Orientadora: Mary Marcondes / Banca: Raimundo Souza Lopes / Banca: Vera Lúcia Fonseca de Camargo Neves / Resumo: A leishmaniose visceral pode ser incluída como uma das causas de miopatia inflamatória em cães, entretanto, pouco se sabe sobre a patogênese da doença no sistema muscular, sendo incriminada muitas vezes apenas à natureza catabólica da enfermidade. O objetivo deste estudo foi avaliar, por meio de imunoistoquímica, a presença de formas amastigotas de Leishmania sp, linfócitos T (CD3+), macrófagos e IgG nos músculos tríceps braquial, extensor carpo radial, bíceps femoral e gastrocnêmio de 23 cães naturalmente acometidos por leishmaniose visceral. Dentre os 92 músculos avaliados,11 (12%) apresentaram marcação antigênica para formas amastigotas de Leishmania sp, 35 (38,1%) para linfócitos T (CD3+), 29 (31,5%) para macrófagos e 14 (12%) para IgG. Os resultados obtidos permitiram concluir que em cães com leishmaniose visceral apresentam imunomarcação para formas amastigotas de Leishmania sp., linfócitos T CD3+, macrófagos e IgG, sugerindo a participação direta do parasito e de uma resposta imune celular e humoral na fisiopatogenia da lesão muscular. / Abstract: Visceral leishmaniasis may be included as a cause of inflammatory myophathy in dogs, however, little is known about the pathogenesis of the disease in the muscular system, which is frequently associated with the catabolic nature of the illness. The purpose of this study was investigate, through immunohistochemistry, the presence of amastigote forms of Leishmania sp, T lymphocytes (CD3+), macrophages and IgG in the muscle triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius of 23 dogs with visceral leishmaniasis. Among 92 evaluated muscles, 11 (12%) presented antigenic marking for amastigote forms of Leishmania sp., 35 (38,1%) for T lymphocites (CD3+), 29 (31,5%) for macrophages and 14 (12%) for IgG. The results of the present experiment led to the conclusion that in dogs with visceral leishmaniasis there may be a straight participation of the parasite and of cellular and humoral immune response in the ethiopatogeny of the muscular injury. / Mestre

Experiments were done to determine the effect of recombinant interleukin 2 (rIL-2) on mononuclear cells (MC) of newborns and adults. MC were tested for (1) ability to lyse Daudi cells in a 51Cr release assay, (2) cell surface markers using monoclonal antibodies and flow cytometer analysis, and (3) cell types as determined by differential cell counts. Without rIL-2 adults show greater cytotoxicity than newborns in vitro. Incubation with rIL-2 dramatically increased the cytotoxicity expressed with cord blood and adult MC showing equivalent responses. Differences in cell surface markers between newborns and adults prior to rIL-2 exposure were in agreement with those previously published. This study did not demonstrate changes in phenotypes after exposure to rIL-2. Slight changes in differential cell counts occurred after increased incubation periods and rIL-2 exposure.

