A instalação imediata de implante dental na região de molares é crítica, devido a grande perda óssea e da discrepância entre a espessura da crista alveolar e a plataforma do implante. Laserfototerapia (LFT) auxilia na reparação óssea, portanto pode acelerar a instalação do implante. Vinte pacientes foram selecionados para o estudo. Dez pacientes foram submetidos à LFT com laser de diodo de GaAlAs (808nm) no transcirúrgico de exodontia de molares, imediatamente após, e em 24h, 48h, 72h, 96h, 7 e 15 dias. As irradiações foram aplicadas no modo contínuo, em contato e pontual (100mW, 0,04cm2, 075,0J/cm2, 30s por ponto, 3J por ponto, em 5 pontos). O grupo controle (n=10) recebeu o mesmo tratamento, no entanto, o equipamento estava desligado. Quarenta dias depois, espécimes do tecido formado no interior dos alvéolos foram coletados para posterior análise por microtomografia (microCT) e histomorfometria. Dados de ambos os grupos foram comparados pelo test t de Student, enquanto aqueles das diferentes avaliações microtomográficas foram comparados pelo teste de correlação de Pearson (p<0.05). O volume relativo do osso, bem como a área relativa do osso foram significativamente maiores (p<0.001) no grupo laser do que no controle. No grupo controle houve correlação negativa significativa entre número e espessura de trabéculas, e entre número e separação entre trabéculas (p<0.01); entre espessura e separação das trabéculas a correlação foi positiva (p<0.01). O grupo laser mostrou correlação negativa significante entre número e espessura de trabéculas (p<0.01). A Laserfototerapia acelerou o reparo ósseo. Baseado na correlação de Pearson foi possível inferir que o grupo laser apresentou uma distribuição trabecular mais homogênea, com trabéculas menos espessas e mais numerosas, o que pode propiciar a redução do tempo para a instalação do implante. / The immediate dental implant placement in the molars region is critical, because of the high amount of bone loss and the discrepancy between the alveolar crest thickness and the dental implant platform. Laserphototherapy (LPT) improves bone repair thus could accelerate the implant placement. Twenty patients were selected for the study. Ten patients were submitted to LPT with GaAlAs diode laser (808nm) during molar extraction, immediately after, 24h, 48h, 72h, 96h, 7 and 15 days. The irradiations were applied in continuous wave, in contact and punctual mode (100mW, 0.04cm2, 075,0J/cm2, 30s per point, 3J per point, in 5 points). The control group (n=10) received the same treatment; however with the power of the laser off. Forty days later samples of the tissue formed inside the sockets were obtained for further microtomography (microCT) and histomorphometry analyses. Data of both groups were compared by the Student t test, whereas those from the different microCT parameters were compared by the Pearson correlation test (p<0.05). The relative bone volume, as well as the relative area were significantly higher (p<0.001) in the lased than the control group. In the control group there were negative correlations between number and thickness, and between number and separation of trabecula (p<0.01); between thickness and separation of trabecula the correlation was positive (p<0.01). The laser group showed significant negative correlation between the number and the thickness of trabecula (p<0.01). Laserphototherapy accelerated bone repair. By the Pearson correlation test it was possible to infer that the lased group presented a more homogeneous trabecula configuration, with thin and numerous trabecula, which would facilite the reduction of time for the installation of the implant.

