O sistema NADPH oxidase humano é responsável pela geração de reativos intermediários do oxigênio e defeitos neste sistema resultam na Doença Granulomatosa Crônica (DGC). Nesta tese de doutorado, investigamos o efeito do IFN-g sobre eventos pós-transcricionais em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. Os dados obtidos sugerem que o uso do IFN-g in vitro interfere no processamento da mensagem causando aumento da expressão de transcritos do gene CYBB e NCF1 em células B-EBV de indivíduos sadios e pacientes DGC analisados. Observamos também que o IFN-g dimunui a expressão dos genes THOC4 NONO, SF3A1, SRRM1 e UPF3A e promove aumento de expressão de SRSF10, SNRPA1 e C2 em células B-EBV de paciente X-DGC secundária a defeitos de splicing. Identificamos que o IFN-g e o TNF-α aumentam a expressão das proteínas envolvidas no processo do splicing. Concluímos que o IFN-g aumenta a expressão de genes importantes para uma resposta eficiente do sistema imunológico, incluindo os do sistema NADPH oxidase, além de promover aumento da expressão de genes e de proteínas relacionados ao processo de splicing, que podem estar relacionados aos efeitos benéficos observados no uso do IFN-g em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. / The human phagocyte NADPH oxidase is responsible for the generation of reactive oxygen intermediates and defects in this system result in Chronic Granulomatous Disease (CGD). In this PhD Thesis, we investigated the effect of IFN-g on post-transcriptional events in normal individuals and patients with X-linked CGD, caused by splicing defects. The obtained data suggests that the use of IFN-g in vitro interferes in the message processing causing an increase of expression of CYBB and NCF1 gene transcripts in B-EBV cells of healthy individuals and analyzed CGD patients. We also observed that IFN-g decreases the expression of THOC4, NONO, SF3A1, SRRM1 and UPF3A, and increases the expression of SRSF10, SNRPA1 and C2 genes in cells from X-CGD patients, due to splicing defects. We identified that IFN-g and TNF-α induce expression of proteins involved in the splicing process. We conclude that IFN-g increases the expression of important genes for an effective immune response, including the NADPH oxidase system genes, and promotes augment of gene and protein expression related to the splicing process, which may be related to the beneficial effects related to the use of IFN-g in CGD patient caused by splicing defects.

La lymphohistiocytose hémophagocytaire (HLH) est une maladie hyper-inflammatoire rare, mais potentiellement mortelle affectant surtout les jeunes enfants. Il existe deux formes de HLH : primaire et secondaire. La HLH primaire est causée par une mutation des gènes impliqués dans la voie cytotoxique médiée par les granules, alors que la HLH secondaire se développe en raison d’une condition préexistante, telle qu’un cancer, une infection chronique ou une transplantation. La HLH est caractérisée par le développement d’inflammation extrême, une production excessive de cytokines inflammatoires ainsi qu’une infiltration tissulaire massive par des lymphocytes T et des macrophages activés. À ce jour, le traitement de la HLH consiste d’abord à réduire l’inflammation à l’aide d’agents de chimiothérapie hautement toxiques et de corticostéroïdes, puis à procéder à une transplantation de cellules souches hématopoïétiques (CSH) chez les patients atteints de HLH primaire, le seul traitement disponible permettant la rémission de ces patients. Toutefois, les thérapies actuelles ne parviennent pas toujours à réduire l’inflammation extrême chez plusieurs patients atteints de HLH, si bien que le taux de mortalité avant la transplantation de CSH est toujours d’environ 20% à 25%. C’est pourquoi le développement de nouveaux traitements anti-inflammatoires, plus efficaces, représenterait une avancée majeure dans le traitement de la HLH. Des études dans des modèles murins avaient déjà démontré que l’utilisation d’anticorps bloquants