVS1-28C~T occurring at a significantly increased frequency in patients with DC compared with population-matched controls (P<O.05). Investigation of the SLC11A 1 gene in individuals presenting with iron overload in the absence of homozygosity for the HFE C282Y mutation, provided further support for the importance of sequence variation in the promoter region of the SLC11A 1 gene in modified risk of iron-related disorders. Genes related to iron homeostasis, including HFE, SLC11A3, HAMP and DCYTB, were investigated in individuals with similar criteria and potential disease-causing mutations were identified in 11% White and 45% Black South African patients. The possible significance of the SLC11A3 and DCYTB genes in iron overload in the Black South African population, and the possible involvement of the DCYTB gene in iron overload in general, are demonstrated for the first time. This study contributed to a better understanding of the function of the SLC11A 1 gene in relation to iron metabolism. The involvement of SLC11A 1 in a range of disease phenotypes including cancer and inflammatory conditions that may involve iron dysregulation, can probably be explained by interaction with external factors such as infectious agents that may affect cellular iron status. Our findings provide both in vivo and in vitro evidence that iron dysregulation mediated by allelic effects of SLC11A 1 may underlie disease susceptibility to infectious and autoimmune conditions. / AFRIKAANSE OPSOMMING: Die opgeloste stof draer familie 11 deel 1 geen (SLC11 A 1) is 'n divalente metaal ioon vervoerder met verskeie pleiotropiese effekte op makrofaagfunksie. Die geen, wat yster reguleer en ook deur sellulêre ystervlakke gereguleer word, is voorheen verbind met verskeie infektiewe en outo-immune siektes. In hierdie studie is in vitro analises, deur middel van die lusiferase verklikker sisteem, asook gevalle-kontrole assosiasie studies gebruik om die rol van SLC11A 1 alleel variasie in pasiënte met diverse siektefenotipes te ondersoek. Vyf verskillende SLC11A 1 promotor variante is geskep vir in vitro studies en gevolg deur transfeksie in U937 en THP-1 sellyne. Die ingevoegde fragmente het twee voorheen beskryfde allele (allele 2 en 3), twee nuwe allele wat in hierdie studie geïdentifiseer is (allele 8 en 9) en In C na T puntmutasie by nukleotied posisie -237 in die teenwoordigheid van alleel 3 ingesluit. Die opvallendste bevinding was die teenoorgestelde effek wat waargeneem is wanneer alleel 3 in die teenwoordigheid van die -237C~ T polimorfisme voorkom, soortgelyk aan alleel 2 uitdrukking. Alhoewel die SLC11A1 geen voorheen geïmpliseer is in yster vervoer, is daar vir die eerste keer aangetoon dat na yster lading, die verskillende allele differensiële uitdrukking van die geen veroorsaak. Verskeie siektes, insluitend slukderm kanker (OC), inflammatoriese dermsiekte (lBO) en oorerflike hemochromatose (HH) (of primêre ysteroorlading), is ondersoek deur middel van assosiasie studies. Betekenisvolle verskille (P<O.05) is waargeneem vir alleel 3 tussen die kontrole- en pasiëntgroepe in al drie siektes wat ondersoek is, maar slegs na stratifikasie volgens die teenwoordigheid van die -237C~ T polimorfisme. Na hersiening van die promotor allele volgens ekspressie profiele verkry met in vitro studies is statisties betekenisvolle assosiasie ook verkry vir alleel 3 met OC en primêre ysteroorlading in vergelyking met die onderskeie populasie kontrolegroepe. Verder is verskeie nuwe variante ook geïdentifiseer in ekson 2 (112G~A, 148deIGACCAGCCC, 157insGACCAGCCCAG) en intron 1 (IVS1- 28C~ T) en 'n statisties betekenisvolle verhoogde frekwensie van variant IVS1- 28C~ T is waargeneem in pasiënte met OC in vergelyking met die populasie kontrolegroep (P<O.05). Die belangrikheid van variasie in die promotor area van die SLC11A 1 geen as 'n modifiserende faktor in ysterverwante siektes, is verder ondersteun deur die SLC11A 1 geen in individue met ysteroorlading in die afwesigheid van homosigositeit vir die HFE C282Y mutasie te ondersoek. Ander gene geassosieerd met yster homeostase, insluitend HFE, SLC11A3, HAMP and DCYTB, is ondersoek in individue met soortgelyke seleksie kriteria en potensiële siekte-verwante mutasies is geïdentifiseer in 11% Wit en 45% Swart Suid-Afrikaanse pasiënte. Die moontlike belang van die SLC11A3 en DCYTB gene in ysteroorlading in die Swart Suid-Afrikaanse populasie en die moontlike betrokkenheid van die DCYTB geen in yster oorlading oor die algemeen, is vir die eerste keer aangetoon. Hierdie studie dra by tot 'n beter insig in die funksie van die SLC11A 1 geen ten opsigte van ystermetabolisme. Die betrokkenheid van SLC11A 1 in 'n reeks siekte fenotipes, wat insluit kanker en inflammatoriese toestande wat verband kan hou met 'n yster wanbalans, kan moontlik verklaar word deur interaksie met eksterne faktore soos infektiewe agente wat die sellulêre yster status kan beïnvloed. Ons bevindinge verskaf beide in vivo en in vitro getuienis dat yster wanbalans, wat bemiddel word deur alleliese effekte van SLC11A1, verantwoordelik mag wees vir vatbaarheid vir infektiewe en outoimmune siekte toestande.

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).

