Orientador: Leonilda Maria Barbosa dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-07T03:41:32Z (GMT). No. of bitstreams: 1 Castro_FabianoRobertode_M.pdf: 943470 bytes, checksum: 271c21deb3b25015f9737d33adf49814 (MD5) Previous issue date: 2006 / Resumo: A Síndrome de Guillain-Barré (SGB) é uma doença desmielinizante do sistema nervoso periférico (SNP). Baseado principalmente nas similaridades clínicas e histopatológicas a Neurite Experimental Auto-imune (EAN) tem sido extensivamente usada como modelo de estudo da SGB. A EAN é uma doença auto-imune, que pode ser experimentalmente induzida em ratos geneticamente suscetíveis através da imunização com os componentes da mielina de nervos periféricos tais como os peptídeos P0 e P2 , ou ainda por transferência adotiva de lifócitos T CD4+ do tipo Th1. Diferentes tentativas de tratamentos para a SGB têm sido estudadas, dentre elas pode-se citar a plasmaferese, o uso de anticorpos monoclonais, administração de corticóides e a imunossupressão global através da administração de intérferon ß. A utilização de venenos totais de serpentes, ou frações deles, já demonstrou bons resultados na tentativa de tratamento de alguns modelos de doenças auto-imunes como a diabetes auto-imune insulino dependente, lúpus e encefalomielite experimental auto-imune (EAE). No presente trabalho foi estudado o efeito de uma fração do veneno da cascavel sul americana Crotalus durissus terrificus (Cdt), a crotapotina, no modelo de EAN. São apresentadas evidências de que tanto a administração intraperitoneal (IP) como a oral de crotapotina reduz significativamente a gravidade da EAN induzida em ratos Lewis, associada a um significativo declínio na resposta proliferativa das células T neuritogênicas, assim como diminuição de infiltrados de células mononucleares no nervo ciático dos os animais / Abstract: Biomedical research in which venom components are being investigated for their potential as novel therapeutic agents has emerged as an interesting option. Crotapotin which is a fraction of the venom of the rattlesnake Crotalus durissus terrificus, has been described as an antinflammatory that acts on the innate arm of the immune response. Here we have demonstrated that intraperitoneal (IP), as well as oral administration of crotapotin significantly reduces the severity of experimental autoimmune neuritis (EAN), an experimental model for Guillain-Barré Syndrome. The reduction of the severity of the disease is associated with a reduction in the mononuclear cells infiltrating in the sciatic nerve and a significant decrease in the lymphocyte proliferative response to neuritogenic peptide / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Lors d’uvéite non infectieuse (UNI), des événements environnementaux vont provoquer une rupture de la tolérance immune périphérique et une activation des cellules résidentes oculaires. Plusieurs données attestent de l’importance du rôle joué par les signaux de danger lors de ces deux phases clefs d’activation pathologique. Si une place capitale a été donnée aux signaux de danger exogènes, notamment microbiens, l’importance des signaux de danger endogènes commence à émerger. A ce titre, les nucléotides constituent une famille importante de signaux de danger endogènes puisque en situation pathologique, ils vont être libérés de façon massive dans l’espace extracellulaire où ils peuvent avoir de nombreux effets en activant des récepteurs P2X et P2Y. Le but de ce travail est d’investiguer si les récepteurs P2Y2 jouent un rôle de récepteurs de danger lors d’UAI. Pour ce faire, nous avons d’abord étudié in vitro l’effet des nucléotides extracellulaires sur la production d’IL-8 (cytokine connue pour son rôle chimiotactique lors d’UNI) par des cellules de l’EPR. Nous avons pu montrer que les nucléotides ATPγS, UTP et UDP, stimulent la sécrétion d’IL-8 tant basale qu’induite par le TNFα en activant la voie intracellulaire d’ERK1/2 via l’activation des récepteurs P2Y2 et P2Y6. Ensuite, in vivo, nous avons comparé le développement d’uvéites autoimmunes expérimentales entre des souris génétiquement déficientes pour le récepteur P2Y2 (P2Y2-/-) et des souris sauvages (P2Y2+/+) et avons pu montrer que le groupe P2Y2-/- était moins affecté par la maladie que le groupe sauvage contrôle. De même, après transfert adoptif de lymphocytes T autoréactifs semi-purifiés, les souris P2Y2-/- étaient moins malades que les souris P2Y2+/+ et le transfert adoptif de lymphocytes T autoréactifs