As infecções são a causa mais freqüente de morbidade e mortalidade observadas tanto aguda como tardiamente nos pacientes receptores de transplante pulmonar, o que pode estar diretamente relacionado a uma deficiência no transporte mucociliar do sistema respiratório. Nosso objetivo foi avaliar a influência de dois fatores envolvidos com o transplante pulmonar sobre o transporte mucociliar de ratos: a secção e anastomose brônquica e a imunossupressão pela ciclosporina A. Setenta e dois ratos foram distribuídos aleatoriamente em cinco grupos de acordo com: i) procedimento operatório e ii) terapia a que seriam submetidos. Os resultados mostram que houve uma diminuição significativa da Freqüência de Batimento Ciliar in situ, da Transportabilidade do Muco in vitro e da Velocidade de Transporte Mucociliar in situ medidos a partir do brônquio principal esquerdo dos ratos tratados com ciclosporina A (p<0,001). A Freqüência de Batimento Ciliar in situ dos brônquios operados mostrou-se diminuída também no grupo tratado com solução salina e sacrificado no 30º dia após a operação (p=0,001). Já a Velocidade de Transporte Mucociliar in situ mostrou uma diminuição significativa em todos os grupos submetidos à secção brônquica (p<0,001). Houve um efeito sinérgico entre a terapia com ciclosporina A e a secção brônquica, causando um prejuízo ao transporte mucociliar ainda maior do que quando analisados isoladamente. Concluímos que a Velocidade de Transporte Mucociliar in situ foi agudamente prejudicada após a secção brônquica e terapia imunossupressora pela ciclosporina A, havendo diminuição da freqüência de batimento dos cílios e alteração das propriedades viscoelásticas do muco respiratório. / Infections are the most common cause of early and late morbidity and mortality in lung transplant recipient, and can be directly related to impaired mucociliary transport. Our aim was to assess the influence of bronchial section and imunossupression on mucociliary transport in rats. Seventy two rats were randomly distributed in five groups according to i) surgical procedure and ii) drug therapy. There was a significant impairment on Ciliary Beating Frequency in situ, Mucus Transportability Rate in vitro and Mucociliary Transport Speed in situ from operated bronchus of cyclosporine A-treated rats (p<0.001). Ciliary Beating Frequency from operated bronchus was also impaired in saline-treated rats that were killed on 30th postoperative day (p=0.001). Mucociliary Transport Speed was impaired in all bronchi underwent to section (p<0.001). We conclude that bronchial section and cyclosporine therapy impaired all factors analyzed. Also there was a synergic effect between cyclosporine therapy and bronchial section on ciliary beating frequency.

L’insuffisance rénale chronique (IRC) est un problème majeur fréquemment rencontré chez les greffés cardiaques. Les inhibiteurs de la calcineurine, pierre angulaire de l’immunosuppression en transplantation d’organes solides, sont considérés comme une des principales causes de dysfonction rénale postgreffe. Plusieurs autres éléments tels que les caractéristiques démographiques, cliniques et génétiques du receveur contribuent également au phénomène, mais il demeure plutôt difficile de déterminer quels sont les patients les plus à risque de développer une IRC après la transplantation. Ainsi, la découverte de nouveaux marqueurs génétiques de dysfonction rénale pourrait un jour mener à l’individualisation de la thérapie immunosuppressive selon le profil génétique de chaque patient. Or, on ne connaît pas les opinions des greffés à l’égard des tests pharmacogénomiques et l’on ne sait pas si celles-ci diffèrent des opinions exprimées par les individus en bonne santé. Cette thèse de doctorat a donc pour objectifs : 1- De décrire l’évolution de la fonction rénale à très long terme après la transplantation et d’identifier les marqueurs démographiques et phénotypiques associés à l’IRC postgreffe cardiaque; 2- D’identifier les marqueurs génétiques associés à la néphrotoxicité induite par les inhibiteurs de la calcineurine; 3- D’évaluer et de comparer les attitudes des patients et des individus en bonne santé par rapport à l’intégration clinique potentielle des marqueurs pharmacogénomiques. Trois projets ont été réalisés pour répondre à ces questions. Le premier repose sur une analyse rétrospective de l’évolution de la fonction rénale chez les patients greffés au sein de notre établissement entre 1983 et 2008. Nous y avons découvert que le déclin de la fonction rénale se poursuit jusqu’à 20 ans après la transplantation cardiaque et que les facteurs de risque d’IRC incluent entre autres l’âge