ciblant l’IFNg (anti-IFNg) ou de ruxolitinib, un inhibiteur de JAK1/2 (« janus activated kinases 1/2 »), pouvait améliorer efficacement, quoique partiellement, les manifestations de la HLH. Nous avons donc émis l’hypothèse que l’utilisation d’une thérapie combinée ciblant des cytokines JAK-dépendantes et indépendantes serait plus efficace qu’une monothérapie pour diminuer les symptômes de la HLH. À l’aide d’un modèle murin déficient pour le gène de la perforine (PKO), nous avons comparé les effets de l’inhibition de l’IL-6 et de l’IL-18, deux cytokines fortement sécrétées dans la HLH, en combinaison avec soit de l’anti-IFNg ou du ruxolitinib. Nous avons aussi vérifié l’efficacité d’une thérapie combinée d’anti-IFNg et de ruxolitinib sur les manifestations de la HLH. Nos travaux ont montré que l’anti-IL-6R et l’anti-IL-18, lorsque combinés avec de l’anti-IFNg ou du ruxolitinib, amélioraient légèrement, mais pas de manière significative, les symptômes de la HLH. Par contre, l’utilisation d’une thérapie combinant du ruxolitinib avec de l’anti-IFNg générait un important effet de synergie sur la résolution des symptômes. Notre étude démontre que l’anti-IFNg et le ruxolitinib, même s’ils sont efficaces par eux-mêmes, devraient être utilisés en combinaison afin de prévenir la progression de la HLH. Cette étude est particulièrement pertinente puisque l’anti-IFNg a récemment été approuvé par la FDA pour le traitement de la HLH tandis que le ruxolitinib est présentement à l’étude chez l’humain. / Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening hyper-inflammatory disease affecting mainly young children. There are two types of HLH : primary and secondary. Primary HLH is caused by defects in genes of the perforin-granzyme cytotoxic pathway, whereas secondary HLH develops following a pre-existing underlying condition, such as a cancer, a chronic infection or a transplantation. HLH is characterized by extreme inflammation, a very high secretion of inflammatory cytokines and a massive tissue infiltration by activated T cells and macrophages. Currently, treatment for HLH consists of reducing the inflammation with a course of highly toxic chemotherapy agents and corticosteroids, followed by hematopoietic stem cell transplantation (HSCT) for patients with primary HLH, which is the only available curative treatment for these patients. However, current therapies often fail to properly manage the extreme inflammation of HLH in many patients, so that the pre-HSCT fatality still stands around 20% to 25%. Thus, the development of new, more potent anti-inflammatory treatments would be a major advance in the treatment of HLH. It had already been shown in relevant mouse models that blocking antibodies targeting IFNg (anti-IFNg) or ruxolitinib, a Janus activated kinase 1/2 (JAK1/2) inhibitor, could efficiently, but partially, improve the pathological manifestations of HLH. Thus, we hypothesized that combined therapies targeting both JAK-dependent and independent cytokines would be more effective than either one alone to reduce the symptoms of this pathology. Using a perforin knock-out (PKO) mouse model, we compared the effects of targeting IL-6 and IL-18, which are highly expressed cytokines during HLH, in combination with either anti-IFNg or ruxolitinib. We also tested the efficacy of a therapy combining anti-IFNg with ruxolitinib on the manifestations of HLH. We found that anti-IL-6R and anti-IL-18, when used in combination with either anti-IFNg or ruxolitinib, showed small, but not significant improvements on the pathological manifestations of HLH. However, combination therapy using ruxolitinib and anti-IFNg showed a major synergistic effect on the resolution of the symptoms of HLH. Our findings support that anti-IFNg and ruxolitinib, although effective independently, should be used in combination to suppress HLH progression. This is particularly relevant since the former was recently approved for treating HLH while the latter is in clinical trials.