Objetivo: Avaliar radiograficamente a prevalência, extensão e severidade das reabsorções radiculares nos dentes adjacentes à área da fissura após enxerto ósseo alveolar secundário. Material e Métodos: Foram utilizadas 1458 radiografias (periapicais, oclusais e panorâmicas) de 200 indivíduos com fissura transforame incisivo (uni e bilateral) submetidos à cirurgia de enxerto ósseo, obtidas do acervo do setor de Radiologia do Hospital de Reabilitação de Anomalias Craniofaciais da Universidade de São Paulo. Resultados: Dos 200 indivíduos avaliados, 33 indivíduos apresentaram reabsorções radiculares externas em algum dente. No total foram observadas 33 reabsorções, 15 estavam presentes nas radiografias pré-enxerto ósseo e 18 somente após o enxerto ósseo. Do total das reabsorções observadas, 30 foram localizadas no terço apical da raiz e 3 no terço cervical, sendo o incisivo central esquerdo o dente mais afetado. Não foram encontradas reabsorções radiculares no terço médio da raiz e nenhuma das reabsorções atingiram más de um terço radicular. Não houve diferença estatisticamente significante entre a idade de realização do enxerto ósseo alveolar e a presença de reabsorção radicular externa; e também em relação ao tratamento ortodôntico. Conclusões: A prevalência de reabsorções radiculares nos dentes adjacentes à área da fissura de pacientes submetidos a enxerto ósseo foi baixa (16,5%). Não foi possível estabelecer uma relação entre a observação de reabsorção radicular de indivíduos submetidos a enxerto ósseo e a presença de tratamento ortodôntico em uma mesma imagem. O terço apical da raiz dos dentes observados foi a localização mais frequente de reabsorções radiculares externas, sendo que nenhuma das reabsorções abarcou mais de um terço da raiz. / Objective: To evaluate radiographically the prevalence, extent and severity of root resorption in teeth adjacent to cleft area after secondary alveolar bone graft. Material and methods: We analyzed 1458 radiographs (periapical, occlusal and panoramic) of 200 patients with complete cleft lip and palate (uni and bilateral) underwent bone graft surgery, obteined from of Radiology department files of the Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo. Results: Of 200 patients, 33 individuals had external root resorption of a tooth. In total 33 resorptions were observed, 15 were present on preoperative bone graft radiographs, and 18 only after the bone graft. Of the total of resorption observed, 30 were localed in the apical third of the root and 3 into the cervical third; left central incisor was the most affected tooth. There were no root resorption in the middle third of the root and none of the resorption reached nore than one third. There was no statistically significant difference between the age of the alveolar bone graft surgery and the presence of external root resorption, and also in relation to orthodontic treatment. Conclusions: The prevalence of the root resorption in teeth adjacent to the cleft area in patients who underwent bone graft was low (16,5%). Unable to establish a relationship between the observation of root resorption in patients undergoing bone grafting and orthodontic treatment presence in the same image. The apical third of the root of the teeth observed was the most common location of external root resorption, and none of resorption spanned more than one third of the root.

A fibrilação atrial (FA) é a arritmia mais comum no pós-operatório de cirurgia cardíaca. Apesar de estar relacionada a alterações estruturais, alguns pacientes, mesmo que sem tais condições, ainda assim, cursam com fibrilação atrial no pós-operatório (FAPO) causando aumento no tempo de internação e custos. Estudos recentes vem ampliando o conhecimento sobre pequenos fragmentos de RNA, chamados de microRNAs (miRNAs) que podem interferir diretamente no aparecimento de algumas doenças na área cardiovascular. O objetivo do presente estudo é: 1) comparar a expressão dos miRNAs 1, 23 e 26 entre pacientes com e sem FAPO; 2) comparar nos grupos a expressão destes miRNAs entre os período pré e pós-cirúrgico; 3)comparar a expressão dos genes GJA1, KCNJ2, CACNB1, CACNA1C e KCNN3 entre os tempos pré e pós-cirúrgico no grupo FAPO; 4) comparar estes últimos genes no tecido atrial; 5) comparar os genes relacionados à produção de interleucinas (IL)-1, 6 e fator de necrose tumoral alfa (TNF?) entre os grupos e entre os tempos pré e pós-cirúrgico; 6)avaliar as características clínicas e evolutivas da população estudada. Pacientes submetidos à cirurgia de revascularização miocárdica foram submetidos à coleta de 20ml de sangue pré e pós-cirurgia bem como fragmento