avancé, le sexe féminin, la dysfonction rénale prégreffe, l’hypertension, l’hyperglycémie et l’utilisation de la prednisone. Le deuxième projet est une étude pharmacogénomique s’intéressant aux déterminants génétiques de la néphrotoxicité induite par les inhibiteurs de la calcineurine. Elle nous a permis d’illustrer pour la première fois qu’un polymorphisme génétique lié à PRKCB (gène codant pour la protéine kinase C-β) est associé avec la fonction rénale des patients greffés cardiaques, alors que cela n’est probablement pas le cas pour les polymorphismes de TGFB1 (gène codant pour le transforming growth factor-β1). La troisième section de cette thèse rapporte les résultats d’un questionnaire dont le but était de comparer les attitudes envers les tests pharmacogénomiques parmi un groupe de personnes en bonne santé, de patients greffés cardiaques et de patients souffrant d’insuffisance cardiaque. Cette étude a démontré que, bien que l’enthousiasme pour la pharmacogénomique soit partagé par tous ces individus, les craintes liées à la confidentialité et aux répercussions potentielles sur l’emploi et les assurances sont plus prononcées chez les personnes en bonne santé. En résumé, les travaux issus de cette thèse ont révélé que l’identification précoce des patients greffés cardiaques les plus susceptibles de présenter une détérioration de la fonction rénale ainsi que l’adoption d’une approche thérapeutique individualisée reposant notamment sur les applications cliniques de la pharmacogénomique pourraient éventuellement permettre de freiner cette complication postgreffe. / Chronic kidney disease (CKD) is a major problem frequently observed in cardiac transplant recipients. Calcineurin inhibitors, which have become the cornerstone of immunosuppressive treatments in solid organ transplantation, are considered a major cause of post-transplant renal dysfunction. Several other factors such as recipients’ demographic, clinical and genetic characteristics also contribute to this phenomenon, but it remains rather difficult to determine which patients present the highest risk of CKD after transplantation. Discovery of new genetic markers of renal dysfunction could one day lead to individualization of immunosuppressive therapy according to each patient’s genetic profile. However, transplant patients’ opinions towards pharmacogenomic testing remain unknown, and it is unclear whether these differ from healthy individuals’ opinions. This doctoral thesis thus aims: 1- To describe the very long-term evolution of renal function after transplantation and to identify demographic and phenotypic markers associated with postheart transplant CKD; 2- To identify genetic markers associated with calcineurin inhibitor-induced nephrotoxicity; 3- To assess and to compare the attitudes of patients and healthy individuals concerning the potential integration of pharmacogenomic markers in clinical practice. Three projects have been conducted to answer these questions. The first one relies on a retrospective analysis of the evolution of renal function in patients who received a heart transplantation at our institution between 1983 and 2008. We discovered that deterioration of renal function continues up to 20 years after transplant and that risk factors of CKD include, among others, advanced age, female gender, pretransplant renal dysfunction, hypertension, hyperglycemia and use of prednisone. The second project is a pharmacogenomic study looking at genetic determinants of calcineurin inhibitor-induced nephrotoxicity. We were able to illustrate for the first time that a genetic polymorphism related to PRKCB (gene encoding protein kinase C-β) is associated with renal function in heart transplant patients, whereas it is probably not the case for polymorphisms in TGFB1 (gene encoding transforming growth factor-β1). The third section of this thesis reports the results of a questionnaire whose purpose was to compare the attitudes towards pharmacogenomic testing in a group of healthy volunteers, cardiac transplant recipients and heart failure patients. This study demonstrated that, although enthusiasm regarding pharmacogenomics is shared equally among these individuals, preoccupations related to confidentiality and potential impacts on employment and insurance are more important in healthy volunteers. In summary, the work presented in this thesis showed that early identification of heart transplant patients who are most likely to develop renal dysfunction as well as adoption of an individualized therapeutic approach involving clinical applications of pharmacogenomics could potentially help to prevent this post-transplant complication.

