La BPCO est une affection de l’appareil respiratoire associée à une réponse inflammatoire chronique. La BPCO associe une bronchite chronique, avec obstruction des voies aériennes, et un emphysème caractérisé par la destruction du parenchyme pulmonaire. Si l’exposition au tabac est le facteur de risque principal de la BPCO, plusieurs observations cliniques sont en faveur du rôle du vieillissement dans la pathogénèse de la maladie. La dégradation des fibres élastiques du poumon en peptides solubles d’élastine (PE) est une caractéristique constante de la BPCO, et le vieillissement physiologique du poumon est associé à une augmentation de son activité élastinolytique. Nous avons montré dans un travail précédent que l’instillation de PE à des souris induit un emphysème aigu. La relation existant entre l’élastolyse et le vieillissement conforte l’hypothèse que le vieillissement pourrait représenter un risque majeur de la BPCO par le biais de production de PE. Le travail de thèse présenté dans ce manuscrit a porté sur l’étude comparative des paramètres histologiques, inflammatoires et immunitaires liés à l’emphysème induit par les PE dans des souris jeunes et des souris âgées. Les résultats obtenus montrent que l’emphysème est d’apparition plus précoce chez les souris âgées et que cette précocité s’accompagne d’une augmentation de la production de PE et de clones T CD28- mémoires spécifiques des PE et du processus d’immunosénescence. / Chronic obstructive pulmonary disease (COPD) is a progressive and irreversible inflammatory state of the lung. COPD includes chronic bronchitis, with obstruction of small airways, and emphysema, characterized by the destruction of lung parenchyma. Although cigarette smoke exposure is the best known risk factor for COPD development, several clinical observations support the hypothesis that aging also play a key role in the pathogenesis of COPD. Degradation of lung elastin fibers, generating soluble elastin peptides (EP), is a feature of COPD development, and normal physiological aging of the lung is associated with increased elastolysis. We previously demonstrated in mice that endotracheal instillation of EP resulted in an acute emphysema establishment. The positive association between lung elastin breakdown and aging is consistent with the hypothesis that aging is a major risk factor of COPD through EP production. The work done during this thesis focused on the comparative study of histological, inflammatory and immune parameters related to PE-induced emphysema in young and elderly mice. The results we obtained show that emphysema development is earlier in the elderly mice and that this earliness is associated with an increase of EP-specific CD28- senescent memory T cell clones.

O objetivo deste trabalho foi de avaliar em Modelo animal, coelhos (raça Nova Zelândia) divididos em três grupos randomizados, (jovem com 60 dias (G1), um idoso com aterosclerose e ingestão de colesterol (G2) e um idoso com aterosclerose e sem ingestão do colesterol (G3)) se, a dieta rica em colesterol e a idade, causam lesões de aterosclerose e placas ateroscleróticas nos animais, alterações nos comprimentos dos dentes, aumento ou diminuição dos espaços periapicais dos dentes, perda óssea alveolar na maxila e mandíbula. Através da metodologia descrita e após as análises histológicas e morfológicas, verificou-se diferença estatisticamente significante, nas variáveis dos comprimentos dos dentes 1º prémolares superiores entre os grupos G3 versus G1 p<5%. médias de 1247,88 (p=0,017) e G3 versus G2, com diferença das médias de 1190,85 (p=0,025) ou seja o comprimento dos dentes fora diferente no grupo G2. Com relação à variável, espessura do osso alveolar, não ocorreu significância estatística, porém tendências de que este esteja sendo alterado. Com relação a variável espessura do espaço periapical fora verificado significância estatística com p=0,017 em relação ao G1, na região dos 1º Pré-molares, apresentando a diferença das médias (403,42) a favor do G2, ou seja aumento do espaço periapical. A variável da área de placa de aterosclerose, correlacionada, com os comprimentos dos dentes e espessura do osso alveolar, também teve resultado significante na região 1º e 2º pré-molares entre G2 e G3. com p=0,025 e r=0,476. Confirmando assim a correlação da aterosclerose, dietas hiperclesterolêmicas e as alterações da cavidade bucal e estrutura de sustentação dos dentes. / The aim of this study is, to evaluate on animal model (New Zealand rabbits), on different and randomized groups (G1 a young group, G2 an older rabbit, with atherosclerosis and ingesting hipercholesterolemic fat diet and another group G3, just an older group) investigated than the hipercholesterolemic fat diet induced atherosclerotic lesions and plaques in ascendant aorta, bone loss in maxillary and jaw, alterations on length of the tooth, alterations on tooth periapical spaces. By the methodology described and aplicated, before the histological and morphological analysis, was verified significant statistical variance in the length of the first premolar tooth and second premolar on maxillary region, the length of this tooth is reduced in G2 (p<5%). The medias are 1247,88 (p=0,017) and 1190,85 (p=0,025). Analyzing another variable on the alveolar bone thickness, was not found any statistical significance, but tendencies that this event may occur. According to another variable of the thickness found on the periapical space, it was verified a significant variable statistic such as p=0,017 related on G1, on first premolar region, suggesting a different statistic, between medias, favoring G2 meaning the increase of periapical space. The variable on the atherosclerosis plaque area, related to the teeth length and alveolar bone thickness, also showed significant results on first and second premolars regions between G2 and G3, with p=0,025 and r=0,476. That confirms the atherosclerosis correlations and hipercholestrolemic fat diet, also alterations in oral cavity and teeth implantation structure.

