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

The presence of the purine nucleotide inosine monophosphate (IMP) was studied in direct relationship to the development and expression of cell mediated immunity in guinea pigs using DNCB or Histoplasma capsulatum as sensitizing antigens. The IMP content of T-cell enriched lymphocytic lysates was measured by isocratic high pressure liquid chromatography (HPLC). Intracellular IMP levels of cells from homologously skin tested sensitized animals were significantly increased one day after skin testing when compared to the concentrations found in these cells during the period following sensitization. Concurrent with these observations were the findings that the absolute lymphocyte counts and histoplasmin stimulated in vitro blastogenic responses increased following sensitization while the PHA-induced proliferative response decreased slightly. One day after skin testing, when IMP levels had increased, there was a slight decrease in lymphocyte numbers and a marked decrease in the PHA response. Cells collected at this time and cultured in vitro with histoplasmin responded with increased levels of protein production and increased IMP levels. These data suggest (1) the proliferative response of cells from sensitized animals appears to be associated with lower levels of intracellular IMP, and (2) sensitized cells stimulated in vivo with antigen appear to have characteristically higher IMP concentrations.

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<p>The larva of the fruit fly Drosophila melanogaster is an excellent model to study the molecular control of innate cellular immune responses. Cellular responses take place, and can be studied, following infestation of the wasp Leptopilina boulardi. This response includes proliferation and activation (differentiation) of the blood cells (hemocytes). In a successful anti-parasitic response, an immune-induced lineage of hemocytes, the lamellocytes, forms a cellular capsule covering and killing the foreign intruder. I will in this thesis present data about the finding and characterization of a novel marker that is expressed specifically in the hemocytes, the Hemese gene. I furthermore describe the construction of a useful tool, the transgenic Hemese-Gal4 fly, which enables blood cell specific expression of any gene of interest. By using the Hemese-Gal4 fly in a directed screen, I have found that a surprisingly large number of genes, that in turn are members of seemingly diverse signaling pathways, are able to induce a cellular response. In many cases their expression is also associated with a blood cell tumor phenotype. Overexpression of certain genes, such as hopscotch (a Drosophila Jak homologue) and hemipterous (a c-jun kinase kinase) lead to the formation of lamellocytes. Other genes may control the cell number, such as Egfr and Ras, as their expression produced a massive in increase the numbers of hemocytes. A third group of genes, including, e.g. Alk, Rac1 and Pvr give a mixed response, promoting both hemocyte proliferation and activation. Surprisingly, the suppression of WNT signaling in hemocytes lead to hemocyte activation. In one case, with a UAS-Pvr dominant negative construct, we observe a reduction of the circulating blood cells in uninfested larva. The expression of DN-Pvr additionally contributes to reduce encapsulation rates in larvae subjected to Leptopilina infestation. In conclusion: the control of blood cells in larval hematopoiesis, and during parasitic wasp attacks, is complex and may involve multiple pathways. In a broader sense, the gene functions found in the directed screen may have implications also for understanding the molecular control of mammalian myeloid lineage blood cells.</p>

No description available.

No description available.

No description available.

The larva of the fruit fly Drosophila melanogaster is an excellent model to study the molecular control of innate cellular immune responses. Cellular responses take place, and can be studied, following infestation of the wasp Leptopilina boulardi. This response includes proliferation and activation (differentiation) of the blood cells (hemocytes). In a successful anti-parasitic response, an immune-induced lineage of hemocytes, the lamellocytes, forms a cellular capsule covering and killing the foreign intruder. I will in this thesis present data about the finding and characterization of a novel marker that is expressed specifically in the hemocytes, the Hemese gene. I furthermore describe the construction of a useful tool, the transgenic Hemese-Gal4 fly, which enables blood cell specific expression of any gene of interest. By using the Hemese-Gal4 fly in a directed screen, I have found that a surprisingly large number of genes, that in turn are members of seemingly diverse signaling pathways, are able to induce a cellular response. In many cases their expression is also associated with a blood cell tumor phenotype. Overexpression of certain genes, such as hopscotch (a Drosophila Jak homologue) and hemipterous (a c-jun kinase kinase) lead to the formation of lamellocytes. Other genes may control the cell number, such as Egfr and Ras, as their expression produced a massive in increase the numbers of hemocytes. A third group of genes, including, e.g. Alk, Rac1 and Pvr give a mixed response, promoting both hemocyte proliferation and activation. Surprisingly, the suppression of WNT signaling in hemocytes lead to hemocyte activation. In one case, with a UAS-Pvr dominant negative construct, we observe a reduction of the circulating blood cells in uninfested larva. The expression of DN-Pvr additionally contributes to reduce encapsulation rates in larvae subjected to Leptopilina infestation. In conclusion: the control of blood cells in larval hematopoiesis, and during parasitic wasp attacks, is complex and may involve multiple pathways. In a broader sense, the gene functions found in the directed screen may have implications also for understanding the molecular control of mammalian myeloid lineage blood cells.