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.

Made available in DSpace on 2016-01-26T12:51:33Z (GMT). No. of bitstreams: 1 sueliaparecidaalves_dissert.pdf: 2026768 bytes, checksum: ebfbac0fbe0f9c10bf7c714736c82a22 (MD5) Previous issue date: 2012-01-23 / Chronic nephrotoxicity induced by cyclosporine A (CsA) is characterized by renal dysfunction and irreversible tubulointerstitial fibrosis. Associated with the use of CsA, the habit of smoking is a risk factor that reduces the glomerular filtration rate, renal vascular resistance increases, deteriorating renal function and also causes morphological changes such as glomerulosclerosis and tubulointerstitial damage. Objective: The aim of this study was to assess the effects of previous cigarette smoke exposure on CsA-induced renal functional and structural changes in a consistent experimental model of chronic CsA nephrotoxicity. Methods: Wistar male Munich-Wistar, 180-200 g were divided into four groups of 10 animals (CsA/SC, CsA/S, VH/SC and VH/S) were exposed to cigarette smoke (Smoke chamber - SC) or sham procedure (chamber without smoke cigarettes - S) for 10 minutes twice a day for 20 weeks, with three cigarettes each exposure. Received a low sodium diet from 16 th to 20 th week and at week 17 was administered to CsA (2.5 mg/kg/day subcutaneously) or vehicle (VH) for 28 days. After 24 hours the last injection, the animals were anesthetized and the following studies: renal glomerular filtration - RGF (inulin clearance), renal blood flow - RBF and renal vascular resistance - RVR (Doppler ultrasound), renal histology (% System Merz), CsA blood level (radioimunoensaio, ng / ml) and immunohistochemistry for &#945;-SMA, vimentin and nitrotyrosine. Statistical analysis: Results are presented as mean ± SD. ANOVA + Newman-Keuls test or Kruskal-Wallis test + Dunn. Statistical significance was set at P < 0.05. Results: The group that used CsA decreased in GFR (CsA/SC 0,39 ± 0,03 vs VH 0,83 ± 0,15 vs CsA/S 0,53 ± 0,05; ***p<0,001), the RBF (CsA/SC 3,9 ± 0,4 vs VH 6,7 ± 1,0 vs CsA/S 3,8 ± 0,6; p < 0,001) and increased RVR (CsA/SC 26 ± 2 vs VH 17 ± 3 vs CsA/S 27 ± 3; p < 0,001). There was also an increase of interstitial fibrosis (CsA/SC 17 ± 3 vs VH 3 ± 2 e CsA/S 21 ± 8 vs VH 3 ± 2; p<0,001), expression of tubulointerstitial &#945;-SMA (CsA/SC 1,8 ± 0,3 vs VH 0,8 ± 0,3 vs CsA/S 1,7 ± 0,4; p<0,001) and periglomerular &#945;-SMA (CsA/SC 1,0 ± 0,4 vs VH 0,3 ± 0,3, p < 0,001, CsA/S 0,5 ± 0,1 vs VH 0,3 ± 0,1; p<0,05) and expression of vimentin (CsA/SC 0,4 ± 0,4 vs VH 0,0 ± 0,0; p < 0,05 e CsA/S 0,5 ± 0,6 vs VH 0,0 ± 0,0; p < 0,01) and nitrotyrosine (CsA/SC 3 ± 0,3 vs VH 2,3 ± 0,4; p<0,01). With the use of tobacco, worsened in the fall of GFR (CsA/SC 0,4 ± 0,03 vs CsA/S 0,5 ± 0,0; *p<0,05), increased expression of &#945;-SMA periglomerular (CsA/SC 1,0 ± 0,4 vs CsA/S 0,5 ± 0,1; p<0,01) and nitrotyrosine (CsA/SC 3 ± 0,3 vs