Dissertation (PhD)--Stellenbosch University, 2007. / ENGLISH ABSTRACT: This research investigated human candidate genes for susceptibility to tuberculosis and the effect of various factors on time to sputum conversion in the admixed South African Coloured (SAC) population. Population stratification was formally tested and excluded. Population based casecontrol studies were the primary analysis method with a variety of genotyping methods. Candidate polymorphisms in RANTES, CCR5, CCR2 and SDF1, were not associated with tuberculosis susceptibility. Initially the RANTES polymorphism -403 was found to be associated with tuberculosis susceptibility but after the testing of additional samples the association was lost, illustrating the challenges with association studies. The C-type lectins DC-SIGN, encoded by the gene CD209, and L-SIGN are important pathogen-recognition receptors of the human innate immune response. Both lectins have been shown to interact with Mycobacterium tuberculosis. CD209 promoter polymorphisms, -336 and - 871, were both found to be associated with tuberculosis susceptibility. The haplotype containing CD209 -871G and -336A was strongly associated with the control group. The CD209 -336A allele has been found to be associated with increased DC-SIGN expression, which may be the underlying reason for an increased efficiency of host phagocytes. Susceptibility to tuberculosis in mice has recently been attributed to the Ipr1 gene. Eight polymorphisms in the human homologue, SP110, were investigated, including two novel polymorphisms. No significant associations were found with any of the polymorphisms investigated, including two polymorphisms that were previously found to be associated with tuberculosis susceptibility in West African populations. A cohort of 249 cases from a longitudinal study of first time pulmonary tuberculosis patients was available. The cohort was used to investigate if the vitamin D receptor gene (VDR) polymorphisms FokI, ApaI and TaqI were associated with tuberculosis susceptibility or time to sputum conversion, and to investigate other clinical and demographic factors affecting the rate of response to treatment. No association between the VDR genotype and tuberculosis was found in the case-control study. The cohort allowed for a reliable conversion time to be determined for smear (n=220) and culture (n=222). Analysis was carried out to determine which factors, including VDR FokI, ApaI, and TaqI genotypes, contribute to faster mycobacterial resolution in sputum. This was done by survival curves and Cox regression models. The results indicate that the extent of disease at diagnosis was predictive of both smear and culture conversion times in the final models. Smoking status and VDR genotype contributed independently to smear conversion time, with ApaI ‘AA’ and TaqI ‘T’ containing genotypes being predictive of a faster response to tuberculosis therapy. We can conclude that the time taken for an individual to convert to sputum negativity while on DOTS therapy, can be independently predicted by the VDR genotype. This may have implications for future immunomodulatory therapies. Identifying what contributes to susceptibility to tuberculosis will provide us with a better understanding of the human immune response to tuberculosis which may lead to the development of accurately targeted therapeutics and vaccines. / AFRIKAANSE OPSOMMING: Kandidaatgene vir die vatbaarheid vir tuberkulose en die effek van verskeie faktore op sputum oorgangstyd was in hierdie navorsingsstudie ondersoek in die Suid-Afrikaanse Kleurlingbevolking (SAC). Dié bevolking was ook getoets vir populasie-stratifikasie, waarvan daar geen bewyse gevind is nie. Populasiegebaseerde pasiënt-kontrole studies was die primêre metode van analise en verskeie genotipering metodes was gebruik. Polimorfismes in kandidaatgene soos RANTES, CCR5, CCR2 en SDF1 was nie met die vatbaarheid van tuberkulose geassosieer nie. Oorspronklik was daar ‘n assosiasie met die RANTES -403 polimorfisme, maar met die genotipering van addisionele individue het die assosiasie verdwyn. Resultate verkry vir die polimorfisme illustreer die uitdagings waaraan assosiasie studies onderworpe is. Die C-tipe lektiene DC-SIGN, wat gekodeer word deur CD209, en L-SIGN is belangrike patogeen herkenningsreseptore in die aangebore immuunreaksie. Interaksies tussen beide lektiene en Mycobacterium tuberculosis is voorheen gerapporteer. Die CD209 promoter polimorfismes, -336 en -871, was met die vatbaarheid van tuberkulose geassosieer. ‘n Haplotipe bestaande uit die CD209 -871G en -336A allele was sterk met die kontrole groep geassosieer. Die CD209 -336A alleel was geassosieer met ‘n toename in die DC-SIGN proteïen vlakke, wat moontlik ‘n onderliggende rede is vir die toename in die effektiwiteit van die gasheer se fagosiete. Vatbaarheid vir tuberkulose is onlangs toegeskryf aan die Ipr1 geen in muise. Agt polimorfismes, insluitend 2 voorheen onbekendes, was in die mens homoloog SP110 bestudeer. Geen positiewe beduidende assosiasie was met enige van die polimorfismes gevind nie ten spyte van die feit dat twee van hierdie polimorfismes voorheen met tuberkulose vatbaarheid geassosieer was in bevolkings van Wes-Afrika. ‘n