semi purifiés de souris P2Y2-/- induisait moins de maladie que le transfert adoptif de cellules contrôles. En accord avec ces dernières données, nous avons mis en évidence que les LT autoréactifs semi-purifiés issus des souris P2Y2-/- immunisées proliféraient moins et sécrétaient moins de cytokines proinflammatoires que ceux issus des souris P2Y2+/+. Une série de co-cultures nous a permis de démontrer que ce déficit de prolifération provenait d’un défaut au niveau des CPA des souris P2Y2-/-. En conclusion, notre travail de thèse a mis en évidence que la stimulation des récepteurs P2Y augmente l’activation de l’EPR et des lymphocytes T autoréactifs, favorisant ainsi le développement d’UNI. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Les uvéites non infectieuses sont considérées actuellement comme une des plus importantes cause de déficience visuelle dans la population des jeunes adultes. Les uvéites non infectieuses sont des atteintes inflammatoires de la rétine et de l’uvée et sont généralement considérées comme autoimmunes et initiées par la perte de la tolérance immune aux protéines rétiniennes. Elles sont orchestrées d’une part, systémiquement par deux sous populations lymphocytaires dont la signature cytokinique est l’IFNγ (Th1) et l’IL-17 (Th17) et d’autre part, localement, par l’activation du tissu rétinien. Néanmoins, la vision systémique actuelle est plus complexe et fait intervenir une activation pathologique de l’immunité innée, donnant une composante d’autoinflammation aux uvéites non infectieuses. En plus de ce volet systémique, de nombreux travaux attestent de l’importance de l’activation des cellules rétiniennes dans le développement d’uvéites non infectieuses. Loin de jouer un rôle passif durant la maladie, elles vont être stimulées par une série de molécules pro-inflammatoires et ainsi permettre le recrutement et l’activation de cellules immunocompétentes. <p>Notre travail de thèse s’inscrit précisément dans ce contexte du rôle de l’activation des cellules rétiniennes et plus spécifiquement de celles de la barrière hémato rétinienne (BHR) dans le développement d’uvéite non infectieuses.<p>Lors de ce travail, nous avons tout d’abord caractérisé in vivo, dans deux modèles expérimentaux, l’expression de la molécule d’adhésion VCAM-1 (Vascular Adhesion Molecule) sur les cellules de la BHR. VCAM-1 est une molécule d’adhésion qui facilite l’extravasation des leucocytes du sang vers les tissus. Nous avons montré que VCAM-1 n’est pas exprimé dans l’œil sain mais est induit progressivement lors de la maladie et que l’intensité et l’extension de son expression étaient dépendantes de la sévérité de la maladie. Par ailleurs, nous avons montré que VCAM-1 pouvait être induit sur l’ensemble des cellules de la BHR. <p>Nous avons ensuite analysé in vitro, sur les cellules de l’EPR (Epithélium Pigmentaire Rétinien) qui forment la partie externe de la BHR, les effets antagonistes du TNFα sur l’induction des molécules de CMH de classe II par l’IFNγ. Durant le processus inflammatoire, l’EPR est la cible d’un ensemble de cytokines secrétées par les cellules inflammatoires. Il a été donc intéressant d’étudier les effets d’autres cytokines présentes lors de l’inflammation sur l’induction du CMHII par l’IFNγ au niveau de l’EPR. Nous avons démontré que le TNFα inhibe l’expression du CMH II induit par l’IFNγ sur les ARPE par régulation négative du CIITA (Class II Transactivator). Comme l’activation des lymphocytes T par les cellules de l’EPR dépend de leur niveau d’expression du CMH II, notre étude soutient l’idée que le TNFα possède des propriétés immunomodulatrices sur l’activation de ces cellules, et participe ainsi à la phase de résolution de l’inflammation.<p>Enfin, nous avons étudié les effets du blocage de l’activation des cellules rétiniennes par l’IFNγ en surexprimant le gène SOCS1 (Suppressor Of Cytokine Signaling) in vivo et in vitro.<p>Nous avons surexprimé le gène SOCS1 au niveau rétinien et étudier l’effet de cette surexpression sur le développement de l’UAE. L’analyse des grading clinique n’a pas montré de différence significative entre les yeux injectés par l’AAV2-SOCS1 versus l’AAV2-EGFP contrôle. Afin de normaliser par rapport à la diversité inter-individuelle de la maladie, nous avons calculé pour chaque souris un ratio des grades cliniques de l’œil injecté sur l’œil non-injecté. L’analyse de la moyenne de ces ratios montre un effet à la limite de la significativité entre le groupe SOCS1 et le groupe EGFP en terme de grades cliniques. La différence devient par contre significative lorsque l’analyse de ces ratios est faite sur les grades histologiques. Nos expériences mènent donc plutôt à la conclusion que l’expression de SOCS1, médié par injection intravitréenne de l’AAV2 ne protège globalement pas les yeux du développement d’une UAE.