Development of a suitable animal model of AIDS is much needed in AIDS research to study infection and pathogenesis as well as to evaluate methods of prevention and treatment of HIV infection. Small animals such as rodents are attractive candidates for AIDS research due to the availability of various inbred and genetically engineered strains, extensive knowledge or their immune system, especially in mice, and the relative ease of breeding and maintaining animal colonies. Transgenic small animal models carrying entire HIV genome or selected genes have been instrumental to understand functions of HIV genes in vivo and their role in HIV pathogenesis. The type of cells in which HIV genes are expressed seems to be an import prerequisite for the study of HIV gene functions in transgenic mice. Mice constitutively expressing the entire HIV-1 genome or HIV-1 nef gene in CD4 + T cells and in the cells of macrophage/dendritic lineage develop an AIDS-like disease very similar to AIDS disease in humans. Similarly, expression of Nef in adult mice, using inducible system, results in the AIDS-like disease. This disease is characterized by thymic atrophy, impaired thymocyte maturation, loss of CD4+ T cells, increased activation and turnover of T cells, which can occur in the absence of lymphypenia, and non-lymphoid organ disease involving the lungs and kidneys. Susceptibility of adult mice to the pathological effects of Nef suggests that the AIDS-like disease in the constitutively expressing Nef Tg mice is not due to developmental defects caused by early expression of Nef. This model highlights the important role of Nef in HIV-1 pathogenesis. The high similarity in the disease in these Tg mice with human AIDS strongly suggest that these mice are a relevant model to study AIDS. This study further evidence that mouse cells can support functions of Nef and these Tg mice represent a unique model to study Nef functions in vivo in the context of the primary immune system. Moreover, the inducible Nef Tg model has given us the ability to control the level and time of expression of Nef which was impossible to do in the previously reported constitutive Nef Tg mouse models. These mice will be useful to study immune reconstitution since Nef expression can be turned off after withdrawal from dox.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online as viewed 8/06/2009 through Digital Dissertations.

Made available in DSpace on 2015-07-22T20:50:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-29 / Introducao: A inflamacao tem sido relacionada a varias doencas neurodegenerativas e dados clinicos e experimentais sugerem uma funcao crucial nos processos inflamatorios no desenvolvimento da epilepsia, em particular, nos mecanismos geradores de crises (ictogenese) e na transformacao de uma rede neuronal normal a uma rede geradora de crises. A lovastatina, substancia usada na reducao da sintese do colesterol, tambem esta relacionada com a resposta inflamatoria, podendo modular a producao de citocinas e diminuir e o estresse oxidativo. Objetivos: O presente estudo teve como objetivo analisar a acao da lovastatina no desenvolvimento das diferentes fases do modelo de epilepsia, induzido por pilocarpina em ratos. Metodos: Ratos Wistar machos foram analisados nos 3 periodos do modelo da pilocarpina (350mg/kg) fases: aguda (24h), silenciosa (15 dias) e cronica (30 dias apos a 1.a crise espontanea) e para cada periodo do modelo usamos 4 grupos de animais: salina, lovastatina (Lova), pilocarpina (Pilo) e pilocarpina+lovastatina (Pilo+Lova). O tratamento com lovastatina (20 mg/kg) se iniciou 2 h apos o inicio do estado de mal epileptico e foi administrada por 15 dias, duas vezes ao dia nos animais do grupo silencioso e cronico. O cerebro foi processado para realizacao de PCR em tempo real e imuno-histoquimica de IL-1ƒÀ, IL-6, TNF-ƒ¿, IL-10, receptor B1 e B2 de cininas e quantificacao de aminoacidos no hipocampo. Alem disso, o tecido hipocampal foi processado para as tecnicas de Nissl e Neo-Timm modificado. Alem disso, foi medida a temperatura corporea na fase aguda, duracao do periodo silencioso e frequencia de crises na fase cronica. Resultados: O tratamento com a lovastatina no grupo Pilo+Lova mostrou diminuicao da expressao de RNAm e das proteinas IL-1ƒÀ e TNF-ƒ¿ nas 3 fases do modelo