Versameling van 249 TB pasiënte van ‘n longitudinale studie was beskikbaar. Dié groep was gebruik om polimorfismes FokI, ApaI and TaqI in die vitamien D reseptor geen (VDR) te bestudeer ten opsigte van vatbaarheid vir tuberkulose of sputum oorgangstyd sowel as ander kliniese en demografiese faktore wat die tempo van respons op behandeling kan affekteer. In hierdie studie was daar geen assosiasie gevind tussen die ontwikkeling van tuberkulose en die VDR genotipes nie. Die bepaling van ‘n betroubare oorgangstyd vir beide smeer en kultuur van die groep was moontlik. Analises was uitgevoer om te bepaal watter faktore bydrae tot vinniger resolusie van Mycobacteria in sputum. Resultate verkry het aangedui dat die aard van die siekte tydens diagnose voorspelbaar was van die oorgangstye van beide smeer en kultuur in die finale modelle. Die rookstatus van individue sowel as die VDR genotipes het onafhanklik bygedrae tot die oorgangstyd van die smeer, met ApaI ‘AA’ en TaqI ‘T’ bevattende genotipes wat ‘n vinniger reaksie op tuberkulose behandeling voorspel het. Ter opsomming, die tyd wat dit ‘n individu op DOTS terapie neem om na sputum negatief oor te gaan kan onafhanklik deur die VDR genotipe voorspel word. Dit kan moontlik implikasies hê vir ander immunomodulerende terapië in die toekoms. Die identifisering van faktore wat bydra tot die vatbaarheid van turberkulose sal ons in staat stel om ‘n beter begrip te hê van die immuunrespons teen tuberkulose wat moontlik kan lei tot die ontwikkeling van akkurate behandelings en inentings.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Children in socioeconomically disadvantaged communities in the Western Cape and elsewhere in South Africa are frequently infected by Trichuris trichiura. Although albendazole and mebendazole are listed as essential drugs for treatment of humans, it is well known that trichuriasis is relatively refractory to anthelmintic treatment. There are some reports that mebendazole is more effective against Trichuris and it is available as generics that are relatively cheap. On the other hand, albendazole is better for hookworm and may have some effect against Giardia duodenalis, which is common in the same communities. Moreover, albendazole is used in a deworming programme in KwaZulu-Natal, at a dose o f400 mg stat, given once or twice a year depending on the health region in that province. In terms of diagnosis, infection by intestinal helminthiasis can be determined and monitored by simple, non-invasive, sustainable and cost effective methods. The epidemiological significance of high prevalence extends far beyond the worms per se because they are an index of environmental conditions that pose a risk of several other diseases. These include infection by organisms that can cause epidemics of enteric disease. These facts pertain within a deteriorating milieu in terms of human ecology, because the informal sector of the population is burgeoning under dynamic forces that include urbanisation, migration, poverty and disease. The study reported in this thesis had three main objectives within the context and concepts of the realities described in the previous two paragraphs. The first was to review and consider all information on trichuriasis that could be detected in the literature, and to relate the result to South African needs. The second was to test efficacy of albendazole against trichuriasis in children from a community where it is the predominant worm infection, by means of a well designed and controlled study. Finally, it was necessary to consider other results associated with treatment. These included possible drug resistance and effects on growth, eosinophilia, iron status and toxicity. The literature survey established that South Africa lags behind many other developing countries in defining and addressing the problem of helminthiasis as a whole. The conventional epidemiology of trichuriasis as described in the literature is based mainly on studies in the West Indies. Surveys completed recently in the Western Cape Province of South Africa confirm some of the epidemiological concepts, with two notable exceptions. First, the age-related prevalence peaked in children who were 14 years old in a suburb of Cape Town. This is older that in West Indian children. The second difference is probably more important and was detected in children at schools serving the informal sector o f Khayelitsha. This is densely populated and the sanitation is often not effective and is sometimes totally lacking. The distribution of egg counts in 316 Khayelitsha children was not overdispersed to low counts, which is perceived as invariably the situation in the West Indies. In the Khayelitsha survey, approximately 25% of children had more than 10 000 eggs per g of faeces. The randomised controlled treatment trial tested a series of four albendazole treatments, at doses of400, 800 and 1200 mg (given as 400 mg/day), repeated at intervals of approximately four months in matched groups of children. Results indicate that mass deworming programmes in South Africa should not use albendazole at a dose o f400 mg stat for control of infection by Trichuris trichiura. The package insert of the product tested (Zentel®, SmithKline Beecham) recommends that "in heavy mixed infestation involving Trichuris, a single daily dose may be inadequate and the dose may be given for three consecutive days". This statement is not accurate because even when infection by Trichuris was not intense, as defined internationally in terms of egg counts per g of faeces, and it was the only helminth present, the cure rate achieved by repeated doses of 400 mg of albendazole was not satisfactory. Moreover, 48% (15/31) of treated children remained continuously infected, although egg counts were clearly reduced. This result demonstrates that continuous use of a dose of 400 mg which is the maximum stat dose permitted in South Africa, is likely to facilitate development of true genetic resistance to albendazole by T. trichiura. Doses of 800 and 1200 mg were more efficacious and continuous infection reduced to 21% (9/43) and 2.5% (1/39), respectively. A dose of 800 mg for routine use in mass deworming programmes, as 400 mg/day, can be recommended on the basis of sufficiently efficacious treatment, simplification of compliance, and reduction of cost. The frequency of treatment within such programmes should probably be three times per year when prevalence of severe infection exceeds 10% ("severe infection" is defined internationally as an egg count of more that 10 000 per g of faeces). When severe infection is less frequent, treatment could be less often, but more definitive research of this aspect is necessary under local conditions. There was some evidence that incidence increased seasonally during summer and autumn in the community concerned. A range of other results was recorded. The possibility of genetic resistance to albendazole by Trichuris trichiura was not excluded. There was significant reduction of eosinophiha during treatment with albendazole. This may have immunological implications for incidence, prevention and progression of other diseases. Treatment appeared to be beneficial in terms of growth and iron status, but there was not sufficient statistical power to confirm this. No evidence of toxicity at the highest dose (1200 mg given as 400 mg/day for three days) was detected. / AFRIKAANSE OPSOMMING: Kinders in sosio-ekonomiese agterblewe gemeenskappe in die Wes-Kaap en ook elders in Suid-Afrika toon gereelde infeksies met Trichuris trichiura. Albendazool en mebendazool word gelys as noodsaaklike medikasie vir menslike behandeling, maar dit is wel bekend dat Trichuris relatief moeilik is omte behandel. Daar word gerapporteer dat mebendazool meer effektief is teen Trichuris en maklik beskikbaar is as goedkoper generiese medikasie. Albendazool, aan die ander kant, is effektief teen haakwurm en kan ook ‘n effek het teen Giardia duodenalis, wat voorkom in dieselfde gemeenskappe. ‘n Dosis van 400 mg albendazool, een of twee keer per jaar, word huidelik gebruik in ‘n onwurmingsprogram in KwaZulu-Natal. Die frekwensie van behandeling word bepaal deur die gesondheids streek binne die provinsie. In terme van diagnose, kan infeksie deur intestinale helminte bepaal en gemonitor word deur eenvoudige, nie-invallende, koste effektiewe maniere. Die epidemiologiese betekenis van ‘n hoe voorkoms strek verder as net die wurms omdat hulle ‘n indeks is van omgewings besoedeling wat kan dui op die risiko van verskeie ander siektes. Dit sluit in infeksies deur organismes wat epidemies van enteriese siektes veroorsaak. Hierdie feite pas binne die agteruitgang in terme van menslike ekologie, want die informele sektor van die bevolking is onder dinamiese druk wat verstedeliking, migrasie armoede en siektes insluit. Die studie, waarvan verslag gegee word in hierdie tesis, het drie vemame objektiewe binne die konteks en konsepte van die realiteite soos beskryf in die vorige twee paragrawe. Die eerste objektief was om alle informasie aangaande trichuriase in die literatuur in oorsig te neem en dit in verband te bring met Suid-Afrikaanse behoeftes. Die tweede was om die werksaamheid van albendazool teen trichuriase te toets in kinders van ‘n gemeenskap waar wurm infeksies oorheers, deur middel van ‘n goed beplande en gekontroleerde studie. Laastens was dit nodig om ander resultate, geassosieer met behandeling, in ag te neem. Dit sluit in moontlike weerstand teen die medikasie en die effek op groei, ysterstatus en toksisiteit. Die literatuur oorsig toon dat Suid-Afrika ‘n agterstand het met baie ander ontwikkelende lande om die omslag van die probleem van helminte te bepaal en ook om dit aan te pak. Die konvensionele epidemiologie van trichuriase, soos beskryf in die literatuur, is gegrond op studies in die Wes-Indies. Opnames wat onlangs in die Wes-Kaap gedoen is, bevestig sekere epidemiologiese konsepte met twee vemame uitsonderings. Die eerste is die ouderdoms verwante voorkoms wat ‘n hoogtepunt bereik in 14 jarige kinders in ‘n voorstad van Kaapstad. Dit is ouer as in die Wes-Indiese kinders. Die tweede verskil is miskien meer belangrik en was vasgestel in skool kinders in ‘n informele sektor van Khayelitsha. Dit is ‘n dig bewoonde area met oneffektiewe of ‘n totale gebrek aan sanitasie. Die verspreiding van eier tellings in 316 Khayelitsha kinders was nie oor-versprei tot lae tellings nie, wat