de tecido atrial. Um total de 143 pacientes compuseram os grupos: FAPO (24 pacientes), controle genético (24 pacientes) e controle total (97 pacientes + 24 grupo controle genético). Do ponto de vista clínico observou-se maior idade, tempo de anóxia, tempo de internação em terapia intensiva e hospitalar no grupo FAPO. A análise genética revelou menor expressão do miRNA-23 no grupo FAPO (p=0,02). A comparação entre os períodos pré e pós-cirúrgico revelou redução dos três miRNAs no tempo pós-cirúrgico (p<0,05) e dos genes relacionados às proteínas de canal (p<0,05). A comparação no tecido não evidenciou alterações entre os grupos. Os genes relacionados ás citocinas revelaram redução no período pós-cirúrgico (p<0,05) em ambos os grupos. Concluiu-se que o miRNA-23 pode ter implicação no surgimento da FAPO e outros miRNAs não estudados devem estar envolvidos neste processo uma vez que houve redução de outros genes de canais relacionados ao aparecimento de FAPO. / Atrial fibrillation (AF) is the most common arrhythmia after cardiac surgery. AF is related to cardiac structural changes although a group of patients still remains developing post-operative atrial fibrillation (FAPO) even without those changes, leading to more days in the hospital and costs. Recent studies showed that short fragments of RNA, called microRNA (miRNA) can contribute to the development of several diseases in the cardiovascular area. The aim of this study is to 1) compare the expression of miRNA-1, 23 and 26 between the group with and without FAPO; 2) compare, in the FAPO group, the expression of these miRNAs in the pre and post-surgery periods; 3) compare the expression of GJA1, KCNJ2, CACNB1, CACNA1C e KCNN3 genes between the pre and post-surgery periods; 4) compare this genes in atrial tissue; 5) compare the genes related to inflammation cytokines as interleukin(IL)-1, 6 and alpha tumoral necrosis factor between the groups in the pre and post-surgery periods; 6) evaluate clinical and evaluative patterns of the study population. Twenty milliliters of blood samples in the pre and post-operative periods and an atrial fragment were extracted from patients submitted to coronary artery bypass graft surgery. A total of 143 patients were divided in the FAPO group (24 patients), genetic control group (24 patients) and a total control (97 + 24 genetic control patients). The clinical analysis showed bigger age and clamp-time, more days in the intensive care unit and hospital in the FAPO group. The genetic analysis revealed less expression of miRNA-23 in the FAPO group (p=0.02). The comparison between the pre and post-surgery periods showed reduction in the three studied miRNAs (p<0.05) and reduction in the genes related to the production of the membrane protein channel sites. The comparisons in the atrial tissue didn´t show any difference in the study groups. The cytokines showed post-surgery reduction (p<0.05) in both groups. The conclusion is that miRNA-23 can be implicated in FAPO as others miRNAs not studied can also be, once there was a significative reduction in the genes related to FAPO development.

O transplante (Tx) de ilhotas pancreáticas (IP) é uma atraente alternativa para o tratamento do diabetes melito tipo 1. No entanto, para evitar a rejeição há necessidade de imunossupressão. Uma nova idéia de tolerância surge a partir do paradoxo imunológico, onde a mãe, imunologicamente competente, não rejeita o embrião durante a gravidez. Uma das hipóteses é que células da placenta expressam a molécula IDO, a qual protege o embrião do ataque imunológico materno. O objetivo do estudo foi analisar o efeito da indução da expressão da IDO em IP em transplante experimental de IP. Para tanto, as seguintes etapas de padronização foram necessárias. Etapa 1: Padronização da perfusão e digestão do tecido pancreático de rato e determinação do método para a purificação das IP, comparando-se diferentes gradientes de densidade: descontínuo de Ficoll, contínuo de Ficoll e contínuo de iodixanol. Foi demonstrado que o gradiente contínuo de iodixanol fornece maior pureza e maior número de IP íntegras e funcionais. Etapa 2: Padronização do Tx experimental de IP sob a cápsula renal para avaliação do número mínimo de IP transplantadas para reverter o diabetes induzido por estreptozotocina, definido como glicemia >300mg/Kg. Foram transplantadas entre 200 a 3.000 IP por experimento. A rejeição das IP foi analisada pela sobrevida das IP (permanência da glicemia <300mg/dL), tanto em Tx isogênico (Lewis-Lewis) como em alogênico (Sprague-Dawley-Lewis). Para reverter o diabetes foram necessárias no mínimo 2.500 IP. No transplante entre ratos