Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Objectif De nombreux travaux ont souligné le rôle de la Cyclosporine A (CsA) dans la prévention des lésions d'ischémie-reperfusion (I/R) mais aucun n'a été effectué sur poumons isolés de grands mammifères. Notre objectif était de mesurer pour la première fois les effets de la CsA sur les lésions d'I/R dans un modèle de poumons porcins reperfusés ex vivo, en évaluant plusieurs doses de CsA pour différents temps d'ischémie. Méthodes L'expérimentation A a été conduite sur 4 groupes de 8 paires de poumons chacune : un groupe contrôle et 3 groupes recevant différentes concentrations de CsA (1, 10 ou 30 μM) au moment de l'ischémie et au début de la reperfusion, après 2 heures d'ischémie. L'expérimentation B a été conduite sur 3 groupes de 5 paires de poumons chacune. Les poumons de chaque paire étaient séparés juste après le début de l'ischémie. Les premiers poumons étaient évalués après une ischémie de 2 heures (jour 0), sans CsA. Les seconds poumons étaient évalués après une ischémie de 24 heures (jour 1), soit sans soit avec CsA (1 ou 5 μM), administrée le cas échéant au début de la reperfusion. Résultats La CsA augmentait le rapport PO2/FiO2 avec un effet dose mais augmentait également la pression artérielle pulmonaire, la pression capillaire et les résistances vasculaires pulmonaires, à 10 et 30 μM mais pas à 1 ni 5 μM. Les poumons qui recevaient 30 μM de CsA affichaient des concentrations élevées en cytokines pro-inflammatoires. La concentration en RAGE (receptor for advanced glycation endproducts) dans le lavage broncho-alvéolaire diminuait avec la CsA à J1 en comparaison à J0. Conclusions Lors de l'I/R pulmonaire, les bénéfices cellulaires des doses élevées de CsA sont contrebalancés par ses effets hémodynamiques sur la microvascularisation. A faibles doses, la CsA semble améliorer la fonction pulmonaire.

Le muscle strié squelettique subit de graves lésions d'ischémie-reperfusion (IR) au cours de la progression de l'artériopathie oblitérante des membres inférieurs et lors d'interventions chirurgicales qui nécessitent l'interruption transitoire du flux sanguin dans les artères des membres. Dans ce contexte, nos objectifs étaient de mettre à profit deux modèles expérimentaux d'IR des membres inférieurs par clampage aortique et garrotage unilatéral pour : ° tester l'efficacité d'une alternative médicamenteuse au postconditionnement ischémique par l'utilisation de la cyclosporine A (CsA). En se liant à la cyclophiline D, la CsA empêche l'ouverture du pore de transition mitochondrial (mPTP) à un niveau très distal de la cascade d'évènements qui conduit à la nécrose après IR. ° déterminer de quelle façon deux comorbidités fréquemment retrouvées chez des patients souffrant d'atteinte artérielle (le diabète et l'âge) influencent l'effet de la cyclosporine. Avec les protocoles de conditionnement et aux doses que nous avons utilisées, la cyclosporine a des effets différents sur les conséquences musculaires de l'ischémie-reperfusion des membres inférieurs, dépendant de la pathologie sous-jacente des animaux étudiés. Il semble intéressant d'étudier l'effet dose-réponse de la cyclosporine A pour déterminer l'intervalle thérapeutique optimal, celui-ci pouvant être différent chez l'animal sain et pathologique. D'autre part, étant donné l'importance considérable du stress oxydant chez les animaux diabétiques et sénescents, la co-administration de cyclosporine et d'un antioxydant au moment de la reperfusion pourrait rétablir une protection.