<p>Cette absence d’effet peut avoir comme explication que l’injection intravitréenne conduit à une infection relativement limitée des cellules rétiniennes impliquées dans le développement de l’UAE. Il se pourrait également que le niveau d’expression de la protéine SOCS1 soit trop faible pour obtenir un effet protecteur ou que la surexpression de SOCS1 affecte uniquement l’activation des cellules de la rétine par l’IFNγ mais pas par d’autres cytokines telles le TNFα, l’IL-17, ou l’IL-22 qui jouent aussi un rôle important dans le développement d’UAE. C’est cette dernière hypothèse que nous avons choisi d’investiguer in vitro. Nos résultats montrent que la surexpression de ce même gène SOCS1 dans les cellules d’EPR a un effet inhibiteur sur leur activation par l’IFNγ mais pas par le TNFα.<p>Ce travail met tout d’abord en évidence l’importante expression, in vivo, de VCAM1 par les cellules de la BHR lors d’UAE et in vitro les effets antagonistes du TNFα et de l’IFNγ sur la régulation de l’expression de molécules du CMHII à la surface de l’EPR. Nos expériences démontrent que la surexpression du gène SOCS1 après injection intravitréenne du vecteur AAV-CAG-SOCS1 n’a que peu d’effet sur le développement de la maladie. Par ailleurs, la surexpression de ce même gène SOCS1 dans les cellules d’EPR a un effet inhibiteur sur leur activation par l’IFNγ mais pas par le TNFα et l’IL-17.<p> / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Background Chronic myeloid leukemia (CML) is a relatively rare hematological malignancy with a constant incidence of approximately 90 new cases each year in Sweden (0.9 cases/100 000 inhabitants). The etiology is largely unknown but high doses of ionizing radiation are a known but rare risk factor. The treatment options were for a long time limited to chemotherapies i.e. hydroxyurea and busulfan, interferon’s and allogeneic hematopoietic stem cell transplantation and the median survival were only about four years. Since the beginning of the 21st century a new way of treating CML has been introduced, the tyrosine kinase inhibitors (TKI), leading to a rapid decrease in leukemic cells and symptoms. Due to the TKIs, the overall 5-year survival is nowadays approximately 85 % and CML patients have time to develop other diseases, including other malignancies. The aims of this thesis was to investigate the present and future prevalence of CML and the prevalence of other malignancies prior and subsequent to the diagnosis of CML, malignancies among first-degree relatives of persons with CML. In addition, the incidence of autoimmune and chronic inflammatory diseases among patients with CML was also investigated.   Methods From the Swedish CML register, data over nearly all Swedish CML patients from 2002 and forward were obtained for paper II-IV. For paper I, the Swedish cancer register was used to identify all Swedish CML patients since 1970 and the Swedish cause of death register was used to identify an eventual date of death for these patients. With a constant incidence and the relative survival rates for CML patients between 2006 and 2012 as a model, the present and future prevalence was calculated. For paper II-IV, data from the Swedish cancer register was used to identify other malignancies than CML. For paper II, information about autoimmune and chronic inflammatory diseases was retrieved from the Swedish national patient register. For paper II and IV, five controls matched for year of birth, gender and county of residence were randomly selected from the Swedish register of the total population. To calculate odds ratio (OR), conditional logistic regression was used. To calculate the risk of a second malignancy for paper III, Standardized incidence ratio (SIR) was used. In paper IV, first-degree relatives (parents, siblings and offsprings) for both cases and controls were retrieved from the Swedish multi-Generation Register, where persons born later than 1932 and registered in Sweden at some time since 1961 are registered.   