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

Os tumores cutâneos e subcutâneos em cães representam um terço das neoplasias nessa espécie. O carcinoma de células escamosas (CCE) é uma neoplasia maligna com origem no epitélio estratificado escamoso da pele e de outras superfícies mucosas. A etiologia do CCE em cães não é bem definida, porém é descrito que a exposição aos raios solares é um importante fator para seu desenvolvimento. O CCE, frequentemente, é precedido pela ceratose actínica (CA). Este trabalho tem como objetivo comparar aspectos clínicos, morfológicos e moleculares da ceratose actínica e carcinoma de células escamosas da pele da região abdominal ventral de cães. Foram coletadas dez amostras de pele apresentando CCE e 9 amostras de pele apresentando CA da região abdominal ventral de caninos domésticos. As amostras foram fixadas em formol a 10%, e rotineiramente processadas para inclusão em parafina. Os cortes histológicos de 5?m foram submetidos à coloração de Hematoxilina e Eosina, para análise histopatológica, e às marcações, por imuno-histoquímica, de E-caderina, p53, Ciclo-oxigenase-2, pancitoqueratina AE1/AE2, vimentina, 5 metilcitosina e Ki67. As expressão de E-caderina, p53, ciclo-oxigenase-2, pancitoqueratina AE1/AE e vimentina foram avaliadas qualititivamente nas 9 amostras de CA e 10 amostras CCE, respectivamente. A metilação global do DNA e a proliferação celular (Ki67) foram quantificadas nos cortes histológicos de CA e CCE por meio da contagem de núcleos de células epiteliais marcados positiva ou negativamente. Os dados foram analisados por meio de testes uni e multivariados. Os cães machos da raça American PitBull Terrier de pelagem branca foram os mais acometidos pela CA e CCE, com média de idade de 8,1 e 8,4 anos, respectivamente. Não houve associação significativa na expressão de p53, COX-2 e vimentina na CA e CCE. Houve aumento no número de células com o núcleo marcado positivamente para Ki-67 e 5 metilcitosina em CCE comparado a CA. Observou-se, também, marcação heterogênea de Ecaderina em CCE. Constatou-se também diminuição na expressão de pancitoqueratina AE1/AE3 quando há invasão linfática e maior expressão de 5 metilcitosina em cães mais idosos, no CCE. Estes achados sugerem que há diferenças entre CA e CCE em relação aos parâmetros estudados, que condizem com a transformação maligna da afecção / Cutaneous and subcutaneous tumors in dogs account for a third of neoplasms in this species. Squamous cell carcinoma (SCC) is a malignant neoplasm that originates in the stratified squamous epithelium of the skin and other mucosal surfaces. The etiology of SCC is not well defined, however it is described that exposure to the sunlight is an important factor for its development. SCC is often preceded by actinic keratosis (AK). This work aims to compare clinical, morphological and molecular aspects of actinic keratosis and squamous cell carcinoma of the skin of the ventral abdominal region of dogs. Ten samples of SCC and 9 skin samples showing AK of the ventral abdominal region of domestic canines were collected. As samples were fixed in 10% formaldehyde, and routinely processed for inclusion in paraffin. Histological sections of 5?m were submitted to staining of Hematoxylin and Eosin for histopathological analysis, and to immunohistochemistry of E-cadherin, p53, Cyclooxygenase-2, pancytokeratin AE1/AE2, vimentin, 5 methylcytosine and Ki-67. The expression of E-cadherin, p53, cyclooxygenase-2, pancytokeratin AE1 / AE and vimentin were qualitatively evaluated in the 9 AK samples and 10 SCC samples, respectively. Overall DNA methylation and cell proliferation (Ki67) were quantified in the histological sections of AK and SCC by counting nuclei of positively