Notamos tambem reducao nos niveis do receptor B1 e B2 de cininas na fase aguda e de IL-6 nas fases aguda e silenciosa do modelo. Houve um aumento da expressão de IL-10 na fase crônica e não houve alteração nos níveis dos aminoácidos no hipocampo dos animais desse grupo, quando comparado ao grupo Pilo. Foi observada uma normalização da temperatura corpórea dos ratos submetidos ao SE e tratados com lovastatina. Não houve diferença significativa entre o grupo Pilo e Pilo+Lova na duração da fase silenciosa e na freqüência de crises. Foi observada uma preservação de neurônios em CA1 e também uma diminuição no brotamento de fibras musgosas no grupo Pilo+Lova, quando comparado ao grupo Pilo, na fase crônica do modelo. Conclusão: O presente estudo demonstrou que o tratamento com a lovastatina diminuiu diversos parâmetros importantes relacionados com o dano neuronal induzido pelo SE, no hipocampo de ratos nas diferentes fases do modelo experimental de epilepsia induzido pela pilocarpina. / Introduction: Inflammation has been associated with several neurodegenerative diseases and experimental and clinical data suggest a crucial role in inflammatory processes in the development of epilepsy, particularly in seizure-generating mechanisms (ictogenesis) and transformation of a normal neuronal network into a network generating seizures. Lovastatin, a drug used in the reduction of cholesterol synthesis, is also related to the inflammatory response and can modulate cytokine production reducing the oxidative stress. Objectives: This study aimed to analyze the action of lovastatin in different stages of development model of epilepsy induced by pilocarpine in rats. Methods: Male Wistar rats were analyzed in three periods of the pilocarpine-induced epilepsy (350mg/kg) into phases: acute (24h), silent (15 days) and chronic (30 days after the 1st spontaneous seizure) and for each period of this model we used 4 groups of animals: saline-treated, lovastatin (Lova), pilocarpine (Pilo) and pilocarpine + lovastatin (Pilo+ Lova). Treatment with lovastatin (20 mg / kg) begun 2 h after the onset of status epilepticus (SE) and was administered for 15 days, twice a day the animals in the silent and chronic phases. The brain was processed for performing real-time PCR and immunohistochemistry of IL-1ƒÀ, IL-6, TNF-ƒ¿, IL-10 and kinin B1 and B2 receptors and quantification of amino acids in the hippocampus. Besides, the hippocampal tissue was processed for Nissl techniques and Neo-Timm. In addition, body temperature was measured in the acute phase and the duration of the silent period and seizure frequency in chronic phase was analyzed. Results: Treatment with lovastatin in Pilo + Lova group showed decreased expression of mRNA and proteins IL-1ƒÀ and TNF-ƒ¿ in the three phases of this model, We also noted reduction of kinin B1 and B2 receptor in the acute and IL-6 into acute and silent periods. There was an increased expression of IL-10 in the chronic phase of this model. There was no change in amino acids levels in the hippocampus of rats from Pilo+Lova group when compared to Pilo group. We observed a normalization of body temperature of rats subjected to SE and treated with lovastatin. There was no significant difference between the group Pilo and Pilo + Lova on the duration of the silent phase and in seizure frequency. We observed a preservation of neurons in CA1 and also a reduction of mossy fiber sprouting in Pilo+ Lova group as compared to the Pilo group in the chronic phase of the model. Conclusion: This study demonstrated that treatment with lovastatin decreased number of important parameters related to the neuronal damage induced by SE in the hippocampus of rats at different stages of the experimental model of epilepsy induced by pilocarpine. / TEDE / BV UNIFESP: Teses e dissertações