No período de 1981-2010, foram estudados, retrospectivamente, 54 casos de criptococose em pacientes com transplante de órgão sólido, identificados no Laboratório de Micologia da Santa Casa Complexo Hospitalar, Porto Alegre, RS. A criptococose ocorreu em 31 transplantados de rim, 13 de fígado, 7 de pulmão, 2 de pâncreas e rim, e 1 de coração. A idade média foi de 47,91 ± 13,98 (12-76 anos). Um total de 38 pacientes do sexo masculino (70,4%). As manifestações clínicas mais frequentes (54 pacientes) foram febre, cefaléia, vômito, tosse e estado mental alterado. Os achados radiográficos mais comuns no tórax, em 27 pacientes, foram nódulo, consolidação, cavitação e derrame pleural, sendo 10 com comprometimento pulmonar comprovado. Trinta e quatro apresentavam acometimento do sistema nervoso central, 7 tinham envolvimento cutâneo, e 4 em outros locais. O liquor, sangue e urina, respectivamente, contribuíram para o diagnóstico microbiológico com maior frequência. A maioria das infecções, nesta série de pacientes com criptococose, foi causada por Cryptococcus neoformans (92,7%). Pela primeira vez na literatura, documentamos C. gattii em pacientes com transplante de pulmão. Finalmente, quanto ao regime imunossupressor primário utilizado, houve maior mortalidade entre os pacientes que usaram o regime terapêutico baseado em ciclosporina e menor naqueles que usaram tacrolimus. / In the period of 1981 to 2010, 54 cases of cryptococcosis in patients with solid organ transplantation indetified at Mycology Laboratory in Santa Casa Hospital Complex, Porto Alegre, RS, were retrospectively studied. Cryptococcois occured in 31 kidney, 13 liver, 7 lung, 2 kidney-pancreas, and 1 heart transplant. The mean age was 47.3 years old (range, 12-76; SD 13.98). A total of 38 patients were male (70.4%). The most frequent clinical manifestation (54 patients) was fever, headache, vomiting, cough and altered mental status. The most common chest radiographic fidings, in 27 patients, were nodules, masses, consolidation, cavitation, and pleural effusion, 10 with proved pulmonary involvement. Thirty four patients had central nervous system involvement, 7 with cutaneous involvement, and 4 at other sites. The cerebospinal fluid, blood, and urine had the highest yield for the microbiologic diagnosis, respectivelly. Nearly all infections in this series of patients with cryptococcosis involved Cryptococcus neoformans (92.7%). By the first time in the literature, we documented C. gattii in lung transplant patients. Finally, considering the type of primary immunosupressive agent used, there was a higher mortality rate on patients with cyclosporine based therapy, and lowest in those with tacrolimus.

No período de 1981-2010, foram estudados, retrospectivamente, 54 casos de criptococose em pacientes com transplante de órgão sólido, identificados no Laboratório de Micologia da Santa Casa Complexo Hospitalar, Porto Alegre, RS. A criptococose ocorreu em 31 transplantados de rim, 13 de fígado, 7 de pulmão, 2 de pâncreas e rim, e 1 de coração. A idade média foi de 47,91 ± 13,98 (12-76 anos). Um total de 38 pacientes do sexo masculino (70,4%). As manifestações clínicas mais frequentes (54 pacientes) foram febre, cefaléia, vômito, tosse e estado mental alterado. Os achados radiográficos mais comuns no tórax, em 27 pacientes, foram nódulo, consolidação, cavitação e derrame pleural, sendo 10 com comprometimento pulmonar comprovado. Trinta e quatro apresentavam acometimento do sistema nervoso central, 7 tinham envolvimento cutâneo, e 4 em outros locais. O liquor, sangue e urina, respectivamente, contribuíram para o diagnóstico microbiológico com maior frequência. A maioria das infecções, nesta série de pacientes com criptococose, foi causada por Cryptococcus neoformans (92,7%). Pela primeira vez na literatura, documentamos C. gattii em pacientes com transplante de pulmão. Finalmente, quanto ao regime imunossupressor primário utilizado, houve maior mortalidade entre os pacientes que usaram o regime terapêutico baseado em ciclosporina e menor naqueles que usaram tacrolimus. / In the period of 1981 to 2010, 54 cases of cryptococcosis in patients with solid organ transplantation indetified at Mycology Laboratory in Santa Casa Hospital Complex, Porto Alegre, RS, were retrospectively studied. Cryptococcois occured in 31 kidney, 13 liver, 7 lung, 2 kidney-pancreas, and 1 heart transplant. The mean age was 47.3 years old (range, 12-76; SD 13.98). A total of 38 patients were male (70.4%). The most frequent clinical manifestation (54 patients) was fever, headache, vomiting, cough and altered mental status. The most common chest radiographic fidings, in 27 patients, were nodules, masses, consolidation, cavitation, and pleural effusion, 10 with proved pulmonary involvement. Thirty four patients had central nervous system involvement, 7 with cutaneous involvement, and 4 at other sites. The cerebospinal fluid, blood, and urine had the highest yield for the microbiologic diagnosis, respectivelly. Nearly all infections in this series of patients with cryptococcosis involved Cryptococcus neoformans (92.7%). By the first time in the literature, we documented C. gattii in lung transplant patients. Finally, considering the type of primary immunosupressive agent used, there was a higher mortality rate on patients with cyclosporine based therapy, and lowest in those with tacrolimus.