It is now clear that human and animal exposure to estrogenic compound occurs through several sources. This include: i) naturally occurring endogenous estrogens, ii) exogenous or intentional estrogens for prophylactic (e.g. oral contraceptive) and therapeutic (e.g. as replacement therapy for ovulation in nulliparous women and in menopausal women, and in some men suffering from prostate cancer) purposes, iii) accidental via estrogenic chemical exposure (e.g. pesticides, industrial byproducts) and phytoestrogens (e.g. soybeans). It has long been recognized that estrogen, a female sex hormone, functions not only on the reproductive system, but also on various other systems including the immune system. Estrogens are thought to be of both physiologic and pathologic importance. Female in general, have better immune capabilities than males, a phenomenon attributed to the action of sex hormones on the immune system. There is also a female-gender bias in susceptibility to autoimmune diseases. Estrogens have been linked either directly or indirectly to the etiology and pathogenesis of various female-predominant autoimmune diseases. Estrogens have also been linked to the onset of cancer, and conditions where the immune system often malfunctions. Estrogen affects the functions of both B and T cells, possibly by regulating such factors as lymphokine gene expression and/or cellular death by apoptosis. However, the functioning of both B and T cells under the influence of long-term exposure to estrogen has not been fully understood. The primary aim of this thesis was to investigate the effect of long-term exposure to 17β-estradiol on lymphokine and bcl-2/fas (proto-oncogenes) mRNA expression. We evaluated the effects of estrogen on the expression of genes for lymphokines, which are essential for the immune response. It is hypothesized that estrogen may regulate the immune system by modifying the expression of lymphokine genes and/or genes that regulate apoptosis. The results demonstrated that long-term 17β-estradiol exposure reduced the viability of lymphocytes when compared to lymphocytes from placebo-treated mice. IL-2 and IFN-g mRNA was consistently higher in ConA-stimulated lymphocytes from estrogen-treated mice (P < 0.05). The mRNA for TGF-β₁ lymphokine was also increased but was not consistent at all time points of incubation. The expression of IL-4 mRNA was not noticeably affected by estrogen treatment of mice. Long-term exposure to 17β-estradiol appear to have some influence on the mRNA expression of proto-oncogenes fas and bcl-2 in splenic and thymic T lymphocytes. There was a trend of increased bcl-2 mRNA expression in estrogen-treated mice compared to placebo-treated mice, whereas the mRNA expression of fas gene appeared to be lower compared to controls. Overall, these findings suggest that 17β-estradiol may selectively influence lymphokine and proto-oncogene mRNA expression. These results suggest that the one mode of modulation of the immune response by 17β-estradiol may be through alterations in the lymphokine and proto-oncogene expression. Since estrogen-treatment markedly induces atrophy of the thymus and diminishes the cellularity of the lymphoid organs (e.g. Spleen), it became necessary to perform multiple assays on the same cells, particularly lymphokine and apoptosis gene expression. A secondary objective of this thesis was to investigate whether lymphocytes, which have undergone proliferation in Lympho-Pro™ assay (Alamar Blue assay), could be utilized for further analysis. In this regard, we found that a non-radioactive assay that utilizes Alamar Blue had significant advantages over the conventional ³H-thymidine incorporation assay. By using cells from estrogen and placebo-treated mice in the Alamar Blue assay, we found that this assay not only allowed determination of lymphocyte proliferation, but also the assessment of mRNA expression, cytogenetics, apoptosis and immunophenotyping of the same lymphocytes. / Master of Science

No description available.

No description available.

No description available.

