INTRODUÇÃO: Toxocaríase é uma infecção parasitária de distribuição global, causada pela fase larval de Toxocara spp. Os hospedeiros naturais são cães e gatos, nos quais o parasita completa o ciclo chegando a fase adulta. Outros hospedeiros podem ser infectados pela fase larval do parasita, após ingestão de ovos embrionados do solo, mãos contaminadas, fomites, ou ingestão de carne ou vísceras de animais infectados. Em hospedeiros paratênicos o parasita não completa o ciclo, invadindo em estágio larval vísceras ou outros tecidos, onde podem sobreviver e induzir a patologia. O presente estudo teve como objetivo caracterizar o hamster (Mesocricetus auratus), como modelo experimental de toxocaríase, inicialmente através do estudo das lesões histopatológicas em fígado, pulmão e rim. A caracterização da resposta imunológica do modelo, foi feita através do estudo de citocinas envolvidas nas respostas Th1 e Th2, e foi sugerida uma correlação entre alterações glomerulares e depósitos de complexos antígenos-anticorpo pré-formados na circulação. MÉTODOS: Hamsters foram inoculados com ovos embrionados de Toxocara canis, e mantidos no biotério do Instituto de Medicina Tropical de São Paulo. O estudo histopatológico foi desenvolvido utilizando-se cortes parafinados corados por hematoxilina e eosina. Para detecção de antígenos nos tecidos foram realizadas reações imunohistoquímicas, utilizando-se anticorpo monoclonal e policlonal anti- Toxocara canis. Utilizando-se o soro dos animais infectados e animais controle, foi realizada pesquisa de antígeno e anticorpo por ELISA. Para pesquisa de imunoglobulinas IgG e IgM e complemento, foram utilizados cortes congelados de rins para realização de reação de Imunofluorescência. Fragmentos de rins foram incluídos para utilização em microscopia eletrônica, para detecção de antígenos de toxocara e de imune complexos. Para caracterização de resposta imunológica foram estudadas citocinas envolvidas na resposta Th1 e Th2 por técnica de RT-PCR. RESULTADOS: Os achados histopatológicos demonstraram desde o início da infecção, presença de larvas em maior número no fígado, seguido de pulmão e raramente rins. Em fígado remanescentes larvares foram visualizados cercados por reação inflamatória granulomatosa. Logo no início da infecção foi encontrado pneumonite intersticial e intraalveolar focal, e lesão renal com glomérulo apresentando hiperplasia focal de células mesangiais (glomerulite mesangio-proliferativa). Houve marcação de antígenos em todos os grupos de animais infectados, tanto pelo anticorpo monoclonal, como pelo policlonal. Depósitos de imunoglobulinas e complemento foram marcados em glomérulo por imunofluorescência A análise dos soros por ELISA, demonstrou na pesquisa de anticorpos aumento gradativo no decorrer da infecção, acompanhado de diminuição de antígenos. Depósitos de antígenos em glomérulos foram detectados por microscopia imonoeletrônica. No RT-PCR foi detectado aumento significativo do nível de IL-4, com tendência de elevação de IL-10 e IFN-?. CONCLUSÃO: O hamster demonstrou ser um modelo experimental eficiente para toxocaríase. Entretanto este modelo é mais adequado para infecções de curto prazo. A resposta imunológica avaliada por RT-PCR, com elevado nível da expressão de IL-4, sugere uma resposta Th2, mas a tendência de aumento de IL-10 e IFN-? poderia sinalizar uma resposta mista Th1 e Th2. Achados de depósitos de imunoglobulinas no glomérulo sugerem a possibilidade de que as manifestações renais com síndrome nefrótica em humanos possa vir a ter como base a toxocaríase / INTRODUCTION: Toxocariasis is a parasitic infection of global distribution, caused by the larval stage of Toxocara spp. The natural hosts are dogs and cats, in which the parasite completes the cycle reaching adulthood. Other hosts can be infected with the larval stage of the parasite, after ingestion of embryonated eggs from the soil, contaminated hands, fomites, or ingestion of meat or viscera of infected animals. In paratenics hosts the parasite not complete the cycle, encroaching on larval stage in viscera or other tissues where they can survive and induce pathology. The present study aimed to characterize the hamster, Mesocricetus auratus, as experimental model of toxocariasis, initially through the study of histopathological lesions in the liver, lung and kidney. The characterization of immune response model, was made through the study of cytokines Th1 and Th2 responses involved, and a correlation was suggested between glomerular changes and antibody-antigen complexes deposits preformed in the circulation. METHODS: Hamsters were inoculated with embryonated eggs of Toxocara canis, and kept in the bioterium of the Institute of Tropical Medicine of the São Paulo. The histopathologic study was developed using paraffin slides stained by hematoxylin and eosin. For detection of antigens in tissues immunohistochemistry reactions were performed using monoclonal and polyclonal anti-Toxocara canis sera. Using the serum of infected and control animals, search has been carried out of antigen and antibody by ELISA. For the search of immunoglobulins IgG, IgM and complement, were used slides prepared from frozen fragments of kidneys and a immunofluorescence reaction. Fragments of kidneys were included for electron microscopy to detect antigens of Toxocara and immune complexes. For characterization of Th1 and Th2 response cytokines involved were detected by RT-PCR technique. RESULTS: Histopathological findings demonstrated since the beginning