andersins die geval in die Wes-Indies was. In die Khayelitsha studie het ongeveer 25% van kinders meer as 10 000 epg stoelgang. Die gekontrolleerde ewekansigheidstoets soos bespreek in hierdie tesis het ‘n reeks van vier albendazool behandelings, by dosisse van 400, 800 en 1200 mg (gegee as 400 mg/dag), herhaal by pouses van ongeveer vier maande in gelyke groepe van kinders, ge-evalueer. Die resultate toon dat massa ontwurmings programme in Suid-Afrika nie die 400 mg dosis vir kontrole van Trichuris trichiura moet gebruik nie. Die pakkie insetsel van die produk wat getoets was (Zentel®, SmithKline Beecham) dui aan dat in swaar gemengde infeksies waar Trichuris betrokke is, ‘n enkele dosis onvoldoende is en dat die dosis vir drie agtereenvolgende dae gegee mag word. Hierdie stelling is onakkuraat want al is die infeksie deur Trichuris nie swaar nie, soos intemasionaal gedefinieer in terme van eier telling per gram stoelgang, en al is dit die enigste intestinale wurm teenwoordig, is die genesingsyfer wat behaal is met herhaalde dossise van 400 mg albendazool nie bevredigend nie. Inteendeel, 48% (15/31) van behandelde kinders was aanhoudend besmet, al was eier tellings duidelik verminder. Hierdie resultaat wys dat herhaaldelike gebruik van ‘n 400 mg dosis, wat die maksimim dosis toelaatbaar in Suid-Afrika is, genetiese weerstand van albendazool deur Trichuris trichiura kan fasiliteer. Die 800 mg en 1200 mg dosis was meer doeltreffend en het herhaalde infeksie verminder tot 21 % (9/43) en 2.5% (1/39) onderskeidelik. Die 800 mg dosis as 400 mg/dag word aanbeveel vir roetine gebruik in massa ontwurmings programme. Dit is meer doeltreffend, behandeling word vereenvoudig en koste word verminder. Die frekwensie van behandeling binne sulke programme moet waarskynlik drie keer per jaar wees wanneer die prevalensie van swaar infeksies meer as 10% is (“swaar infeksie” word intemasionaal gedefinieer, wanneer eier tellings meer as 10 000 eiers per g stoelgang is). As swaar infeksies minder voorkom, kan behandeling minder gereeld wees, maar meer bepaalde navorsing onder plaaslike kondisies is nodig. Daar is sekere bewysse dat insidensie seisoenaal verminder, tydens somer en herfs, in hierdie gemeenskap. ‘n Reeks van ander resultate was ook aangeteken. Die moontlikheid van genetiese weerstand van Trichuris trichiura teen albendazool word nie uitgesluit nie. Daar was ‘n aanmerklike vermindering van eosinophillia tydens behandeling met albendazool. Dit mag immunologiese implikasies vir insidensie, voorkoming en voortgang van ander siektes voorspel. Dit blyk of behandeling groei en yster status bevoordeel het, maar daar was nie genoegsame statistiese mag om dit te bevestig nie. Geen bewys van vergiftiging by die hoogste dosis (1200 mg gegee as 400 mg/dag vir drie dae) is waargeneem nie.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Prostate cancer (CaP) has the highest incidence of any malignancy affecting South African males. The aetiology of prostate carcinoma indicate that ethnicity is one of the most important risk factors. The causes of these ethnic differences are unknown but presumably involve both environmental and genetic factors. Carcinoma of the prostate is androgen dependent, and it has been suggested that variations in androgen metabolism and synthesis may affect an individuals' risk. Therefore, genes involved in these pathways are candidates for determining CaP susceptibility. In this study two candidate genes in the androgen biosynthetic and metabolic pathway were analysed, viz., the androgen receptor gene (AR), involved in androgen transport and transcriptional activation, and the cytochrome p450c17a gene (CYP17), important for testosterone biosynthesis. Comprehensive mutation detection assays were designed (appropriate for analysis of archival paraffin-embedded material) for almost the entire coding region (excluding polymorphic repeat sequences), and including all splice site junctions of the AR gene, as well as the entire coding region of CYP17. The aim of this study was thus to determine the type and frequencies of genetic variants of these androgen metabolism genes within the diverse South African population, and to determine if the observed ethnic variation in the incidence and progression of CaP can be explained by ethnic-based genetic differences. For high sensitivity mutation detection, the most powerful of the pre-screening methods was used, namely denaturing gradient gel electrophoresis (DGGE). 