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The disabling effects of central nervous system (CNS) tumors are out of proportion to their low incidence. Theyâre second only to stroke as neurologic mortality causes. Brain metastases are the commonest intracranial tumors in adults, almost 10 times more frequent than primary brain tumors. Their diagnosis and treatment have met significant advances, although much more research about drug resistance and new treatment modalities are needed. New and even better brain tumor animal models will help to evaluate novel drug regimens and adjuvant therapies for CNS neoplasms. In the present work, the author presents a simple and easily reproducible brain tumor model utilizing the tumor cell line W256 transplanted to Wistar rats. They tested a drug widely used for palliative treatment of tumoral brain edema (dexamethasone), for survival impact. They also have tested the effects of a drug newly proposed as multidrug resistance reversal agent (cyclosporin â CS). Wistar albino rats had stereotaxic intracranial tumor inoculation after the surgical installation of a permanent canulla on the area of interest (right subfrontal caudate). The brain tumor model, as a model of metastatic brain disease, was successful, with reproducible tumor growth (95%), low incidence of extracranial tumor implantation (21% local, no distant metastasis) and few evidence of surgical site infection (21%). The median survival of the animals was 12.5 days (control), 13 days (CS vehicle treated), 11 days (CS treated), 9.5 and 9 days (dexamethasone 0.3 and 3.0 mg/kg/day). These differences were not significant, although the survival rates on the 12th day post-inoculation have showed a significant survival decrease for the case of dexamethasone 3,0 mg/kg/day (p < 0.05), but not for CS treatment (Fischerâs Exact Test). The estimated tumor volume was 17.08  6.7 mm3 (control) and 12.61  3.6 mm3 (CS treatment, not significant, Studentâs t-test). The tumor volume in the 9th day post-inoculation was estimated in 67,25  19,8 mm3. The doubling time was 24.25 h. This model behaved as an undifferentiated tumor, with local invasiveness features compared with that of primary brain tumors. It fits well, in this way, for the study of tumor cell migration on CNS parenchyma. Phenomena like neuronal degeneration, neuron cell edema and death, and gliosis, as well as perivascular cell infiltrates, were seen frequently. One could find, also, neoangiogenesis, satellite tumor growth, and tumor cell migration in normal brain parenchyma. Besides heavy parenchymatous infiltration, it was also disclosed markedly tumor cell migration along white matter tracts, such as callosal fibers and infiltration in the Virchow-Robins perivascular space. The model presents as a dual brain tumor and leptomeningeal carcinomatosis model. It could be used for the study and treatment test in the scenario of these two pathologies. The intracerebral tumor growth induced peripheral blood neutrophil count elevation (ANOVA, p < 0.01), higher chance for neutrophilia (Fischerâs Exact Test, p < 0.01), higher chance for lymphopenia (Fischerâs Exact Test, p < 0.01) and brain weight increase (Studentâs t-test, p < 0.001) comparing to control. There was no significant change in any of the other hematologic, biochemical and biological parameters tested. CS treatment did not alter any of the tests, as compared to non-treated brain tumor animals. The only exception was the mean animal weight on the first week post-inoculation (ANOVA, p < 0.05). CS, in this way, was responsible for an early cachexia in the brain tumor inoculated animals. CS treatment of brain tumor animals did show non-significant effects indicating a volume (26%) and weight tumor decrease, and tumor infiltrating neutrophil increase (odds ratio - OR = 5.6). This indicates the necessity to further study morphologically and functionally the local inflammation in brain tumor inoculated animals, as well the effects of CS administration. In conclusion, the W256 intracerebral tumor model is simple, easily performed, reproducible and of great potential utility. In this model, tumor inoculation can lead to hematologic and biologic modifications in the experimental animals. CS could apparently lead to early tumor caquexia in this tumor model. However, CS treatment did not modify the survival chance of the brain tumor animals, in sharp contrast to dexamethasone 3.0mg/kg/day, a much-used drug in the treatment of brain tumors, which decreased the animal survival. / Os importantes efeitos incapacitantes dos tumores do sistema nervoso central (SNC) sÃo desproporcionais a sua baixa incidÃncia. Mesmo assim, entre as doenÃas neurolÃgicas, ficam atrÃs apenas dos acidentes vasculares do SNC como causa de morte. MetÃstases cerebrais constituem os tumores intracranianos mais comuns