isogênicos (n=6) não houve rejeição das IP. Já no transplante entre animais alogênicos (n=12), as IP apresentaram uma curta sobrevida pós-Tx (11±1 dias; p<0,01 vs. Tx isogênico). Dez dias pós-Tx, houve um grande infiltrado de macrófagos e linfócitos T no enxerto alogênico e uma diminuição significativa da expressão de insulina (p<0,001 vs. Tx isogênico). Etapa 3: Construção do vetor de expressão para IDO. A partir de RNA extraído de placenta de rata no 10º dia de gestação, foi amplificada a seqüência completa do cDNA para IDO, utilizando-se RT-PCR. Em seguida, o cDNA para IDO foi inserido em vetor de expressão (vetor-IDO). Etapa 4: Transfecção do vetor-IDO nas IP. O vetor-IDO foi introduzido nas IP através de lipofecção (Lipofectamina 2000), testando-se diferentes concentrações do vetor-IDO (0, 0,5, 1 e 10 ng/uL) e diferentes períodos de incubação (1h, 15h e 24h). A expressão de IDO nas IP foi confirmada por RT-PCR e imuno-histoquímica. A incubação com 10 ng/uL de vetor-IDO durante 24h foi eficaz para induzir a expressão de IDO nas IP, confirmada a nível de RNAm (RT-PCR) e de proteína (imuno-histoquímica). A eficiência da transfecção em nível funcional foi confirmada pela degradação de triptofano em cultura (dosagem de triptofano por HPLC). Etapa 5: Onze transplantes alogênicos (Sprague-Dawley-Lewis) com IP transfectadas com vetor-IDO foram realizados para analisar o efeito da IDO. Três animais foram sacrificados para análise de imuno-histoquímica e 8 animais foram acompanhados por 45 dias. A sobrevida das IP transfectadas com vetor-IDO foi significativamente maior comparada com a sobrevida de IP não-transfectadas (p<0,01). O estudo conclui que a expressão da IDO protege as IP aumentando a sobrevida das IP. / Transplantation (Tx) of pancreatic islets (PI) is an attractive alternative of treatment for type 1 diabetes mellitus. However, continuous immunossupression is necessary in order to avoid allograft rejection. A new idea of tolerance is based on the immunological paradox, during pregnancy, in that the mother, immunologically competent, does not reject the semi-allogeneic fetus. The hypothesis is that the placenta produces IDO molecules, which protect the embryos against the maternal immunologic attack. The aim of this study was to analyze the effect of the induction of the IDO expression into PI in an experimental model of PI transplantation. The following steps for standardization were necessary. Step 1: Besides the standardization of the rat pancreas perfusion and digestion, the best method for purification of the PI was determined, comparing several density gradients: Ficoll discontinuous, Ficoll continuous and iodixanol continuous. The iodixanol continuous gradient was able to provide high purity and a high number of intact and functional PI. Step 2: The transplantation of the PI between rats was established determining the minimal number of PI to reverse the diabetes (glycemia > 300mg/dL) induced by streptozotocin. In addition, the rejection was analyzed by PI survival (time with glycemia <300mg/dL) in syngeneic (Lewis-Lewis) and allogeneic (Sprague-Dawley-Lewis) transplantation. To reverse the diabetes at least 2,500 PI were necessary. Transplantation between syngenic rats (n=6) disclosed no rejection of the PI. In the allogeneic transplantation (n=12), the PI had a short survival (11±1 days). Ten days post-Tx, a higher number of macrophages and T lymphocytes were observed in the grafts, accompanied by very low insulin expression. Step 3: The expression vector for IDO was constructed from RNA extracted from rat placenta. RT-PCR was carried out to amplify the IDO cDNA, which was inserted into expression vector (IDO vector). Step 4: The IDO vector was introduced into PI through lipofection (Lipofectamine 2000) analyzing several concentrations of the IDO vector (0, 0.5, 1.0 and 10 ng/uL) and several periods of incubation (1h, 15h e 24h). The IDO expression in PI was confirmed by RT-PCR and immunohistochemistry. The incubation with 10 ng/uL of IDO vector during 24h was efficient to induce IDO expression in PI. The function of the IDO was confirmed by tryptofan degradation in culture (measurement of tryptofan by HPLC). Step 5: Eleven allogenic transplants (Sprague-Dawley to Lewis) of PI expressing IDO were performed to analyze the effect of the IDO in the rejection. Eight animals were accompanied for 45 days, whereas three were sacrificed after 10 days for immunohistochemistry analysis. Finally, the survival of the PI expressing IDO was significantly higher than nontransfected PI. The study concludes that the induction of the IDO into PI protects the PI increasing the PI survival.