or negatively labeled epithelial cells. The data were analyzed by means of uni and multivariate tests. Male dogs of the American PitBull Terrier breed were the most affected by AK and SCC, with an average age of 8.1 and 8.4 years, respectively. There was no significant association in the expression of p53, COX-2 and vimentin in CA and CCE. There was an increase in the number of cells with the nucleus positively labeled for Ki-67 and 5 methylcytosine in CCE compared to CA. There was also a heterogeneous labeling of Ecaderin in CCE. It was also observed a decrease in the expression of pancytokeratin AE1/AE3 when there is lymphatic invasion and greater expression of 5 methylcytosine in older dogs in the ECC. These findings suggest that there are differences between CA and SCC in relation to the studied parameters, which are consistent with the malignant transformation of the disease

Die Signalübertragung durch Wnt/beta-Catenin stellt einen der wichtigsten Signalwege während der Embryogenese sowie im adulten Organismus dar. Die homologen Gerüstproteine Conductin und Axin stehen im Mittelpunkt eines zentralen Multiproteinkomplexes, der im Zytoplasma für die Regulation des Wnt/beta-Catenin-Signalwegs verantwortlich ist. In der vorliegenden Arbeit habe ich eine kombinierte genetische Analyse von Conductin und Axin in der Maus durchgeführt. Dazu habe ich mit Hilfe der Technik der homologen Rekombination das Conductin-Gen deletiert und ein Reportergen unter die Kontrolle des endogenen Conductin-Promotors gebracht. Im Weiteren habe ich festgestellt, dass der gemeinsame Verlust von Conductin und einem Axin-Allel (Con-/-;Ax+/-) zu Holoprosenzephalie (HPE) führt, die durch schwere kraniofazialen und Vorderhirndefekten in der Maus charakterisiert ist. Dabei zeigte die detaillierte Analyse eine genetische Interaktion des Wnt-Signalwegs mit dem Shh-Signalweg. Störungen im Shh-Signalweg sind auch beim Menschen für die Ausbildung von HPE verantwortlich gemacht worden. Daneben führt die gleichzeitige Abwesenheit von Conductin und Axin (Con-/-;Ax-/-) zum Verlust der anterior-posterioren Achse in einem frühen Entwicklungsstadium und zum Absterben der Embryonen nach dem Tag 6,5 der Entwicklung. Die vorliegende Arbeit zeigt, wie Unterschiede in der biologischen Bedeutung zweier funktionell redundanter Faktoren mit genetischen Methoden in der Maus aufgeklärt werden können. / The Wnt/beta-Catenin pathway represents one of the most important signaling cascades during development as well as in the adult organism. The homologous scaffolding proteins Conductin and Axin are the backbone of a central multi protein complex that is responsible for the tight regulation of the Wnt/beta-Catenin pathway in the cytoplasm. In the present study I have performed a combined genetic analysis of Conductin and Axin in the mouse. To this end I have deleted the Conductin gene by homologous recombination in embryonic stem cells and inserted a reporter gene under the control of the endogenous Conductin promoter. I could show that the simultaneous loss of Conductin and one Axin allele (Con-/-;Ax+/-) causes Holoprosencephaly (HPE), which is characterized by severe craniofacial and forebrain defects in the mouse. The detailed analysis of the mutant mice reveals a genetic interaction of Wnt and Shh signaling and defective Shh signaling has previously been implicated in the formation of HPE in human patients. Moreover, complete absence of both Conductin and Axin (Con-/-;Ax-/-) leads to loss of the anterior-posterior axis early in development and death of the embryos after E6.5. The present study exemplifies how differences in the biological function of two mechanistically redundant factors can be studied by genetic means in the mouse.