do adulto, ocorrendo atà 10 vezes mais freqÃentemente que tumores primÃrios. AvanÃos significativos ocorreram em seu diagnÃstico e tratamento, embora mais pesquisa sobre os fenÃmenos que diminuem o efeito de drogas em metÃstases cerebrais e tratamentos eficazes para estas patologias sejam cada vez mais necessÃrios. O desenvolvimento de melhores modelos animais de tumores do SNC serà necessÃrio para a avaliaÃÃo in vivo de novas formas de quimioterapia (QT) e terapia adjuvante para tumores cerebrais. No presente trabalho, o autor objetivou desenvolver um modelo de tumor cerebral simples e de fÃcil reproduÃÃo utilizando a linhagem W256, alÃm de testar o efeito na sobrevida animal de uma droga largamente usada para o tratamento de efeitos secundÃrios a edema cerebral (dexametasona). O autor tambÃm testou uma droga envolvida numa nova proposta de reversÃo de multirresistÃncia a drogas anti-neoplÃsicas em tumores cerebrais (ciclosporina â CS). Ratos albinos (Wistar) tiveram o tumor inoculado atravÃs de estereotaxia, apÃs a instalaÃÃo cirÃrgica de uma cÃnula no ponto escolhido (caudato subfrontal direito). O modelo de tumor implantado no cÃrebro de ratos, simulando uma metÃstase cerebral, mostrou-se bem sucedido e reprodutÃvel (95% de crescimento tumoral), com baixa incidÃncia de disseminaÃÃo tumoral extracraniana local (21%), baixa evidÃncia de infecÃÃo local (21%), ausÃncia de metÃstases à distÃncia e ausÃncia de sinais de infecÃÃo sistÃmica. Os animais sobreviveram uma mediana de 12,5 dias (grupo controle), 13 dias (tratados com veÃculo da CS), 11 dias (tratados com CS), 9,5 e 9 dias (dexametasona 0,3 e 3,0 mg/kg/dia, respectivamente). As diferenÃas entre estas medianas nÃo foram significantes (teste de Kruskal-Wallis), embora as diferenÃas entre as taxas de sobrevida no 12o dia apÃs a inoculaÃÃo tenham mostrado reduÃÃo significante no grupo que recebeu dexametasona 3,0 mg/kg/dia (p < 0,05), mas nÃo no grupo tratado com CS (teste de Fischer). O volume tumoral estimado (VTE) no sÃtimo dia pÃs-inoculaÃÃo (7DPI) foi de 17,08  6,7 mm3 no controle e 12,61 3,6 mm3 apÃs tratamento com CS, sem diferenÃa significante (teste t-Student). O VTE no 9DPI de animais do grupo Tumor foi de 67,25  19,8 mm3. O tempo de duplicaÃÃo foi de 24,25 h. O modelo comportou-se como um tumor de caracterÃsticas indiferenciadas, apresentando invasividade local comparada à de tumores primÃrios do SNC, prestando-se ao estudo da migraÃÃo de cÃlulas tumorais no SNC. Observaram-se fenÃmenos como degeneraÃÃo neuronal hidrÃpica, edema celular neuronal, sinais de morte celular neuronal e gliose, alÃm da presenÃa de infiltrados celulares tumorais e inflamatÃrios perivasculares. Observaram-se, tambÃm, neoformaÃÃo vascular, formaÃÃo de nÃdulos tumorais satÃlites ao tumor principal e migraÃÃo celular tumoral no parÃnquima cerebral normal. Observou-se, alÃm da infiltraÃÃo parenquimatosa, marcante migraÃÃo celular tumoral ao longo de tratos de substÃncia branca (corpo caloso) e ao longo dos espaÃos perivasculares de Virchow-Robins. O modelo apresenta-se como um misto de tumor cerebral intraparenquimatoso e carcinomatose leptomenÃngea, podendo ser utilizado para estudar o comportamento e testar formas de tratamento para ambas as patologias. O crescimento tumoral intracerebral induziu aumento do nÃmero de neutrÃfilos no sangue perifÃrico (ANOVA, p < 0,01), maior chance de apresentar neutrofilia (teste de Fischer, p < 0,01), maior chance de apresentar linfopenia (teste de Fischer, p < 0,01) e aumento do peso dos cÃrebros dos animais experimentais (teste t-Student, p < 0,001) em relaÃÃo ao controle. Nenhum dos outros valores hematolÃgicos, bioquÃmicos e biolÃgicos foi alterado de maneira significante. O tratamento de animais inoculados com tumor com a CS, nÃo alterou nenhuma das medidas hematolÃgicas, bioquÃmicas ou biolÃgicas em relaÃÃo aos animais inoculados com tumor e nÃo tratados, exceto o peso dos animais na primeira semana apÃs inoculaÃÃo tumoral (ANOVA, p < 0,05). A CS, dessa forma, induziu significantemente uma caquexia precoce nos animais inoculados com tumor cerebral. O tratamento com CS de animais inoculados com tumor mostrou tendÃncias nÃo significantes a diminuir volume (26%) e massa (7%) tumorais e aumentar nÃmero de neutrÃfilos infiltrantes de tumor (razÃo de chance - RC = 5,6) e necrose tumoral, indicando a necessidade de posteriores estudos para caracterizar morfolÃgica e funcionalmente a resposta inflamatÃria local em animais inoculados com tumor e a influÃncia da CS neste processo, alÃm do efeito da CS na angiogÃnese tumoral. Concluindo, o modelo de W256 intracerebral mostrou-se simples, de fÃcil execuÃÃo, reprodutÃvel e Ãtil. Neste modelo, a inoculaÃÃo tumoral induz modificaÃÃes hematolÃgicas e biolÃgicas nos animais. A CS pareceu exarcebar a caquexia tumoral neste modelo. A CS, todavia, nÃo alterou a chance de sobrevida de animais inoculados com tumor cerebral, ao contrÃrio da dexametasona 3,0 mg/kg/dia, que reduziu esta chance. A CS, assim, parece ser mais segura neste modelo tumoral que uma droga largamente utilizada para tratamento de pacientes com metÃstase cerebral.