CsA/S 2 ± 0,2; p<0,05). Conclusion: In conclusion, CS aggravated significantly CsA-induced GFR impairment, periglomerular structural lesion and oxidative stress in a rat model of CsA nephrotoxicity. / A nefrotoxicidade crônica induzida pela ciclosporina A (CsA) é caracterizada por disfunção renal e fibrose tubulointersticial irreversível. Associado ao uso da CsA, o hábito de fumar é um fator de risco que reduz a taxa de filtração glomerular, aumenta a resistência vascular renal, deteriora a função renal e causa alterações morfológicas como glomeruloesclerose e dano tubulointersticial. Objetivo: Avaliar os efeitos da exposição prévia à fumaça do cigarro sobre as alterações renais funcionais e estruturais induzidas pela CsA em um modelo experimental de nefrotoxicidade crônica. Material e Método: Ratos machos, Munich-Wistar, 180-200 g, divididos em quatro grupos de 10 animais (CsA/F, CsA/S, VH/F e VH/S), foram expostos à fumaça de cigarros (câmara de Fumo - F) ou procedimento SHAM (câmara de fumo sem os cigarros - S) por 10 minutos, duas vezes/dia, durante 20 semanas, com três cigarros cada exposição. Receberam dieta hipossódica da 16ª a 20ª semana, e na 17ª semana foi administrada a CsA (2,5mg/Kg/dia; subcutânea) ou o veículo (VH), por 28 dias. Após 24 horas da última injeção, os animais foram anestesiados e os seguintes estudos realizados: filtração glomerular renal - FGR (clearance de inulina); fluxo sanguíneo renal FSR e resistência vascular renal - RVR (Ultra-som Doppler); histologia renal (% de fibrose, Sistema de Merz), nível de CsA sanguínea (radioimunoensaio, ng/ml) e imuno-histoquímica para &#945;-SMA, nitrotirosina e vimentina. Na análise estatística foi utilizado ANOVA + teste Newman-Keuls ou Kruskal-Wallis + teste Dunn. Resultados: Os grupos que utilizaram CsA apresentaram queda na FGR (CsA/F 0,39 ± 0,03 vs VH 0,83 ± 0,15 vs CsA/S 0,53 ± 0,05; ***p<0,001), no FSR (CsA/F 3,9 ± 0,4 vs VH 6,7 ± 1,0 vs CsA/S 3,8 ± 0,6; p < 0,001) e aumento da RVR (CsA/F 26 ± 2 vs VH 17 ± 3 vs CsA/S 27 ± 3; p < 0,001), aumento da fibrose intersticial (CsA/F 17 ± 3 vs VH 3 ± 2 e CsA/S 21 ± 8 vs VH 3 ± 2; p<0,001), da expressão de &#945;-SMA túbulo intersticial (CsA/F 1,8 ± 0,3 vs VH 0,8 ± 0,3 vs CsA/S 1,7 ± 0,4; p<0,001) e periglomerular (CsA/F 1,0 ± 0,4 vs VH 0,3 ± 0,3, p < 0,001, CsA/S 0,5 ± 0,1 vs VH 0,3 ± 0,1; p<0,05), e da expressão da nitrotirosina (CsA/F 3 ± 0,3 vs VH 2,3 ± 0,4; p<0,01) e vimentina (CsA/F 0,4 ± 0,4 vs VH 0,0 ± 0,0; p < 0,05 e CsA/S 0,5 ± 0,6 vs VH 0,0 ± 0,0; p < 0,01). Com a utilização do fumo, houve piora na queda da FGR (CsA/F 0,4 ± 0,03 vs CsA/S 0,5 ± 0,0; *p<0,05), aumento da expressão da &#945;-SMA periglomerular (CsA/F 1,0 ± 0,4 vs CsA/S 0,5 ± 0,1; p<0,01) e da nitrotirosina (CsA/F 3 ± 0,3 vs CsA/S 2 ± 0,2; p<0,05). Conclusões: O fumo agrava significativamente o declínio da FGR, a lesão estrutural renal e o estresse oxidativo causados pela CsA.