INTRODUÇÃO: A conjuntivite alérgica é a forma mais comum de doença alérgica que afeta o olho. Neste trabalho, desenvolvemos um modelo murino reprodutível e simular a doença humana, para possibilitar o estudo dos mecanismos fisiopatológicos da conjuntivite alérgica crônica. MÉTODOS: Imunizamos os camundongos BALB/c e C57Bl/6 com extrato do ácaro Dermatophagoides pteronyssinus (Dpt). Foi realizada a dissecção dos linfonodos ilíacos e para-aórticos, e a enucleação dos olhos. O plasma obtido pela punção cardíaca foi utilizado para a dosagem de IgE e IgG totais e específicas para Dpt. Os olhos enucleados foram enviados para estudo anátomo-patológico da conjuntiva e córnea. RESULTADOS: 1) Houve uma diferença estatisticamente significante entre as duas linhagens (BALB/c e C57Bl/6) para os grupos imunizados com 5 ?g e 500 ?g na gradação clínica e histopatológica, dosagens de IgE Total e Específica, proliferação de linfócitos específica para Dpt e IgG Específica, e na dosagem das IL-5, IL-8 e IL-13; 2) Os níveis de IgG Total não se mostraram significantes para as duas linhagens nos grupos imunizados com 5 ?g e 500 ?g; 3) Os níveis de IL-4 e IL-10 tiveram uma diferença significante nos animais da linhagem BALB/c imunizados com 5 ?g e 500 ?g, mas não nos camundongos da linhagem C57BI/6; 4) Os níveis de IFN-? foram maiores nos camundongos C57BI/6 que receberam as menores quantidades de antígeno. Porém nos camundongos BALB/c o fenômeno foi o inverso; 5) O exame histológico revelou afilamento corneano, infiltrado linfocítico corneano e conjuntival, degeneração da conjuntiva e úlceras de córnea nos animais que obtiveram as maiores gradações clínicas da doença (camundongos BALB/c imunizados com 500 ?g de Dpt e camundongos C57Bl/6 imunizados com 5 ?g. CONCLUSÃO: Desenvolveu-se um modelo simples e reprodutível de conjuntivite alérgica crônica do Dermatophagoides pteronyssinus depois de repetidas exposições ao antígeno, o qual apresenta manifestações clínicas similares à doença humana, e serve como modelo de estudo dos mecanismos imunológicos envolvidos no desenvolvimento da doença / INTRODUCTION: Allergic conjunctivitis is the most common form of allergic disease that affects the eye. In this study we developed a reproducible mouse model and simulated human disease to enable the study of physiopathologic mechanisms of chronic allergic conjunctivitis. METHODS: We immunized BALB/c and C57B1/6 mice with Dermatophagoides Pteronyssinus (Dpt) dust mite extract. The iliac and paraaortic lymph nodes were dissected and the eyes were enucleated. The plasma obtained by cardiac puncture was used to measure Total and Specific IgE and IgG and Dpt-specific. Lymph node cells were used to measure Dpt specific proliferation cytokine detection in the culture supernatant. Eyes were enucleated for histopathological analysis of the conjunctiva and cornea. RESULTS: 1) There was a statistically significant difference between the 2 strains (BALB/c and C57B1/6) for the 2 groups immunized with 5?g and 500?g in the clinical and histopathological score, Total and Specific IgE dosages, proliferation Dpt-specific lymphocytes, dust mite Specific IgG, and in the levels of IL-5, IL-8 and IL-13; 2) The level of Total IgG was not significantly different between the 2 lineages in the groups immunized with 5?g and 500?g; 3) The levels of IL-4 and IL-10 showed a significant difference in BALB/c mice sensitized with 5?g and 500?g, but not in C57B1/6 mice; 4) The IFN-? levels were higher in C57B1/6 mice that received the smallest quantity of antigen. But among BALB/c mice the phenomenon was inversed; 5) The histological examination revealed that there was a tapering of the cornea, lymphocytic infiltration of the cornea and conjunctiva, conjunctival degeneration and corneal ulcers in the animals that developed the highest clinical scores of disease (BALB/c immunized with 500 ug of Dpt and C57Bl/6 immunized with 5 ?g of Dpt). Conclusion: A simple and reproducible model of chronic allergic conjunctivitis to Dermatophagoide pteronyssinus was developed after repeated exposure to the allergen, which exhibit similar clinical manifestations as human disease, therefore serving as a template to study the immunological mechanisms involved in the development of disease

