Дисертації з теми "Human Immunological aspects"

The objective of this study was to investigate the effect of recombinant human interleukin-1beta (rHIL-1beta) and interleukin-8 (rHIL-8) on bovine neutrophil migration and degranulation. An in vitro co-culture system was used to study bovine neutrophil migration. This simulative system allowed studying neutrophil migration across endothelium (bovine aorta endothelial cells), extracellular matrix (ECM), and epithelium (MAC-T) in the correct sequences and directions. Quantification of neutrophil migration was carried out by assaying the activity of myeloperoxidase, a major enzyme of neutrophils. Degranulation of azurophilic, specific, and tertiary granules was studied by measuring releases of myeloperoxidase, lactoferrin, and gelatinase, respectively. The results showed that bovine neutrophils were able to migrate across the simulative co-culture system in response to zymosan activated serum. Recombinant HIL-8 was demonstrated to have a dose-dependent effect on bovine neutrophil migration. Furthermore, rHIL-8 had a dose-dependent effect directly on degranulation of azurophilic and specific granules, but not on tertiary granules. On the other hand, rHIL-1beta only had a significant effect on degranulation of azurophilic granules when the concentration of 100 ng/ml was used. The dose effect of rHIL-1beta on specific degranulation was much stronger. Moreover, the effect of 100 ng/ml rHIL-1beta was augmented when the rHIL-1beta containing solution was preincubated with MAC-T monolayers for four hours. This indicated that MAC-T cells might generate other degranulating factors in response to the stimulation of rHIL-1beta. These MAC-T-derived degranulating factors did not have effect on the release of tertiary granule contents.

The human pancreatic cancer cell line T3M4, is known to produce transforming growth factor-alpha (TGF-alpha); as well as overexpress the receptor for this ligand, epidermal growth factor (EGF) receptor. TGF-alpha messenger RNA (mRNA) levels were assayed using northern blot, after addition of epidermal growth factor or TGF-alpha. The level of TGF-alpha mRNA was found to increase 2-fold at 2 hours and then return to near basal levels at 10 hours, after treatment with either ligand. Both ligands were also equipotent in a 2 hour dose response assay, with half maximal stimulation seen at 1 nM and maximal stimulation reached at 4 nM. Furthermore, there appeared to be a 2-fold increase in TGF-alpha transcription as determined by nuclear runoff experiments. Induction of TGF-alpha mRNA coupled with the overexpression of the EGF receptor, may result in a potent autocrine cycle; which may be found in other cancers.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007.<br>The infectious disease tuberculosis (TB) is one of the leading causes of death worldwide. The idea that infectious diseases are the most important driving force in natural selection and that they sustain frequent polymorphisms in the human genome was formally suggested by Haldane in 1949. This hypothesis implicated the human genetic component in the response to infectious disease. Today the involvement of host genetics in TB has been proven unequivocally and, together with environmental factors (e.g. nutrition and crowding) and the causative bacterium, Mycobacterium tuberculosis (M.tuberculosis), may influence the outcome of disease. As is evident, TB is a complex disease and the implication for studying genetic susceptibility is that a number of genes will be involved. Interferon gamma (IFN-7) is the major macrophage-activating cytokine during infection with M.tuberculosis and its role has been well established in animal models and in humans. This cytokine is produced by activated T helper 1 (Th1) cells. These Th1 responses can best deal with intracellular pathogens such as M.tuberculosis. We selected twelve candidate genes based on the hypothesis that genes which regulate the production of IFN-7 may influence TB susceptibility. We also selected polymorphisms from 27 other candidate genes, which may affect immunological pathways involved in TB, to investigate as susceptibility factors based on the following hypotheses: 1) granulomatous diseases can share susceptibility genes; 2) gene expression studies done by DNA-array analysis experiments may reveal TB susceptibility genes; 3) genomewide linkage studies in TB can determine susceptibility loci and genes in this region are possibly susceptibility factors; and 4) functional susceptibility polymorphisms in genes involved in immune-mediated diseases other than TB may contribute to susceptibility to TB. This research tested the association of 136 genetic polymorphisms in 39 potentially important genes with TB in the South African Coloured population. Well-designed case-control association studies were used and we attempted to replicate these findings in an independent sample set using family-based case-control designs (transmission disequilibrium tests (TDTs)). In addition, haplotypes and linkage disequilibrium (LD) in the candidate genes were also investigated. During the case-control analyses we found significant associations for 6 single nucleotide polymorphisms (SNPs) in the following genes: SH2 domain protein 1A, tolllike receptor 2, class II major histocompatibility complex transactivator, interleukin 1 receptor antagonist, runt-related transcription factor 1 and tumour necrosis factor superfamily, member 1B. Discrepant results were obtained during the TDT analyses. The number of families available was small and for this reason we cannot conclude that the case-control results were spurious. We also tested the association of haplotypes with TB. Haplotypes in the interleukin 12, beta (IL12B) and toll-like receptor 4 genes were nominally associated with TB in both the case-control and TDT analyses. We observed strong LD for the genes in the South African Coloured population. In total 17 novel SNPs were identified and one novel allele was found for a microsatellite in IL12B. This research contributes to the increasing amount of information available on genes involved in TB susceptibility, which in the future may help to predict high risk individuals.