INTRODUÇÃO: O papel da linfangiogênese no transplante renal em humanos é desconhecido até o momento. As poucas publicações disponíveis acerca do assunto revelam resultados controversos. O presente estudo visa a avaliar a influência dos vasos linfáticos sobre aspectos clínicos e patológicos no transplante renal. MÉTODOS: Biópsias de indicação clínica de pacientes submetidos a transplante renal com enxertos oriundos de doadores falecidos na Unidade de Transplante Renal do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo de janeiro de 2007 a dezembro de 2009 foram selecionadas e os dados clínicos destes pacientes foram coletados do banco de dados. Estas biópsias foram classificadas de acordo com a Classificação de Banff. Reação imuno-histoquímica foi empregada para identificar vasos linfáticos, linfócitos T, linfócitos B e macrófagos. Análise histomorfométrica foi empregada para quantificar estes quatros elementos e a fibrose intersticial cortical. RESULTADOS: A presença de vasos linfáticos foi significativamente mais intensa em biópsias com rejeição aguda mediada por linfócitos T e com distúrbios infecciosos (nefropatia do poliomavírus e pielonefrite), quando comparadas à expressão de linfáticos com biópsias sem rejeição e com biópsias com fibrose intersticial e atrofia tubular de etiologia indeterminada. Biópsias com expressão de vasos linfáticos apresentaram escores semiquantitativos da Classificação de Banff mais altos. Os linfócitos B túbulo-intersticiais apresentaram maior concentração em amostra com presença de vasos linfáticos. A linfangiogênese não demonstrou influência sobre desfechos clínicos relevantes, como função renal e sobrevida do enxerto. CONCLUSÃO: O presente estudo associa a linfangiogênese com distúrbios inflamatórios túbulo-intersticiais do enxerto (rejeição aguda mediada por linfócitos T e infecções) e com infiltrado de linfócitos B. No entanto, a expressão de linfáticos não foi associada à influência sobre a função e a sobrevida do enxerto / INTRODUCTION: The role of lymphangiogenesis in human kidney allograft is currently unknown. Controversial results have arisen from few publications available. This study intends to evaluate the influence of lymphatics on relevant clinical and pathological aspects of renal transplantation. METHODS: Clinically indicated biopsies from patients who underwent renal transplantation with allografts from deceased donors at the Renal Transplantation Unit of the Clinics Hospital of the University of São Paulo Medical School from January of 2007 to December of 2009 were selected and clinical data of these patients were retrieved from the database. These biopsies were classified according to the Banff Classification. Immunohistochemistry was used to identify lymphatic vessels, T lymphocytes, B lymphocytes, and macrophages. Morphometric analysis was employed to quantify their expression and cortical interstitial fibrosis. RESULTS: Lymphatic vessel formation was significantly higher in biopsies with acute T-cell mediated rejection and infectious disorders (polyomavirus-associated nephropathy and pyelonephritis) compared with no rejection and interstitial fibrosis and tubular atrophy without evidence of any specific etiology. Biopsies with expression of lymphatics presented higher levels of semiquantitative scores of the Banff Classification. B lymphocytes infiltrate was more intense in biopsies with lymphatics compared with those without the vessels. Lymphangiogenesis had no effect on important clinical parameters examined (graft function and graft survival two years post transplant). CONCLUSION: The present results associate lymphangiogenesis with kidney allograft tubulointerstitial inflammation (ATCMR and infectious disorders) and with B lymphocytes infiltrate. However, the presence of lymphatic vessels was not associated with any influence on graft function and survival