Foi realizado um estudo prospectivo em dez crianças (média de idade de apresentação da doença, 3.0 ± 1.6 anos) usando ciclosporina (CSA) uma microemulsão utilizada no tratamento da síndrome nefrótica idiopática (SNI) - com função renal normal avaliada pelo clearance de creatinina estimado pela estatura, que apresentavam o C0 (nível do vale) entre 50 e 150 ng/ml e que encontraram remissão completa da doença com a CSA. O objetivo foi comparar os parâmetros farmacocinéticos da CSA entre crianças com SNI em remissão ou descompensadas da doença. O perfil farmacocinético da CSA foi avaliado através da área sob a curva de 12 horas (AUC012) utilizando sete pontos. Esse procedimento foi realizado no mesmo paciente durante a remissão e a descompensação da doença com a mesma dose de CSA em mg/kg. A AUC012 foi calculada através de régua trapezoidal. Todos os pontos da curva e a AUC04 simplificada foram comparados com a AUC012. Não foi encontrada diferença significativa em quaisquer pontos da curva nos dois estados da doença, nem mesmo quando normalizados pela dose em mg/kg. Nosso estudo mostrou que a AUC04 foi o principal parâmetro em ambos os estados da doença (remissão e recidiva) quando comparado com a AUC012 (r=0.95 na remissão e r=0.93 na recidiva), e o segundo melhor parâmetro foi o C2 (r=0.86 na remissão e r=0.80 na recidiva). Mais estudos controlados são necessários para reproduzir melhor estes achados e confirmar nossa proposta. Além disso, o C2 necessita ser determinado em crianças com SNI para evitar efeitos adversos enquanto promove eficiência / We report a prospective study in ten children (mean age at presentation, 3.0 ± 1.6 years) using Cyclosporin(CSA) - a microemulsion to treat Idiopathic Nephrotic Syndrome (INS), with normal renal function evaluated by creatinine clearance estimated by stature, who presented C0 (trough level) between 50 and 150 ng/ml and achieved complete remission with CSA. The objective is to compare the pharmacokinetic parameters of CSA between nephrotic children on remission and relapse of the nephrotic state. The pharmacokinetic profile of CSA was evaluated with the 12-hour area under the time-concentration curve (AUC012) using seven time sample points. This procedure was done in the same patient during remission and relapse with the same dose of CSA (mg/kg). The AUC012 was calculated by trapezoidal rule. All points of the curve and the resumed AUC04 were compared with AUC012. We detected no significant differences at any points of the curve during remission and relapse, even when data were normalized by dose (mg/kg). Our study has shown that AUC04 was the main point on both states of the disease, remission and relapse, when compared to AUC012 (r=0.95 on remission and r=0.93 on relapse), and the second parameter found was C2 (r=0.86 on remission and r=0.80 on relapse). More controlled studies are needed to reproduce the findings and confirm this proposition. The target C2 concentration needs to be determined for INS in children to avoid adverse effects while promoting efficacy

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.