Thesis (MNutr)--Stellenbosch University, 2013.<br>ENGLISH ABSTRACT: Background: The Ironman® triathlon is an ultra-endurance event. It has previously been shown that heavy training schedules and racing ultra-endurance events can lead to immune impairment. Evidence supporting the potential role of dietary intake and body composition on immune impairment or upper respiratory tract infections (URTIs) is currently lacking. Aim: To investigate the relationship between dietary intake, body composition and the incidence of URTI in triathletes residing in Port Elizabeth (PE), during training and competition for the Ironman® 2011 triathlon. Method: An observational longitudinal descriptive study with an analytical component was conducted. The study population included triathletes living in PE, who completed an Ironman® distance event one year prior to, and who were training for the April 2011 Ironman®. Habitual dietary intake was assessed with a quantitative food frequency questionnaire; and race dietary strategies with a three day food record. Body composition was determined with anthropometry and the incidence of URTI was assessed with the WURSS-44. A general health screen (SF-36) was also administered. Results: Habitual dietary intake during the three months pre- and post-Ironman® 2011 triathlon was adequate for all nutrients except for carbohydrate intake in female and male participants (pre-Ironman® of 4.0 (1.7) g/kg body weight (BW)/day and 5.4 (1.8) g/kg BW/day; and post-Ironman® 3.0 (1.0) g/kg BW/day and 4.7 (1.5) g/kg BW/day respectively). Carbohydrate-loading strategies were below recommendations with intakes of 6.0 (2.9) and 5.1 (2.5) g/kg BW/day for female and male participants respectively. Race day nutrition strategies were below recommendations for carbohydrate intake. Post-race dietary intake was below recommendations for carbohydrate in the female participants (0.9 (0.5) g/kg BW). Body mass index was 26.6 (3.4) kg/m2 and 26.1 kg/m2 (1.40) for female and male study participants respectively. Body fat percentage was at the upper end for endurance athletes (29.3 (9.4) % and 13.7 (5.1) % for females and males respectively). In this study 25 % of the triathletes (N=20) developed an episode of URTI during the 3 months post-Ironman®. Dietary intake parameters measured three months pre-Ironman® that had a significant influence on URTI were: potassium (p=0.04) and thiamine (p=0.02) and dietary intake parameters measured 3 months post-Ironman® that had a significant influence on URTI were: total protein (p=0.04); isoleucine (p=0.03); leucine (p=0.03); phenylalanine (p=0.03); valine (p=0.02); thiamine (p=0.01); and Beta-tocopherol (p=0.03). Dietary intake parameters measured during the race that had a significant influence on URTI were: selenium (p=0.04); folate (p=0.04) and proline (p=0.02). Body composition did not have a significant influence on URTI. Conclusion: Habitual dietary intake three months pre- and post-Ironman® as well as pre- and post Ironman race strategies were low for carbohydrate. Body composition indicated that athletes were at the upper end associated with endurance sport. There was a relationship found between an episode of URTI and dietary intake.<br>AFRIKAANSE OPSOMMING: Agtergrond: Die Ironman® driekamp is 'n ultra-uithouvermoë kompetisie. Daar is voorheen bewys dat swaar oefening skedules en ultra-uithouvermoë kompetisies kan lei tot ‘n immuungebrek. Daar is tans ‘n tekort aan wetenskaplike bewyse wat die potensiële rol van dieetinname en liggaamsamestelling op immuungebrek of boonste lugweginfeksies ondersoek. Doel: Die doel van die studie was om ondersoek in te stel oor die verhouding tussen dieetinname, liggaamsamestelling en die insidensie van boonste lugweg infeksies in driekamp atlete woonagtig in Port Elizabeth (PE), tydens oefening en deelname aan die Ironman® 2011 driekamp. Metodes: 'n Waargenome, longitudinale beskrywende studie is gedoen met 'n analitiese komponent. Die studiepopulasie het bestaan uit driekampatlete woonagtig in PE, wat 'n Ironman® afstand kompetisie voltooi het een jaar voor en wat oefen vir die April 2011 Ironman® kompetisie. Gewoontelike dieetinname is bepaal met 'n kwantitatiewe voedselfrekwensie vraelys, en dieet strategieë rondom die byeenkoms met 'n drie dag voedselrekord. Liggaamsamestelling is bepaal met antropometrie en die insidensie van boonste lugweg infeksies is bepaal met die WURSS-44. 'n algemene gesondheid vraelys (SF- 36) is ook ingevul. Resultate: Die gewoontelike dieetinname gedurende die drie maande voor- en na-Ironman® 2011 was voldoende vir alle voedingstowwe, behalwe vir koolhidraat-inname in die vroulike en manlike deelnemers (voor Ironman® 4.0 (1.7) g / kg liggaamsmassa (LM) / dag en 5.4 (1.8) g / kg LM / dag, en na Ironman® 3.0 (1.0) g / kg LM / dag en 4.7 (1.5) g / kg LM / dag onderskeidelik). Koolhidraatlading strategieë was ontoereikend met innames van 6.0 (2.9) en 5.1 (2.5) g / kg BW / dag vir vroulike en manlike deelnemers onderskeidelik. Die inname op die dag van die byeenkoms was onvoldoende vir koolhidraat. Die dieetinname na die byeenkoms was onvoldoende vir koolhidraat inname in die vroulike deelnemers (0.9 (0.5) g / kg LM). Die liggaamsmassa-indeks was 26.6 (3.4) kg/m2 en 26.1 (1.4) kg/m2 vir vroulike en manlike deelnemers onderskeidelik. Persentasie liggaamsvet was aan die boonste grens geassosieer met uithouvermoë oefening atlete 29.3 (9.4) % en 13.7 (5.1) % vir vrouens en mans onderskeidelik. Die insidense van boonste lugweg infeksies was 25% (N=20) gedurende die drie maande na Ironman®. Dieetinname paramters wat gemeet was drie maande voor Ironman® wat beduidende beïnvloed met boonste lugweginfeksies getoon het, was, kalium (p=0.04) en tiamien (p=0.02) en die dieetinname parameters wat drie maande na Ironman® gemeet is en betekenisvolle beïnvloed getoon het met boonste lugweginfeksies was, totale proteïen (p=0.04); isoleusien (p=0.03), leusien (p=0.03), fenielalanien (p=0.03), valien (p=0.02), tiamien (p=0.01), en B-tocopherol (p=0.03). Die dieetinname parameters wat gemeet was tydens die wedloop wat beduidende beïnvloed met boonste lugweginfeksies getoon het na Ironman® 2011 was, selenium (p=0.04), folaat (p=0.04) en prolien (p=0.02). Die antropometriese parameters gemeet het nie beïnvloed op boonste lugweginfeksies gehad nie. Gevolgtrekking: Die gewoontelike dieetinname drie maande voor- en na Ironman® sowel as voor- en na Ironman® kompetisie strategieë was onvoldoende vir koolhidrate. Liggaamsamestelling het aangedui dat atlete aan die boonste grens geassosieer met uithouvermoë oefening geval het. Daar was beduidende beïnvloed gevind tussen dieetinname en boonste lugweginfeksies.

<p>Onchocerciasis, or river blindness, is a parasitic disease that affects more than 20 million people globally. The induction of pathology is directly related to the presence and destruction of the microfilarial stages (mf) of this filarial nematode. The disease presents clinically with a wide spectrum of dermal and ocular manifestations, the basis of the variation is believed to involve the immune system. The clinical presentations of infected hosts relate to the intensity of the reactions against the parasite. Anti-microfilarial drugs are also thought to somehow involve the immune system in their pharmacological action. In this study we have investigated some of the factors that might contribute to the pathogenesis, with the aim of gaining a better understanding of the role of immune response in these host inflammatory reactions to <i>Onchocerca volvulus</i> parasite. In the first study we have highlighted the clinically most severe form of dermal onchocerciasis, known as reactive onchocercal dermatitis (ROD), one that is often ignored and has not been properly identified. This form has special characteristics and important biological information that could greatly assist the general understanding of the disease as a whole. Amongst the three major foci of the disease in the study country, Sudan, the prevalence of ROD was found to be associated with different environmental and epidemiological characteristics; strikingly higher in the hypo-endemic areas. Including ROD cases in the prevalence will upgrade the level of endemicity of a locality, and often bring patients much in need of treatment into mass treatment programs that currently only treat localities with medium to high levels of endemicity. In the following research studies, we tried to address the immunological characteristics of the clinically different onchocerciasis patients. Then we also investigated the role of genetic polymorphism in the gene encoding receptor that links innate and adaptive immunity, namely, FcγRIIa.</p><p>Patients with either of two major forms of the clinical spectrum-mild and severe dermatopathology were studied by assaying the antigen-driven proliferation of peripheral blood mononuclear cells and the ability of patients’ serum antibodies to promote cytoadherence activity to mf <i>in vitro</i>. Immune responses of those with severe skin disease were found to be stronger compared with the mild dermatopathology group. Mectizan® treatment was followed by an increase in immune responsiveness in those with initially poor responses. Thus the degree of dermatopathology is related to the host’s immune response against mf and immunocompetence may be necessary for Mectizan® to clear the infection efficiently.</p><p>The infection has also been associated with increased levels of circulating immune complexes (CIC) containing parasite antigens and a cytokine response that involves both pro-and anti-inflammatory cytokines. Our fourth paper investigated the effect of IC from the <i>O. volvulus</i> infected patients on the production of pro-and anti-inflammatory cytokines. CIC were increased in all patients studied. The precipitate from plasma treated with polyethylene glycol (PEG) were added to peripheral blood mononuclear cell (PBMC) cultures, and the levels of IL-10, tumor necrosis factor TNF-α, IL-1β and their endogenous antagonists soluble TNF-Rp75 and IL-1-receptor antagonist (IL-1ra) were measured. A significant induction of all cytokines measured occurred in the onchocerciasis patients compared to healthy controls. However, the IL-1ra level was suppressed. The suppression of the production of IL-1ra suggests that the IC containing antigens may have a selectively suppressive effect on the production of this anti-inflammatory cytokine; thus implicating its possible role in counteracting inflammatory responses associated with the disease, and suggesting a potential therapeutic significance. </p><p>FcgRIIa receptors are involved in many important biological responses, and considered as important mediators of inflammation. A polymorphism in the gene encoding this receptor, that is either arginine (R) or histidine (H) at position 131, affects the binding to the different IgG subclasses. We therefore hypothesized that this polymorphism might be one of the underlying mechanisms to the varied clinical presentations seen in this disease. FcgRIIa genotyping was carried out by gene specific polymerase chain reaction (PCR) and allele-specific restriction enzyme digestion of DNA from clinically characterized patients. The genotype R/R frequencies were found to be significantly higher among patients with the severe form of the disease (including ROD), and it was particularly associated with one tribe (Masaleet) compared to Fulani. Moreover, the H allele was found to be associated with lower risk of developing the severe form. As no significant difference was seen between onchocerciasis cases and controls, the study also implies that this polymorphism influences protection from developing the severe form rather than being related to protection from the infection. </p>