Toxoplasma gondii, an obligate intracellular protozoan parasite, establishes a latent, chronic infection by forming cysts preferentially in the brain after replication of tachyzoites in various organs during the acute stage of infection. Chronic infection with T. gondii is one of the most common parasitic diseases in humans. The immune system is required for maintaining the latency of chronic infection. Reactivation of infection can occur in immunocompromised individuals, such as AIDS patients, which results in the development of life-threatening toxoplasmic encephalitis (TE). IFN-gamma-dependent, cell mediated immune responses play an essential role in preventing the reactivation of chronic infection of T. gondii in the brain. In my dissertation study, we examined the mechanisms of IFN-gamma-mediated prevention of TE by using models of reactivation of chronic infection in BALB/c mice. This strain of mouse is genetically resistant to T. gondii infection and establishes a latent chronic infection as do immunocompetent humans, and therefore provides an excellet model for this purpose. Our laboratory previously demonstrated that both T cells and IFN-gamma-producing non-T cells are required for genetic resistance of BALB/c mice against development of TE. However, the function of T cells required for the resistance is still unclear. Therefore, in the present study, we examined whether IFN-gamma production or perforin-mediated cytotoxicity of T cells play an important role in their protective activity against TE. Immune T cells were obtained from infected IFN-gamma-knockout (IFN-g-/-), perforin-knockout (PO), and wild-type (WT) BALB/c mice, and transferred into infected, sulfadiazine-treated athymic nude mice which lack T cells but have IFN-gamma-producing non-T cells. Control nude mice that had not received any T cells developed severe TE due to reactivation of infection and died after discontinuation of sulfadiazine treatment. Animals that had received immune T cells from either PO or WT mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-gamma-/- mice developed severe TE and died as early as control nude mice. T cells obtained from spleens of the animals that had received either PO or WT T cells both produced large amounts of IFN-gamma following stimulation with T. gondii antigens in vitro. In addition, the amounts of IFN-gamma mRNA expressed in the brains of PO T-cell recipients did not differ from those of WT T-cell recipients. These results indicate that IFN-gamma production, but not perforin-mediated cytotoxic activity, by T cells is required for prevention of TE in genetically resistant BALB/c mice. In our attempt to identify a T cell population(s) that produces IFN-gamma in the brain and plays an important role for prevention of TE, we analyzed T cell receptor (TCR) Vb chain usage in T cells expressing IFN-gamma in the brains of infected BALB/c mice. We found T cells bearing TCR V beta8 chain to be the most frequent IFN-g-producing population in the brains of infected animals. To examine the role of IFN-gamma production by this T cell population for prevention of TE, V beta8+ immune T cells purified from spleens of infected BALB/c and IFN-g-/- mice were transferred into infected, sulfadiazine-treated athymic nude mice. After discontinuation of sulfadiazine treatment, control nude mice that had not received any T cells and animals that had received Vb8+ T cells from IFN-g-/- mice all died due to reactivation of infection (TE). In contrast, animals that had received the cells from WT mice survived. These results indicate that IFN-gamma production by Vb8+ T cells in the absence of any other T cell population can prevent reactivation of infection. Thus, V beta8+ T cells play a crucial role in genetic resistance of BALB/c mice to TE through their production of IFN-gamma. When V beta8+ immune T cells were divided into CD4+ and CD8+ subsets, a potent protective activity was observed only in the CD8+ subset whereas a combination of both subsets provided greater protection than did the CD8+Vb8+ population alone. These results indicate that CD8+ subset of V beta8+ T cells is a major afferent limb of IFN-gamma-mediated resistance of BALB/c mice against TE, although the CD4+ subset of the T cell population works additively or synergistically with the CD8+V beta8+ population. T cells need to enter into the brains of infected mice to demonstrate their protective activity against TE. This migration is mediated, in part, by endothelial adhesion molecules. Since IFN-gamma is essential for preventing reactivation of chronic infection with this parasite in the brain, we examined whether this cytokine plays an important