of the infection the presence of larvae in greater numbers in the liver, followed by lung and rarely kidneys. In the liver larval remnants were surrounded by a granulomatous inflammatory reaction. Early in the infection was found interstitial pneumonitis with intraalveolar focal inflammatory infiltrate and renal injury with glomerulus showing mesangial cell focal hyperplasia (mesangioproliferative glomerulonephritis). There were the presence of antigens in all groups of animals infected detected by both the monoclonal and polyclonal antibodies. Deposits of immunoglobulin and complement were present in glomerulus by immunofluorescence analysis. ELISA, showed that the presence of antibodies increased gradually in the course of infection, accompanied by progressive diminution of antigens. Clusters of antigen/s were detected by immunoelectron microscopy. RT-PCR showed a significant increase of IL-4, with a tendency of increase of IL- 10 and IFN-?. CONCLUSION: The hamster has proved to be an efficient experimental model for toxocariasis. However this model is best suited for short-term infections. The immune response evaluated by RT-PCR, with high level of expression of IL-4, suggests a Th2 response, but the trend of increase of IL-10 and IFN-? might suggest a Th1 and Th2 mixed response. Findings of immunoglobulin deposits in glomeruli suggests the possibility that the renal manifestations with nephrotic syndrome in humans might have, in certain circunstances, as a basis the toxocariasis

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-10-06T17:20:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Denise Maria do Nascimento Costa.pdf: 2568349 bytes, checksum: 97687424c47175731885cd254c815ad4 (MD5) / Made available in DSpace on 2016-10-06T17:20:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Denise Maria do Nascimento Costa.pdf: 2568349 bytes, checksum: 97687424c47175731885cd254c815ad4 (MD5) Previous issue date: 2016-07-21 / Introdução: Glomerulopatia membranosa (GM) é uma causa de síndrome nefrótica cuja etiologia pode ser primária (GMP) ou secundária, dentre estas é frequente o Lúpus eritematoso sistêmico (LES). Trata-se de uma doença imunologicamente mediada, caracterizada pela deposição de imunocomplexos no espaço subepitelial glomerular. A maioria dos antígenos envolvidos identificados são alvos da imunoglobulina G4 (IgG4), subclasse predominante em imunofluorescências renais na GMP, em contraste com a GM secundária a LES (GMS) na qual IgG1, IgG2 e IgG3 prevalecem. Apesar da IgG4 ser um subtipo de imunoglobulina com baixa capacidade de ativação do complemento, há várias evidências deste envolvimento na GMP. Esses dados, em conjunto com achados de depósitos glomerulares de lectina ligadora de manose (MBL), um dos principais componentes da via das lectinas do complemento, podem sugerir que tanto a via da lectina como a IgG4 estão envolvidas nesta patologia. Sabe-se ainda que o desenvolvimento de GMP também está associado a alterações genéticas. Entretanto, a etiopatogenia da GMP ainda não é totalmente conhecida e estudos para avaliação gênica do MBL2 e dosagem sérica de IgG em GM são escassos. Assim, foi realizado este estudo com o objetivo de avaliar a frequência de polimorfismos do gene MBL2 em portadores de GM, comparados a indivíduos saudáveis. Um segundo objetivo foi comparar pacientes com GMP e GMS quanto a diferenças do percentual de IgG4 sérico em relação a IgG (%IgG4) e da frequência de polimorfismos do MBL2. Métodos: Estudo realizado entre 2014 e 2015, em Pernambuco - Brasil. A amostra incluiu 60 pacientes adultos com diagnóstico histopatológico de GMP ou GMS. Outras causas de GM secundárias foram excluídas. Foram avaliados 35 pacientes com GMP e 24 com GMS, e um grupo controle (GC), formado por 101 indivíduos saudáveis. Resultados: O alelo mutante O do gene MBL2 foi mais frequente no grupo com GM comparados aos GC (42% x 22%; p < 0,001). A heterozigose A/O, em relação ao genótipo A/A, predominou entre os pacientes comparados ao GC, associando-se a GM com OR = 11,16 (95% IC = 4,77 - 28,41). À análise comparativa entre os pacientes com GMP e GMS, não houve diferença das frequências dos polimorfismos genéticos entre os grupos. O grupo GMP apresentou menor mediana de IgG sérica total (p = 0,008) e maior %IgG4 (p = 0,016), comparado ao grupo GMS. Nível sérico de IgG4 não diferiu significativamente entre os grupos GMP e GMS (p = 0,289). Conclusão: O polimorfismo do éxon 1 do gene MBL2 associou-se à GM, comparado a indivíduos saudáveis, porém sem diferença entre as etiologias avaliadas. Já o %IgG4 sérico foi maior na GMP em relação a GMS. Estes resultados sugerem que esta mutação genética possa conferir maior vulnerabilidade a GMP e que o %IgG4 sérico possa ser utilizado como marcador adicional para diagnóstico diferencial entre as duas etiologias da GM. / Introduction: Membranous glomerulopathy (MG) is a cause of nephrotic syndrome whose etiology may be primary (PMG) or secondary, wich is frequent systemic lupus erythematosus (SLE). It is an immune-mediated disease characterized by the deposition of immune complexes in the glomerular subepithelial space. Most of the identified antigens are targets to immunoglobulin IgG4, most common subclass in renal immunofluorescence in GMP, in contrast to the SLE secondary MG (SMG) in which IgG1, IgG2 and IgG3 prevail. Although IgG4 is a immunoglobulin subtype with low complement activation capacity, there is abundant evidence of this involvement in PMG. These data, together with