Submitted in complete fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Immunomodulation using plants is of primary interest in scientific communities because it provides an alternative to conventional chemotherapy for a wide range of diseases. It is based on the ability of the plants to effectively modulate immune functions, thus being able to promote positive health and maintain the body’s resistance to infection. This research is aimed to evaluate the immunomodulatory potential of fourteen traditional leafy vegetables from Kwa-Zulu Natal, South Africa on human peripheral blood mononuclear cells (PBMC). In this study the methanolic and aqueous extracts were screened for lymphocyte proliferation using the MTT assay. The cytokine response was evaluated by measuring the secretion of interleukin 10 (IL-10) and interferon-gamma (IFN-γ) using the ELISA assay. The subpopulation of T cells viz., CD4+, CD8+, NK and B cells were measured by flow cytometry. Most of the methanolic extracts stimulated PBMC’s whilst a few suppressed lymphocyte proliferation. Most of the aqueous extracts were inactive. The methanolic extracts of Amaranthus hybridus and Centella asiatica stimulated PBMC’s and showed an increase in IFN-γ secretion and the CD8+ cytotoxic T cells and B cells. Thus, they induced the Tc-1 immune response and stimulated cell mediated immunity. The methanolic extracts of Asystasia gangetica, Bidens pilosa, Emex australis, Justicia flava Momordica balsamina, Oxygonum sinuatum, Senna occidentalis and Sonchus oleraceous and the aqueous extracts of Amaranthus spinosus and Asystasia gangetica, Ceratotheca triloba, Oxygonum sinuatum, Physalis viscosa and Sonchus oleaceous stimulated PBMC’s and showed an increase in IL-10 secretion and the CD8+ cytotoxic T cells and B cells. Thus, they induced the Tc-2 immune response and stimulated humoral immunity. Also, the methanolic extracts of Amaranthus spinosus and Ceratotheca triloba and the aqueous extracts of Bidens pilosa and Justicia flava increased both IL-10 and IFN-γ secretion and the CD8+ vii cytotoxic T cells indicating the stimulation of both the Tc1 and Tc2 cytokine profiles. The elevated secretion of IFN-γ and IL-10 caused by the extracts can be attributed to the CD8+ cytotoxic T cells and B cells. The findings of this study show that leafy vegetables hold promise as immunomodulatory candidates. They may enhance cell-mediated immune functions by a pro-inflammatory response whilst some can promote humoral immune functions by means of an anti-inflammatory response. Further investigation should be considered on the effect of the extracts on other immune parameters. / National Research Foundation

Recently, cholesterol metabolism has been shown to modulate the infection of several viruses and there is growing evidence that inflammatory response to infection also modulates lipid metabolism. However little is known about the role of inflammatory processes in modulating lipid metabolism and their consequences for the viral infection. This study investigates host-lipid viral interaction pathways using mouse cytomegalovirus, a large double-stranded DNA genome, which represents one of the few models for a natural infection of its natural host. In this study, transcriptomic and lipidomic profiling of macrophages shows that there is a specific coordinated regulation of the sterol pathways upon viral infection or treatment with IFNγ or β (but not TNFα, IL1β or IL6) resulting in the decrease of free cellular cholesterol. Furthermore, we show that pharmacological and RNAi inhibition of the sterol pathway augments protection against infection in vitro and in vivo and we identified that the prenylation branch of the sterol metabolic network was involved in the protective response. Finally, we show that genetic knock out of IFNβ results in a partial reduction while genetic knock out of Ifnar1 completely abolishes the reduction of the sterol biosynthetic activity upon infection. Overall these results support a role for part of the sterol metabolic network in protective immunity and show that type 1 IFN signalling is both necessary and sufficient for reducing the sterol metabolic network upon infection; thereby linking the sterol pathway with IFN defence responses.