role in expression of lymphocyte and endothelial adhesion molecules and recruitment of T cells into the brain during chronic infection with T. gondii using IFN-g-/- and WT BALB/c mice. Although the number of cerebral vessels expressing intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) increased in both WT and IFN-g-/- mice following infection, there were more VCAM-1+ vessels in brains of infected WT than infected IFN-g-/- mice; in contrast, numbers of ICAM-1+ vessels did not differ between strains. We did not detect endothelial E-selectin, P-selectin, MAdCAM-1 or PNAd in any of the brains. Significantly fewer CD8+ T cells were recruited into brains of infected IFN-g-/- than WT mice. Treatment of infected IFN-g-/- mice with recombinant IFN-gamma restored the expression of VCAM-1 on their cerebral vessels and recruitment of CD8+ T cells into their brains, confirming an importance of this cytokine for up-regulation of VCAM-1 expression and CD8+ T cell trafficking. In infected WT and IFN-g-/- animals, almost all cerebral CD8+ T cells had an effector/memory phenotype (LFA-1high, CD44high and CD62Lneg) and approximately 38% were positive for a4b1 integrin (the ligand for VCAM-1). In adoptive transfer of immune spleen cells, pre-treatment of the cells with a monoclonal antibody against a4 integrin markedly inhibited recruitment of CD8+ T cells into the brain of chronically infected wild-type mice. These results indicate that IFN-g-induced expression of endothelial VCAM-1 and its binding to a4b1 integrin on CD8+ T cells is important for recruitment of the T cells into the brain during the chronic stage of T. gondii infection. Since we found strong expression of ICAM-1 on endothelia and LFA-1 on T cells in the brains of infected mice, LFA-1/ICAM-1 interaction, in addition to a4b1 integrin/VCAM-1 interaction, may also be involved in this process. As mentioned earlier, CD8+ T cells are crucial for prevention of TE in BALB/c mice. Therefore, IFN-gamma-mediated expression of VCAM-1 and its binding to a4b1 integrin for recruitment of CD8+ T cells may play a critical role in genetic resistance of BALB/c mice to development of TE. / Ph. D.

No description available.

VSR Dukkipati (2007). A search for genetic factors influencing immune responses to Mycobacterium avium subspecies paratuberculosis. Doctoral thesis, Massey University, Palmerston North, New Zealand. A study was conducted to identify associations between genetic markers and immune responses in Australian fine-wool Merino sheep to a killed Mycobacterium avium subspecies paratuberculosis (Map) vaccine (GudairTM). Blood samples and immune response data (antibody and interferon gamma, IFN-gamma results) were obtained from 934 sheep from a longterm Map vaccination trial undertaken on three independent properties in New South Wales, Australia. Blood samples were genotyped for eight microsatellite markers that included four (DYMS1, OLADRW, OLADRB and SMHCC1) from the Ovar-Mhc region, two each from the SLC11A1 (OVINRA1 and OVINRA2) and IFN-gamma (o(IFN)gamma and OarKP6) gene regions. Vaccination with GudairTM induced strong antibody and IFN-gamma responses as early as two weeks post-vaccination. Between-property differences in magnitude and trend of immune responses, concomitant with season of vaccination and magnitude of natural infection prevalent in individual flocks, were evident. Immune responses in controls on all the three properties remained consistently low, except for slightly elevated IFN-gamma levels at a few time points in controls of properties 2 and 3, concomitant with exposure to natural infection. There were only 2 alleles and 3 genotypes for marker o(IFN)gamma but other loci exhibited extensive polymorphisms, the most occurring at OLADRW which had 42 alleles and 137 genotypes. Heterozygosities varied between 33% (OVINRA2) and 87% (SMHCC1), while polymorphic information contents ranged from 0.31 (o(IFN)gamma) to 0.88 (OLADRW). Genotypes at loci DYMS1, OLADRB, SMHCC1, OVINRA1 and o(IFN)gamma were in Hardy- Weinberg equilibrium (HWE), while those at OarKP6 were in HWE only when rare alleles (<1.0% frequency) were pooled with the closest size class. Departure from HWE, resulting from possible preferential amplification of alleles in heterozygotes, was evident at OLADRW and OVINRA2. Associations between immune responses and genetic polymorphisms at the marker loci were examined by analysing both genotypic and allelic affects. The study revealed several genotypes/alleles at different marker loci to be significantly associated with antibody and IFN-gamma responses to vaccination with GudairTM. However, the majority of those effects were inconsistent