Thesis (PhD)--Stellenbosch University, 2013.<br>ENGLISH ABSTRACT: The immune response against M. tuberculosis is multifactorial, involving a network of innate and adaptive immune responses. Characterization of the immune response, a clear understanding of the dynamics and interplay of different arms of the immune response and the identification of infection-stage specific biomarkers are critical to allow the development of better tools for combating tuberculosis. In an attempt to identify such biomarkers, we studied pulmonary tuberculosis patients and their contacts in Addis Ababa, Ethiopia as part of EDCTP and BMGF funded tuberculosis projects by using multiplex techniques. We analysed 45 genes using the Multiplex Ligation Dependent Probe Amplification (MLPA) technique and the expression of IL-4δ2, BLR1, MARCO, CCL-19, IL7R, Bcl2, FcyR1A, MMP9, and LTF genes discriminate TB cases from their healthy contacts. FoxP3, TGFß1 and CCL-19 discriminate latently infected from uninfected contacts. Single genes predict with an area under the Receiver Operating Characteristic (ROC) curve of 0.68 to 0.85 while a combination of genes identified up to 95% of the different groups. Similarly, the multiplex analysis of cytokines and chemokines also showed that single or combinations of plasma cytokines and chemokines discriminate between different clinical groups accurately. The median plasma level of EGF, fractalkine, IFN-y, IL-4, MCP-3 and IP-10 is significantly different (p<0.05) in active tuberculosis and non active tuberculosis infection and the median plasma levels of IFN-y, IL-4, MCP-3, MIP-1ß and IP-10 were significantly different (p<0.05) before and after treatment. We also found a significant difference (p<0.05) in plasma levels of cytokines of patients infected with the different lineages and different families of the modern lineage. The plasma level of IL-4 was significantly higher in patients infected with lineage 3 (p<0.05) as compared to lineage 4 and the CAS familyinfected patients had a higher plasma level of IL-4 (P<0.05) as compared to patients infected with H and T families but there was no difference between H and T families. We identified genes and cytokines which had been reported from other studies in different settings and we believe that these molecules are very promising biomarkers for classifying active tuberculosis, latent infection, absence of infection and treated infection. These markers may be suitable for the development of clinically useful tools but require further validation and qualification in different populations and in larger studies.<br>AFRIKAANSE OPSOMMING: Die immuunrespons teen M. tuberculosis is multifaktoriaal en betrek ‘n netwerk van niespesifieke and spesifieke immuunresponse. Karakterisering van die immuunrespons, ‘n duidelike insig in die dinamika en tussenspel deur die verskillende arms van die immuunrespons en die identifikasie van spesifieke biomerkers is krities belangrik om die ontwikkeling van nuwe hulpmiddels teen tuberkulose te bevorder. In ‘n poging om sulke biomerkers te identifiseer het ons pulmonale tuberkulose pasiënte en hulle kontakte in Addis Ababa, Etiopië, as deel van die EDCTP en BMGF befondste tuberkulose projekte bestudeer met multipleks tegnieke. Ons het 45 gene analiseer met ‘Multiplex Ligation Dependent Probe Amplification (MLPA)’ en gevind dat die geenuitdrukking van IL-4•2, BLR1, MARCO, CCL-19, IL7R, Bcl2, Fc•R1A, MMP9, en LTF TB pasiënte van hulle kontakte onderskei. FoxP3, TGF•1 en CCL-19 onderskei tussen latent infekteerde en ongeïnfekteerde kontakte. Enkele gene voorspel met ‘n area onder die ‘Receiver Operating Characteristic (ROC)’ kurwe van 0.68 tot 0.85 terwyl die kombinasie van gene 95% van die verskillende groepe identifiseer. Soortgelyk het multipleks analise van sitokiene en chemokiene verskillende kliniese groepe akkuraat van mekaar onderskei. Die mediane plasmavlakke van EGF, fractalkine, IFN-•, IL-4, MCP-3 en IP-10 is beduidend verskillend (p<0.05) in aktiewe tuberkulose en nie-aktiewe tuberkulose infeksie en die mediane plasmavlak van IFN-•, IL-4, MCP-3, MIP-1• en IP-10 was beduidend verskillend voor en na behandeling. Ons het ook beduidende verskille (p<0.05) in plasmavlakke van sitokiene in pasiënte gevind wat infekteer is met verskillende stamme and verskillende families van die moderne stamme. Die plasmavlak van IL-4 was beduidend hoër in pasiënte wat infekteer is met stam 3 (p<0.05) teenoor stam 4 en die CAS familie-infekteerde pasiënte het ‘n hoër plasmavlak van IL-4 (p<0.05) teenoor pasiënte met H en T familie infeksie hoewel daar geen versikke was tussen die H en T families nie. Ons het gene en sitokiene identifiseer wat deur ander werkers onder verskillende omstandighede ook beskryf is en ons glo dat hierdie molekules baie belowende biomerkers is om aktiewe tuberkulose, latent tuberkulose, die afwesigheid van infeksie en behandelde infeksie van mekaar te onderskei. Hierdie merkers mag toepaslik wees vir die ontwikkeling van bruikbare kliniese hulpmiddele maar benodig verdere validasie en kwalifikasie in verskillende populasiegroepe en in groter studies.<br>Bill and Melinda Gates Foundation (BMGF)<br>European and Developing Countries Clinical Trials Partnership (EDCTP)<br>African European Tuberculosis Consortium (AE TBC).