O tratamento de pacientes com doenças inflamatórias oculares crônicas frequentemente implica no uso prolongado de drogas anti-inflamatórias sistêmicas como, corticosteroides e outros imunossupressores, podendo acarretar efeitos colaterais importantes. O uso local destas drogas pode contribuir para aumentar seus efeitos desejáveis e reduzir os efeitos colaterais. Implantes intraoculares biodegradáveis são capazes de disponibilizar o fármaco diretamente na cavidade vítrea em doses terapêuticas por período prolongado. O copolímero do ácido lático e glicólico (PLGA) é um clássico exemplo entre os polímeros sintéticos biodegradáveis aplicados em sistemas de liberação de fármacos devido à sua biocompatibilidade e ausência de toxicidade em testes in vivo. A ciclosporina A (CsA) é um imunossupressor largamente usado na clínica médica, e também tem sido empregada no tratamento de várias doenças inflamatórias intraoculares. O objetivo deste estudo foi avaliar a farmacocinética vítrea da CsA, quando aplicada por meio de implante biodegradável de PLGA intravítreo na concentração de 350 µg em olhos de coelhos, assim como avaliar a ocorrência de toxicidade retiniana causada pela presença intraocular do sistema de liberação de fármacos por meio de eletrorretinografia (ERG) e histopatologia. Dos sessenta coelhos que foram utilizados neste estudo, 38 receberam o implante intravítreo de PLGA contendo CsA e 22 somente os veículos. Somente o olho direito dos coelhos foi analisado na pesquisa. O estudo teve duração de oito semanas. Quatro coelhos do grupo CsA e dois do grupo controle foram sacrificados semanalmente para a coleta do vítreo e posterior estudo farmacocinético. Quatro animais de cada grupo foram escolhidos para terem a pressão intraocular aferida semanalmente. Seis coelhos foram submetidos a ERG no início e ao final do estudo, sendo então sacrificados, e os olhos processados para estudos histológicos da retina. O período inferido de permanência da CsA na cavidade vítrea foi de 17 semanas. Nos dois grupos, com e sem CsA, não foram observadas alterações histológicas na retina, entretanto houve importante redução da onda b nas fases escotópicas da ERG no grupo CsA, indicando toxicidade na via dos bastonetes após as oito semanas de seguimento. Em resumo, estes resultados mostraram que a CsA aplicada por meio de implantes oculares de PLGA na dose de 350 µg não causa alterações histológicas da retina, mas provoca um padrão exclusivo de diminuição da onda b. Em estudos futuros, seria interessante avaliar os efeitos de implantes contendo concentrações inferiores a 350 µg de CsA, e também, veículos que permitam que sua liberação seja mais lenta, evitando-se, assim, a toxicidade observada nos ERGs e confirmar sua aplicabilidade clínica como alternativa interessante para o tratamento de doenças oculares inflamatórias crônicas. / Treatment of patients with chronic inflammatory ocular diseases often involves the use of systemic anti-inflammatory drugs such as corticosteroids and other immunosuppressive agents for a long period of time, which may cause significant systemic side effects. Intraocular use of these drugs may help to improve their local beneficial effects and reduce systemic adverse effects. Biodegradable intraocular implants are able to deliver drugs directly into the vitreous cavity in therapeutic doses for an extended period of time. Poly-lactic-co-glycolic acid (PLGA) is a good example of synthetic biodegradable polymers used in ocular drug delivery systems due to its biocompatibility and absence of toxicity. Cyclosporine A (CsA) is a largely used immunossupressor, and it has also been employed for treatment of various intraocular inflammatory diseases. The objective of this work was to evaluate the pharmacokinetics of CsA, when applied in biodegradable PLGA intravitreal implants in rabbit eye and its retina toxicity by electroretinography and histopathology. Right eyes of sixty rabbits were used on this study, 38 received the PLGA implant containing 350 µg of CsA, and 22 the implant without the drug and were followed during 8 weeks. Four animals of CsA group and 2 of control group were sacrificed weekly to have their vitreous samples collected for subsequent pharmacokinetic study. Four animals from each group were chosen to have intraocular pressure measured weekly. Six animals of each group underwent electroretinography tests at baseline and at the end of the study. Then they were sacrificed and had their eyes processed for histological studies of the retina. It was hypothetically calculated that CsA would take 17 weeks to be completely delivery by this system. Histologically the retina did not show alterations in both groups, but there was a significant reduction in the b wave of the scotopic ERG phases in the CsA group indicating toxicity of the rods pathway after 8 weeks of follow-up. In summary, PLGA implants with 350 µg of CsA does not cause retinal histological changes, but decreases b wave amplitude. In future studies it would be interesting to test lower concentrations of CsA using this delivery system to decrease possible toxicity and to guarantee its clinical applicability.