Law Wai Keung. / Thesis (M.Sc.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 98-108). / Abstracts in English and Chinese. / Abstract --- p.v / 摘要 --- p.viii / Acknowledgement --- p.x / List of Abbreviations --- p.i / Index of tables --- p.xiv / Index of figures --- p.xv / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Literature review --- p.3 / Chapter 2.1 --- Immunosuppression in Organ Transplantation --- p.3 / Chapter 2.2 --- Mechanism of Graft Rejection --- p.4 / Chapter 2.3 --- Conventional immunosuppressive drugs --- p.4 / Chapter 2.3.1 --- Corticosteriod --- p.6 / Chapter 2.3.2 --- Azathioprine --- p.6 / Chapter 2.3.3 --- Polyclonal antilymphocyte globulin and OKT3 --- p.7 / Chapter 2.4 --- Cyclosporine A (CsA) --- p.8 / Chapter 2.4.1 --- Mechanisms of CsA --- p.8 / Chapter 2.4.2 --- Pharmacokinetics of CsA --- p.10 / Chapter 2.4.2.1 --- Absorption --- p.10 / Chapter 2.4.2.2 --- Distribution --- p.11 / Chapter 2.4.2.3 --- Metabolism and elimination --- p.11 / Chapter 2.4.2.4 --- Toxicity --- p.12 / Chapter 2.4.3 --- Therapeutic drug monitoring of CsA --- p.13 / Chapter 2.4.3.1 --- CsA trough monitoring --- p.13 / Chapter 2.4.3.2 --- Full AUC monitoring --- p.15 / Chapter 2.4.3.3 --- Limited sampling strategy --- p.16 / Chapter 2.4.3.4 --- Two-hour post dose CsA level monitoring --- p.20 / Chapter 2.4.4 --- Conventional techniques of measuring cyclosporine concentration --- p.23 / Chapter 2.4.4.1 --- High performance liquid chromatography --- p.23 / Chapter 2.4.4.2 --- Non-specific immunoassays --- p.25 / Chapter 2.4.4.3 --- Specific radioimmunoassays --- p.26 / Chapter 2.4.4.4 --- Specific fluorescent polarization immunoassay --- p.26 / Chapter 2.4.4.5 --- Enzyme multiplied immunoassay technique --- p.28 / Chapter 2.4.4.6 --- Cloned enzyme donor immunoassay --- p.29 / Chapter 2.4.4.7 --- Summary for conventional techniques --- p.29 / Chapter 2.5 --- Liquid chromatography mass spectrometry for CsA measurement --- p.30 / Chapter 2.5.1 --- Main components of MS --- p.31 / Chapter 2.5.1.1 --- Specific interfaces to LC --- p.31 / Chapter 2.5.1.2 --- Mass analyzer --- p.33 / Chapter 2.5.1.3 --- Electron multiplier --- p.36 / Chapter 2.5.2 --- Sample preparation for LC-MS/MS for CsA measurement --- p.36 / Chapter 2.5.2.1 --- Liquid-liquid extraction --- p.37 / Chapter 2.5.2.2 --- Solid phase extraction --- p.38 / Chapter 2.5.2.3 --- Column switching --- p.39 / Chapter 2.5.2.4 --- Dilute and shoot --- p.40 / Chapter 2.5.3 --- LC-MS/MS for CsA measurement --- p.40 / Chapter 2.6 --- Summary --- p.42 / Chapter 3. --- Aim of study --- p.43 / Chapter 4. --- Materials and methods --- p.44 / Chapter 4.1 --- Materials --- p.44 / Chapter 4.1.1 --- Chemicals --- p.44 / Chapter 4.1.2 --- Equipment --- p.44 / Chapter 4.1.3 --- Reagent preparation for CsA analysis --- p.45 / Chapter 4.2 --- Methods --- p.48 / Chapter 4.2.1 --- Immunoassay --- p.48 / Chapter 4.2.2 --- Operation of tandem mass spectrometer --- p.48 / Chapter 4.2.2.1 --- Optimization of cone voltage --- p.50 / Chapter 4.2.2.2 --- Optimization of collision energy --- p.50 / Chapter 4.2.3 --- Optimization of LC-MS/MS --- p.51 / Chapter 4.2.3.1 --- Deproteinization procedures of whole blood --- p.52 / Chapter 4.2.3.2 --- Optimization of mobile phase flow rate --- p.52 / Chapter 4.2.3.3 --- Optimization of source temperature --- p.53 / Chapter 4.2.3.4 --- Optimization of the drying gas flow rate --- p.53 / Chapter 4.2.4 --- Matrix interference on MS/MS response --- p.53 / Chapter 4.2.5 --- Analytical performance of CsA on LC-MS/MS --- p.54 / Chapter 4.2.5.1 --- Linearity study --- p.54 / Chapter 4.2.5.2 --- Precision performance --- p.54 / Chapter 4.2.5.3 --- Accuracy performance --- p.54 / Chapter 4.2.5.4 --- The lowest detection limit of the CsA analysis --- p.55 / Chapter 4.2.5.5 --- Correlation study of the CsA analysis --- p.55 / Chapter 4.3 --- CsA pharmacokinetic studies in Chinese patients --- p.56 / Chapter 4.3.1 --- Determining the time point of CsA correlating better with AUC --- p.56 / Chapter 4.3.1.1 --- Patient and method --- p.56 / Chapter 