glomerular deposits of mannose-binding lectin (MBL), a major component of the lectin pathway of complement, may suggest that both the lectin pathway and IgG4 are involved in this pathology. It is also known that the development of PMG is associated with genetic alterations. As the pathogenesis of PMG is not yet fully known, and studies for genetic evaluation of MBL2 and serum IgG in MG are scarce, this study was conducted to evaluate the frequency of MBL2 gene polymorphisms in patients with MG, compared to healthy subjects. A second objective was to compare patients with PMG and SMG with respect to the percentage of serum IgG4 (IgG4%) and frequency MBL2 polymorphisms. Methods: This study was conducted between 2014 and 2015 in Pernambuco - Brazil. The sample included 60 adult patients with histopathologic diagnosis of PMG or SMG. Other causes of secondary MG were excluded. Thity five patients with PMG and 24 with SMG were evaluated, compared to a control group (CG) of 101 healthy subjects. Results: The mutant allele O was more frequent in the MG population compared to CG (42% vs. 22%; p <0.001). The heterozygous A/O, compared to genotype A/A, predominated among patients compared to the control group, and was associated with MG (OR = 11.16; 95% CI = 4.77 to 28.41). In the comparative analysis between patients with PMG and SMG, there was no difference in the frequency of genetic polymorphisms between groups. The PMG group had lower median total serum IgG (p = 0.008) and higher IgG4% (p = 0.016) compared to the SMG group. Serum IgG4 did not differ significantly between the groups PMG and SMG (p = 0,289). Conclusion: The polymorphism of exon 1 MBL2 gene was associated with MG, compared to healthy subjects, but no difference between the assessed etiologies. Serum IgG4% was higher in PMG relative to SMG. These results suggest that this gene mutation can confer increased vulnerability to PMG and the serum IgG4% may be used as an additional marker for the differential diagnosis between the two etiologies MG.

O Lúpus Eritematoso Sistêmico (LES) é uma doença auto-imune caracterizada pela deposição de imunocomplexos nos tecidos. O clareamento de imunocomplexos está comprometido no LES, contribuindo para a patogênese da nefrite lúpica. Os receptores Fcg (FcgR) participam do clareamento dos imunocomplexos contendo IgG, pois se ligam à porção Fc desta molécula. O FcgRIIa é um receptor que tem dois alelos co-dominantemente expressos, o R131 e o H131, os quais diferem na sua eficiência em se ligar a subclasses de IgG. Células que expressam o homozigoto FcgRIIa-H/H131 são as únicas que se ligam eficientemente a imunocomplexos contendo IgG2, enquanto as que expressam FcgRIIa-R/R131 o fazem de forma menos eficaz. Este polimorfismo tem sido descrito como fator de risco para nefrite lúpica, embora ainda haja controvérsias. O propósito do nosso estudo foi o de analisar, em uma população de nefrite lúpica e em outra de glomerulopatias primárias, a associação entre o genótipo FcgRIIa-R/R131 e a gravidade da doença renal na sua instalação (definida pelo momento da biópsia renal) e ao final do seguimento, bem como possíveis relações com aspectos histológicos renais. A genotipagem do receptor FcgRIIa foi realizada em 76 pacientes com nefrite lúpica e 63 com glomerulopatias primárias através da extração do DNA genômico, seguido de reação de polimerização em cadeia (PCR) e nested PCR, utilizando-se primers específicos. Os pacientes foram avaliados por parâmetros clínicos e laboratoriais. Setenta e um pacientes com nefrite lúpica realizaram biópsia renal, enquanto 5 que já se encontravam em hemodiálise não a realizaram. Pacientes com glomerulonefrite membranoproliferativa, nefropatia da IgA e glomerulonefrite proliferativa mesangial foram agrupados como glomerulopatias proliferativas enquanto os com glomeruloesclerose segmentar e focal, glomerulopatia de lesões mínimas ou glomerulonefrite membranosa foram agrupados como glomerulopatias não proliferativas. O homozigoto FcgRIIa-R/R131 foi mais prevalente no grupo com nefrite lúpica (42,1% de R/R131 e 14,5% de H/H131) em relação ao grupo com glomerulopatias primárias (23,8% de R/R131 e 23,8% de H/H131), dado este estatisticamente significativo (p<0.05). Houve segregação do genótipo FcgRIIa- R/R131 nos pacientes com nefrite lúpica quando comparados aos com glomerulopatias não proliferativas, mas não quando comparados aos com glomerulopatias proliferativas (p<0.05). Não houve diferença na distribuição genotípica do receptor FcgRIIa em relação a classe histológica de nefrite lúpica, tampouco em relação aos que evoluíram ou não para insuficiência renal (Pcr = 1,4mg/dl ao final do seguimento). Um aumento na frequência do genótipo FcgRIIa- R/R131 foi encontrado nos pacientes com nefrite lúpica apresentando níveis mais elevados de FAN (FAN>1/100) e consumo de complemento C3 (p<0,05), mas não naqueles com presença de anticorpos anti-dsDNA ou anti-fosfolípide (p>0,05). Estes achados sugerem que uma distribuição anormal dos genótipos do receptor FcgRIIa com predomínio do homozigoto R/R131 é um fator importante que pode influenciar o desenvolvimento de nefrite lúpica e de glomerulopatias proliferativas. O genótipo FcgRIIa-R/R131 também está relacionado com maior atividade lúpica (FAN>1/100 e consumo de C3) em pacientes brasileiros. / Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by tissue deposition of immune complexes. Immune complex clearance is impaired in SLE, contributing to the pathogenesis of lupus nephritis. Fcg