Thesis (MSc MedSc)--Stellenbosch University, 2013.<br>ENGLISH ABSTRACT: Background The QuantiFERON In-Tube (QFT IT) assay is an Interferon-gamma release assay (IGRA) which is currently used to detect Mycobacterium tuberculosis (M. tb) infection. It however cannot differentiate between latent infection and active tuberculosis (TB) disease. In an attempt to improve this tool to accurately diagnose active TB, the release of a variety of markers should be assessed in combination with Interferon gamma (IFN- γ). Luminex analysis was previously done on QFT plasma and promising candidates were identified which could be of great value in treatment response studies. IFN-γ ELISpot, are not only used to detect M.tb infection, but is also implicated in vaccine trails to assess immunogenicity. The IFN-γ ELISpot and flow cytometry are the most common assays to assess these phenomena during clinical trials. Our aim therefore was to develop a multi platform immune analysis assay using the QFT IT system. Study design and method The first approach of this study was to optimize the QFT IT assay for flow cytometry applications. The following questions formed part of the optimization study: How does the QFT whole blood assay (QFT-WBA) compare to the currently used WBA? Is antigen re-stimulation required after the initial incubation time and for how long should cells be re-stimulated in the presence of Brefeldin A? The second approach was to use the optimized QFT-WBA for community controls (CTRL), household contacts (HHC) and TB cases, which were recruited from the high TB incidence areas Ravensmead, Uitsig and Elsies River. The infection status of each participant was determined by IFN-γ ELISA and Luminex analysis was performed to measured wide range of cytokine expression. In addition immune cell markers like CD14, CD4, CD8, CD19, and T cell receptor gamma delta (TCRγδ) were characterized; polyfunctional characteristics (IFN-γ, Tumor necrosis factor-alpha (TNF-α) and Interleukin-2 (IL-2)) and proliferation (Ki-67+) of T cells determined by flow cytometry. Results After stimulating the whole blood of the study participants for 22 hours with the M. tb specific antigens, early secreted antigenic target 6 kDa (ESAT-6), culture filtrate protein-10 kDa (CFP-10) and TB7.7 the levels of TNF-α producing CD4 T cells were elevated in TB cases compared to HHCs. After stimulating the whole blood for 6 days TNF-α producing T cells declined in TB cases and HHC showed a higher expression. CD40L+CD4+ (p=0.0225) was increased in HHC while IL-9+CD8+ (0.3230) was decreased in HHC compared to TB cases. Other markers such as IL-5(AG-NIL), IL-13(Ag- NIL), FGF basicAg, GM-CSFNIL, VEGFNIL/(Ag-NIL), MIP-1βAg and MCP-1Ag/(Ag-NIL) showed significant differences between HHC and TB cases. Conclusions The responses in the QFT-based assay were generally comparable to the WBA that is routinely used. The differences of TNF-α expression seen in QFT-WBA and QFTLPA could be explained by the fact that effector T cell responses were measured in the short term assay and the central memory T cell responses in the long term assay. Our study therefore shows that the QFT-based tests can be used to simultaneously assess a wide range of immunological markers and not only IFN-γ expression.<br>AFRIKAANSE OPSOMMING: Agtergrond Die QuantiFERON In Tube (QFT IT) toets is ‘n Interferon-gamma vrystellingstoets (IGRA) wat huidiglik dien as ‘n maatstaf van Mycobacterium tuberculosis (M. tb) infeksie. Hierdie toets kan egter nie onderskei tussen latente infeksie en aktiewe tuberkulose (TB) nie. ‘n Noemenswaardige verbetering in die vermoë van hierdie toets om aktiewe TB te diagnoseer, berus op die studie van ‘n verskeidenheid vrygestelde merkers, insluitend Interferon gamma (IFN-γ). In vorige Luminex studies op QFT plasma, is belowende kandidate geïdentifiseer wat van groot waarde kan wees vir studies wat fokus op die reaksie tot behandeling. Die IFN-γ ELISpot dien nie net as ‘n maatstaf van M.tb infeksie nie, maar word ook in vaksienproewe betrek om die aard van immuniteit te ondersoek. Die IFN-γ ELISpot toets sowel as vloeisitometriese toetse, is van die mees algemene toetse om hierdie verskynsels te meet, tydens kliniese proewe. Die doel van hierdie studie was dus om die QFT IT sisteem te ontwikkel as ‘n basis vir ‘n multiplatform immunologiese analiseringstoets. Studie ontwerp en metode Die inleidende benadering van hierdie studie was die optimisering van die QFT IT toets, vir vloeisitometrie doeleindes. Die volgende vrae het deel uitgemaak van die optimiseringstudie: Hoe vergelyk die QFT heelbloedtoets (QFT-WBA) met huidige WBAs wat in gebruik is? Word meermalige antigeenstimulasies benodig na die oorspronklike inkubasieperiode en hoe lank moet die tydperk wees vir sellulêre opvolgstimulasie, in die teenwoordigheid van Brefeldin A? As ‘n tweede benadering, was om die geoptimiseerde QFT-WBA te gebruik vir gemeenskapskontroles (CTRL), huishoudelike kontakte (HHC) en TB gevalle. Al drie hierdie groepe was opgeneem uit Ravensmead, Uitsig en Elsies Rivier, areas met betreklik hoë vlakke van TB infeksie. Elke persoon in die studie se vlak van infeksie is vasgestel met behulp van die IFN-γ ELISA en Luminex analiese was uitgevoer, om ‘n wye verskeidenheid uitdrukkingsvlakke van sitokiene te meet. Dies meer, was immuunselmerkers soos CD14, CD4, CD8, CD19 en T sel reseptor gamma delta (TCRγδ) gekarakteriseer. Meervuldige funskionele karakteristieke (IFN-γ, Tumor nekrose faktor-alpha (TNF-α) en Interleukin-2 (IL-2)) en vermenigvuldiging van T-selle, was vasgestel deur middel van vloeisitometrie. Resultate Nadat die heelbloed van studiedeelnemers gestimuleers was met M. tb spesifieke antigene, vroeë afskeidings antigeniese teiken 6kDa (ESAT-6), kultuurfiltraatproteïn 10kDa (CFP-10) en TB7.7, vir 22 uur, was gevind dat vlakke van TNF-α produserende CD4 T selle hoër was in TB pasïente, in vergelyking met HHCs. Nadat die heelbloed vir 6 dae gestimuleer was, het die vlak van TNF-α produserende T-selle afgeneem in TB pasïente, terwyl dit hoër was in HCC. CD40L+CD4+ (p=0.0225) het hoër vlakke bereik in HHC, terwyl IL-9+CD8+ (0.3230) vlakke afgeneem het, in vergelyking met TB pasïente. Ander merkers soos,onder andere, IL-5(AG-NIL), IL-13(Ag-NIL), FGF basicAg, GMCSFNIL, VEGFNIL/(Ag-NIL), MIP-1βAg and MCP-1Ag/(Ag-NIL), het noemenswaardige verskille geopenbaar tussen HHC en TB pasïente.