Abstract The aim of this work was to describe the clinical features and clinical course of Henoch-Schönlein purpura (HSP) in a prospective setting, to compare the efficacy of cyclosporine A (CyA) and methylprednisolone (MP) pulses for the treatment of severe HSP nephritis (HSN) and to study the effect of prophylactic prednisone treatment given at disease onset on the long-term outcome. A total of 223 children with newly diagnosed HSP were followed up prospectively for 6 months. Patients with severe HSN also had extrarenal symptoms more frequently during this time. Protein loss via the intestine was more common than previously described, occurring in 3% of the patients. HSN developed in the early course of the disease. The results suggest that weekly urine dipstick tests are indicated for 2 months after HSP onset and individually for over 6 months in cases of HSN or HSP recurrences. Prednisone did not affect the frequency or timing of the appearance of HSN. The efficacy of CyA and MP treatments was evaluated in a trial with a mean follow-up time of 6 years involving 24 paediatric patients (11 CyA, 13 MP), 15 of whom were randomized and 9 were treated according to the given protocol without randomization. Oral CyA was not inferior to intravenous MP pulses and proved to be an efficient, safe steroid-sparing treatment for severe HSN. All the CyA-treated patients achieved remission of nephrotic-range proteinuria within 3 months, while remission was achieved more slowly in the MP group and only in 6/13 (46%) with the initial treatment. There was no difference in the renal biopsy findings two years after initiation of the therapy. The 8-year outcome of HSP was assessed by means of a health questionnaire in 160 (94%) of the 171 former patients in the randomized placebo-controlled prednisone trial and in 138 (81%) with urine analysis and measurement of blood pressure. HSP carried a good prognosis, although skin relapses occurred up to a decade after the initial onset and could be accompanied by late-onset nephritis. Hypertension and/or renal abnormalities were recorded in 13% of the patients, being more frequent in those with an initial occurrence of HSN (OR 4.3, p=0.009, 95% CI 1.4–14.0) and warranting long-term follow-up of HSN patients. Early prednisone treatment did not affect the long-term outcome of HSP and should not be routinely used. / Tiivistelmä Väitöskirjan tarkoituksena oli kuvata Henoch-Schönleinin purppuran (HSP) oireita ja taudinkulkua, verrata siklosporiini A:n (CyA) ja metyyliprednisolonipulssihoidon (MP-pulssihoidon) tehoa vaikean HSP-nefriitin (HSN) hoidossa ja selvittää taudin alussa annetun prednisonihoidon vaikutusta pitkäaikaisennusteeseen. Taudinkulkua seurattiin prospektiivisesti 6 kuukauden ajan diagnoosista 223 lapsipotilaan aineistossa. Potilailla, joilla oli vaikea HSN, esiintyi myös muita oireita pitempään. Proteiinin menetystä suolistoon esiintyi 3 %:lla, mikä on aiemmin kuvattua yleisempää. HSN ilmaantui taudin alkuvaiheessa. Tutkimustulosten perusteella viikoittainen virtsanäytteiden seuranta riittää 2 kuukauden ajan taudin alusta. Seuranta-aikaa tulee pidentää yksilöllisesti yli 6 kuukauden, jos potilaalla todetaan HSN tai HSP uusiutuu. Prednisonilla ei todettu olevan vaikutusta HSN:n yleisyyteen tai ilmaantumisaikaan. CyA- ja MP-hoitojen tehoa vaikeaan HSN:n seurattiin 24 lapsipotilaan aineistossa (11 CyA, 13 MP) 6 vuoden ajan. Potilaista 15 satunnaistettiin hoitoryhmiin ja 9 hoidettiin tutkimussuunnitelman mukaan ilman satunnaistamista. Suun kautta otettu CyA vaikutti hoidoista tehokkaammalta, sillä kaikilla potilailla nefroottistasoinen valkuaisvirtsaisuus hävisi kolmessa kuukaudessa. MP-hoitoa saaneista vain 6/13 (46 %) pääsi remissioon MP-hoidolla ja hekin CyA-hoidettuja hitaammin. Kaksi vuotta tutkimuksen alusta otettujen munuaisbiopsioiden histologisissa löydöksissä ei ollut eroa ryhmien välillä. Varhaisen kortisonihoidon pitkäaikaisvaikutuksia arvioitiin 8 vuotta lumekontrolloidun prednisonihoitotutkimuksen jälkeen, jolloin aiemman tutkimuksen 171 potilaasta 160 (94 %) vastasi terveyskyselyyn ja 138 (81 %) osallistui virtsa-analyysin ja verenpaineen mittauksen sisältäneeseen seurantatutkimukseen. HSP:n ennuste oli hyvä, vaikka taudin iho-oireet saattoivat uusia jopa 10 vuoden ajan ja taudin uusiutumisen yhteydessä saattoi ilmaantua myöhäinen HSN. Kohonnut verenpaine ja/tai valkuais-/verivirtsaisuus todettiin 13 %:lla. Ne olivat yleisempiä potilailla, joilla oli ollut HSN taudin alkuvaiheessa (OR 4.3, p=0.009, 95 % CI 1.4–14.0). Siten HSN-potilaiden pitkäaikaisseuranta on tarpeen. Varhaisella kortisonihoidolla ei ollut vaikutusta taudin ennusteeseen, minkä vuoksi kortisonia tulee käyttää HSP-potilaiden hoidossa vain harkiten.