receptors (FcgR) participate in the clearance of the immune complexes containing immunoglobulin G, because they bind the Fc domain of this molecule. The FcgRIIa receptor has two co dominant alleles, R131 and H131. They differ in their efficiency to bind IgG subclasses. Cells expressing the homozygote FcgRIIa-H/H131 are the only ones, which bind efficiently immune complexes containing IgG2, whereas those expressing FcgRIIa-R/R131 do not. This polymorphism has been described as a risk factor for lupus nephritis. However, reports are still controversial. This study aims to establish the role of FcgRIIa polymorphism in the severity and prognosis of lupus nephritis compared to primary glomerulopathies, and whether it is related to histological findings or not. In 76 patients with lupus nephritis and 63 patients with primary glomerulopathies, genotyping of the FcgRIIa receptor was performed with standard PCR, followed by nested PCR using specific primers. The same patients were assessed according to clinical and laboratory patterns. Seventy-one patients with lupus nephritis underwent biopsy, while five did not since they were already under dialysis. Patients diagnosed as membranoproliferative glomerulonephritis, IgA glomerulonephritis and mesangial proliferative glomerulonephritis were grouped as proliferative glomerulopathies, while those with focal segmental glomerulosclerosis, membranous glomerulonephritis and minimal change disease were grouped as nonproliferative glomerulopathies. The homozygous FcgRIIa-R/R131 was more prevalent in lupus nephritis (42,1% being R/R131 and 14,5% H/H131) than in glomerulopathies (23,8% being R/R131 and 23,8% H/H131). These data were statistically significant (p<0.05). A segregation of the FcgRIIa-R/R131 genotype was found in patients with lupus nephritis compared to nonproliferative glomerulopathies, but not when compared to proliferative glomerulopathies (p<0.05). No relation was found between genotype distribution and histological class or renal insufficiency (end-study serum creatinine = 1.4 mg/dl). The genotype R/R131 was more prevalent in lupus nephritis patients presenting complement 3 (C3) consumption and higher antinuclear factor (ANF) titers, but not in those with antidouble- stranded DNA or antiphospholipid antibodies (p>0.05). We concluded that a skewed distribution of the FcgRIIa genotypes with R/R131 predominance may contribute to the development of lupus nephritis and proliferative glomerulopathy. In Brazilian patients, this polymorphism is also related to more intense lupus activity (ANF > 1/100 and C3 consumption).

L‟infection par le VIH-1, chez les patients, affecte principalement le système immunitaire et conduit à une destruction graduelle des lymphocytes T CD4 et, par conséquent, entraîne un état d‟immunodéficience. Cette immunodéficience permet l'établissement d‟infections opportunistes qui sont responsables de manifestations cliniques associées au Sida. Ces patients peuvent aussi développer des lymphomes, lésions du système nerveux central et une atteinte rénale. L'ampleur et la sévérité des conditions associées observées chez les patients infectés par le VIH-1 ne peuvent être imputées seulement au processus infectieux et à la déplétion des cellules T CD4+. Ceci suggère que les produits des gènes de régulation pourraient avoir des effets cytopathogènes. Cependant, les études sur la physiopathogenèse induite par le VIH ou ses différents gènes ont été difficiles à mener en raison de l'absence d'animaux de laboratoire infectés par ce virus. Ceux-ci auraient pu aider à disséquer le rôle des différents composants du génome viral et les mécanismes pathogénétiques impliqués. Pour pallier cette contrainte, nous avons produit le premier modèle de souris transgéniques pour le gène vpu. Vpu code pour une phosphoprotéine membranaire avec plusieurs fonctions connues. Elle participe au relargage des virions à la surface cellulaire, induit la dégradation des CD4, induit la régulation négative des CMH-1, augmente la susceptibilité à la mort cellulaire des lymphocytes T infectés par le VIH et favorise la réplication virale en empêchant les mécanismes antiviraux cellulaires. Dans ce travail, nous avons caractérisé pathologiquement un modèle de souris transgéniques porteuses du gène vpu du VIH-1. Nos résultats démontrent que l‟expression de vpu chez les souris transgéniques induit le développement spontané d‟une hyperplasie lymphoïde pansystémique, une splénomégalie avec une hyperplasie lymphoïde folliculaire évoluant en lésions prémalignes et malignes qui présentent certaines similarités avec la maladie de Castleman et une iv glomérulonéphrite mesangioproliférative qui rappelle certaines altérations de néphropathie associée au VIH chez les patients infectés. L‟ensemble des altérations démontre que les souris Tg/vpu développent une activation chronique et non spécifique du système immunitaire. Dans cette activation immunitaire, une dérégulation de l‟IL-6 et une hyperplasie du réseau de cellules métallophiliques pourraient être impliquées. D‟autres résultats obtenus sur les évaluations du fonctionnement du système immunitaire de la rate et du thymus mettent en évidence une susceptibilité augmentée des lymphocytes des tissus lymphoïdes aux effets apoptotiques de la dexaméthasone et des lipopolysaccharides et un retard dans le repeuplement par les cellules d‟organes lymphoïdes ainsi qu‟une réaction inflammatoire (Schwartzman) exacerbée et des anomalies dans la réaction d‟hypersensibilité retardée