4.3.1.2 --- Statistical analysis --- p.57 / Chapter 4.3.2 --- "Intra-individual variability of CO, C1 and C2" --- p.57 / Chapter 4.3.2.1 --- Patient and method --- p.57 / Chapter 4.3.2.2 --- Statistical analysis --- p.57 / Chapter 5. --- Results and discussion --- p.59 / Chapter 5.1 --- Optimization of MS parameters --- p.5 9 / Chapter 5.1.1 --- Optimization of cone voltage --- p.61 / Chapter 5.1.2 --- Optimization of collision energy --- p.63 / Chapter 5.2 --- Optimization of LC-MS/MS --- p.63 / Chapter 5.2.1 --- Optimization of mobile phase flow rate --- p.63 / Chapter 5.2.2 --- Optimization of ion source temperature and drying gas flow rate --- p.67 / Chapter 5.3 --- Matrix interference on MS/MS response --- p.69 / Chapter 5.4 --- Analytical performances of CsA on LC-MS/MS method --- p.71 / Chapter 5.4.1 --- Linearity --- p.71 / Chapter 5.4.2 --- Precision performance --- p.71 / Chapter 5.4.3 --- Accuracy performance --- p.72 / Chapter 5.4.4 --- The lowest limit of detection --- p.73 / Chapter 5.4.5 --- Correlation study of the CsA analysis --- p.80 / Chapter 5.5 --- The correlation between CsA at different point and AUCo-6 --- p.84 / Chapter 5.6 --- "Intra-individual variability of CO, C1 and C2" --- p.88 / Chapter 5.7 --- Therapeutic ranges of C2 --- p.90 / Chapter 5.8 --- Practical consideration for C2 measurement by LC-MS/MS method --- p.94 / Chapter 6. --- Conclusions --- p.97 / References --- p.98

Foi realizado um estudo prospectivo em dez crianças (média de idade de apresentação da doença, 3.0 ± 1.6 anos) usando ciclosporina (CSA) uma microemulsão utilizada no tratamento da síndrome nefrótica idiopática (SNI) - com função renal normal avaliada pelo clearance de creatinina estimado pela estatura, que apresentavam o C0 (nível do vale) entre 50 e 150 ng/ml e que encontraram remissão completa da doença com a CSA. O objetivo foi comparar os parâmetros farmacocinéticos da CSA entre crianças com SNI em remissão ou descompensadas da doença. O perfil farmacocinético da CSA foi avaliado através da área sob a curva de 12 horas (AUC012) utilizando sete pontos. Esse procedimento foi realizado no mesmo paciente durante a remissão e a descompensação da doença com a mesma dose de CSA em mg/kg. A AUC012 foi calculada através de régua trapezoidal. Todos os pontos da curva e a AUC04 simplificada foram comparados com a AUC012. Não foi encontrada diferença significativa em quaisquer pontos da curva nos dois estados da doença, nem mesmo quando normalizados pela dose em mg/kg. Nosso estudo mostrou que a AUC04 foi o principal parâmetro em ambos os estados da doença (remissão e recidiva) quando comparado com a AUC012 (r=0.95 na remissão e r=0.93 na recidiva), e o segundo melhor parâmetro foi o C2 (r=0.86 na remissão e r=0.80 na recidiva). Mais estudos controlados são necessários para reproduzir melhor estes achados e confirmar nossa proposta. Além disso, o C2 necessita ser determinado em crianças com SNI para evitar efeitos adversos enquanto promove eficiência / We report a prospective study in ten children (mean age at presentation, 3.0 ± 1.6 years) using Cyclosporin(CSA) - a microemulsion to treat Idiopathic Nephrotic Syndrome (INS), with normal renal function evaluated by creatinine clearance estimated by stature, who presented C0 (trough level) between 50 and 150 ng/ml and achieved complete remission with CSA. The objective is to compare the pharmacokinetic parameters of CSA between nephrotic children on remission and relapse of the nephrotic state. The pharmacokinetic profile of CSA was evaluated with the 12-hour area under the time-concentration