20 CaP and 25 control benign prostatic hyperplasia (BPH) tissue samples were screened in order to identify possible mutations. Blood samples from the same patients were analysed in order to determine whether mutations are germline and therefore present in all cells of the body. Additional blood samples from the Western Province Blood Transfusion Service (WPBTS) (Refer to section 2.1.2, Table) were also analysed in order to determine the frequency of identified polymorphisms within the general population. Certain polymorphisms were further analysed in paraffin-embedded wax material (exclusively from Blacks) to determine the distribution of these polymorphisms in the Black population. Direct sequencing of mutant-containing DNA fragments was performed to determine the exact location and nature of mutation. Using the AR- DGGE assay 4 novel mutations were identified as well as a previously reported codon 211 (E211) polymorphism. With the CYP17- DGGE assay, 3 novel single nucleotide polymorphisms (SNPs) were detected. Three base variants occured, in codons 36 (L36), 46 (H46) and 65 (S65), as well as intronic substitutions in intron 4 (IVS+58G4C) and intron 6 (IVS-25C7A). Frequencies of SNPs were measured in the CaP and BPH samples. In conclusion, the identified polymorphisms could be used as markers in determining CaP susceptibility and may thus facilitate the identification of individuals with a high- or low-risk of developing carcinoma of the prostate. / AFRIKAANSE OPSOMMING: Prostaatkanker vertoon die hoogste voorkoms van enige kwaardaardigheid wat Suid-Afrikaanse mans aantas. Die etiologie van prostaatkarsinoom dui aan dat etnisiteit een van die mees belangrike risikofaktore is. Oorsake van hierdie etniese verskille is onbekend, maar vermoedelik is omgewing en genetiese faktore albei betrokke. Karsinoom van die prostaat is androgeenafhanklik en daar is voorgestel dat variasies in androgeenmetabolisme en androgeensintese 'n persoon se risiko mag affekteer. Gevolglik, is gene betrokke in hierdie paaie kandidate vir die bepaling van prostaatkanker vatbaarheid. In hierdie studie het ons twee kandidaat gene in die androgeen biosintetiese en metaboliese pad geanaliseer, naamlik, die androgeen reseptor geen (AR), betrokke in androgeen vervoer en aktivering van transkripsie, en die sitokroom p450c17a geen (CYP17), belangrik vir testosteroon biosintese. Ons het omvattende mutasie-bespeurings-essai-sisteme ontwikkel (ook uitvoerbaar op argivale paraffien-bewaarde materiaal), wat amper vir die hele koderende streek van die AR geen gebruik kan word (uitsluitend herhalende polimorfiese reekse) en wat alle splytpunt-aansluitings van die AR geen insluit, asook vir die hele koderende streek van CYP17. Die doel van hierdie studie was dus om die tipe en frekwensies van genetiese variante van androgeen metabolisme gene in ons diverse Suid-Afrikaanse bevolking te bepaal, en om vas te stel of die waarneembare etniese wisseling in die insidensie en vordering van prostaatkanker verstaan kan word deur etnies gebaseerde genetiese verskille. Die mees sensitiewe tegniek wat tans beskikbaar is vir vooraf-sifting vir onbekende mutasies is gekies, naamlik denaturerende gradiënt gel elektroforese (DGGE). Om moontlike mutasies op te spoor, het ons 20 prostaatkanker en 25 benijne prostaathiperplasie (BPH) monsters geanaliseer. Analise was gedoen op bloedmonsters van dieselfde pasiënte om vas te stel of kiemlyn mutasies (in alle liggaamselle) teenwoordig is. Bykomstige bloedmonsters (van die Westelike Provinsie Bloedoortappingsdiens) is ook geanaliseer om die frekwensie van bespeurde polimorfismes in die algemene bevolking te bepaal. Argivale paraffien-bewaarde materiaal (eksklusief van Swartes) is ook geanaliseer om die verspreiding van sekere polimorfismes in die Swart bevolking te bepaal. Direkte DNA volgorde bepaling van mutante DNA fragmente is uitgevoer om die ligging en tipe van mutasies te bepaal. Met die toepassing van ons AR-DGGE mutasiesisteem het ons 4 nuwe mutasies ontdek asook 'n kodon 211 (E211) polimorfisme wat voorheen gevind is. Vyf enkel nukleotied polimorfismes is met die CYP17-DGGE mutasiesisteem opgespoor. Die polimorfismes sluit in: drie basis veranderinge wat voorkom in kodons 36 (L36), 46 (H46) en 65 (S65), asook introniese substitutisies in intron 4 (IVS+58G4C) en intron 6 (IVS-25C7 A). Frekwensies van die polimorfismes was bereken in die prostaatkanker en BPH monsters. Die resultate aangebied in hierdie tesis dui aan dat die gevonde polimorfismes as merkers gebruik kan word om prostaatkanker vatbaarheid te bepaal en daardeur individue te identifiseer met 'n hoë of lae risiko vir prostaatkarsinoom ontwikkeling.

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: New agents for the diagnosis, prevention and treatment of tuberculosis are urgently required. Yet, despite extensive tuberculosis research over recent years, no new drugs, vaccines or diagnostics have been identified to date. It is widely speculated that the major obstacle to the identification of new therapies is the lack of understanding of the hostpathogen interaction. This study has investigated whether patterns of antigen expression correlate with molecular epidemiological data and strain virulence through the analysis of protein expression and antigen recognition profiles of different M tuberculosis clinical isolates. Using polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, and Western blotting, protein expression and antigen recognition by two genotypically different clinical strains that differed in their frequency in the study population have been compared. In addition to differences in protein expression and antigen recognition between the clinical strains and the reference strain H37Rv, protein expression differences between the clinical strains themselves were observed which may relate to strain frequency and virulence. Differential protein expression by M tuberculosis strains, may explain the