Les pathologies vasculaires placentaires (PVP), prééclampsie (PE) et retard de croissance intra-utérin (RCIU), sont une des causes majeures de morbidité et mortalité maternelles et périnatales dans les pays développés. Un défaut de vascularisation utéro-placentaire est présent dans ces deux pathologies. Le Doppler énergie 3D (3DPD) est un outil facile à utiliser en clinique, les indices quantitatifs calculés à partir de volume utéro-placentaire semblent prometteurs pour dépistage de la PE. Le but principal de cette thèse est d’évaluer cette technique sur le plan de sa valeur discriminative entre situation physiologique et pathologique, ainsi que sur le plan de ses limites techniques en clinique et sur modèles animaux. La performance des indices 3DPD au 1er trimestre pour dépister la PE et/ou RCIU a été étudiée dans une étude prospective multicentrique. Une évaluation de la corrélation entre les indices 3DPD et les indices morphométrique a été ensuite réalisée chez les femmes enceintes à la fin de grossesse. Une corrélation entre le phénotype des villosités placentaire et la qualité de la perfusion placentaire a été mise en évidence. À la fin, l’intérêt de la technique a été démontré dans un modèle de RCIU chez brebis gestante. / Placental vascular pathologies (PVP), preeclampsia (PE) and intra uterine growth restriction (IUGR), are a major cause of maternal and perinatal morbidity and mortality in developed countries. A default of utero-placental vascularization is present in these two pathologies. 3D Power Doppler (3DPD) is an easy tool to use in clinic, its quantitative indices calculated from obtained utero-placental volume seem promising for PE prediction. The major objective of this thesis was to evaluate the discriminative value of this technique between physiological and pathological situations, as well as its limitations in clinical and animal models. The screening performance of PE and/or IUGR by 3DPD indices in the first trimester was studied in a prospective multicenter study. An evaluation of the correlation between 3DPD indices and morphometric indices was then carried out among pregnant women at term. At the end, the interest of the technique was demonstrated in an ewe model of IUGR (treated with testosterone).

Der Effekt des Cagedesigns, von Carrier-Systemen und Wachstumsfaktoren auf die intervertebrale Spondylodese am Tiermodell der Schafshalswirbelsäule wurde in einer anatomisch-biomechanischen, einer rein biomechanischen und sechs tierexperimentellen Teilstudien untersucht. Cages weisen eine designspezifische in vitro Primärstabilität auf. Jedoch besteht kein direkter Zusammenhang zwischen der in vitro Primärstabilität und der in vivo Sekundärstabilität der Implantate. Daher sind rein biomechanische in vitro Untersuchungen nicht dazu geeignet das Einheilungsverhalten eines Cages zu prognostizieren. Die Ergebnisse dieser Studien zeigen auch, dass die Auflagefläche für das Sinterungsverhalten eines Cages in vivo nur von untergeordneter Bedeutung ist. Hingegen sind das "stress-shielding" und die Volumen-bezogene-Steifigkeit eines Cages für das Einheilungsverhalten des Implantates in vivo entscheidend. In den vorliegenden Untersuchungen konnte gezeigt werden, dass die biodegradierbare PDLLA-Beschichtung intervertebraler Implantate eine sichere, einfache und effektive Applikation von Wachstumsfaktoren im Intervertebralraum gewährleistet. Darüber hinaus konnte gezeigt werden, dass der PDLLA-Carrier dem Kollagen-Carrier in seiner Effektivität signifikant überlegen ist. Nebenwirkungen der PDLLA-Beschichtung konnten in diesen Untersuchungen nicht ermittelt werden. Die Applikation einer geeigneten Dosis von IGF-I und TGF-(1 mittels PDLLA-beschichtetem Cage ist in der Lage, die intervertebrale Spondylodese signifikant zu stimulieren. Des weiteren konnte demonstriert werden, dass die verwendeten Wachstumsfaktoren IGF-I und TGF-(1 eine signifikante Verbesserung der intervertebralen Fusionsergebnisse im Vergleich zu autologem Knochenmaterial ermöglichen und gleichzeitig die Entnahme dieses Knochenmaterials überflüssig machen. Die Applikation von IGF-I und TGF-(1 mittels PDLLA-Carrier zeigte außerdem im Vergleich zum bisherigen Standard BMP-2 appliziert mittels Kollagen-Carrier signifikant bessere Fusionsergebnisse. Lokale oder systemische Nebenwirkungen konnten in den bisherigen Untersuchungen für die verwendeten Wachstumsfaktorendosen nicht ermittelt werden. Weiterführende Untersuchungen müssen belegen, ob die kombinierte lokale Applikation von IGF-I und TGF-(1 mittels PDLLA-beschichtetem Cage auch beim Menschen zu einer signifikanten Verbesserung der Ergebnisse der zervikalen Spondylodese führen können. / Anatomical, biomechanical and animal experimental studies were carried out to determine the effect of cage design, carrier systems and growth factors on interbody fusion in a sheep cervical spine model. Primary stability of cages was influenced by the implant design. However, there was no direct correlation between primary and secondary stability of cages. The dimension of the endplate-implant-contact area had no significant influence on subsidence of cages. Yet, stress-shielding and volume-related-stiffness significantly influenced bone matrix formation inside the cage. Biodegradable PDLLA-coating of cages allowed a safe, simple and effective intervertebral application of growth factors. The PDLLA-carrier was more effective than the collagen sponge carrier. No side effects were associated with the use of the PDLLA-carrier. An adequate dose of IGF-I and TGF-(1 applied by a PDLLA-coated interbody fusion cage stimulated interbody bone matrix formation. In comparison to autologous bone grafts the local application of IGF-I and TGF-(1 demonstrated significantly better fusion results and eliminated donor site morbidity. Further, in comparison to the golden standard BMP-2 applied by a collagen sponge carrier the combined application of IGF-I and TGF-(1 by a PDLLA-carrier significantly increased fusion results. No local or systemic side effects were associated with the use of the growth factors. Further studies have to determine, if the local combined application of IGF-I and TGF-(1 by a PDLLA-coated interbody cage increases fusion results in the human cervical spine.