L’allogreffe de cellules souches hématopoïétiques est un traitement curatif pour de nombreuses maladies hématologiques. Cependant, le succès de cette procédure est entravé par la maladie du Greffon-contre-l’Hôte (GVH), une complication potentiellement fatale induite par l’attaque des tissues de l’hôte par les cellules T alloreactives du donneur. La GVH a une prévalence de 40 à 80%, selon les caractéristiques de la transplantation. La GVH est la cause principale de mortalité post-greffe en dehors des rechutes et, en dépit des nombreuses avancées dans le domaine de l’allogreffe, la physiopathologie de cette maladie est encore mal comprise à ce jour, en particulier chez l’homme. Dans cette étude, nous nous sommes concentrés sur la caractérisation phénotypique et moléculaire du système immunitaire des patients greffés et de leurs donneurs. L’objectif de l’étude est de déterminer les facteurs liés à la reconstitution du système immunitaire et au développement de la GVH aigüe. Nous avons collecté le sang de 101 couples donneur /receveur apparentés HLA-identique provenant de trois cohortes indépendantes : avant transplantation pour les donneurs, à l’apparition des premiers symptômes chez les patients atteint de la GVH aigüe et 30 ou 90 jours post-greffe pour les patients non atteints. Nous avons caractérisé le profil cellulaire de ces échantillons par cytométrie spectrale et analysé leur profil transcriptomique.L’immunophenotypage des patients nous a permis de montrer que la reconstitution du compartiment des cellules T est incomplète, avec une inversion du rapport CD4/CD8 après la greffe. De plus, le compartiment cellulaire T est enrichi en cellules possédant un phénotype effecteur/mémoire tandis que le réservoir des cellules T naïves est appauvri. La reconstitution des cellules NK est caractérisée par un enrichissement du sous-type CD56bright et celle des monocytes par l’enrichissement des cellules CD16+. A l’apparition de la GVH, une augmentation de la fréquence des cellules possédant un phénotype semblable aux cellules souches mémoire est observée, par rapport aux patients non-atteints. Ces cellules pourraient représenter un réservoir cellulaire de la maladie, maintenant la production de cellules T alloréactives. Le profilage transcriptomique montre que les cellules T du donneur réagissent à l'environnement de l’hôte en acquérant un phénotype activé, avec une surexpression des gènes associés à l’activation, l’adhésion, la migration et les fonctions effectrices. Au commencement de la maladie, on observe que les cellules T surexpriment des gènes médiateurs de l’inflammation ainsi que des gènes impliqués dans la transduction du signal cytokinique et la migration cellulaire. Nos données démontrent que l’analyse de la distribution des sous-types cellulaires par cytométrie associée au profilage transcriptomique peut contribuer à élucider les mécanismes impliqués dans la reconstitution immunitaire et dans le développement de la GVH humaine<br>Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematologic malignancies. However, its success is hindered by graft-versus-host disease (GVHD), a potentially fatal complication deriving from alloreactive donor T cells attacking recipient tissues. Acute GVHD (aGVHD) prevalence lies between 40 and 80% depending on transplant characteristics. GVHD is the main cause of non-relapse morbidity and mortality after HSCT and despite the advances in the field, disease processes in humans remain poorly understood.In this study we investigated the phenotypic and molecular characteristics of immune cells in patients after HSCT and in their HLA-identical sibling donors, with the goal of defining immune parameters associated with the recovery of donor-derived immunity and with the development of acute GVHD. We analysed 101 donor-recipient pairs in three independent cohorts for which blood was collected from the donors before transplantation and for the recipients either at aGVHD onset, before any treatment, or at day 30 or 90 post-HSCT for recipients that did not develop GVHD. On the donors’ and recipients’ samples we performed cellular profiling using spectral flow cytometry as well as gene expression analysis.Immunophenotyping reveals an incomplete reconstitution of the T cell compartment in the recipients, with an inversion of the CD4/CD8 ratio, both at one and three months after HSCT. Moreover, the reconstituting T cell compartment is characterized by a shift in the effector/memory phenotype of these cells, with a parallel depletion of the naïve T cell pool. NK cell reconstitution is characterized by an expansion of the CD56bright subset, while monocytes undergo an expansion of CD16+ cells. At aGVHD onset recipients have an increase of cells with a T stem cell memory-like (TSCM-like) phenotype compared to recipients without aGVHD. These cells may represent a cellular reservoir for GVHD, maintaining the production of alloreactive T cells in the presence of host persistent antigens. Molecular profiling shows that donor T cells react to the environment of the host by acquiring an activated phenotype, with upregulation of genes associated with T cell activation, adhesion, migration and effector functions. T cell transcriptome profiling at aGVHD onset shows upregulation of inflammatory mediators as well as genes involved in cytokine signal transduction, cell migration and cell trafficking.Our data demonstrate that comprehensive analysis of the distribution of different immune cell subsets with flow cytometry together with gene expression profiling can contribute to elucidate the processes involved in immune reconstitution and acute GVHD development in humans. In the future, studies with new technologies will hopefully bring insights into the mechanisms underlying GVHD development that will help design new preventive and therapeutic strategies to be applied in the clinics