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-17T13:31:54Z No. of bitstreams: 1 paulacarolinadesouzachandretti.pdf: 2793966 bytes, checksum: 462b8d77041bf22a342c174823ca57ef (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-28T14:16:58Z (GMT) No. of bitstreams: 1 paulacarolinadesouzachandretti.pdf: 2793966 bytes, checksum: 462b8d77041bf22a342c174823ca57ef (MD5) / Made available in DSpace on 2016-06-28T14:16:58Z (GMT). No. of bitstreams: 1 paulacarolinadesouzachandretti.pdf: 2793966 bytes, checksum: 462b8d77041bf22a342c174823ca57ef (MD5) Previous issue date: 2014-05-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os enxertos ósseos autógenos onlay são largamente utilizados na reconstrução de rebordos alveolares atróficos. Recentemente, o etilcianoacrilato tem sido sugerido para a fixação desses enxertos. O objetivo deste estudo foi realizar uma análise histológica qualitativa e semiquantitativa dos enxertos ósseos autógenos em calota de ratos fixados com adesivo etilcianoacrilato e com parafuso de titânio durante 30 dias. Quatorze ratos da espécie Rattus novergicos albinus (Wistar) foram submetidos a enxertos ósseos autógenos parietais onlay, fixados com etilcianoacrilato (Superbond® Flex Gel® - SB, n = 7), apenas nas bordas e parafuso de titânio no centro (n = 7). Após 30 dias, os animais foram eutanasiados e removidas às amostras para análise histológica. Depois da descalcificação das peças, estas foram coradas com hematoxilina e eosina. O seguinte aspecto foi analisado em ambos os grupos: vitalidade do enxerto pela presença de osteócitos. Na interface, foi avaliada a presença do adesivo (SB), presença de reação tecidual com formação de matriz osteoide, presença de reabsorção óssea, formação de capilares, infiltrado inflamatório, proliferação fibroblástica, presença de osteoblastos, osteócitos e osteoclastos. Os resultados foram submetidos à análise estatística realizada pelo software SPSS Statistic 15.0 ® e também foram realizados testes não paramétricos. Foi usado o teste de Mann-Whitney e o teste Exato de Fisher para comparação intergrupos e, para comparações intragrupos, foi usado o teste de Wilcoxon e o teste de McNemar (p-valor< 0,05) e todos os testes foram bicaudais. Nas avaliações qualitativas, não foram encontradas diferenças estatisticamente significativas. Foi obtida diferença significativa na comparação da avaliação quantitativa intergrupo: PT apresentou mais osteócitos na região central (p= 0,035); em SB, foi observada maior inflamação (p= 0,030) e mais osteoclastos (p= 0,048). Quando se compararam as regiões centrais e as extremidades dos enxertos, o grupo PT demostrou um processo de neoformação óssea mais adiantado, principalmente na região central. Nenhum enxerto se apresentou osteointegrado no grupo SB com 30 dias e, no PT, havia pontos de soldadura do enxerto ao osso receptor, mas não uma completa incorporação do enxerto ao leito receptor. / Autogenous onlay bone grafts are widely used in the reconstruction of atrophic alveolar ridges. Recently, etilcianoacrilate was suggestesfor fixing autogenous bone onlay graft. The aim of this study was to perform a qualitative and semiquantitative histological analysis of autogenous bone grafts in the calvaria of rats fixed with etilcianoacrilate adhesive and titanium screw for 30 days. Fourteen Rattus novergicos Albinus (Wistar) underwent onlay parietal bone autografts fixed with etilcianoacrilate (Superbond Gel ® Flex ® - SB, n = 7) only in the edges and titanium screw in the center (n = 7). After 30 days, the animals were euthanized and the samples removed for histological analysis. After decalcification, the specimes were stained with hematoxylin and eosin. The graft vitality to the presence of osteocytes was analyses in the both groups. The interface was evaluated for the presence of: the adhesive (SB), tissue reaction with formation of osteoid matrix, bone resorption, formation of capillary, inflammatory infiltration, fibroblast proliferation, osteoblasts, osteocytes and osteoclasts. Statistical analysis of the results by SPSS Statistic 15.0 ® software and nonparametric tests were performed. Mann-Whitney and Fisher's exact test were used for comparison between groups. Wilcoxon test and McNemar were used for intragroup comparisons (p - value < 0.05). In qualitative assessments, no statistically significant differences were found. A significant difference was found in the intergroup comparison for the quantitative evaluation: the central region of the PT group presented more osteocytes (p = 0.035), increased inflammation (p = 0.030) and more osteoclasts (p = 0.048) were observed in the edges of the SB group. When comparing the central regions and the edges of the grafts, PT group demonstrated more bone formation in the central region. The SB groups showed no osseointegration on the graft. At 30 days in the PT group the graft was only partially integrated to the host bed.