Results Prevalence and survival As shown in paper I, the 5-year overall survival for CML patients increased remarkably from 0.18 to 0.82 between 1970 and 2012. The prevalence increased from 3.9 to 11.9 per 100 000 inhabitants in Sweden between 1985 and 2012. By assuming no further improvements in relative survival as compared to the survival rates between 2006 and 2012, the prevalence by 2060 is expected to increase to 22.0 per 100 000 inhabitants. This corresponds to 2 587 CML patients as compared to 1 137 CML patients in 2012.   Malignancies, autoimmune and chronic inflammatory diseases prior to CML In study II, more than 45 000 person-years of follow-up were evaluated in 984 CML patients diagnosed between 2002 and 2012. With an OR of 1.47 (95 % CI 1.20–1.82) and 1.55 (95 % CI 1.21–1.98), respectively, the prevalence of prior malignancies and autoimmune diseases were significantly increased as compared to matched controls. On the other hand, no association between CML and chronic inflammatory diseases was shown.   Second malignancies In 868 CML patients, diagnosed between 2002 and 2011, 52 malignancies were observed in the Swedish cancer register, as shown in paper III. When compared to expected rates in the background population, a significantly increased risk of second malignancies with a SIR of 1.52 (95 % CI 1.13–1.99) was shown. When looking at specific cancer types, gastrointestinal as well as nose and throat cancer were significantly increased.   Familial aggregation of malignancies 984 CML patients were identified in paper IV. However, 184 had a birth date prior to 1932, subsequently only 800 patients were analyzed. Among them, 4 287 first-degree relatives were identified, compared to 20 930 first-degree relatives of the matched controls. 611 malignancies were retrieved; no significant increase of malignancies in first-degree relatives of CML patients was shown (OR 1.06; 95 % CI: 0.96–1.16).   Conclusion Since CML patients nowadays have a high survival rate, the calculations in this thesis shows that the prevalence of CML will almost double by 2060. CML patients have an increased risk of developing malignancies prior and subsequent to the diagnosis of CML, suggesting a hereditary or acquired predisposition to develop cancer. Since there is no familial aggregation of malignancies in CML patients, a hereditary predisposition to develop cancer is unlikely to be part of the pathogenesis of CML, leaving an acquired predisposition more likely.

Pharmacogenetics (PGx) and personalised medicine are new disciplines that, gathering the existing knowledge about the genetic and phenotypic factors that underpin drug response, aim to deliver more targeted therapies that avoid the existing problems of adverse drug reactions or lack of drug efficacy. PGx and personalised medicine imply a shift in the way drugs are prescribed, as they require introducing diagnostic tools and implementing pre-screening mechanisms that assess patients' susceptibility to new or existing drugs. The direct benefit is an improvement in drug safety and/or efficacy. However, neither pharmacogenetics nor personalised medicine, are widely used in clinical practice. Both technologies face a number of controversies that hamper their widespread use in clinical practice. This thesis investigates the scientific; technological; social; economic; regulatory and ethical implications of PGx and personalised medicine, to understand the enablers and barriers that drive the process of technology diffusion in three conditions: autoimmune diseases, acute lymphoblastic leukaemia and non-small cell lung cancer.The thesis uses concepts of the sociology of science and a qualitative approach, to explore the arguments for and against the use of the technology by different actors (pharmaceutical and biotechnology companies, researchers, clinicians, regulators and patient organisations). The core of this analysis lies in the understanding of how, diagnostic testing (TPMT testing in the case of autoimmune diseases, acute lymphoblastic leukaemia, and EGFR testing in the case of non-small-cell lung cancer) may affect the existing drug development and service delivery mechanisms, with a particular focus on the user-producer interactions and feedback mechanisms that underpin diffusion of medical innovations and technological change in medicine.The thesis concludes by identifying gaps in knowledge and common issues among TPMT and EGFR testing, which might be used, in the future, to inform policy on how to improve PGx service delivery through a public Health System such as the NHS.