L'infection par le cytomégalovirus humain (HCMV) est une cause majeure de mortalité chez les patients immunodéprimés et représente la première cause d'infection congénitale. HCMV est un virus complexe qui s'est adapté au système immunitaire humain en développant de multiples mécanismes d'évasion. L'infection primaire à HCMV est associée à une réplication virale prolongée avant l'établissement de la latence. Il a été montré que cette intense réplication lors de la phase initiale de l'infection était associée à une épuisement fonctionnel des lymphocytes T CD4 spécifiques du virus. Alors que les anticorps jouent un rôle dans la limitation de la dissémination virale et la prévention de l'infection à HCMV, les réponses des lymphocytes B sont peu caractérisées. Dans le présent travail, nous avons étudié l'impact de l'infection à HCMV sur le phénotype et la fonctionnalité des sous-populations de LB du sang circulant chez une cohorte de femme enceintes avec une primo-infection par HCMV en utilisant comme contrôles des sujets sains séropositifs et séronégatifs pour HCMV ainsi que des femmes enceintes séronégatives. Nous montrons que l'infection primaire par HCMV induit une expansion significative et prolongée de deux sous-populations de LB :les LB mémoires activés (CD27+CD21low) et mémoires atypiques (CD27-CD21low), précédemment décrites lors d'infection chroniques. Les LB mémoires atypiques démontrent des signes d'épuisement fonctionnel comme en témoigne une expression élevée de récepteurs inhibant le BCR et une moindre réponse à la stimulation in vitro mesurée par la production de TNF-α. Les expansions de ces deux sous-populations sont corrélées entre elles et liées à la virémie. Ces résultats contribuent à la compréhension de la régulation des réponses des LB lors d'infections virales, en montrant que l'épuisement fonctionnel de LB, précédemment décrit lors d'infections chroniques, peut également survenir lors d'infections primaires.<p>Dans un deuxième temps, nous avons étudié l'acquisition des réponses B mémoires spécifiques de HCMV dirigées contre la principale glycoprotéine de surface, la glycoprotéine B (gB), et deux polypeptides du tégument. Lors de l'infection primaire par HCMV, la production d'anticorps neutralisant le virus, dirigés contre les glycoprotéines d'enveloppe, est retardée par rapport aux anticorps dirigés contre le tégument qui sont non neutralisant. Nous montrons que le phénotype des LB mémoires spécifiques de gB est différent de celui des LB mémoires spécifiques du tégument. La majorité des LB mémoires spécifiques de gB exprime un phénotype CD27+CD21+ alors que la majorité de ceux du tégument exprime le phénotype CD27+CD21low. Nous montrons par la suite chez des sujets sains que ces deux sous-populations de LB mémoires présentent des différences phénotypiques, au niveau de l'expression de récepteurs liés au "trafficking" cellulaire ainsi qu'au niveau de la fonctionnalité. Les LB mémoires CD21low, contrairement au LB mémoires CD21high, expriment des taux bas des récepteurs CXCR5 et CCR7, qui permettent la migration vers les centres germinatifs, mais des taux élevés de CD11c promouvant la migration vers les tissus périphériques. Après stimulation in vitro, les LB mémoires CD21low vont avoir une capacité de production d'immunoglobulines immédiate mais une réponse proliférative plus faible comparée aux LB mémoires CD21+. Nous démontrons la relevance de cette division des LB mémoires sur base de l'expression du CD21 dans un modèle de vaccination de rappel contre la toxoïde tétanique (TT). Après rappel, nous observons une expansion significative de LB mémoires spécifiques de la TT exprimant un phénotype CD27+CD21lowCXCR5lowCD11chigh. Nous proposons ainsi un nouveau mécanisme de manipulation des réponses humorales par des pathogènes qui se traduit par une limitation de l'induction de réponses B effectrices. Nos travaux permettraient également une meilleure approche des réponses B mémoires physiologiques chez l'homme en proposant une classification des LB mémoires basées sur leur fonctionnalité et leur phénotype.<p><p>Human cytomegalovirus (HCMV) infection is a major cause of mortality in immunocompromised patients and is the first cause of congenital infection worldwide. HCMV is a complex virus that has developed multiples immune evasions mechanisms during its co-evolution with mankind. Although often asymptomatic, primary HCMV infection is associated with an intense and prolonged viral replication. It has been previously shown that this intense viral replication is associated with functional exhaustion of virus-specific CD4+ T cells. Although neutralizing antibodies limits viral dissemination and play a role in the prevention of HCMV infection, B cell responses during HCMV infection have been poorly studied so far.<p>In this work, we have studied the impact of HCMV infection on the phenotype and functionality of peripheral-blood B cell subsets in a cohort of pregnant women with a primary HCMV infection. Controls were healthy seronegative and seropositive HCMV donors and HCMV seronegative pregnant women. We show that primary HCMV infection induces a significant and prolonged expansion of two B-cell subsets, previously described in chronic infections :activated memory B cells (MBC) (CD27+CD21low) and atypical MBC (CD27-CD21low). Atypical MBC display signs of functional exhaustion with increased expression of inhibitory receptors and a lower response to in vitro stimulation as assessed by TNF-α production. Expansion of these two subsets are correlated and higher in subjects with detectable viremia. These results contribute to the understanding of the regulation of B cell responses during viral infections and indicate that B cell exhaustion, previously described during chronic infections, can be observed in primary infection.<p>Next, we have characterized the acquisition of HCMV-specific B cell responses directed against envelope glycoprotein B (gB) and two tegument polypeptides (pp150 and pp52). During primary HCMV infection, the production of neutralizing antibodies targeting envelope glycoproteins is delayed when compared to non-neutralizing anti-tegument antibodies. We show that gB and tegument-specific MBC have distinct phenotype during primary HCMV infection. The majority of gB-specific MBC have a CD27+CD21+ phenotype while the majority of tegument-specific MBC have a CD27+CD21low phenotype. We show that CD27+CD21+ and CD27+CD21low MBC express different pattern of chemokine receptors pattern but also have distinct functionality. CD27+CD21low MBC, on the contrary to CD27+CD21+ MBC, express low levels of CXCR5 and CCR7 that favor migration to lymph nodes and germinal centers but express high levels of CD11c that promotes migration to inflammatory tissues.<p>In vitro stimulation of sorted subsets of healthy individuals indicates that CD27+CD21low MBC have higher capacity of immediate immunoglobulin production but a lower proliferative potential as compared to CD27+CD21+ MBC. We further show the relevance of a division of MBC subsets based on CD21 expression in a model of TT booster immunization. Following booster immunization, a significant expansion of TT-specific MBC expressing the phenotype CD27+CD21lowCXCR5lowCD11chigh is observed. <p>We propose that HCMV manipulates the host humoral response by limiting the induction of gB-specific CD27+CD21low "effector" MBC. Our work also indicates that human MBC physiological responses should be studied according to their respective phenotype and functions.<p><br>Doctorat en Sciences médicales<br>info:eu-repo/semantics/nonPublished

Bibliography: leaves 154-185<br>iv, 185 leaves, [15] leaves of plates : ill. (some col.) ; 30 cm.<br>Title page, contents and abstract only. The complete thesis in print form is available from the University Library.<br>Thesis (Ph.D.)--University of Adelaide, Dept. of Science, 1987

Indiana University-Purdue University Indianapolis (IUPUI)<br>Vaccinia virus (VV) is the prototypical member of the orthopoxvirus genus of the Poxviridae family, and is currently being evaluated as a vector for vaccine development and cancer cell-targeting therapy. Despite the importance of studying poxvirus effects on the human immune system, reports of the direct interactions between poxviruses and primary human leukocytes (PHLs) are limited. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias towards binding and infecting monocytes among PHLs. VV binding strongly co-localized with lipid rafts on the surface of these cell types, even when lipid rafts were relocated to the cell uropods upon cell polarization. In humans, monocytic and professional antigen-presenting cells (APCs) have so far only been reported to exhibit abortive infections with VV. We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, were permissive to VV replication. The majority of virions produced in MDMs were extracellular enveloped virions (EEV). Visualization of infected MDMs revealed the formation of VV factories, actin tails, virion-associated branching structures and cell linkages, indicating that infected MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. Classical activation of MDMs by LPS plus IFN-γ stimulation caused no effect on VV replication, whereas alternative activation of MDMs by IL-10 or LPS plus IL-1β treatment significantly decreased VV production. The IL-10-mediated suppression of VV replication was largely due to STAT3 activation, as a STAT3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data indicate that PHL subsets express and share VV protein receptors enriched in lipid rafts. We also demonstrate that primary human macrophages are permissive to VV replication. After infection, MDMs produced EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread.

Indole-3-carbinol (I3C) is a naturally occurring plant alkaloid, found in significant concentrations in cruciferous vegetables such as broccoli and Brussels sprouts. I3C is an unstable compound that undergoes rapid oligomerization in an acidic environment to form higher order condensation products (I3C-ACPs), such as 3-3'-diindolylmethane (DIM). Both I3C and DIM are marketed as dietary supplements and are under investigation as potential chemopreventive agents, despite limited data on the effects of chronic exposure. Previous studies have demonstrated that the chemopreventive potential of I3C and DIM in animal studies is dependent on species, strain, tissue and timing of treatment relative to carcinogen exposure, and long-term post-initiation exposure can even promote tumors. The majority of biological effects from I3C are the result of the abilities DIM and other I3C-ACPs to bind to the aryl hydrocarbon receptor and the subsequent induction of phase I and phase II enzymes. Phase I and phase II enzyme induction in many cases leads to protection from carcinogens by increasing the rate of metabolism and excretion but in some cases enhances carcinogenicity by increasing the rate of bioactivation. It has been demonstrated that modulation of enzyme levels can also result in altered metabolism of compounds that could affect efficacy and toxicity of pharmaceuticals and xenobiotics. The current work utilizes chronic dietary I3C and DIM exposures in rodent models to further elucidate the effect these compounds might have on health, drug metabolism and carcinogenesis. The reduced weight of Fischer 344 rats treated with 2500 ppm I3C for 1 year may be indicative of adverse effects but toxicity was not confirmed by blood chemistry or histopathological examination. Furthermore, no toxicity was observed after a comparable treatment of Sprague-Dawley rats. As observed after acute and sub-chronic exposures to I3C and DIM, we documented significant induction of cytochrome P450 enzymes and a related modification to drug metabolism in liver slice incubations. Evidence is also provided that may suggest that tumor modulation in mice may occur through an estrogenic mechanism. Further studies should be completed to determine the potential for similar responses in humans.<br>Graduation date: 2004