expérimentale. Ce modèle transgénique reproduit plusieurs anomalies rencontrées chez les patients infectés par le VIH et ouvre de nouvelles hypothèses sur la pathogenèse de l‟infection par le VIH. / The HIV-1 infection results in a severe immunodepression due to an increasing depletion of the CD4+T lymphocytes and in a neurodegeneration that leads to a motor/cognitive complex dysfunction. In addition to these pathological alterations, patients with AIDS may also develop additional complications including neoplasms, nephropathy, hematological abnormalities and opportunistic infections. None of these complications can be accounted for solely by the loss of CD4+T lymphocytes and several investigators have started to suspect the regulatory and accessory genes of possibly playing a role in the AIDS physiopathogenesis. However, studies on the pathological effects contributed by these genes have been hampered by the lack of laboratory animals susceptible to HIV-1 infection. These could have helped to dissect out the role of the different components of the viral genome and their pathogenetic mechanisms. To overcome this obstacle, several investigators have developed transgenic mice carrying either HIV-1 individual genes or partially deleted genome. The vpu gene, which is unique to HIV-1, encodes a membrane phosphoprotein with several known functions as determined by in vitro studies. It participates in virions release at the cell surface, induces CD4 degradation, MHC-1 downregulation, increases apoptosis susceptibility of infected T cells and promotes viral replication by inhibiting cellular antiviral mechanisms. In order to investigate the contribution of vpu to the complex pathogenesis associated with HIV-1 infection, our laboratory developed a transgenic mouse model carrying vpu under the RSV LTR promoter. Here, we described our results of the clinical and pathological investigation of this unique animal model. Our results demonstrate that the expression of vpu in the transgenic mice induces a pansystemic lymphoid hyperplasia, splenomegaly with follicular lymphoid hyperplasia that progress to premalignant and malignant lesions, with striking characteristics of Castelman like-disease, and a mesangial vi hyperplasia variant of HIVAN frequently observed in adult and pediatric AIDS. All lesions observed in this mouse model suggest that the vpu carrying mice develop a nonspecific chronic activation of the immune system with hyperplasia of perivascular metallophillic cells and deregulation of the IL-6 expression within the hyperplastic follicles. Following treatment with dexamethazone and LPS, we observed an increased apoptosis susceptibility of the lymphocytes of the spleen and thymus with slow lymphocyte repopulation of these lymphoid organs. In addition, the transgenic mice developed an exacerbated reaction to the induced Schwartzman reaction and demonstrated a more delayed experimental DTH reaction. Thus, this transgenic model reproduces several abnormalities present in HIV patients. It represents a very interesting and valuable model for studies on the pathology contributed by the vpu gene in the absence of the cumulative effects of the other HIV-1 genes and might help define new possible functions and mechanisms of vpu in the HIV-1 pathogenesis.

O anticorpo anti-proteína P ribossomal é um dos marcadores sorológicos do lúpus eritematoso sistêmico, previamente associado a glomerulonefrite lúpica classe V (ISN-RPS). Neste trabalho foi avaliado o prognóstico renal em pacientes que possuem positividade para este anticorpo. Sessenta pacientes foram avaliados para parâmetros de sobrevida renal. Onze pacientes (18%) apresentaram positividade sorológica exclusiva para anticorpo anti-P ribossomal e vinte e oito pacientes (47%) para anti-dsDNA. Ao final do período de seguimento, foi observado que os pacientes anti-P positivos apresentaram uma maior sobrevida renal (11,0 ± 4,5 vs. 9,2 ± 4,5 anos, p=0,03) quando comparados aqueles anti-P negativos, assim como menor frequência de necessidade de terapia substitutiva renal (0 vs. 35% p = 0,025). Pacientes anti-P positivos apresentaram também maior frequência de classe V (91% vs. 31%, p < 0.001) e menor incidência de alterações proliferativas (45% vs. 82%, p = 0,021) na avaliação da biópsia renal quando comparados aos pacientes sem a positividade para este anticorpo. Os dados reforçam a hipótese de que o anticorpo anti-P é um marcador útil de um melhor prognóstico renal em pacientes portadores de lúpus eritematoso sistêmico / Antibodies to ribossomal P proteins are one of the serologic markers of systemic lupus erythematosus, previously described as associated to class V lupus glomerulonephritis (ISN-RPS). Our study assessed renal prognosis in patients with anti-P antibodies. Sixty consecutive SLE patients with biopsyproven nephritis (2004 ISN/RPS) were evaluated for renal survival parameters. Eleven patients (18%) had exclusive anti-P positivity and 28 (47%) patients anti-dsDNA. The post-biopsy follow-up analysis demonstrated that anti-P positive patients disclosed better renal survival (11.0 ± 4.5 vs. 9.2 ± 4.5 years, p = 0.03) as well as lower frequency of patients requiring dialysis (0 vs. 35% p = 0.025). The frequency of class V nephritis was higher in anti-P positive patients (91% vs. 31%, p < 0.001) and the occurrence of proliferative lesions at biopsy was lower in these patients (45% vs. 82%, p=0.021). Our data supports the notion that anti-P antibody is a valuable marker to predict a better long-term renal outcome in lupus patients