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Chronic HIV-1 infection is characterized by immune activation and dysregulation of immune homeostasis, which impacts on multiple immune cell types. The B-cell compartment, which plays an important role in the producing neutralizing antibodies, is also dysregulated in HIV- 1 infection. In this study we investigated peripheral blood B-cell subset distribution, and changes in expression of cellular activation, inhibition, and apoptosis signaling markers in both untreated chronic HIV-1 infected individuals and healthy uninfected controls. The neuropeptide immune modulator, vasoactive intestinal peptide (VIP) is known to selectively down-regulate activation of CD4+ T-cells in various disease settings including HIV-1, however to our knowledge, no studies have investigated the effect of VIP inhibition on B-cell activation. Materials & Methods: A total of 21 HIV+ve (CD4 count >250 cells/µl), and 19 HIV-ve individuals were recruited from the Emavundleni voluntary testing and counseling clinic in Crossroads, Western Province, South Africa. Whole blood was stained to distinguish B-cell subsets (activated memory (AM: CD21-CD27+), resting memory (RM: CD21+CD27+), mature naïve (MN: CD21+CD27-), or tissue-like memory (TLM: CD21loCD27lo). In addition expression of markers of B-cell activation (CD126, CD86, CD38, CD284, CD287), inhibition (CD72, CD85j, CD300a, CD305, CD307d), and apoptosis signaling (CD95), was assessed ex vivo by flow cytometry (BD FACSCanto II). For determination of functional responsiveness isolated B-cells (RosetteSep, Stemcell Technologies) were cultured for 18h (37°C, 5%CO2) without stimulation or stimulated with TLR ligands (LPS or R848). Stimulation experiments were also performed in the presence or absence of VIP. Results: Chronic HIV-1 infection affected B-cell subset distribution. The percentage (%) of TLM was increased by 59.24%, and %RM was decreased by 22.73% (both p<0.01). Total expression of the VIP receptor VPAC2 was decreased by 47.35% (p=0.0296). Subsets had a mixed phenotype ex vivo; HIV infection upregulated CD38 (by 59.56%, p=0.0004), CD72 (by 60.70%, p=0.0396), CD307d (by 68.63%, p=0.0015) on AM, while RM B-cells had increased expression of TLR4 (by 107.04%, p=0.0057) and TLR7 (by 208.14%, p=0.0199). TLM B cells (i.e. exhausted phenotype) displayed upregulated TLR7 (by 550%, p=0.0128) and CD307d (by 72.40% p=0.045) expression. MN B-cells had increased CD72 expression (by 70.98%, p=0.0026). R848 upregulated CD86 expression by 42.20% on AM (p<0.01), and by 56.06% on RM B-cells (p<0.01), which was significantly downregulated with VIP inhibition (both p<0.05). Similarly, CD95 expression on RM, TLM, and MN B-cells increased by 31.10% (p<0.001), 21.46% (p<0.01), and 39.92% (p<0.01) with R848 stimulation respectively, which was also significantly downregulated with VIP inhibition. Conclusion: These data indicate that B-cells in untreated HIV infection display increased levels of activation, and also the potential for increased susceptibility to apoptosis as evidenced by increased FAS (CD95) expression. VIP significantly down-regulated markers of activation, inhibition, and apoptosis signaling. Dysregulation of B-cells is thus apparent in asymptomatic stable chronic HIV-1 infection, which may impact on both inefficient neutralizing antibody production and hypergammaglobulinemia. The ability of VIP to prevent stimulationassociated marker upregulation may indicate that VIP is a potential therapeutic agent. Its immuno-modulatory properties were demonstrated to limit B-cell hyperactivation, and selectively down-regulate apoptosis and mark it out for further investigation. / AFRIKAANSE OPSOMMING: Inleiding: Immunaktivering en ongekoppelde immuun-homeostase is kenmerke van chroniese MIVinfeksie. Ons het perifere bloed B-sel subgroep-verspreiding, en veranderinge in die uitdrukking van merkers van aktivering, inhibisie, en apoptose in 'n onbehandelde MIV-1 besmettende groep ondersoek (in vergelyk met 'n gesonde onbesmettende kontrole). Die immuun-moduleerder, vasoaktiewe intestinale peptied (VIP) is bekend om aktivasie van geaktiveerde CD4+ T-selle te verminder, maar tot ons kennis, is daar geen studies wat die effek van VIP-inhibisie op B-sel aktivering ondersoek het, in die konteks van MIV-1 infeksie. Materiaal & Metodes: MIV+we individue (CD4-telling >250 selle/µl) , en MIVwe kontroles is gewerf uit die vrywillige toetsing en berading Emavundleni kliniek, Crossroads, Westelike Provinsie, Suid-Afrika. Bsel subgroepe is gedefinieer as geaktiveerde geheue (AM: CD21- CD27+ ), rusende geheue (RM: CD21+ CD27+ ), volwasse naïef (MN: CD21+ CD27- ), of weefsel-agtige geheue (TLM: CD21loCD27lo). Merkers van aktivering (CD126, CD86, CD38, CD284, CD287), inhibisie (CD72, CD85j, CD300a, CD305, CD307d), en apoptose signalering (CD95) is via vloeisitometrie (BD FACSCanto II) op B-selle ex vivo en ook op geïsoleerde B-selle (RosetteSep, Cell Technologies) ondersoek. Vir die bepaling van funksionele responsiwiteit, geïsoleerde B-selle (RosetteSep, StemCell Technologies) was vir 18h (37°C, 5%CO2) gekweek, sonder stimulasie of gestimuleer met TLR ligande (LPS of R848). Stimulasie eksperimente het ook in die teenwoordigheid of afwesigheid van VIP plaasgevind. Resultate: Chroniese MIV-1 infeksie het B-sel subset verspreiding geraak. Die persentasie (%) van TLM is verhoog deur 59,24%, en% RM het met 22.73% afgeneem (beide p <0,01). Totale uitdrukking van die VIP reseptor VPAC2 het met 47,35% afgeneem (p = 0,0296). Subgroepe het 'n gemengde ex vivo fenotipe; MIV-infeksie het CD38 (deur 59,56%, p=0,0004), CD72 (deur 60,70%, p=0,0396), CD307d (deur 68,63%, p=0,0015) op AM verhoog, terwyl RM Bselle het verhoogde uitdrukking van TLR4 (deur 107,04%, p=0,0057) en TLR7 (deur 208,14%, p=0,0199). TLM B-selle (die uitgeputtende fenotiep) het verhoogde TLR7 (deur 550%, p=0,0128) en CD307d (deur 72,40% p=0.045) uitdrukking gewys. MN B-selle het verhoogde uitdrukking van CD72 (deur 70,98%, p = 0,0026). R848 het CD86 uitdrukking op AM deur 42,20%, en op RM deur 56,06% toegeneem (beide p <0,01). Dit het met VIP inhibisie beduidend afgeneem (beide p <0.05). CD95 uitdrukking was soortgelyk verhoog op RM, TLM, en MN B-selle met 31.10% (p <0.001), 21,46% (p <0,01), en 39,92% (p <0,01) met R848 stimulasie. Al drie het beduidend afgeneem met VIP inhibisie. Gevolgtrekking: Hierdie data dui daarop dat B-selle in onbehandelde MIV-infeksie vertoon verhoogde aktiveringsvlakke, en ook die potensiaal vir verhoogde vatbaarheid vir apoptose soos bewys deur verhoogde uitdrukking van FAS (CD95). VIP het beduidend merkers van aktivering, inhibisie, en apoptose af-gereguleer. Wanfunksie van B-selle is dus in asimptomatiese stabiele chroniese MIV-1 infeksie duidelik, wat impak kan hê op beide oneffektiewe neutraliserende teenliggaampie produksie, en hiepergammaglobulinimie. Die vermoë van VIP stimulasie-verwante merker opregulasie te voorkom kan aandui dat VIP 'n potensiële terapeutiese agent is. VIP se immuno-moduleerende eiendomme is gedemonstreer om Bsel hieperaktiveering te beperk, en selektief apoptose afreguleer, en merk dit vir verdere ondersoek.

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 (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The evaluation of the immune responses in peripheral blood and at the site of disease of people with differential outcomes following M.tb exposure will lead to the discovery of host biomarkers that will increase our understanding of the protective and non-protective immune responses against the bacterium. The main study consisted of a number of pilot studies and the objectives of the studies were: (1) To determine the background and stimulated whole blood cytokine profiles of children and adults of the community; (2) to establish biomarker profiles in whole blood of children with different M.tb infection phenotypes; (3) to investigate the anti-mycobacterial whole blood immune responses in HIV infected and uninfected children; (4) and to investigate the role of the innate immune system during human tuberculosis disease. The study designs were as follow: (1) Adults and children were enrolled in order to determine cytokine profiles in the community. Whole blood was stimulated with BCG and ESAT-6 or left unstimulated. Eighteen cytokines were measured in supernatants of each condition. Progression to active tuberculosis in the years after study participation was assessed by searching for patient entries in the tuberculosis register. (2) Children with known tuberculosis exposure in their households and with M.tb infection as assessed through interferon-ã release assays, children with exposure but no infection and a control group with no exposure and no infection were investigated. Whole blood was stimulated in QuantiFeron tubes overnight and 21 cytokines were measured in antigen stimulated and unstimulated supernatants by multiplex cytokine arrays. (3) HIV infected and uninfected children were enrolled in a hospital based study. Whole blood interferon-ã responses against specific mycobacterial antigens were investigated in a diluted 7 day whole blood assay and compared to QuantiFeron supernatants from the same participants. (4) Tuberculosis diseased adults were enrolled before the onset of