La ciclosporine est un traitement immunosuppresseur très utilisé : elle inhibe l'activation des lymphocytes T via la calcineurine. Sa néphrotoxicité limite son utilisation : la ciclosporine induit une augmentation de la vasoconstriction ainsi qu'une augmentation de la réponse des cellules musculaires lisses vasculaires (CMLV) aux agents vasoactifs. Le récepteur minéralocorticoïde (RM), au-delà de ses effets sur la réabsorption sodée, agit sur le tonus vasculaire en modulant la réponse des cellules (endothéliales et musculaires lisses) vasculaires aux agents vasoactifs. Notre hypothèse était que le RM pouvait participer à l'action vasoconstrictrice de la ciclosporine ; son inactivation pourrait limiter la néphrotoxicité de la ciclosporine. Deux modèles de souris ont été invalidés génétiquement pour le RM : dans les cellules endothéliales et les CMLV (KO-RM CMLV). Seules les souris KO-RM CMLV étaient protégées contre la néphrotoxicité de la ciclosporine. Ces effets impliquent une action sur le tonus vasculaire rénal. L'antagonisme pharmacologique du RM (par le canrénoate) administré per os confère la même protection. En revanche, la néphrotoxicité induite par le tacrolimus (une autre anticalcineurine) n'est pas prévenue par l'antagonisme du RM. Utiliser un antagoniste sélectif du RM (l'éplérénone) pourrait prévenir la néphrotoxicité de la ciclosporine. Nous avons prouvé sa bonne tolérance en association à la ciclosporine chez les patients transplantés et insuffisants rénaux chroniques. Une kaliémie supérieure à 4,35mmol/L à l'initiation indique un sur-risque de développer une hyperkaliémie. L'efficacité reste à démontrer au cours d'un essai prospectif et randomisé. / Cyclosporine A (cyclo) is a widely used drug in kidney transplantation: its anticalcineurin actioninhibits T lymphocytes activation and prevents allograft rejection. Despite a huge benefit on graftsurvival, cyclo exerts a side effect that limits its use: nephrotoxicity. Vasculotoxicity appears to becentral: cyclo enhances renal vasoconstriction by altering vasoactive factors and vascular smoothmuscle cells (VSMC) response to vasoactive factors. Beyond its effects on sodium reabsorption,Mineralocorticoid Receptor (MR) acts on vascular tone by modulating both endothelial and VSMCresponses to vasoactive factors. Our working hypothesis was that MR could participate to cycloinducedvasoconstriction and that MR inactivating (pharmacologically or genetically) could alleviatecyclo-induced nephrotoxicity. Two genetically MR-knock out (MR-KO) were generated: inendothelial or VSMC. Only VSMC MR-KO mice were protected from cyclo-induced nephrotoxicity.We also show that such an effect was mediated by vascular tone modulation. This prevention was alsoconferred by the systemic pharmacological antagonism of MR (by canrenoate) in mice but not duringnephrotoxicity induced by tacrolimus (another anticalcineurine drug used in kidney transplantation).Then, we proposed to use MR pharmacological antagonism in humans (by eplerenone) during kidneytransplantation. We first had to prove its safety in such a population. Among 31 cyclo-treated patients,only 9 developed hyperkalemia (>5mmol/L) and none presented serious side effect. We propose akalemia higher than 4.35mmol/L at baseline to be the marker of a higher risk of developinghyperkalemia under treatment. The efficiency of eplerenone to prevent/alleviate cyclo-inducednephrotoxicity during kidney transplantation should be tested during a randomized controlled trial.