INTRODUÇÃO: Apesar do amplo uso da medida da saturação central de oxigênio como meta terapêutica em pacientes de terapia intensiva, diferenças absolutas em relação à saturação venosa mista existem. As causas desses gradientes, bem como o comportamento das mesmas ao longo do tempo nas doenças graves não foram completamente esclarecidas. Considerando que a maioria das intervenções atualmente empregadas para reverter desequilíbrios de oxigenação tecidual presentes nos pacientes graves é direcionada, direta ou indiretamente, ao coração; a situação particular de elevada taxa de extração de oxigênio basal do miocárdio e a ausência de ferramentas de monitorização do impacto miocárdico dessas intervenções, o presente estudo, diante da possibilidade teórica da participação do efluente do seio coronário nessas diferenças centrais para pulmonares, não só para a saturação de oxigênio (SO2), analisou o comportamento da SO2, pressão parcial de dióxido de carbono (PCO2), lactato e glicose, em diferentes modelos de hipóxia e compartimentos vasculares, com ênfase na avaliação do metabolismo miocárdico e seu impacto nos gradientes centrais para pulmonares. MÉTODOS: 37 porcos, machos, com peso em torno de 35 Kg, sedados e ventilados mecanicamente, foram estudados após indução de quatro diferentes tipos de injúria hipóxica (hipóxia anêmica, estagnante, hipóxica e sepse), sendo 8 animais por grupo e mais 5 controles. Além de variáveis hemodinâmicas e de oxigenação, SO2, PCO2, lactato e glicose foram medidos, em diferentes momentos, em 9 compartimentos vasculares distintos, incluindo o seio coronário (artéria femoral, veia cava inferior e superior, átrio direito, ventrículo direito, artéria pulmonar, veia suprahepática direita e veia porta). PRINCIPAIS RESULTADOS: As concentrações de O2, lactato e glicose no efluente do seio coronário apresentaram padrões distintos entre os grupos: troca de substrato energético de lactato por glicose nos grupos hipóxia hipóxica e anêmica, aumento no consumo de ambos os substratos na sepse e ausência de tendência clara no grupo da hipóxia estagnante. Os gradientes de PCO2 entre seio coronário e artéria femoral mantiveram-se estáveis com tendência de alargamento tardio em todos os modelos. Na análise dos demais gradientes regionais, o seio coronário apresentou a menor SO2 do organismo, as menores concentrações de lactato, os maiores níveis de PCO2, e esses padrões variaram ao longo do tempo. Mesma tendência evolutiva foi percebida entre os gradientes centrais para pulmonares de O2, lactato, CO2 e glicose e a medida desses mesmos parâmetros no seio coronário. CONCLUSÕES: As concentrações de O2, lactato e glicose no efluente do seio coronário estão relacionadas ao tipo de injúria e não apenas à disponibilidade de substrato energético. Padrões de gravidade, comuns às fases tardias de todos os grupos, puderam ser identificados: qualquer redução da SO2 coronariana; incremento do metabolismo de glicose; produção de lactato pelo miocárdio e surgimento de igualdade ou inferioridade dos níveis da PCO2 coronariana em relação aos valores dos demais compartimentos vasculares do organismo (independentemente da trajetória). A tendência dos gradientes de PCO2 transmiocárdicos seguiu a do débito cardíaco e, certamente, deve refletir fluxo coronariano. A análise dos gradientes regionais se mostrou capaz de permitir a avaliação de contextos orgânicos regionais específicos, como na avaliação do metabolismo hepático, na qual foi possível demonstrar que na hipóxia, a produção de glicose hepática é mantida até o óbito, diferentemente do padrão descrito para a sepse. Por fim, com a análise dos dados do grupo sepse, foi possível demonstrar que: a) assim como os gradientes centrais para pulmonares de SO2 e lactato já foram descritos, gradientes de glicose e PCO2 também existem; b) o seio coronário participa, significativamente, na formação desses gradientes de lactato, CO2 e glicose / INTRODUCTION: Despite of the widespread use of the central venous oxygen saturation measurement as a therapeutic goal in critically ill patients, absolute differences between this measurement and the mixed venous oxygen exist. Causes of these differences, as well the behavior of these gradients in critical illness, are not completely understood. Considering current therapeutic interventions aimed to reverse tissue oxygenation impairment are mediated by increases in cardiac output; the particular scenario in which the heart is not physiologically able to further increase oxygen extraction and the absence of tools to monitoring the myocardium impact of those interventions, the present study, facing the theoretical possibility of the coronary sinus effluent participation in those central to mixed venous differences, has analyzed the oxygen saturation (SO2), carbon dioxide partial pressure (PCO2), lactate and glucose concentrations behaviors