treatment and innate and adaptive cell populations were investigated upon start of treatment and at treatment end. In addition, pleural effusion fluid was collected from tuberculosis and cancer patients and innate cell populations further investigated. The studies were performed in Cape Town, South Africa and included Tygerberg Academic Hospital and the surrounding neighbourhoods of Ravensmead, Uitsig and Elsies River. The main findings of the studies included: (1) We showed age related cytokine differences in our study community. Tuberculosis progressors had significantly higher levels of IL-10 in the unstimulated sample several years before the onset of tuberculosis disease. (2) Cytokines that distinguished best between children with tuberculosis infection and no infections were all cytokines that correlated with interferon-ã (interferon-ã was used to make the classification of M.tb infected and uninfected). Higher IL-1â and lower IL-17 levels in children with tuberculosis exposure without subsequent M.tb infection compared to children with no exposure were shown. (3) HIV infected children showed better responses after 7 day whole blood antigen stimulation compared to the overnight stimulation in QuantiFeron tubes. TB10.4 stimulation in HIV infected TST positive children gave higher interferon-ã responses than ESAT-6 and CFP-10. (4) The presence of myeloid derived suppressor cells is shown during tuberculosis disease circulating in peripheral blood. Upon treatment a decrease in the population is observed. No differences were seen in the myeloid derived suppressor cell frequencies between tuberculosis and cancer patients, however significantly lower frequencies were seen in healthy controls. The immune response against M.tb is complex and interactions between the different cell types are essential to control and fight infection and disease. In this thesis we presented new biomarkers that play important roles during different stages of M.tb pathogenesis from exposure to infection and even during disease. These may shed light on mechanisms of protection against M.tb, relevant to development of tuberculosis diagnostics and vaccine strategies. Combinations of multiple biomarkers including cytokines and chemokines and cell subsets are required to characterize biosignatures relevant to the diagnosis of tuberculosis infection and disease. / AFRIKAANSE OPSOMMING: Deur die immuunreaksie te ondersoek, in heelbloed en in die setel van infeksie, in mense met verskillende uitkomste van M.tb blootstelling sal lei tot die ontdekking van biologiese merkers en sal bydra tot ons begrip van die beskermde en nie-beskermde immuunreaksies teen die bakterium. Die hoofstudie het bestaan uit ‘n aantal loodsstudies en die doel van die studies was: (1) Om die sitokienprofiele in gestimuleerde heelbloed, asook agtergrond waardes, van kinders en volwassenes te bepaal, in die gemeenskap; (2) om die profiele van biologiese merkers in heelbloed van kinders met verskillende M.tb infeksie fenotipes te bepaal; (3) om die anti-mykobakteriële immuunreaksies in heelbloed by MIV geïnfekteerde en nie-geïnfekteerde kinders te bepaal; (4) om ondersoek in te stel na die doel van die aangebore immuunsisteem tydens tuberkulose siekte. Die studie ontwerpe was soos volg: (1) Volwassenes en kinders het deelgeneem aan die ondersoek van sitokienprofiele in die gemeenskap. Heelbloed is gestimuleer met BCG en ESAT-6 of is ongestimuleerd gelaat. Agtien sitokiene is gemeet in die bo-stand verkry van elke kondisie. Mense wat aktiewe tuberkulose siekte in die jare na die studie ontwikkel het, is geïdentifiseer deur die pastiëntinligting in die tuberkulose-register. (2) Kinders met gedokumenteerde huishoudelike tuberkulose blootstelling en met M.tb infeksie, soos bepaal deur vrygelate interferon-ã toetse, kinders met blootstelling maar geen infeksie en ‘n kontrole groep met geen blootstelling en geen infeksie, is ondersoek. Heelbloed is gestimuleer in die QuantiFeron buise oornag en 21 sitokiene is gemeet in die antigeen gestimuleerde en ongestimuleerde bostande deur die multiplex sitokienpaneel. (3) MIV geïnfekteerde en nie-geïnfekteerde kinders het deelgeneem aan ‘n hospitaal baseerde studie. Heelbloed interferon-ã reaksies teen spesifieke mykobakteriële antigene is bestudeer in ‘n verdunde 7 dag heelbloed toets en vergelyk met die QuantiFeron bostande van dieselfde deelnemers. (4) Siek tuberkulose volwassenes wat nie op behandeling is nie, het deelgeneem. Die aangebore en verworwe selpopulasies is