Η Ιδιοπαθής Μεμβρανώδης Σπειραματονεφρίτιδα (ΙΜΣ) ή νεφροπάθεια, η πιο συχνή αιτία νεφρωσικού συνδρόμου στους ενήλικες, συνήθως αντιμετωπίζεται με τη χορήγηση κορτικοειδών και κυτταροτοξικών φαρμάκων ή κυκλοσπορίνης (cyclosporine-A, CsA). Σκοπός της μελέτης ήταν η εκτίμηση της αποτελεσματικότητας της μακροχρόνιας χορήγησης CsA στην πρόκληση ύφεσης του νεφρωσικού συνδρόμου και των ιστολογικών αλλοιώσεων σε επαναληπτικές βιοψίες νεφρού μετά τη χορήγηση του δυνητικά νεφροτοξικού αυτού φαρμάκου. Επιπλέον, εκτιμήθηκε η αποτελεσματικότητα του Mycophenolate Mofetil (MMF) ως σχήματος θεραπείας της ΙΜΣ σε μικρό αριθμό ασθενών και η προγνωστική αξία των επιπέδων του αυξητικού παράγοντα TGF-β1 στα ούρα και στο πλάσμα ως δεικτών εξέλιξης της νόσου. Μελετήθηκαν 32 ασθενείς με ΙΜΣ οι οποίοι εμφάνιζαν νεφρωσικό σύνδρομο και είχαν ικανοποιητική νεφρική λειτουργία κατά τη διάγνωση της νόσου και στους οποίους χορηγήθηκε συνδυασμός πρεδνιζολόνης και CsA. Παρατηρήθηκε πλήρης ύφεση του νεφρωσικού συνδρόμου σε 18 (56%) και μερική ύφεση σε 10 ασθενείς (31%) μετά από 12 μήνες θεραπείας (συνολικά στο 87% των ασθενών). Επεισόδια υποτροπών παρατηρήθηκαν στο 39% και 60% των ασθενών με πλήρη ή μερική ύφεση αντίστοιχα, και πολλαπλές υποτροπές στο 25% των ασθενών, οι οποίοι παρουσίασαν βαθμιαία μείωση της απαντητικότητας στη CsA και επιδείνωση της νεφρικής λειτουργίας. Επαναληπτική βιοψία νεφρού έγινε σε 18 ασθενείς με ύφεση του νεφρωσικού συνδρόμου μετά από 24 μήνες θεραπείας για να εκτιμηθεί η δραστηριότητα της νόσου και οι πιθανές ιστολογικές αλλοιώσεις σε πλαίσια τοξικότητας από κυκλοσπορίνη. Στις επαναληπτικές βιοψίες παρατηρήθηκαν: εξέλιξη του σταδίου της νόσου, επιδείνωση της σπειραματοσκλήρυνσης και της διαμεσοσωληναριακής βλάβης στο 60% των ασθενών. Δεν παρατηρήθηκαν χαρακτηριστικές αλλοιώσεις νεφροτοξικότητας από την κυκλοσπορίνη. Η βαρύτητα των ιστολογικών αλλαγών συσχετίστηκε με το χρονικό διάστημα που είχε παρέλθει από την πρώτη βιοψία νεφρού (r = 0.452, p < 0.05) και θεωρήθηκε ως φυσική εξέλιξη της νόσου. Ικανοποιητικά αποτελέσματα διαπιστώθηκαν από τη χορήγηση Mycophenolate Mofetil σε 6 ασθενείς με ΙΜΣ, στους οποίους το MMF χρησιμοποιήθηκε σε συνδυασμό με μικρή δόση πρεδνιζολόνης, είτε λόγω ανθεκτικότητας του νεφρωσικού συνδρόμου στην CsA, είτε ως αρχική θεραπεία σε περιπτώσεις αντένδειξης στην χορήγηση CsA. Ύφεση του νεφρωσικού συνδρόμου παρατηρήθηκε σε 4 από τους 6 ασθενείς. Τα επίπεδα του TGF-β1 στα ούρα ασθενών με ΙΜΣ και λευκωματουρία ήταν σημαντικά υψηλότερα συγκριτικά με αυτά υγιών εθελοντών και ασθενών με άλλες σπειραματοπάθειες που δεν παρουσίαζαν λευκωματουρία και μειώθηκαν σημαντικά μετά από χορήγηση κορτικοειδών και κυκλοσπορίνης. Η συγκέντρωση του TGF-β1 στο πλάσμα δε διέφερε σημαντικά μεταξύ υγιών εθελοντών και ασθενών με ΙΜΣ και νεφρωσικό σύνδρομο, καθώς και μεταξύ ασθενών με ή χωρίς ύφεση της λευκωματουρίας μετά από τη θεραπευτική αγωγή. / Idiopathic membranous nephropathy (IMN), the most common cause of nephrotic syndrome in adults, is usually treated with a combination of corticosteroids with cytotoxic drugs or cyclosporin A (CsA). The aim of this study was the estimation of the effectiveness of long-term use of CsA in the remission and relapse rate of nephrotic syndrome along with histological changes in repeat renal biopsies after treatment with this potentially nephrotoxic drug, and the evaluation of Mycophenolate Mofetil (MMF) as a treatment regimen for IMN. In addition, urinary and plasma TGF-β1 levels were evaluated as markers of progression of kidney disease. Thirty-two nephrotic patients with well-preserved renal function treated by prednisolone and CsA were studied. Complete remission of nephrotic syndrome was observed in 18 (56%) and partial remission in 10 patients (31%) after 12 months of treatment (total 87%). Relapses were observed in 39% and 60% of patients with complete and partial remission, respectively, and multiple relapses in 25% of patients, who showed gradual unresponsiveness to CsA and decline of renal function. A repeat biopsy was performed in 18 patients with remission of nephrotic syndrome, after 24 months of treatment, to estimate the activity of the disease and features of CsA toxicity. Progression of the stage of the disease, more severe glomerulosclerosis and tubulointerstitial injury were recognized in 60% of patients in repeat renal biopsies. Features of CsA nephrotoxicity were not observed. The severity of histological changes was related to the time elapsed from the first biopsy (r = 0.452, P < 0.05). MMF was proved effective in a small number of nephrotic patients with IMN and well-preserved renal function. MMF in combination with small dose of prednisolone was given in 6 patients with either persistent nephrotic syndrome to CsA or as initial therapy because of contraindication to CsA administration. Remission of nephrotic syndrome was observed in 4 out of 6 MMF treated patients. Urinary and plasma TGF-β1 levels were examined as markers of progression of the disease. TGF-β1 levels in the urine of patients with proteinuria were significantly higher compared with those of healthy individuals and patients with other types of nephropathy without proteinuria. Furthermore, urinary TGF-β1 of nephrotic patients with membranous nephropathy significantly reduced after treatment with CsA and corticosteroids. Plasma TGF-β1 levels showed no difference between patients and healthy subjects as well as between patients with and without remission of proteinuria after treatment.