Lung transplantation is the only therapeutic approach for patients presenting end-stage pulmonary failure. Despite progress made in organ preservation and immunosuppression, primary graft dysfunction and obliterative bronchiolitis still hamper short-term and long-term outcomes, respectively. Interleukin-17 recently emerged as a major actor in several immuno-inflammatory disorders. Clinical and experimental evidence also suggest the implication of interleukin-17 or type 17 CD4+ T cells in lung rejection. We therefore investigated the contribution of this cytokine to graft pathology in a murine model of tracheal transplantation that recapitulates pathological features of lung rejection including the development of obliterative airway disease.<p>We first demonstrated that interleukin-17 contributes to inflammatory lesions in the early phase post-transplantation. Interleukin-17 was found to be produced by &61543;&61540;+ T cells and CD4+ T cells infiltrating the graft and interleukin-17 neutralization significantly reduced the development of epithelial lesions together with inhibition of interleukin-6 and heat-shock-protein 70 gene transcription.<p>We then investigated the contribution of interleukin-17 to obliterative airway disease. Although interleukin-17 did not play a dominant role in absence of immunosuppression, it was found to contribute to airway pathology in animals receiving cyclosporin A. Under this treatment, we first observed dramatic changes in the composition of the lymphocyte populations infiltrating the graft: the numbers of CD8+ T cells producing interferon-&61543; and type 1 CD4+ T cells were dramatically decreased while the numbers of type 17, and also type 2 CD4+ T cells were unaffected. The pathological relevance of these findings was first demonstrated by the prolongation of graft survival afforded by the depletion of CD4+ T cells in cyclosporin A-treated animals. Furthermore, graft rejection was also delayed in mice genetically deficient in either interleukin-17 or interleukin-4, providing evidence that type 17 and type 2 CD4+ T cells actively contribute to graft rejection in cyclosporin A-treated recipients. On the other hand, parallel experiments in interferon-&61543;-deficient mice revealed that interferon-& / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

This work discusses the role of CT in the etection and quantification of chronic allograft rejection in patients after lung transplantation and provides solutions to the technical challenges involved with this approach. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

La rapamycine est un inhibiteur de la voie mTOR dont la propriété immunosuppressive est reconnue pour son action préférentielle sur les lymphocytes T effecteurs et sa capacité à favoriser l’expansion de lymphocytes T régulateurs (Treg) et d’autres cellules immunosuppressives. En transplantation hépatique, la rapamycine a été retirée de l’arsenal thérapeutique dans le traitement de novo du rejet aigu, suite aux résultats obtenus et décrits au sein du « Rapamune Liver Transplant Study Group » qui a conclu à une forte incidence de thromboses de l’artère hépatique. Des travaux récents montrent que l’introduction retardée d’une immunosuppression après une greffe permet de créer, dans certains cas, une fenêtre d’opportunité pour freiner la réponse allogénique initiée. Cette nouvelle configuration pourrait remettre en de question l’utilisation en jeu des inhibiteurs de mTOR en transplantation hépatique. L’objectif de ma thèse a été d’évaluer l’efficacité d’un traitement court et retardé par la rapamycine dans un modèle de greffe hépatique immunogène chez le rat. Notre étude montre qu’un traitement de 8 jours débuté à J4 post transplantation permet de prolonger de manière significative la survie des animaux greffés en améliorant la fonction des greffons. De plus, l’analyse cellulaire des organes lymphoïdes secondaires, montre une prédominance de cellules myéloïdes suppressives (MDSC) et de lymphocytes CD8+CD45RClow à un stade précoce chez les animaux tolérants et dont le taux reste stable à un stade tardif sans modifications majeures du pourcentage de Treg CD4+CD25+Foxp3+ . Ainsi, nos résultats suggèrent un effet protecteur de la rapamycine lorsqu’elle est administrée de façon retardée et courte dans un modèle de greffe hépatique et dont le mécanisme d’induction de tolérance ne semble pas être dépendant des Treg. Cette approche permet de reconsidérer la place de la rapamycine en transplantation hépatique et suggère d’évaluer quelles sont les populations cellulaires impliquées dans le maintien de tolérance. / Rapamycin is an mTOR pathway inhibitor with immunosuppressive property recognized for its preferential action on effectors T cells and its ability to promote the expansion of regulatory T cells (Treg) and other immunosuppressive cells. In liver transplantation, rapamycin has been removed from the therapeutic arsenal for the treatment of de novo acute rejection, following the results of "Rapamune Liver Transplant Study Group" which concluded of a high incidence of thrombosis of the hepatic artery. Recent studies show that the delayed introduction of immunosuppression following transplantation creates, in some