But de l’étudeL’atrésie intestinale est une anomalie congénitale définie par une perte de la continuité digestive. Malgré une restauration chirurgicale précoce de cette continuité, surviennent durant les premiers mois de vie des troubles de la motilité digestive et des surinfections bactériennes chez un tiers des patients. Ces troubles fonctionnels étaient attribués jusque là principalement à des altérations du système nerveux entérique. Le but de cette étude était de confirmer cette hypothèse mais également d’élargir le champ des explorations aux autres composants du tube digestif.Matériel et méthodesLe modèle animal de l’atrésie chez le rat initialement décrit dans notre équipe a été utilisé pour caractériser les anomalies d’expression génique par transcriptomique. L’étude portait également sur la maturation digestive chez des fœtus de rat contrôle entre un stade de développement embryonnaire E15 et E21. Des modifications en amont et en aval de l’obstruction ont été étudiées en prélevant deux segments successifs de 1 cm par cette approche globale transcriptomique puis précisées par RT-PCRq et confirmées par des techniques immunohistochimiques et de microscopie électronique. RésultatsChez les fœtus témoins, l’expression génique montre une décroissance physiologique pour le SNE et une augmentation pour les systèmes neuroendocrine et épithélial de E15 à E21. Concernant les fœtus avec atrésie, les modifications concernent quasi exclusivement le segment d’amont avec une augmentation du calibre intestinal, de l’épaisseur musculaire et une accélération globale de la maturation. Une redistribution des sous types neuronaux est constatée dans le segment d’amont ainsi qu’une augmentation de l’expression du système neuro endocrine. Pour ces deux systèmes, le segment d’aval est peu modifié. Des modifications importantes du système épithélial sont observées en amont comme en aval avec pour conséquence probable une altération de la barrière intestinale et du système anti infectieux.ConclusionCes résultats montrent que les changements prédominent dans le segment en amont de l’atrésie alors que le segment d’aval était parfois considéré comme le plus pathologique. De plus, il a été retrouvé des changements inattendus du système neuroendocrine et épithélial qui sous tendent une implication non exclusive du SNE. D’autres recherches sont nécessaires pour confirmer ces données et les exploiter dans une démarche thérapeutique. / Aim of the studyIntestinal atresia is a rare congenital affection with postoperative motility disorders, leading sometimes to death. Previous related studies mainly focused on enteric nervous system (ENS) alterations as it was identified to cause abnormal peristalsis. The aim of the study was to expertise the underlying pathological conditions of intestinal atresia using a global approach, before focusing on ENS and neuroendocrine cells in order to precise the presumptive involvement of the different layers of the intestinal wall.MethodsPreliminary transcriptomic approach was elected to screen global gene expression involved in intestinal development and atresia-linked disorders in the rat model previously described by our team. Rat embryos were assigned to atretic group and controls embryos at different stages of development ED15, ED17, ED19 and ED21. Two successive intestine samples of 1 cm were harvested in the proximal segment and in the distal one. The pattern of gene expression was further assessed by immunohistochemistry, electron microscopy and RT-qPCR. Main resultsA physiological decrease in gene expression for enteric nervous system markers and an increase for neuroendocrine and epithelial system was observed on controls from stages ED15 to ED21. Regarding affected embryos, structural modifications concerned the proximal segment with increased muscular layer and a significant disruption including global accelerated maturation was observed in the proximal segment with increased gene expression of neuroendocrine system. Distal segment was comparable to controls for the two systems. Important modifications were noted concerning the epithelial system with consequent abnormalities of the gut barrier and anti infectious functions.ConclusionsFetal intestinal obstruction results in a disrupted gut development predominant in the proximal segment. The distal segment and the ENS were poorly concerned by theses changes. Neuroendocrine and epithelial cells underwent significant unexpected changes, supporting the evidence that ENS do not play an exclusive role in the pathways of intestinal motility disorders.