L‟infection par le VIH-1, chez les patients, affecte principalement le système immunitaire et conduit à une destruction graduelle des lymphocytes T CD4 et, par conséquent, entraîne un état d‟immunodéficience. Cette immunodéficience permet l'établissement d‟infections opportunistes qui sont responsables de manifestations cliniques associées au Sida. Ces patients peuvent aussi développer des lymphomes, lésions du système nerveux central et une atteinte rénale. L'ampleur et la sévérité des conditions associées observées chez les patients infectés par le VIH-1 ne peuvent être imputées seulement au processus infectieux et à la déplétion des cellules T CD4+. Ceci suggère que les produits des gènes de régulation pourraient avoir des effets cytopathogènes. Cependant, les études sur la physiopathogenèse induite par le VIH ou ses différents gènes ont été difficiles à mener en raison de l'absence d'animaux de laboratoire infectés par ce virus. Ceux-ci auraient pu aider à disséquer le rôle des différents composants du génome viral et les mécanismes pathogénétiques impliqués. Pour pallier cette contrainte, nous avons produit le premier modèle de souris transgéniques pour le gène vpu. Vpu code pour une phosphoprotéine membranaire avec plusieurs fonctions connues. Elle participe au relargage des virions à la surface cellulaire, induit la dégradation des CD4, induit la régulation négative des CMH-1, augmente la susceptibilité à la mort cellulaire des lymphocytes T infectés par le VIH et favorise la réplication virale en empêchant les mécanismes antiviraux cellulaires. Dans ce travail, nous avons caractérisé pathologiquement un modèle de souris transgéniques porteuses du gène vpu du VIH-1. Nos résultats démontrent que l‟expression de vpu chez les souris transgéniques induit le développement spontané d‟une hyperplasie lymphoïde pansystémique, une splénomégalie avec une hyperplasie lymphoïde folliculaire évoluant en lésions prémalignes et malignes qui présentent certaines similarités avec la maladie de Castleman et une iv glomérulonéphrite mesangioproliférative qui rappelle certaines altérations de néphropathie associée au VIH chez les patients infectés. L‟ensemble des altérations démontre que les souris Tg/vpu développent une activation chronique et non spécifique du système immunitaire. Dans cette activation immunitaire, une dérégulation de l‟IL-6 et une hyperplasie du réseau de cellules métallophiliques pourraient être impliquées. D‟autres résultats obtenus sur les évaluations du fonctionnement du système immunitaire de la rate et du thymus mettent en évidence une susceptibilité augmentée des lymphocytes des tissus lymphoïdes aux effets apoptotiques de la dexaméthasone et des lipopolysaccharides et un retard dans le repeuplement par les cellules d‟organes lymphoïdes ainsi qu‟une réaction inflammatoire (Schwartzman) exacerbée et des anomalies dans la réaction d‟hypersensibilité retardée expérimentale. Ce modèle transgénique reproduit plusieurs anomalies rencontrées chez les patients infectés par le VIH et ouvre de nouvelles hypothèses sur la pathogenèse de l‟infection par le VIH. / The HIV-1 infection results in a severe immunodepression due to an increasing depletion of the CD4+T lymphocytes and in a neurodegeneration that leads to a motor/cognitive complex dysfunction. In addition to these pathological alterations, patients with AIDS may also develop additional complications including neoplasms, nephropathy, hematological abnormalities and opportunistic infections. None of these complications can be accounted for solely by the loss of CD4+T lymphocytes and several investigators have started to suspect the regulatory and accessory genes of possibly playing a role in the AIDS physiopathogenesis. However, studies on the pathological effects contributed by these genes have been hampered by the lack of laboratory animals susceptible to HIV-1 infection. These could have helped to dissect out the role of the different components of the viral genome and their pathogenetic mechanisms. To overcome this obstacle, several investigators have developed transgenic mice carrying either HIV-1 individual genes or partially deleted genome. The vpu gene, which is unique to HIV-1, encodes a membrane phosphoprotein with several known functions as determined by in vitro studies. It participates in virions release at the cell surface, induces CD4 degradation, MHC-1 downregulation, increases apoptosis susceptibility of infected T cells and promotes viral replication by inhibiting cellular antiviral mechanisms. In order to investigate the contribution of vpu to the complex pathogenesis associated with HIV-1 infection, our laboratory developed a transgenic mouse model carrying vpu under the RSV LTR promoter. Here, we described our results of the clinical and pathological investigation of this unique animal model. Our results demonstrate that the expression of