over time, in different models of tissue hypoxia and in different vascular sites. Emphasis on the myocardial energetic metabolism and its impact over central to mixed venous gradients was placed. METHODS: 37 pigs, males, weighting about 35 Kg, sedated and mechanically ventilated, were studied after induction of four different hypoxic injury models (sepsis, and anemic, stagnant, hypoxic hypoxia), eight for group and five controls. In addition to hemodynamic and oxygen variables, SO2, PCO2, lactate and glucose were measured in different phases, in 9 distinct vascular sites, including coronary sinus (femoral artery, inferior and superior vena cava, right atria, right ventricle, pulmonary artery, right suprahepatic vein and portal vein). MAIN RESULTS: Concentrations of O2, lactate and glucose in the coronary sinus effluent presented distinctive patterns among groups: shift from lactate to glucose consumption in hypoxic hypoxia and anemic hypoxia groups, increase in both glucose and lactate consumption in sepsis and absence of clear trend in stagnant hypoxia group. PCO2 gradients from systemic artery to coronary sinus presented late enlargement trend in all groups. In the regional gradients analysis comparisons, coronary sinus presented the lowest SO2, the lowest lactate concentrations, the highest PCO2 levels, and these patterns changed over time. Similar evolution trends were observed between central to mixed venous O2, PCO2, lactate and glucose gradients and the same parameters measured in coronary sinus. CONCLUSIONS: Different concentrations of O2, PCO2, lactate and glucose in coronary sinus are related to the type of hypoxic injury and not only to energetic substrate availability. Severity-related patterns, common to all groups in late phases, were identified: any reduction of coronary SO2, shift to glucose consumption, net lactate myocardial production and equality or inferiority of PCO2 levels related to other vascular compartments (independently of trend). Trends in transmyocardial PCO2 gradients followed cardiac output ones and, certainly, should mirror coronary blood flow. Regional gradients analysis showed suitable to explore specific regional metabolic settings, as in the described example of liver metabolism, in which production of glucose were maintained in all phases by this organ in hypoxic hypoxia groups, differently from the impaired production described in literature for sepsis. At last, data from sepsis group have showed: a) as to the previously known central to mixed venous SO2 and lactate gradients, PCO2 and glucose gradients also exist; b) coronary sinus has participated significantly in formation of central to mixed venous lactate, PCO2 and glucose gradients

INTRODUÇÃO: A microanastomose vascular é um componente importante na cirurgia de transferência livre de tecidos. Atualmente, a técnica de sutura convencional ainda é considerada o padrão ouro, no entanto, ela apresenta alguns inconvenientes por ser tecnicamente difícil, consumir muito tempo e ter uma longa curva de aprendizado. Na busca de uma técnica mais fácil e rápida, métodos alternativos de anastomose são estudados incluindo a cola de fibrina. Apesar dos bons resultados publicados, a sua aceitação na prática clínica ainda é limitada. Controvérsias a cerca de sua trombogenicidade e resistência mecânica geram dúvidas em relação a sua segurança. A ausência de um modelo experimental mais fidedigno impede que os potenciais benefícios de sua aplicação clínica sejam apreciados. O objetivo deste estudo é esclarecer essas controvérsias e estudar os benefícios da aplicação da cola de fibrina em um ambiente que simule a prática clínica. MÉTODOS: O modelo experimental utilizado foi a transferência livre de um retalho inguinal para a região cervical anterior. A circulação do retalho era restaurada através de microanastomoses vasculares entre as artérias femoral e carótida (término-lateral) e entre as veias femoral e jugular externa (término-terminal). Utilizamos 20 coelhos que foram divididos em dois grupos (n= 10) de acordo com a técnica de sutura empregada: Grupo I (sutura convencional) e Grupo II (sutura com cola). RESULTADOS: A aplicação da cola de fibrina reduziu significativamente o número de pontos necessários para se completar as anastomoses, 4 pontos a menos nas artérias e 4,5 