bepaal aan die begin van behandeling asook voor die einde van behandeling. Verder is pluralevog van tuberkulose en kanker pastiënte bestudeer vir aangebore selpopulasies. Die studies is uitgevoer in Kaapstad, Suid-Afrika en sluit in Tygerberg Akademiese Hospitaal en die gemeenskappe van Ravensmead, Uitsig en Elsiesrivier. Die hoofbevindinge van die studies sluit in: (1) Ons het gewys dat daar ouderdomsverwante sitokien verskille in die studie gemeenskap is. Mense wat tuberkulose siekte ontwikkel het, het beduidende hoër vlakke van IL-10 in die ongestimuleerde monsters getoon ‘n paar jaar voor die begin van die siekte. (2) Sitokiene wat die beste onderskeiding gewys het tussen infeksie en geen infeksie was sitokiene wat ook korrelasie getoon het met interferon-ã (interferon-ã is gebruik om die klassifikasie te maak van M.tb infeksie of geen infeksie). Hoër IL-1â en laer IL-17 vlakke in kinders met tuberkulose blootstelling en sonder M.tb infeksie, is gewys wanneer dit vergelyk is met kinders sonder blootsteling. (3) MIV geïfekteerde kinders het beter reaksies getoon na 7 dag heelbloed antigeen stimulasie as met die oornag stimulasie in QuantiFeron buise. TB10.4 stimulasie in MIV geïnfekteerde TST positiewe kinders het hoër interferon-ã reaksies getoon as na stimulasie met ESAT-6 en CFP-10. (4) Die teenwoordigheid van miloïed afgeleide onderdrukkende selle in heelbloed, is getoon tydens tuberkulose siekte. Na behandeling is ‘n afname in die populasie gesien. Geen verskille is gesien in die aantal miloïed afgeleide onderdrukkende selle tussen tuberkulose en kanker pastiënte nie, alhoewel beduidende laer getalle is waargeneem in gesonde kontrole deelnemers. Die immuunreaksie teen M.tb is kompleks en interaksies tussen die verskillende seltipes is belangrik om infeksie en siekte te kontroleer en te beveg. In die tesis het ons nuwe biologiese merkers geïdentifiseer wat belangrike funksies het, tydens die verskillende stadiums van M.tb patogenesiteit, van blootstelling tot infeksie asook tydens siekte. Dit kan gebruik word as biologiese merkers betrokke by die immuunreaksie teen M.tb en sal bydra tot die diagnose van tuberkulose infeksie en siekte.

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

The JAK/STAT pathway is a non-receptor tyrosine kinase signaling pathway that is well conserved and highly re-utilized in many mammalian and Drosophila developmental processes. Compared to dozens of ligands and receptors in mammalian JAK/STAT, Drosophila JAK/STAT pathway is simpler with one receptor and three ligands, Upd, Upd2 and Upd3, which have similar amino acid sequences. Previous literature shows that upd and upd2 exhibit the same dynamic striped expression pattern in embryos and have semi-redundant functions during embryogenesis. Do Upd and Upd3 also have redundant functions? To answer this question, the functions of Upd3 in Drosophila development were investigated in this dissertation. In addition, the coordinate expression mechanism of upd and upd3 in eye discs was also analyzed. To study the functions of Upd3 in development, the expression pattern of upd3 was examined and detected in larval eye discs, wing discs, haltere discs, lymph glands and adult ovaries with in situ hybridization to upd3 mRNA and an upd3 reporter line. Consistent with the expression pattern, the loss of function mutants of upd3 exhibit small eyes, outstretched wings, downward extended halteres and reduced circulating blood cell concentration, demonstrating the roles of Upd3 in these tissues’ development. However, functions of Upd3 in other aspects of immune response were not detected. To investigate the mechanism of the coordinate expression of upd and upd3, the genetic and molecular relationship of upd, upd3 and os was dissected. The os alleles, oso, oss and os1, are a group of classical alleles which display outstretched wings, small eyes, or both, respectively. The genetic complementation tests of upd, upd3 and os showed that both upd and upd3 failed to complement os while upd complemented upd3, suggesting functions of both upd and upd3 are affected in os alleles. Consistent with the genetic tests, the expression of upd and upd3 in eye discs is lost in os allele. Molecularly, putative enhancer regions are deleted at the 5’ end of upd3 in os alleles. Hence, a transcriptional co-regulation model of upd and upd3 is proposed in which upd and upd3 share a common cis-regulatory region, lesions of which cause the os phenotype.

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