Background. Islet amyloid polypeptide (IAPP, also known as amylin) is a 37-amino acid peptide principally co-secreted with insulin from the beta-cells of the pancreatic islets. Some of the physiological actions of human amylin (hIAPP) include glucose regulation, suppression of appetite and stimulation of renal sodium and water reabsorption. Amylin deficiency and diminished post-prandial amylin response have been reported in advanced stages of type 1 and type 2 diabetes. In autopsy specimens of type 2 diabetes, amyloid is found in 40--90% of cases. During the characterization of islet morphology of aged hIAPP transgenic mice, I observed pathological features suggestive of immune dysregulation. Review of literature also suggested possible immuno-modulating functions of human amylin in in vitro experiments. Since autoimmunity and innate immunity are implicated in aging and diabetes, I explored the immunological role of amylin with particular focus on CD4+CD25+ T regulatory cells and toll-like receptors (TLR) which are known mediators of autoimmunity and innate immunity respectively.<br>Conclusions. Human amylin may play an important role in modulating immunity mainly through stimulating CD4+CD25+ Treg cells, decreasing PLN and altering expression of TLR-4 and cytokines. If these findings are confirmed in in vivo model, human amylin has the potential to become a novel and promising therapy to prevent and reverse autoimmune disease such as autoimmune type 1 diabetes.<br>Hypothesis. Human amylin may have immunomodulating effects which may have implications on pathogenesis of autoimmune type 1 diabetes.<br>Materials and methods. Male hemizygous hIAPP transgenic mice (n=32) and their nontransgenic littermates (n=20) were fed with normal chow and studied longitudinally up to 18 months of age with measurement of plasma insulin, glucose and amylin at regular intervals. Detailed oral glucose tolerance test, intra-peritoneal insulin tolerance test, insulin and amylin protein expression were examined at 3, 7, 12 and 18 months of age. Histological changes of pancreas and spleen including changes in CD4+CD25+ T regulatory cells and cytokines were examined at 12 and 18 months.<br>Objectives. (1) I systemically characterized the morphological, functional and immune regulatory role of human amylin in aged hIAPP transgenic mice which include metabolic profiles, plasma levels of amylin and insulin as well as morphological changes of pancreatic lymph nodes (PLN). (2) I then examined splenic expression of TLR-4 associated changes in cytokines (TNF-alpha, TGF-beta, and IL-6). (3) I also examined the expression level of receptor activity modifying proteins (RAMPs) in pancreas and spleen. (4) I finished by investigating the role of human amylin on stimulating CD4+CD25+ T regulatory (Treg) cells in hIAPP transgenic mice and peripheral blood monocytes (PBMC) from healthy subjects.<br>Results. (1) With aging, the hIAPP transgenic mice demonstrated increased plasma amylin, decreased plasma insulin, reduced insulin to amylin ratio and improved insulin sensitivity (p&lt;0.05). (2) The aged hIAPP transgenic mice showed changes in immune function as indicated by: (a) Reduced number and size of PLN (p&lt;0.05). (b) Decreased expression level of TLR-4 in splenocytes (p&lt;0.05). (c) Increased expression of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) protein but decreased level of IL-6 in splenocytes (p&lt;0.05). (3) The changes in the levels of immune cytokines such as IL-1, IL-2, IL-4, IL-10, IL-17, interferon-gamma and GM-CSF were similar between hIAPP transgenic and nontransgenic mice (p>0.05). (4) The levels of RAMP1, RAMP2, and RAMP3 were higher in the spleen of hIAPP transgenic mice than nontransgenic mice (p&lt;0.05). (5) The hIAPP transgenic mice showed higher percentage of CD4+CD25+ Treg cells compared with nontransgenic littermates. Treatment with human amylin, but not rat amylin, increased the percentage of FoxP3+CD4+CD25+ Treg cells in both splenic T lymphocytes of hIAPP transgenic mice and PBMCs of healthy subjects ex vivo (p&lt;0.05).<br>He, Lan<br>Adviser: Juliana C.N. Chan.<br>Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: .<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.<br>Includes bibliographical references (leaves 176-199).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.

Immunologic monitoring is part of the standard care for patients on antiretroviral treatment. Yet, little is known about the routine implementation of immunologic monitoring in Ethiopia. This study assessed the pattern of immunologic monitoring, immunologic response, level of immunologic treatment failure and factors related to it among patients on antiretroviral therapy in selected hospitals in southern Ethiopia. A retrospective longitudinal analytic study was conducted using documents of patients started on antiretroviral therapy. A total of 1,321 documents of patients reviewed revealed timely immunologic monitoring were inadequate. Despite overall adequate immunologic response, the prevalence of immunologic failure was 11.5% (n=147). Having WHO Stage III/IV of the disease and a higher CD4 (cluster differentiation 4) cell count at baseline were identified as risks for immunologic failure. These findings highlight the magnitude of the problem of immunologic failure. Prioritizing monitoring for high risk patients may help in effective utilisation of meager resources<br>Health Studies<br>M.A. (Public Health)

MSc (Biochemistry)<br>Department of Biochemistry<br>Plasmodium falciparum is a unicellular protozoan parasite that causes malaria in humans. The parasite is passed to humans through mosquito bites and migrates to the liver before it infects host erythrocytes. It is at the erythrocytic stage of development that the parasite causes malaria pathology. Malaria is characterized by the modification of host erythrocytes making them cytoadherent. This is as a result of formation of protein complexes (knobs) on the surface of the erythrocyte. The knobs that develop on the surface of the erythrocyte are constituted by proteins of host origin as well as some proteins that the parasite ‘exports’ to the host cell surface. Nearly 550 parasite proteins are thought to be exported to the infected erythrocyte. Amongst the exported proteins is P. falciparum heat shock protein 70-x (PfHsp70-x). Hsp70 proteins are known to maintain protein homeostasis. Thus, the export of PfHsp70-x may be important for maintaining protein homeostasis in the host cell. PfHsp70-x is not essential for parasite survival although is implicated in the development of parasite virulence. This is possibly through its role in facilitating the trafficking of parasite proteins to the erythrocyte as well as supporting the formation of protein complexes that constitute the knobs that develop on the surface of the infected erythrocyte. The main objective of the current study was to investigate protein interaction partners of PfHsp70-x. It is generally believed that PfHsp70-x interacts with various proteins of human and parasite origin. Potential candidate interactors include its protein substrates, Hsp70 co-chaperones such as Hsp40 members, and human Hsp70-Hsp90 organizing protein (hHop). The establishment of the PfHsp70-x interactome would highlight the possible role of PfHsp70-x in the development of malaria pathogenicity. Based on bioinformatics analysis, PfHsp70-x was predicted to interact with some exported P. falciparum Hsp40s, hHop and human Hsp90 (hHsp90). Recombinant forms of PfHsp70-x (full length and a truncated form that lacks the C-terminal EEVN motif implicated in co-chaperone binding) were expressed in E. coli BL21 Star (DE3) cells. Recombinant hHop and hHsp70 were expressed in E. coli JM109 (DE3) cells. The proteins were successfully purified using nickel affinity chromatography. Co-affinity chromatography using recombinant PfHsp70-x and immuno-affinity chromatography using PfHsp70-x specific antibody did not confirm the direct interaction of PfHsp70-x with human Hop. However, the direct interaction of hHop and PfHsp70-x has previously been validated in vitro and the current bioinformatics data support ii the existence of such a complex. PfHsp70-x was not stable in the cell lysate that was prepared and this could explain why its interaction with hHop could not be ascertained. However, taken together the evidence from a previous independent study, and the predicted interaction of PfHsp70-x with human chaperones suggests cooperation of chaperone systems which possibly facilitates the folding and function of parasite proteins that are exported to the infected erythrocyte.<br>NRF