across the three properties. Based on significance and consistency in effects across the three properties, five genotypes (two at DYMS1 and one each at OLADRB, SMHCC1 and OVINRA1) and three alleles (one each at DYMS1, OLADRB and o(IFN)gamma) were considered either ‘probable’ or ‘most likely’ to be associated with low IFN-gamma responses, while a genotype at o(IFN)gamma was considered ‘most likely’ to influence high IFN-gamma responses. An allele at OarKP6 was considered ‘probable’ to be associated with low antibody responses to vaccination. Considering the significance of IFN-gamma responses in protection against Map, it is likely that the identified genotype/alleles influencing IFN-gamma responses to vaccination would also influence immune responses to natural Map infections. However, further studies need to be conducted to determine the role of these marker genotypes/alleles in protection against paratuberculosis under natural infection conditions. Key words: paratuberculosis, OJD, Johne’s disease, sheep, immune response, genetic markers, gene polymorphisms, MHC, SLC11A1, IFN-gamma

VSR Dukkipati (2007). A search for genetic factors influencing immune responses to Mycobacterium avium subspecies paratuberculosis. Doctoral thesis, Massey University, Palmerston North, New Zealand. A study was conducted to identify associations between genetic markers and immune responses in Australian fine-wool Merino sheep to a killed Mycobacterium avium subspecies paratuberculosis (Map) vaccine (GudairTM). Blood samples and immune response data (antibody and interferon gamma, IFN-gamma results) were obtained from 934 sheep from a longterm Map vaccination trial undertaken on three independent properties in New South Wales, Australia. Blood samples were genotyped for eight microsatellite markers that included four (DYMS1, OLADRW, OLADRB and SMHCC1) from the Ovar-Mhc region, two each from the SLC11A1 (OVINRA1 and OVINRA2) and IFN-gamma (o(IFN)gamma and OarKP6) gene regions. Vaccination with GudairTM induced strong antibody and IFN-gamma responses as early as two weeks post-vaccination. Between-property differences in magnitude and trend of immune responses, concomitant with season of vaccination and magnitude of natural infection prevalent in individual flocks, were evident. Immune responses in controls on all the three properties remained consistently low, except for slightly elevated IFN-gamma levels at a few time points in controls of properties 2 and 3, concomitant with exposure to natural infection. There were only 2 alleles and 3 genotypes for marker o(IFN)gamma but other loci exhibited extensive polymorphisms, the most occurring at OLADRW which had 42 alleles and 137 genotypes. Heterozygosities varied between 33% (OVINRA2) and 87% (SMHCC1), while polymorphic information contents ranged from 0.31 (o(IFN)gamma) to 0.88 (OLADRW). Genotypes at loci DYMS1, OLADRB, SMHCC1, OVINRA1 and o(IFN)gamma were in Hardy- Weinberg equilibrium (HWE), while those at OarKP6 were in HWE only when rare alleles (<1.0% frequency) were pooled with the closest size class. Departure from HWE, resulting from possible preferential amplification of alleles in heterozygotes, was evident at OLADRW and OVINRA2. Associations between immune responses and genetic polymorphisms at the marker loci were examined by analysing both genotypic and allelic affects. The study revealed several genotypes/alleles at different marker loci to be significantly associated with antibody and IFN-gamma responses to vaccination with GudairTM. However, the majority of those effects were inconsistent across the three properties. Based on significance and consistency in effects across the three properties, five genotypes (two at DYMS1 and one each at OLADRB, SMHCC1 and OVINRA1) and three alleles (one each at DYMS1, OLADRB and o(IFN)gamma) were considered either ‘probable’ or ‘most likely’ to be associated with low IFN-gamma responses, while a genotype at o(IFN)gamma was considered ‘most likely’ to influence high IFN-gamma responses. An allele at OarKP6 was considered ‘probable’ to be associated with low antibody responses to vaccination. Considering the significance of IFN-gamma responses in protection against Map, it is likely that the identified genotype/alleles influencing IFN-gamma responses to vaccination would also influence immune responses to natural Map infections. However, further studies need to be conducted to determine the role of these marker genotypes/alleles in protection against paratuberculosis under natural infection conditions. Key words: paratuberculosis, OJD, Johne’s disease, sheep, immune response, genetic markers, gene polymorphisms, MHC, SLC11A1, IFN-gamma