vpu in the transgenic mice induces a pansystemic lymphoid hyperplasia, splenomegaly with follicular lymphoid hyperplasia that progress to premalignant and malignant lesions, with striking characteristics of Castelman like-disease, and a mesangial vi hyperplasia variant of HIVAN frequently observed in adult and pediatric AIDS. All lesions observed in this mouse model suggest that the vpu carrying mice develop a nonspecific chronic activation of the immune system with hyperplasia of perivascular metallophillic cells and deregulation of the IL-6 expression within the hyperplastic follicles. Following treatment with dexamethazone and LPS, we observed an increased apoptosis susceptibility of the lymphocytes of the spleen and thymus with slow lymphocyte repopulation of these lymphoid organs. In addition, the transgenic mice developed an exacerbated reaction to the induced Schwartzman reaction and demonstrated a more delayed experimental DTH reaction. Thus, this transgenic model reproduces several abnormalities present in HIV patients. It represents a very interesting and valuable model for studies on the pathology contributed by the vpu gene in the absence of the cumulative effects of the other HIV-1 genes and might help define new possible functions and mechanisms of vpu in the HIV-1 pathogenesis.

O anticorpo anti-proteína P ribossomal é um dos marcadores sorológicos do lúpus eritematoso sistêmico, previamente associado a glomerulonefrite lúpica classe V (ISN-RPS). Neste trabalho foi avaliado o prognóstico renal em pacientes que possuem positividade para este anticorpo. Sessenta pacientes foram avaliados para parâmetros de sobrevida renal. Onze pacientes (18%) apresentaram positividade sorológica exclusiva para anticorpo anti-P ribossomal e vinte e oito pacientes (47%) para anti-dsDNA. Ao final do período de seguimento, foi observado que os pacientes anti-P positivos apresentaram uma maior sobrevida renal (11,0 ± 4,5 vs. 9,2 ± 4,5 anos, p=0,03) quando comparados aqueles anti-P negativos, assim como menor frequência de necessidade de terapia substitutiva renal (0 vs. 35% p = 0,025). Pacientes anti-P positivos apresentaram também maior frequência de classe V (91% vs. 31%, p < 0.001) e menor incidência de alterações proliferativas (45% vs. 82%, p = 0,021) na avaliação da biópsia renal quando comparados aos pacientes sem a positividade para este anticorpo. Os dados reforçam a hipótese de que o anticorpo anti-P é um marcador útil de um melhor prognóstico renal em pacientes portadores de lúpus eritematoso sistêmico / Antibodies to ribossomal P proteins are one of the serologic markers of systemic lupus erythematosus, previously described as associated to class V lupus glomerulonephritis (ISN-RPS). Our study assessed renal prognosis in patients with anti-P antibodies. Sixty consecutive SLE patients with biopsyproven nephritis (2004 ISN/RPS) were evaluated for renal survival parameters. Eleven patients (18%) had exclusive anti-P positivity and 28 (47%) patients anti-dsDNA. The post-biopsy follow-up analysis demonstrated that anti-P positive patients disclosed better renal survival (11.0 ± 4.5 vs. 9.2 ± 4.5 years, p = 0.03) as well as lower frequency of patients requiring dialysis (0 vs. 35% p = 0.025). The frequency of class V nephritis was higher in anti-P positive patients (91% vs. 31%, p < 0.001) and the occurrence of proliferative lesions at biopsy was lower in these patients (45% vs. 82%, p=0.021). Our data supports the notion that anti-P antibody is a valuable marker to predict a better long-term renal outcome in lupus patients

Nephrotic syndrome is characterized by proteinuria, hypoproteinemia, edemas and hyperlipidemia. It occurs in primary (e.g. focal segmental glomerulosclerosis, FSGS or minimal change disease, MCD) and in secondary glomerulopathies (e.g. kidney amyloidosis). In primary forms, great attention is paid to the potential genetic background of the disease and due to new molecular genetic methods genes, whose mutations cause different nephropathies (e.g. ACTN4 or INF2) were identified. The aims of presented doctoral thesis were following. Firstly, to continue the mutational analysis of ACTN4 that was described in the author's diploma thesis in other glomerulopathies. Secondly, to implement the mutational analysis of INF2 and subsequently analyse this gene in patients with FSGS/MCD and in patients from special group characterized by positive family history for end stage renal disease (ESRD) in combination with advanced chronic kidney disease (CKD) or already developed ESRD at the time of diagnosis. Thirdly, mutational analysis of NPHS2 and TRPC6 (methods implemented in laboratory earlier) in selected patients from the special group. Finally, expression analyses of genes important for podocyte function or connected with human immune system. This part also verifies the applicability of NPHS2/SYNPO expression...