Le CD154, est une glycoprotéine transmembranaire de type II appartenant à la famille des TNF (tumor necrosis factor). Il est exprimé de façon inductible et transitoire sur plusieurs types cellulaires. Il existe deux formes du CD154, membranaire (mCD154) et soluble (sCD154) qui forment une structure trimérique biologiquement active. Le CD154 a été initialement identifié pour son rôle important dans la réponse humorale thymodépendante suite à sa liaison avec son récepteur, le CD40. Il a été démontré par la suite, que le CD154 joue un rôle dans différentes réponses inflammatoires dont certaines peuvent conduire à des processus pathogènes de maladies inflammatoires chroniques et auto-immunes. Depuis quelques années, des études ont montré que les intégrines αIIbβ3, αMβ2, αvβ3 et α5β1 sont de nouveaux récepteurs pour le CD154. Elles jouent un rôle important dans le contrôle de nombreux processus cellulaires tels que la prolifération et l’apoptose cellulaire. Cependant, le rôle biologique de l’interaction du CD154 avec ses récepteurs nouvellement identifiés reste peu étudié. Les cellules T sont des cellules clés de la réponse immunitaire, leur survie et leur persistance constituent unesignature caractéristique de nombreuses maladies inflammatoires et auto-immunes. Lors de ces dernières, les cellules T expriment des niveaux élevés d’intégrines β1. Étant donné le rôle attribué aux intégrines et en particulier aux intégrines β1, dans l'inhibition des événements apoptotiques dans les cellules T, nous étions intéressés d’étudier le rôle de l'interaction CD154 / α5β1 dans la promotion de la survie des cellules T. Dans cette étude, nous avons démontré que le sCD154 est capable de se lier spécifiquement à lʼintégrine α5β1 exprimée par les cellules T, et que cette interaction inhibe l'apoptose cellulaire induite par Fas dans les lignées de cellules T suite à lʼinhibition du clivage de la caspase-8. Nous avons également montré que lʼeffet anti-apoptotique induit par l’axe CD154/α5β1 nʼétait pas restreint au Fas seulement, mais était généralisé aux autres récepteurs de mort de la famille des TNF tel que le TRAIL et le TNF. Plus intéressant encore, nos résultats ont montré que la pré-incubation avec le sCD154 des cellules humaines T CD3+ , protège ces dernières de la mort induite par Fas. Enfin, nous avons montré que comme le sCD154, le mCD154 était capable dʼinhiber la mort induite via les différents récepteurs de mort Fas, TRAIL et TNF, mais seulement par un mécanisme cis-dépendant lorsque le CD154 et lʼintégrine α5β1 sont exprimés à la surface d’une même cellule. Ces résultats soulignent l’importance de l'interaction CD154/α5β1 dans la survie des cellules T et de ce fait, dans leurs rôles biologiques. Cette nouvelle observation inculpe davantage le CD154 et ses récepteurs les intégrines dans l’initiation, mais également dans la progression et la persistance des pathologies inflammatoires et auto-immunes en particulier. L'inhibition spécifique de cet axe peut représenter une cible thérapeutique potentielle dans le traitement des pathologies. / CD154 is a type II glycoprotein belonging to the tumor necrosis factors (TNF). It’s expressed in a transitory way on different cell types. There are two forms of CD154, membrane (mCD154) and soluble (sCD154). Both form are biologically active trimeric structure. CD154 has been identified on its role in the thymus dependent humoral response following its binding with its CD40 receptor. It has subsequently been shown that CD154 plays a role in various inflammatory responses, some of which may lead to pathogenic processes of chronic inflammatory and autoimmune diseases. Recently, studies have shown that other αIIbβ3, αMβ2, αvβ3 and α5β1 receptors belonging to the integrin family could interact with CD154. They play an important role in the control of many cellular processes such as cell proliferation and apoptosis. However, the biological role of the interaction of CD154 with its newly identified receptors remains poorly studied. T lymphocytes are key cells in the immune response. Their survival and persistence is a characteristic signature of numerous inflammatory and autoimmune diseases. In these latter, T cells express high levels of β1 integrins. Given the role attributed to integrins and in particular to β1 integrins in the inhibition of apoptotic events in T cells, we were very interested in studying the role of the CD154 / α5β1 interaction in promoting T cell survival. In this study we have demonstrated that sCD154 is able to specifically bind to T-cell expressed α5β1 integrin. This interaction inhibits Fas-induced apoptosis in Jurkat E6.1 and HUT-78 T cell lines following the inhibition of cleavage of caspase-8. We have also shown that the anti-apoptotic effect induced by the CD154 / α5β1 axis was not restricted to Fas, but was generalized to other TNF-like death receptors such as TRAIL and TNF. More interestingly, our results showed that preincubation with sCD154 of previously activated human T CD3+ cells, protects them from Fas-induced death. Finally, we have shown that, like sCD154, mCD154 was able to inhibit death induced by the different death receptors Fas, TRAIL and TNF, but only by a cis-dependent mechanism when CD154 and α5β1 are expressed at the same time surface of the same cells. These results enhance the importance of the CD154 / α5β1 interaction in the survival of T cells and thus in their biological roles. This new observation further induces CD154 and its integrin receptors in initiation, but also in the progression and persistence of inflammatory and autoimmune pathologies, in particular. The specific inhibition of this axis may represent a potential therapeutic target in the treatment of pathologies.