Introdução: A fibrose pulmonar idiopática (FPI) é definida como um tipo de doença fibrosante intersticial crônica de etiologia desconhecida limitada aos pulmões e que apresenta padrão histológico de pneumonia intersticial usual. A prevalência de FPI é estimada em, aproximadamente, 20/100.000 em homens e em 13/100.000 em mulheres, sendo que a idade média do diagnóstico é 67 anos e a sobrevivência média é 2 a 5 anos. Estima-se que 5 milhões de pessoas sejam afetadas em todo o mundo. O tratamento clínico atual está associado com melhora parcial e transitória, com resultados duvidosos ou insatisfatórios. Na abordagem cirúrgica da FPI, tem destaque o transplante pulmonar, cuja realização é rara, devido à escassez de doadores e à limitação das equipes capazes de realizar tais procedimentos. A terapia celular é uma alternativa terapêutica com grande potencial de aplicabilidade na fibrose pulmonar. Objetivos: Utilizar um modelo de fibrose pulmonar induzida pela bleomicina em ratos para investigar os efeitos da terapia com células-tronco mesenquimais (CTM) e o meio condicionado no remodelamento pulmonar com objetivo de elucidar o mecanismo de ação das CTM e meio condicionado, na reversão da fibrose pulmonar. Para tanto, buscamos: 1) padronizar a cultura de células-tronco mesenquimais e meio de cultura; 2) caracterizar o modelo experimental de fibrose pulmonar induzida pela bleomicina por microscopia óptica antes e após o tratamento com CTM e meio de cultura; 3) avaliar a expressão de biomarcadores sorológicos (Fibrinogênio, Fator von Willebrand e PDGF); 4) quantificar a expressão de proteínas relacionadas ao estresse oxidativo (NOS), citocinas pró-inflamatórias e pró-fibróticas (IL-17 e TGF-beta) e pró-angiogênicas (VEGF e endotelina) por imuno-histoquímica; e 5) quantificar a deposição de fibras do colágeno I e V por imunofluorescência. Materiais e Métodos: Utilizou-se um total de 44 ratos Wistar machos albinos com peso médio de 250-300g e 8 semanas de idade. Quatro grupos experimentais foram compostos de 10 animais, que participaram do experimento divididos em três momentos: D0, D10 e eutanásia (D14 ou D21). Em D0, foi realizada a instilação orotraqueal de bleomicina na dose de 1,5 U/kg; em D10, foi realizada a infusão em veia caudal de células-tronco mesenquimais na dose de 106 CTM/Kg ou 200 ?l de meio condicionado. Para o preparo das CTM, foi obtido, em média, 1,2 g de tecido adiposo, procedida a dissociação com colagenase tipo I, sendo que a contagem média de células foi de 3,05 x 106 células linfomononucleares/g de tecido adiposo. Estas células foram cultivadas durante 21 dias em meio Knockout DMEM-F12 suplementado com 10% de soro fetal bovino. Os seguintes três critérios foram utilizados para comprovar o perfil das células-tronco mesenquimais: aderência plástica, expressão de CD90 por citometria de fluxo ( > 90%) e capacidade de diferenciação em três linhagens mesodérmicas. Em D10, um pool destas células alogênicas foi infundido intravenosamente, na veia caudal, a uma concentração de 1 x 106 células/ animal num volume de 200 ul de solução salina. Em D14 ou D21, os animais foram eutanasiados e analisados quanto ao peso e conforme análises microscópico-laboratoriais. As análises histológicas foram realizadas por dois especialistas diferentes em estudo duplo-cego. Os parâmetros de ganho de peso e recuperação microscópica do tecido pulmonar foram analisados em cada grupo. Resultados: Nossos dados mostram que as células-tronco mesenquimais oriundas do tecido adiposo abdominal de ratos Wistar tiveram seu perfil fenotípico, capacidade de adesão plástica e diferenciação em 3 linhagens mesodérmicas estabelecidas inequivocamente, conforme estabelecido internacionalmente pela ISSCR. As células-tronco mesenquimais e o meio condicionado induziram: a recuperação clínica após tratamento; a reversão da inflamação e de fibrose pulmonar induzida pela bleomicina; a reversão da arteriopatia no parênquima pulmonar distal; a redução das concentrações de fibrinogênio, fator von Willebrand e PDGF (marcadores sorológicos); a diminuição da expressão de enzimas oxidantes; a diminuição da expressão de endotelina e a modulação da expressão da proteína de remodelamento (IL-17), da ativação dos fibroblastos TGF-B e da síntese de colágeno. Conclusão: Neste estudo, comprovamos que as terapias com células-tronco mesenquimais derivadas do tecido adiposo e com o meio condicionado foram eficazes na modulação dos processos inflamatórios e fibrogênicos no modelo induzido por bleomicina, agindo na