heterogeneous host humoral immune response and why no fully effective serodiagnostic test has been developed to date. To explore this hypothesis, the potential of serodiagnosis in this community, where patients are infected with a wide variety of genotypically distinct strains, was investigated. IgG levels to three mycobacterial antigens showed that serodiagnosis of TB is possible in this community, despite infection by a wide variety of genotypically different M tuberculosis strains. Disease episode affected antibody levels, suggesting that care should be taken when evaluating serological diagnosis for repeat episode patients. This study has shown that M tuberculosis protein expression is dynamic and that the bacillus presents a hypervariabie array of antigens to the host immune system. It is likely that different antigens become immunodominant as antituberculosis chemotherapy progresses, and that these differentially expressed antigens may be tracked as predictors of treatment outcome. This hypothesis was tested by correlating Ag85-specific IgG with treatment response, as assessed by sputum smear conversion after two months of antimycobacterial chemotherapy. No significant correlation between antibody levels and treatment responses was observed, suggesting that antibodies may not be useful surrogate markers or that the incorrect antibody type or mycobacterial antigen were selected. Results were consistent with previous findings where patient-to-patient variation dictated the host humoral response. The results obtained in this study have demonstrated that although bacteriological factors may influence strain prevalence due to antigen variation and immune evasion, both bacteriological and host factors affect humoral immunity. Differential protein expression by M tuberculosis strains has potentially important implications for serodiagnosis and the development of subunit or DNA vaccines, by suggesting that multi-antigen cocktails should be used. Differential protein expression may also explain why patients do not develop adequate protective immunity and are susceptible to reinfection. / AFRIKAANSE OPSOMMING: Daar is 'n dringende behoefte vir nuwe middels vir die diagnosering, voorkoming en behandeling van tuberkulose. Ondanks intense tuberkulose navorsing gedurende die afgelope paar jaar, is daar geen nuwe tuberkulose medikasie, vaksines of diagnostiese metodes geïdentifiseer nie. Daar word gespekuleer dat die hoof struikelblok vir die identifisering van nuwe medikasie die onkunde oor die tuberkulose patogeen is. Deur die analise van proteien-uitdrukking en antigeen-erkenning profiele van verskillende M. tuberculosis kliniese isolate is daar tydens hierdie studie ondersoek ingestel of die patroon van antigeen uitdrukking korreleer met molekulêre epidemiologiese data and stam-virulensie. Proteien-uitdrukking en antigeen-erkenning deur twee genotipies verskillende kliniese stamme wat verskil in hul frekwensie in die bestudeerde populasie, is vergelyk deur middel van poli-akrielamied gel elektroforese, ensiem-gekoppelde immuunabsorberende analise en Westelike oordrag. Addisoneel tot die verskille in proteienuitdrukking en antigeen-ekenning tussen kliniese stamme en die verwysingstam H37Rv, is daar ook verskille aangedui tussen die kliniese stamme self wat kan dui op stam frekwensie en virulensie. Differensiële proteien-uitdrukking deur M. tuberculosis stamme, kan moontlik die heterogene gasheer se humorale immuunreaksie verduidelik en daarmee saam die rede waarom daar nie tot op hede 'n effektiewe sero-diagnostiese toets ontwikkel is nie. Daar is dus ondersoek ingestel na die potensiaal van sero-diagnose in 'n gemeenskap waar pasiënte geïnfekteer is met 'n wye verskeidenheid genotipiese stamme. Die IgG vlakke van drie mikobakteriële antigene het aangedui dat sero-diagnose van tuberkulose moontlik is in hierdie gemeenskap, ten spyte van infektering deur 'n wye verskeidenheid genotipies-verskillende M. tuberculosis stamme. Die tussenspel van die siekte het teenliggaampie-vlakke beïnvloed wat daarop dui dat daar versigtig moet gelet word tydens die evaluering van serologiese diagnose van geïnfekteerde pasiënte wat voorheen siek was. Hierdie studie toon dat M. tuberculosis proteïen-uitdrukking dinamies is en dat die bacillus 'n groot variëteit van antigene tot die immuun sisteem bied. Dit is moontlik dat verskillende antigene immuun dominant kan word soos wat antituberkulose chemoterapie toeneem, en dat hierdie verskillend-uitgedrukte antigene as 'n gevolg daarvan gebruik kan word as voorspellers vir behandeling. Hierdie hipotese is getoets deur die korrelering van Ag85-spesifieke IgG met die reaksie op behandeling soos geëvalueer deur speeksel-monster verandering na twee maande se anti-mikobakteriële chemoterapie. Daar was geen noemenswaardige korrelasie tussen teenliggaampie vlakke en die reaksie op behandeling nie, wat daarop dui dat die teenliggaampies nie toepaslike surrogaat merkers is nie of dat die verkeerde teenliggaampie-tipe of mikobakteriële antigeen geselekteer is. Hierdie resultate bevestig vorige bevindinge waar pasiënt-tot-pasiënt verskille die gasheer se humorale immuunreaksie gedikteer het. Die resultate wat uit hierdie studie volg dui dat alhoewel bakteriologiese faktore die stam-frekwensie kan beïnvloed as gevolg van antigeen-variasie en immuun-ontduiking, kan beide bakteriologiese en gasheer faktore die humorale immuunreaksie beïnvloed. Differensiële proteiën uitdrukking deur 'n verskeidenheid M. tuberculosis stamme het potensieël belangrike toepassings vir sero-diagnose en die ontwikkeling van subeenheid of DNS vaksines wat impliseer dat multi-antigeen mengsels gebruik moet word. Differensiële proteiën uitdrukking mag ook verduidelik waarom pasiënte nie 'n voldoende beskermende immuniteit opbou nie en sodoende ontvanklik is vir her-infeksie.