cases, a window of opportunity to curb the allogeneic response initiated. This therapeutic approach has restored hope in the future of mTOR inhibitors in liver transplantation. The aim of my thesis was to evaluate the efficacy of a delayed and short course rapamycin treatment in an immunogenic liver transplantation model in rats. Our study shows that treatment of 8 days started at day 4 post-transplantation can significantly prolong the survival of grafted animals by improving the function of the grafts. Cellular analysis of secondary lymphoid compartments shows a predominance of myeloid suppressor cells (MDSC) and CD8+CD45RClow T cells at early stage in tolerant animals and the rate remains stable at a late stage without any major changes in the frequency of CD4+CD25+Foxp3+ Treg. Thus, our preliminary results suggest a protective effect of rapamycin when administered at delayed and short way in a liver transplantation model. Early induction mechanism does not appear to be Treg-dependent. This approach allows to reconsider the place of rapamycin and the cell population involved in the maintenance of tolerance.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) has been a successful cellular therapy for patients suffering from hematological malignancies for many decades; however, the beneficial effects of graft-versus-leukemia (GVL) are classically offset by graft-versus-host disease (GVHD). GVHD occurs when major and/or minor human leukocyte antigen (HLA) mismatches between donor and recipient cause rapid expansion and activation of donor effector T cells (Teffs) resulting in end organ damage to the recipient’s epithelial tissues. Given the lymphoproliferative nature of this disease, the standard treatment option is broad immunosuppression, which can result in primary disease relapse, steroid refractory GVHD, and/or opportunistic infection. A more targeted therapy that can selectively suppress GVH responses with maintained GVL responses would achieve the optimal goal of allo-HCT. Regulatory T cells (Tregs) both natural (nTregs) or induced (iTregs) could be potential cellular therapies for the treatment of GVHD, given their innate suppressive function. Initial clinical trials using nTregs have yielded positive results; however, nTreg cellular therapy has been cumbersome due to the necessity for large scale ex vivo expansion given their low yield within an apheresis product and non-specific suppression. Conversely, iTregs can be generated from naïve T cells thus decreasing ex vivo culture times and can be educated with specific antigen thus providing targeted suppression, but a consensus on their efficacy for GVHD therapy has not been reached. Therefore, we investigated the efficacy of antigen specific iTreg therapy for the prevention of GVHD while maintaining GVL responses. In Chapter 2, we evaluated the effectiveness of monoclonal HY-specific iTregs in GVHD attenuation. We chose HY as a target antigen because it is a naturally processed, ubiquitously expressed minor mismatch antigen carried by only male donors/recipients cited to increase GVHD prevalence when donor and recipient are sex-mismatched. Utilizing HY-transgenic mice in which all T cells recognize HY antigen exclusively, we generated HY specific iTregs which effectively attenuating GVHD in male, but not female recipients in three murine bone marrow transplantation (BMT) models (major mismatch, parent to F1, and miHAg mismatch). We found HY specific iTregs lost stability in female recipients but remained stable and suppressive in male recipients suggesting expression of HY antigen was required for their suppressive function and stability. GVL responses were not compromised with the addition of HY specific iTregs in recipient mice using a pre-established tumor model. Thus, HY-specific iTregs can be generated and suppress GVHD in an antigen-dependent manner while sparing the GVL effect. In Chapter 3, we extend our findings in Chapter 2, which provided proof of principle that antigen specific iTregs effectively control GVHD; however, this therapy has a limited translational potential. Therefore, we generated alloreactive CD4 and CD8 iTregs and evaluated GVHD attenuation and GVL preservation in either full or haplo-MHC mismatched BMT models. We found alloreactive CD4 iTregs significantly suppress lethal GVHD, but completely abrogated the GVL effect against aggressive tumors. Conversely, alloreactive CD8 iTregs moderately attenuated GVHD and possessed direct cytotoxicity against tumor cells. Therefore, to rescue the impaired GVL effect mediated by CD4 iTregs, we established a combinational therapy with CD8 iTregs. Indeed we found combination CD4 and CD8 iTreg therapy significantly suppressed GVHD while sparing GVL responses compared to either CD4 or CD8 singular therapy. Mechanistically, this was achieved by potent suppression of both CD4 and CD8 Teffs coupled with preserved cytolytic molecule expression by both CD8 iTregs and Teffs. Taken together, we propose antigen specific iTreg therapy can effectively attenuate GVHD while preserving GVL responses. We further uncovered unique characteristics of CD4 and CD8 iTregs that can be exploited to achieve the optimal cellular therapy following allo-HCT.