ativação miofibroblástica, e, também, na restauração tecidual e endotelial. Além disso, o meio condicionado se mostrou tão eficiente quanto as células-tronco propriamente ditas, no efetivo remodelamento pulmonar, devendo ser considerado como uma proposta terapêutica viável e inovadora / Introduction: Idiopathic pulmonary fibrosis (IPF) is defined as a type of chronic interstitial fibrosing disease of unknown etiology limited to the lungs and presenting a histological pattern of usual interstitial pneumonia. The prevalence of IPF is estimated at approximately 20/100,000 in men and 13/100,000 in women, with the mean age of diagnosis being 67 years and the average survival is 2 to 5 years. It is estimated that 5 million people are affected worldwide. The current clinical treatment is associated with partial and transient improvement, with dubious or unsatisfactory results. In the surgical approach to IPF, pulmonary transplantation is a prominent feature, which is rare because of the scarcity of donors and the limitation of the teams capable of performing such procedures. Cell therapy is a therapeutic alternative with great potential for applicability in pulmonary fibrosis. Objectives: To use a model of bleomycin-induced lung fibrosis in rats to investigate the effects of mesenchymal stem cell (MSC) therapy and conditioned medium on lung remodeling in order to elucidate the mechanism of action of MSC and conditioned medium, in the reversion of pulmonary fibrosis. To do so, we aim to: 1) standardize the culture of mesenchymal stem cells and conditioned medium; 2) characterize the experimental model of lung fibrosis induced by bleomycin by optical microscopy before and after treatment with MSC and conditioned medium; 3) to evaluate the expression of serological biomarkers (Fibrinogen, Factor von Willebrand and PDGF); 4) to quantify the expression of proteins related to oxidative stress (NOS), pro-inflammatory and pro-fibrotic (IL-17 and TGF-beta) and pro-angiogenic cytokines (VEGF and endothelin) by immunohistochemistry; and 5) to quantify the deposition of collagen I and V fibers by immunofluorescence. Materials and methods: A total of 44 male albino Wistar rats weighing 250-300g and 8 weeks of age were used. Four experimental groups were composed of 10 animals, which participated in the experiment divided in three moments: D0, D10 and euthanasia (D14 or D21). In D0, orotracheal instillation of bleomycin at the dose of 1.5 U/ kg was performed; in D10 caudal vein infusion of mesenchymal stem cells at the dose of 106 MSC/ kg or 200 ul of conditioned medium was performed. To prepare the MSC, a mean of 1.2 g of adipose tissue was obtained, dissociated with type I collagenase and the mean cell count was 3.05 x 106 lymphomonuclear cells / g of adipose tissue. These cells were cultured for 21 days in DMEM-F12 Knockout medium supplemented with 10% fetal bovine serum. The following three criteria were used to prove the profile of mesenchymal stem cells: plastic adherence, expression of CD90 by flow cytometry ( > 90%) and differentiation capacity in three mesodermal lines. In D10, a pool of these allogeneic cells was infused intravenously into the caudal vein at a concentration of 1 x 106 cells / animal in a volume of 200 ul of saline. In D14 or D21 the animals were euthanized and analyzed for weight and microscopic-laboratory analyzes. Histological analyzes were performed by two different specialists in a double-blind study. The parameters of weight gain and microscopic recovery of lung tissue were analyzed in each group. Results: Our data show that the mesenchymal stem cells derived from the abdominal adipose tissue of Wistar rats had their phenotypic profile, plastic adhesion capacity and differentiation in 3 mesodermal lines established unequivocally, as established internationally by ISSCR. Mesenchymal stem cells and conditioned medium induced: clinical recovery after treatment; bleomycin-induced reversal of inflammation and pulmonary fibrosis; the reversal of arteriopathy in the distal pulmonary parenchyma; the reduction of fibrinogen, von Willebrand factor and PDGF concentrations (serologic markers); decreased expression of oxidizing enzymes; the reduction of endothelin expression and the modulation of the expression of the remodeling protein (IL-17), the activation of TGF-B fibroblasts and the synthesis of collagen. Conclusion: In this study we demonstrated that therapies with mesenchymal stem cells derived from adipose tissue and conditioned medium were effective in the modulation of inflammatory and fibrogenic processes in the bleomycin-induced model, acting to modulate myofibroblastic activation and also in tissue restoration and endothelial cells. In addition, the conditioned medium proved to be as efficient as the stem cells themselves, in effective pulmonary remodeling, and should be considered as a viable and innovative therapeutic proposal