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Hypertensive conditions of pregnancy, such as pre-eclampsia, are the principal direct cause of maternal morbidity and mortality and affect up to 10% of first pregnancies worldwide. The placenta is vital in the pathogenesis of pre-eclampsia since the condition only occurs in the presence of placental tissue and the only cure is delivery of the placenta and the fetus. It has been hypothesised that the placenta may be the source of a circulating factor(s), which transports freely in the maternal system, resulting in the multi-systemic and immunological responses that are characteristic of pre-eclampsia. Among the potential "circulating" candidates currently being investigated worldwide, is the tachykinin member, neurokinin B (NKB). The aim of this project was to use a novel approach and investigate the role of Neurokinin B in pre-eclampsia on a genetic level. This would be achieved by bioinformatie characterisation of the neurokinin B (TAC3) and neurokinin B receptor (TACR3) genes. Samples from thirty pre-eclampsia patients (of whom 10 also had abruptio placentae) and twenty control individuals were used for mutation detection analysis involving Multiphor gel electrophoresis and automated sequencing. Three sequence variants were identified in the TAC3 gene and include: (i) 5' UTR variant (-25 c-t); (ii) intronic variant IVS3-53 (t-g) and (iii) 3' UTR variant exon 7 (479, t-c). Only the -25 c-t variant had been reported before (SNP database). A further two variants were identified in the TACR3 gene: (i) exon 3 variant (nt 857, a-t) and (ii) 3' UTR variant, amplicon 5b (nt 1471, t-c), of which the latter had previously been reported in the SNP database. In the analysis of allele and genotype frequencies, only variant homozygosity for TAC3 -25 c-t could be associated with increased risk of pre-eclampsia (RR 3.33, p=0.03). Follow-up work will include extended genotyping in further stratified and larger patient cohorts and transfection studies to assess splicing potential and functional consequences of the mutant alleles. These data represent the first documented mutation screen of the TAC3 and TACR3 genes and report novel variants in patients with pre-eclampsia. This study contributes to the knowledge of neurokinin B as a circulatory molecule and confirms the heterogeneity of pre-eclampsia. / AFRIKAANSE OPSOMMING: Die belangrikste direkte oorsaak van moedersterftes is hipertensiewe toestande in swangerskap, insluitende pre-eklampsie. Hierdie toestande kompliseer wêreldwyd 10% van alle swangerskappe. Die plasenta is kardinaal in die ontwikkeling van die siekte aangesien dit slegs voorkom terwyl die plasenta in-situ is en die simptome opklaar na verlossing van die plasenta. 'n Moontlike hipotese is dat die plasenta 'n sirkulerende agens afskei wat in die moederlike sisteem beland en die uiteenlopende multi-sistemiese simptome en tekens van die siekte veroorsaak, asook aktivering van die immuunsisteem. Een van die moontlike kandidate wat tans wêreldwyd ondersoek word as moontlike sirkulerende agens, is Neurokinien B (NKB), 'n lid van die Tachikinien familie. Die unieke benadering van hierdie projek was om die rol van Neurokinien B in pre-eklampsie te ondersoek op 'n genetiese grondslag. Dit is bereik deur bio-informatiewe karakterisering van die neurokinien B (TAC3) en neurokinien B reseptor (TACR3) en deur mutasie sifting op DNA monsters van 30 pasiënte met pre-eklampsie (waarvan 10 ook abruptio placentae gehad het) en twintig kontrole individue met behulp van Multiphor gel elektroforese en ge-outomatiseerde volgorde bepaling. Drie volgorde variasies is geïdentifiseer in die TAC3 geen en sluit in: (i) 5' UTR variant (-25 c-t); (ii) introniese variant IVS3-53 (t-g) en (iii) 3' UTR variant in ekson 7 (479, t-e). Slegs die -25 c-t variasie is voorheen raporteer (SNP databasis). Nog twee variante is ook gevind in die TACR3 geen: (i) ekson 3 variant (nt 857, a-t) en (ii) 3' UTR variant, amplikon 5b (nt 1471, t-e); hierdie laaste een is al in die SNP databasis raporteer. In 'n analise van genotipe en allele frekwensies is slegs homosigositeit vir variant TAC3 -25 c-t geassosieër met 'n verhoogde risiko vir preeklampsie (RR 3.33, p=0.03). Verdere werk sal nou fokus op die genotipering van groter en gestratifiseerde pasiënt kohorte en transfeksie studies om splitsing potensiaal en funksionele gevolge van mutante allele te ondersoek. Hierdie data is die eerste gedokumenteerde mutasie sifting van die TAC3 en TACR3 gene en verslag word gelewer van unieke variasies in pasiënte met pre-eklampsie.