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

Indiana University-Purdue University Indianapolis (IUPUI) / Polycystic kidney diseases (PKD) are genetic disorders characterized by fluid filled cysts in the kidney tubules and liver bile ducts. There are two forms of PKD, autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). The focus of the studies in this thesis has been on ADPKD. The disease progresses slowly and the fluid-filled cysts grow in size due to increased rates of cell proliferation and fluid secretion into the cyst lumen. The expanding cysts compromise the normal kidney function and result in a decrease of renal function to the point of end-stage renal failure in midlife. Cyst enlargement is due, at least in part, to chloride secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Currently therapy is limited to renal cyst aspiration, dialysis, and eventually renal transplantation after organ failure, thus it has critical to determine possible drug therapies for the treatment of PKD. Previous studies showed that cyst fluid caused a secretory response in cells lining the cysts. We hypothesized that once the cyst have expanded and become so large that they burst or leak, which could also occur due to renal injury or aging, the cyst fluid may stimulate additional cyst growth. Lysophosphatidic Acid (LPA) was determined to be the active component of human cyst fluid, and we investigated the LPA stimulated signaling pathway. Our data suggest that the LPA stimulates chloride and fluid secretion by a combination of CFTR and Calcium-Activated chloride channels (CaCC) and that the two channels may functionally be linked to each other. The secretion is not occurring through a cAMP stimulated pathway, and it is possible that TMEM16A, a CaCC, plays a larger role than previously expected. Previous studies demonstrated that PPARγ agonists, insulin sensitizing drugs used to treat diabetes, inhibit chloride secretion by the collecting duct principal cells by decreasing CFTR synthesis. It was logical therefore to considered PPARγ agonists as long-term treatment for PKD. The first preclinical studied showed that high (20 mg/kg BW) dose pioglitazone, a PPARγ agonist, inhibited cyst growth in the PCK rat model, a slow progressing model, of PKD. To continue to look at the effects of the PPARγ agonists another preclinical study was completed, which tested if there was a class action of PPARγ agonists and if a lower dose was effective in treating the cystic burden. Using the PCK rat model, and another PPARγ agonist, rosiglitazone, a 24 week study was completed using 3 doses (4, 0.4, and 0.04 mg/kg BW). 4 mg/kg BW rosiglitazone is analogous to 20 mg/kg BW pioglitazone. The data indicated that the rosiglitazone is effective in lowering the cystic burden, and importantly the low dose proved to be effective. An additional rat model, the W-WPK rapidly progressing model was used to determine efficacy across multiple models, and to determine if there was a way to track the progress of the disease in a manner analogous to that used in human patients. The animals were treated with pioglitazone using 2 doses (2 and 20 mg/kg BW), and were imaged using CT scans to track the progress of the disease. The data suggest that pioglitazone was not as effective in the W-WPK rat model as it was the PCK rat model. There was a trend however, that low dose PPARγ agonist was as effective ad high dose. Even more important, the CT scans proved to be an effective way to track the progress of the disease in animal models.

Interpretation of Common Used Tumor Markers Affected by Systemic and Inflammatory Diseases Introduction: An examination of tumor markers is often made as a basis for the successful diagnosis and follow-up treatment of patients with malignant tumors. However, are tumor markers truly significant by themselves, or are they just a baseline quantitative expression of value that we use to diagnose a patient as better or worse based on it increasing or decreasing value? Objective: This paper attempts to answer the question of what factors can affect serum protein and mucin markers and thus lead to a misinterpretation of their results. Methods: Tumor markers were determined by isotopic and non-isotopic laboratory analysis methods, using operational protocols of the immunoanalytic laboratory. All methods were checked using internal quality control, and four times a year using an external quality control. Additionally, 16 236 samples were analysed using 3180 probands during the period 2008-2014. Results: We discovered that in premenopausal women, the markers AFP, CA 125 and HE 4 rise during ovulation peak periods while other markers changed minimally or not at all. However, in postmenopausal women, we proved the incidence of a false positivity marker. With women in the 1st and 2nd trimester of pregnancy, the...