pontos a menos nas veias. No Grupo I, a média do tempo de anastomose arterial foi de 17,21 minutos, contra 12,72 minutos no Grupo II. Nas anastomoses venosas, a média de tempo no Grupo I foi de 22,93 minutos, contra 16,57 minutos no Grupo II. A aplicação da cola de fibrina também diminuiu o tempo de isquemia do retalho e o tempo de cirurgia em 11,5 minutos e 15,67 minutos, respectivamente. A taxa de sobrevida do retalho foi de 90% nos dois grupos. CONCLUSÕES: A aplicação da cola de fibrina em microanastomoses vasculares demonstrou ser confiável e eficiente no presente estudo. / INTRODUCTION: Microvascular anastomosis is an important component of the free flap surgical procedure. Currently, the conventional suture is still considered the gold standard technique. However, it presents some problems for being technically demanding, time consuming and with a long learning curve. In looking for an easier and faster technique, alternative methods of anastomosis were studied including the fibrin glue. Despite the good results reported in the literature, its acceptance in the clinical setting is still small Controversies regarding its thrombogenicity and mechanical resistance create some concerns about its safeness. The absence of a more realistic experimental model has not allow a full aprecciation of its potencial benefits in clinical use. The aim of this study is clarify these controversies and demonstrate the advantages of fibrin glue application in an environment that can reproduce the clinical practice. METHODS: A free inguinal flap transfer to the anterior cervical region was used as experimental model. The circulation of the flap was restored by means of microvascular anastomosis between the femoral and carotid arteries (end-to-side) and between the femoral and jugular veins (end-to end). The procedures were performed in 20 rabbits that were divided into two groups (n= 10) according to the anastomosis technique: Group I (conventional) and Group II (fibrin glue). RESULTS: The application of fibrin glue significantly reduced the amount of sutures required to complete the anastomoses: 4 less sutures in the arteries and 4,5 less sutures in the veins. In Group I, the mean arterial anastomosis time was 17,21 minutes against 12,72 minutes in Group II. In the veins, the mean anastomosis time in Group I was 22,93 minutes against 16,57 minutes in Group II. The application of fibrin glue also reduced the flap ischemic time and the total operative time by 11,5 minutes and 15,67 minutes, respectively. The flaps\' survival rate was 90% in both groups. CONCLUSIONS: The application of fibrin glue in microvascular anastomoses was reliable and effective in this study.

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No description available.

Allergic asthma is characterized by airway hyperresponsiveness, inflammation, and a cellular infiltrate dominated by eosinophils. Numerous epidemiological studies have related the exacerbation of allergic asthma with an increase in ambient inhalable particulate matter from air pollutants. This is because inhalable particles efficiently deliver airborne allergens deep into the airways, where they can aggravate allergic asthma symptoms. However, the cellular mechanisms by which inhalable particulate allergens (pAgs) potentiate asthmatic symptoms remain unknown, in part because most in vivo and in vitro studies exploring the pathogenesis of allergic asthma use soluble allergens (sAgs). Using a mouse model of allergic asthma, we found that, compared with their sAg counterparts, pAgs triggered markedly heightened airway hyperresponsiveness and pulmonary eosinophilia in allergen-sensitized mice. Mast cells (MCs) were implicated in this divergent response, as the differences in airway inflammatory responses provoked by the physical nature of the allergens were attenuated in MC-deficient mice. The pAgs were found to mediate MC-dependent responses by enhancing retention of pAg/IgE/FcεRI complexes within lipid raft–enriched, CD63(+) endocytic compartments, which prolonged IgE/FcεRI-initiated signaling and resulted in heightened cytokine responses. These results reveal how the physical attributes of allergens can co-opt MC endocytic circuitry and signaling responses to aggravate pathological responses of allergic asthma in mice. / Dissertation