Introdução: A curcumina é um polifenol presente no rizoma da espécie Curcuma longa L. que tem sido usado há séculos como medicamento anti-inflamatório na medicina asiática. A síndrome nefrótica é classicamente tratada com corticosteroides, uma potente classe antiinflamatória e imunossupressora. O tratamento pode trazer sérios efeitos adversos. Objetivos: Este estudo foi desenhado para avaliar o efeito anti-inflamatório e antiproteinúrico da C. longa na forma de droga vegetal, na lesão renal induzida pela doxorrubicina. Desenho do estudo: Trata-se de um estudo experimental in vivo. Métodos: O efeito anti-inflamatório e anti-proteinúrico da C. longa foi avaliado utilizando-se 4 grupos de ratos Wistar: dois grupos com lesão renal induzida por doxorrubicina (3,5 mg/kg) em dose única endovenosa, sendo um alimentando-se de ração padrão e outro com ração misturada a C. longa (5 mg/g de ração). Outros dois grupos controles sem lesão renal foram alimentados, sendo um com ração padrão e outro com C. longa. Foram coletadas amostras de urina para dosagem de albuminúria a cada 2 semanas. Após 8 semanas os animais foram anestesiados e coletado sangue para dosagem no plasma de creatinina, albumina, sódio, potássio, colesterol e osmolalidade. Nas amostras de urina foram dosados albuminúria, sódio, potássio, osmolalidade e os mediadores inflamatórios proteína quimiotática para monócitos-1 e fator de transformação do crescimento beta urinário. Foi coletado tecido renal para realização de microscopia de luz e de imuno-histoquímica para desmina, vimentina e células ED-1 positivas. Resultados: Após a 8a semana de acompanhamento, o tratamento com C. longa atenuou o aumento do MCP-1 urinário, do TGF-? urinário, da imunomarcação para desmina, vimentina e células ED-1+ nos ratos com lesão renal induzida. Conclusão: Os resultados sugerem que o uso de C. longa em um modelo experimental de lesão renal por doxorrubicina, por 8 semanas, não reduz a albuminúria, mas leva a diminuição dos mediadores inflamatórios renais MCP-1 e TGF-? urinário, além de imunomarcação para desmina, vimentina e células ED-1+ no tecido renal. / Background: Curcumin is a polyphenol present in the rhizome of the species Curcuma longa L., which has been used for centuries as an anti-inflammatory remedy in Asian medicine. Nephrotic syndrome is classically treated with corticosteroids, a potent antiinflammatory and immunosuppressive class. The treatment can cause serious adverse effects. Objectives: This study was designed to evaluate the anti-inflammatory and antiproteinuric effects of C. longa, as powdered dried rhizomes, in renal injury induced by doxorubicin. Study design: This is an in vivo experimental study. Methods: The antiinflammatory and anti-proteinuric effects of C. longa were evaluated in four groups of Wistar rats: two groups with intravenous doxorubicin-induced kidney injury (3.5 mg/kg), one fed with standard food and another with standard food mixed with C. longa (5 mg/g food). Two other control groups without kidney injury were fed, one with standard and one with C. longa-mixed food. Urine samples were collected for albuminuria every 2 weeks. After 8 weeks, the animals were anesthetized and blood was collected for measurement of plasma creatinine, albumin, sodium, potassium, cholesterol and osmolality. In the urine samples, measurements of albuminuria, sodium, potassium, osmolality and inflammatory mediators as monocyte chemoattractant protein-1 and transforming growth factor beta were done. Renal tissue was collected for light microscopy and immunohistochemistry for desmin, vimentin and ED-1 positive cells. Results: After the 8th week of follow-up, treatment with C. longa attenuated the increase of urinary MCP-1, urinary TGF-?, and immunostaining for desmin, vimentin and ED-1+ cells in rats with doxorubicin-induced kidney injury. Conclusion: The results suggest that the use of C. longa in an experimental model of nephrotic syndrome for 8 weeks does not reduce albuminuria, but there is a decrease in the inflammatory mediators urinary MCP- 1, urinary TGF-?, and immunostaining for desmin, vimentin and ED-1+ cells.