curve (AUC012) using seven time sample points. This procedure was done in the same patient during remission and relapse with the same dose of CSA (mg/kg). The AUC012 was calculated by trapezoidal rule. All points of the curve and the resumed AUC04 were compared with AUC012. We detected no significant differences at any points of the curve during remission and relapse, even when data were normalized by dose (mg/kg). Our study has shown that AUC04 was the main point on both states of the disease, remission and relapse, when compared to AUC012 (r=0.95 on remission and r=0.93 on relapse), and the second parameter found was C2 (r=0.86 on remission and r=0.80 on relapse). More controlled studies are needed to reproduce the findings and confirm this proposition. The target C2 concentration needs to be determined for INS in children to avoid adverse effects while promoting efficacy

Το CD40L είναι ένα συνδιεγερτικό μόριο και αποτελεί πρώιμο δείκτη ενεργοποίησης του Τ λεμφοκυττάρου. Υπάρχουν δεδομένα που υποστηρίζουν την υπόθεση ότι τα ενεργοποιημένα Τ λεμφοκύτταρα πιθανώς παίζουν ρόλο στην παθογένεια της ψωριασικής αρθρίτιδας (ΨΑ). Μελετήσαμε συνολικά 12 ασθενείς με ΨΑ, 6 ασθενείς με ρευματοειδή αρθρίτιδα (ΡΑ) και 4 υγιείς εθελοντές. Υπολογίσαμε την έκφραση του CD40L στην επιφάνεια των Τ λεμφοκυττάρων με κυτταρομετρία ροής σε κατάσταση ηρεμίας και μετά από διέγερση με ΡΜΑ/ιονομυκίνη. Επίσης μελετήσαμε την ανασταλτική δράση της κυκλοσπορίνης στην επαγόμενη έκφραση του CD40L. Η έκφραση του CD40L ήταν σημαντικά αυξημένη στην επιφάνεια των Τ λεμφοκυττάρων των ασθενών με ΨΑ, ειδικότερα αυτών με ενεργό νόσο, σε σύγκριση με τους υγιείς εθελοντές και τους ασθενείς με ΡΑ (μέσο ποσοστό των CD3+CD40L+ κυττάρων: 23.74%, 11.59% και 9.57% για τους ασθενείς με ενεργό ΨΑ, ασθενείς με ΡΑ και υγιείς εθελοντές αντίστοιχα). Η αναστολή από την κυκλοσπορίνη της επαγόμενης έκφρασης του CD40L ήταν εξίσου αποτελεσματική και στις 3 ομάδες μελέτης. Συμπερασματικά αναφέρουμε ότι το CD40L υπερεκφράζεται στη επιφάνεια των Τ λεμφοκυττάρων ασθενών με ενεργό ΨΑ μετά από in vitro διέγερση. Το γεγονός αυτό ενισχύει την άποψη ότι ο άξονας CD40- CD40L παίζει σημαντικό ρόλο στη παθογένεια της ΨΑ και κατά συνέπεια θεραπείες που να στοχεύουν εκλεκτικά αυτόν τον άξονα θα μπορούσαν να δοκιμαστούν στην ΨΑ. / CD40L is a costimulatory molecule and an early activation marker of T lymphocytes. Evidence supports the hypothesis that activated T cells may play a role in the pathogenesis of psoriatic arthritis (PsA). We examined the levels of CD40L expression on resting T cells from 12 patients with PsA, 6 patients with rheumatoid arthritis (RA) and 4 healthy volunteers, and following stimulation with phorbol myristate acetate (PMA)/ionomycin. The inhibitory effect of cyclosporine A (CsA) on the induced expression of CD40L was also evaluated. This expression was significantly increased on the cell surface of T cells from patients with PsA, particularly those with active disease, when compared to normal individuals and patients with RA (mean percentages of CD3+ CD40L+ cells: 23.74%, 11.59% and 9.57% for patients with active PsA, patients with RA and healthy volunteers, respectively). CsA-mediated inhibition of CD40L induction was equally effective in all study groups. In conclusion, we report herein that CD40L is overexpressed in patients with active PsA. This may indicate that CD40L with its counter-receptor may be crucially involved in the pathogenesis of PsA. Consequently, therapies specifically targeting this pair may be worth testing.

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