A Ciclosporina A (CsA) é o medicamento de escolha utilizado no controle da rejeição de órgãos em pacientes transplantados. Efeitos adversos associados ao fármaco, como alterações ósseas e o aumento gengival são fatores de risco para a doença periodontal. O objetivo do estudo foi avaliar o efeito da indução de doença periodontal no tecido ósseo, tecido epitelial e tecido conjuntivo de ratos previamente tratados com a CsA. Foram utilizados quarenta ratos Wistar, com 12 semanas de vida, divididos em quatro grupos (n=10): grupo Controle (GC); grupo Ciclosporina A (GCsA), administração de 10mg/kg de CsA durante sessenta dias a partir do início do experimento; grupo Ciclosporina A Ligadura (GCsAL), inserção da ligadura após trinta dias do início do experimento e administração de 10mg/Kg de CsA desde o início do experimento, durante sessenta dias; grupo Ligadura (GL), inserção da ligadura após trinta dias do início do experimento. Os animais foram sacrificados após sessenta dias por meio de perfusão cardíaca para a realização da análise histológica e histomorfométrica do tecido gengival e tecido ósseo, análise radiográfica do suporte ósseo periodontal e da densidade radiográfica e análise bioquímica da Fosfatase Alcalina. Os resultados foram submetidos à análise de variância (ANOVA, Tukey) a 5% e ao teste não paramétrico de Kruskal- Wallis. Os valores médios para GC (60.5 2.22%) e GCsAL (58.1 2.24%) foram equivalentes entre si para o suporte ósseo periodontal e diferentes de GCsA (55.0 4.44%) e GL (54.8 3.11%) (p=0.0007). Os valores médios da densidade radiográfica não apresentaram diferença estatística significativa (p=0.1776). Em relação à fosfatase alcalina, novamente não foi observada diferença significativa entre os grupos (p=0.2806). Os valores médios de células TRAP+ por grupo experimental, não apresentaram diferença estatística significativa (p=0.3995). Os valores médios para GC (0.29 0.03mm2) e GCsA (0.30 0.02mm2) foram equivalentes entre si para a área do ligamento periodontal e diferentes de GCsAL (0.43 0.17mm2) e GL (0.41 0.11mm2) (p=0.3994). Na área total do tecido gengival, os valores médios para GCsA (0.088 0.033mm2) e GL (0.101 0.034mm2) foram equivalentes entre si e diferentes de GC (0.053 0.020mm2) e GCsAL (0.146 0.047mm2) (p=0.000001). Na proporção área do conjuntivo e área total, os valores médios para GC (28.60 8.64%) foi equivalente ao GCsA (32.72 14.13%) e diferente do GCsAL (38.50 10.98%) e GL (37.70 7.49%) (p=0.0093). Em relação à proporção área do epitélio e área total, os valores médios para GC (71.39 8.64%) foi equivalente ao GCsA (67.27 14.13%) e diferente do GCsAL (61.49 10.98%) e GL (63.37 7.44%) (p=0.0142). Na proporção área do epitélio e área do conjuntivo, os valores médios para GC (2.80 1.13) foi equivalente ao GCsA (2.18 1,32) e diferente do GCsAL (1.89 1.17) e GL (1.81 0.80) (p=0.0334). Baseados nestes resultados pode-se concluir que a exposição prévia a CsA não modificou significativamente a evolução da doença periodontal induzida em ratos. / Cyclosporine A (CsA) is the drug of choice used to prevent organ transplant rejection. Side effects associated to the drug, like bone alterations and gingival overgrowth are considered risk factors to periodontal disease. The objective of the present study was to evaluate the effect of periodontal disease on the bone tissue, epitelial tissue and connective tissue of the rats previously treated with CsA. Forty Wistar rats with 12 weeks were divided into four groups: Control Group (CG, n=10); CsA Group (CsAG, n=10), with CsA (10mg/kg) administration during 60 days since the beginning of the experiment; CsA and Ligature Group (CsALG, n=10), with ligature placement at 30 days after the beginning of the experiment with CsA administration during the whole period; and, Ligature Group (LG, n=10), with ligature placement at 30 days after the beginning of the experiment. After blood sample collection for the biochemical analysis of the Alkaline Phosphatase (PA) activity, the animals were sacrificed by intracardiac perfusion at 60 days after the beginning of the experiment. The mandibles were removed for histologic and histometric analyses of the gingival and bone tissues, and radiographic analysis of the alveolar bone support and density. The data were subjected to analysis of variance (ANOVA, Tuckey) at 5% and to the non-parametric test of Kruskal-Wallis. The mean percentage of alveolar bone support for the CG (60.5 2.22%) was similar to the CsALG (58.1 2.24%) and different to the CsAG (55.0 4.44%) and LG (54.8 3.11%) (p=0.0007). Bone density and PA activity were not statistically different among groups (p=0.1776, and p=0.2806, respectively). The mean values for TRAP+ cells were not statistically different among experimental groups (p=0.3995). The mean values of the periodontal ligament area for CG (0.29 0.03mm2) were similar to the CsAG (0.30 0.02mm2) and statistically different to the CsALG (0.43 0.17mm2) and to the LG (0.41 0.11mm2) (p=0.3994). With regards to the total area of the gingival tissue, the mean values for the CsAG (0.088 0.033mm2) and LG (0.101 0.034mm2) were similar between each other and statistically different to the CG (0.053 0.020mm2) and CsALG (0.146 0.047mm2) (p=0.000001). Regarding the proportion of connective tissue area to the total area, the mean value of the CG (28.60 8.64%) was similar to the CsAG (32.72 14.13%) and statistically different to the CsALG (38.50 10.98%) and to the LG (37.70 7.49%) (p=0.0093). In relation to the proportion of the epithelial tissue area to the total area, the mean value for the CG (71.39 8.64%) was similar to the CsAG (67.27 14.13%) and different to the CsALG (61.49 10.98%) and LG (63.37 7.44%) (p=0.0142). In the proportion of epithelial tissue area to the connective tissue area, the mean value for the CG (2.80 1.13) was similar to the CsAG (2.18 1.32) and different to the CsALG (1.89 1.17) and LG (1.81 0.80) (p=0.0334). Based on these results it can be concluded that previous exposure to CsA did not significantly modify the development of periodontal disease induced in rats.