Contexte : Les personnes vivant avec le VIH (PVVIH) ont un risque de maladies auto-immunes 2 fois plus élevé que la population générale. Les corticostéroïdes constituent la pierre angulaire du traitement de ces maladies. Toutefois, ils peuvent favoriser la survenue d’infections. A ce jour, le risque d’infections associé aux corticostéroïdes chez les PVVIH demeure incertain. Méthodes : Nous avons réalisé une étude de cohorte rétrospective de 1991 à 2011, utilisant les données médico-administratives de la régie de l’assurance maladie du Québec. Tous les patients ayant reçu un code diagnostic spécifique au VIH, appariés aux VIH négatifs pour l’âge, le sexe et la date index ont été inclus. L'exposition d'intérêt était l'utilisation de corticostéroïdes pour une durée minimale de 20 jours. L’issue d’intérêt était la survenue d'infections sévères nécessitant une hospitalisation. Les taux d’infection bruts et ajustés en fonction de l’exposition aux corticostéroïdes ont été obtenus au moyen d’un modèle de Poisson avec effet aléatoire et les résultats stratifiés selon le statut du VIH. Résultats : Au total, 4798 PVVIH ont été appariés à 17644 patients VIH négatifs. 1083 (22.6%) PVVIH et 1854(10.5%) VIH négatifs ont été exposés aux corticostéroïdes. La durée moyenne d'exposition était de 4±4.4 mois chez les PVVIH et de 1.6±5.5 mois chez les VIH négatifs. Le ratio de densité d’incidence pour infections sévères associé à l’exposition aux corticostéroïdes était de 2.49 [1.71-3.60] pour les PVVIH et 1.32 [0.71-2.47] pour les VIH négatifs (valeur p interaction=0.18). Les infections sévères les plus fréquentes étaient représentées par les infections pulmonaires (50.4%), suivies des infections du tractus urinaire (26%) et les infections opportunistes (10.5%). Conclusion : Bien que les estimés ponctuels des mesures d’incidence obtenus soient différents dans les deux groupes, laissant suspecter un effet différent des stéroïdes chez les PVVIH, les intervalles de confiance demeurent larges. De ce fait, nous ne pouvons pas conclure à une différence d’effet des corticostéroïdes entre VIH+ et VIH-. Des recherches additionnelles avec des données récentes et de grandes tailles sont nécessaires pour confirmer s’il existe ou non un risque d’infections associé aux corticostéroïdes chez les PVVIH tels que suggérés par les estimés ponctuels, surtout chez ceux dont la maladie est contrôlée. / Background: People living with HIV (PLWH) have been shown to have an increased risk of autoimmune diseases. Corticosteroids are the cornerstone of autoimmune diseases treatment, but their use is associated with an increased risk of infections. It is unclear how HIV status affects the risk of infection associated with corticosteroid use. Methods: We conducted a retrospective cohort study from 1991 to 2011, using a medico-administrative database from Quebec. Medical billing codes were used to identify PLWH, and we matched them on age, sex and index date with up to 4 HIV-negative controls. The exposure of interest was corticosteroid use, defined as a systemic corticosteroid dispensation lasting at least 20 days. The outcome of interest was hospitalization for severe infection. Crude and adjusted incidence rates ratios of infection were obtained using a random effect Poisson model, and results were stratified by HIV status. Results: In total, 4798 PLWH were matched to 17644 HIV-negative controls. 1083 (22.6%) PLWH and 1854 (10.5%) HIV-negative controls received at least one course of corticosteroid. The mean duration of corticosteroid use was 4±4.4 months in PLWH and 1.6±5.5 months in HIV-negative controls. The incidence rate ratio (IRR) for infections associated with corticosteroid use was 2.49 [1.71-3.60] in PLWH and 1.32 [0.71-2.47] in HIV-negative controls (P value for interaction 0.18). The most frequent infections were pulmonary infections (50.4%), followed by urinary tract infections (26%) and opportunistic infections (10.5%). Conclusion: Although the points estimates suggest a more pronounced risk of infection associated with corticosteroid use in PLWH, the confidence interval remains large. Therefore, we cannot infer that there is a difference in the risk of severe infections between PLWH and HIV-negative. However, further research with contemporary data and large sample size are warranted to confirm whether the risk associated with corticosteroid use remains high in PLWH with well-controlled HIV infection.