The study sought to explore and describe the breastfeeding practices of Human Immunodeficiency Virus (HIV) positive postnatal clients’ non-commitment to exclusive breastfeeding in George Mukhari Hospital, South Africa. A non-experimental quantitative design was used. Inferences drawn from the study were that HIV positive clients that opted for exclusive breastfeeding did not commit for fear of transmission of HIV to the baby and exclusive breastfeeding was stopped before the recommended 6 months. Most of the respondents’ partners did not come for counselling. There was lack of emotional support by staff after testing. Health education and emotional support of HIV positive clients has to be intensified.<br>Health Studies<br>M. A. (Health Studies)

Japanese encephalitis virus (JEV) causes viral encephalitis in new born and young adults that is prevalent in different parts of India and other parts of South East Asia with an estimated 6000 deaths per year. JEV is a single stranded RNA virus that belongs to the Flavivirusgenus of the family Flaviviridae. It is a neurotropic virus which infects the central nervous system (CNS). The virus follows a zoonotic life-cycle involving mosquitoes and vertebrates, chiefly pigs and ardeid birds, as amplifying hosts. Humans are dead end hosts. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral leukocytosis in the brain and damage to Blood Brain Barrier (BBB). The exact role of the endothelial cells during CNS infection is still unclear. However, disruption of this endothelial barrier has been shown to be an important step in entry of the virus into the brain. Humoral and cell mediated immune responses during JEV infection have been intensively investigated. Previous studies from our lab have shown the activation of cytotoxic T-cells (CTLs) upon JEV infection. MHC molecules play pivotal role in eliciting both adaptive (T-cells) and innate (NK cells) immune response against viral invasion. Many viruses such as HIV, MCMV, HCMV, AdV and EBV have been found to decrease MHC expression upon infection. On the contrary, flaviviruses like West Nile Virus (WNV) have been found to increase MHC-I and MHC-II expression. More recently, data from our lab has shown that JEV infection can lead to upregulation of mouse non-classical MHC class Ib molecules like Qb1, Qa1 and T-10 along with classical MHC molecules. Non-classical MHC molecules are important components of the innate and adaptive immune systems. Non-classical MHC molecules differ from their classical MHC class I counterparts by their limited polymorphism, restricted tissue distribution and lower levels of cell surface expression. Human classical MHC class I molecules are HLA-A, -B and –C while non-classical MHC Class Ib molecules are HLA-E, -G and –F. HLA-E, the human homologue of the mouse non-classical MHC molecule, Qa-1b has been shown to be the ligand for the inhibitory NK, NKG2A/CD94 and may bridge innate and adaptive immune responses. In this thesis, we have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecule, HLA-E in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial like cell line ECV304 (ECV), human glioblastoma cell line U87MG and human foreskin fibroblast cells (HFF). We observed an upregulation of classical HLA molecules and HLA-E mRNA in endothelial and fibroblast cells upon JEV infection. This mRNA increase also resulted in upregulation of cell surface classical HLA molecules and HLA-E in HFF cells but not in both the human endothelial cell lines, ECV and HBMECs. Release of soluble classical HLA molecules upon cytokine treatment has been a long known phenomenon. Recently HLA-E has also been shown to be released as a 37 kDa protein from endothelial cells upon cytokine treatments. Our study suggests that JEV mediated upregulation of classical HLA and HLA-E upregulation leads to release of both Classical HLA molecules and HLA-E as soluble forms in the human endothelial cell lines, ECV and HBMEC. This shedding of sHLA-E from human endothelial cells was found to be mediated by matrix metalloproteinase (MMP) proteolytic activity. MMP-9, a protease implicated in release of sHLA molecules was also found to be upregulated upon JEV infection only in endothelial cell lines but not in HFF cells. Our study provides evidence that the JEV mediated solubilisation of HLA-E could be mediated by MMP-9. Further, we have tried to understand the role of the MAPK pathway and NF-κB pathway in the process of HLA-E solubilisation by using specific inhibitors of these pathways during JEV infection of ECV cells. Our data suggests that release of sHLA-E is dependent on p38 and JNK pathways while ERK 1/2 and NF-κB pathway only had a minor role to play in this process. Treatment of endothelial cells with TNF-α, IL-1β and IFN-γ is known to result in release of sHLA-E. In addition to TNF-α and IFNtreatment, we observed that activating agents like poly (I:C), LPS and PMA also resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Treatment of endothelial cells with IFN-β, a type-I interferon also led to release of sHLA-E. IFN-γ, a type II interferon and TNF-α are known to show additive increase in solubilisation of HLA-E. We studied the interaction between type I interferon, IFN-β and TNF-α with regard to shedding of sHLA- E. Both IFNand TNF, when present together caused an additive increase in the shedding of sHLA-E. These two cytokines were also found to potentiate the HLA-E and MMP-9 mRNA expression. Hence, our data suggest that these two cytokines could be working conjunctly to release HLA-E, when these two cytokines are present together as in the case of virus infection of endothelial cells. HLA-E is known to be a ligand for NKG2A/CD94 inhibitory receptors present on NK and a subset of T cells. Previous reports have suggested that NKG2A/CD94 mediated signaling events could inhibit ERK 1/2 phosphorylation leading to inhibition of NK cell activation. IL-2 mediated ERK 1/2 phosphorylation is known to play a very important role in maintenance and activation of NK cells. We studied the effects of sHLA-E that was released, either by JEV infection or IFN-γ treatment on IL-2 mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. The soluble HLA-E that was released upon JEV infection was functionally active since it inhibited IL-2 and PMA induced phosphorylation of ERK 1/2 in NKL and Nishi cells. Virus infected or IFN-γ treated ECV cell culture supernatants containing sHLA-E was also found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. CD25 is a component of the high affinity IL-2 receptor and hence could play an important role in proliferation and activation of NK cells. sHLA-E was also found to inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit the proliferation and activation of NK cells. In summary, we found that establishment of JEV infection and production of cytokines like IFN-β, TNF-α, IL-6 along with MMP-9 in human endothelial cells. These cytokines may also indirectly lead to the reported damage and leukocyte infiltration across infected and uninfected vicinal endothelial cells. The increased surface expression of HLA-E in fibroblast and release of sHLA and sHLA-E molecules from endothelial cells may have an important immunoregulatory role. HLA-E is an inhibitory ligand for NKG2A/CD94 positive CD8+ T and NK cells. Hence our finding that sHLA-E can inhibit NK cell proliferation suggests an immune evasive strategy by JEV.