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Kidney transplantation is the best treatment for patients with end-stage renal failure. The main problem of kidney transplantation is however the development of a cellular and antibody-mediated (humoral) rejection. During the last decade, thanks to the advanced immunosuppression, prognosis of survival and function of transplanted organs has significantly improved. Nevertheless, humoral rejection remains very serious obstacle in high-risk patients, because it can permanently damage the graft. Therefore, before transplantation it is necessary to stratify patients into high and low risk groups for development of antibody-mediated rejection. Current immunogenetic tests performed before transplantation include, in addition to HLA typing, detection of panel-reactive antibodies. However, this test does not provide information about B cells which participate in the humoral response of the kidney recipient. Therefore, in the presented thesis we studied B cell reactivity and its regulation in transplanted patients. In this retrospective analysis we measured levels of the B cell activating factor, a cytokine regulating the function of B lymphocytes (BAFF). Current reports suggest that BAFF could serve as a marker of humoral rejection. Furthermore, we focused on B lymphocytes and their capacity to produce...

Le virus de l‘hépatite E (VHE) est endémique mondialement. Dans les pays en voie de développement, le VHE est une cause majeure d'hépatite aiguë et l'hépatite E est une maladie du péril fécal, transmise par la consommation d'eau contaminée. Le taux de mortalité de l'hépatite E aiguë en situation d'épidémie est estimé entre 0,2 et 4%. Dans les pays développés, l'hépatite E d'origine autochtone a émergé au début du XXIème siècle et un réservoir porcin à l'origine de transmissions zoonotiques du VHE a été établi. En 2008, pour la première fois, des formes chroniques de l'hépatite E ont été décrites révélant que le VHE était également une cause d'hépatite chronique et de cirrhose. Ces nouvelles formes d'hépatite E ont été rapportées chez des patients immunodéprimés pour transplantation d'organes solides, par le virus de l'immunodéficience humaine ou par des hémopathies malignes ou les traitements de ces hémopathies. Les travaux de cette Thèse ont eu pour objectifs de décrire les aspects cliniques puis épidémiologiques de l'hépatite E, d'étudier certains aspects immunologiques de l'hôte et certains aspects virologiques des VHE dans le but de comprendre les mécanismes conduisant au développement d'une hépatite E chronique dans la population des patients transplantés d'un rein suivis au CHU de Marseille. Nous avons décrit dans une étude rétrospective les caractéristiques et l'histoire naturelle de 16 infections autochtones diagnostiquées devant une hépatite inexpliquée. Nous avons décrit une évolution majoritairement chronique de l'hépatite E et son potentiel cirrhogène. / Hepatitis E virus (HEV) is endemic worldwide. In developing countries, HEV is a major cause of acute hepatitis and hepatitis E is a disease transmitted by the faecal-oral route through contaminated water. The estimated mortality rate of acute hepatitis E in the setting of outbreaks ranges from 0.2 to 4%. In developed countries, hepatitis E of autochthonous origin emerged at the beginning of twenty-first century and a porcine reservoir for HEV has been established that is a source for zoonotic transmission. In 2008, for the first time, chronic forms of hepatitis E have been reported showing that HEV was also a cause of chronic hepatitis and cirrhosis. These new forms of hepatitis E have been reported in immunocompromised patients due to solid organ transplantation, infection by human immunodeficiency virus, hematological malignancies or treatment of these malignancies. The objectives of this Thesis have been to describe the clinical and epidemiological features of hepatitis E, to examine some immunological aspects of the host and some virological aspects of HEV in order to understand the mechanisms leading to the development of chronic hepatitis E in the population of kidney transplant recipients followed at the University Hospital of Marseille. In a retrospective study, we described the characteristics and natural history of 16 HEV infections diagnosed in patients presenting with unexplained hepatitis. We described that hepatitis E progressed the most frequently towards chronicity and possibly towards liver cirrhosis. We showed that after dose reduction of immunosuppressants, more than half of the chronic infections resolved.

Title: Functional disorders of the musculoskeletal system on individuals after kindey transplantation Defining of the problem: Renal transplantation is a method to treat patients with renal failure,which is associated to a prolonged survival of patients compared to a dialysis method (Viklický et al., 2008). After a successful renal transplantation the renal function is restored and complications caused by dialysis treatment disappear . However, complications due to the transplant, not only in terms of surgical intervention, but also from the point of a lifelong immunosuppressive therapy remain at risk. Functional changes on the locomotor system can occur as a concequence of a long-term renal disease or kidney transplant. This could further reduce the quality of life on patients due to self-sufficiency, being the main role of the locomotor system. Objectives: The aim of this study is to valuate functional disorders of the musculoskeletal system on individuals after kindey transplantation up to one year after a transplant. Methods: This thesis is elaborated in the form of an analytical - experimental study in which 2 groups of selected individuals were tested. Testing was conducted by using suitable tests focused on functional disorders of the locomotor system and parameters in each group and time...

INTRODUÇÃO E OBJETIVO: A disfunção erétil (DE) em pacientes transplantados renais é frequente. A modalidade de tratamento ideal não deve interferir com a função do enxerto. A terapia de onda de choque extracorpórea de baixa intensidade (TOCE) tem atraído interesse devido às suas propriedades angiogênicas e mostrou resultados interessantes quando usada para tratar pacientes com doença cardiovascular e em homens com DE. Nosso objetivo é estudar a eficácia e segurança da TOCE para o tratamento da DE de provável etiologia vascular em homens transplantados renais. MÉTODOS: Foram selecionados 20 homens (média de idade = 53,7 anos, 46 a 61 anos) que foram submetidos a transplante de rim há pelo menos 6 meses e com DE há pelo menos 6 meses. O estudo foi duplo-cego, realizado em um único centro, prospectivo, randomizado e placebo-controlado. Dez pacientes foram randomizados para o grupo de terapia placebo e 10 pacientes para o grupo TOCE .O protocolo da TOCE consistiu em 2 sessões de tratamento por semana durante 3 semanas. O tratamento placebo foi realizado utilizando o mesmo dispositivo, porém com substituição do probe eficaz por um sem emissão de energia, mas que proporcionava um som e uma sensação de pulso durante o tratamento. A avaliação pré-tratamento e do seguimento foi realizada com o Índice Internacional de Função Erétil-5 (IIEF-5) e Escore de Rigidez Peniana (EHS) após 1, 3 e 12 meses. Ultra-sonografia Doppler do pênis com fármaco ereção foi realizada antes da terapia e após o tratamento (3 meses). RESULTADOS: Não houve diferença entre estes dois grupos no escore basal do IIEF-5 e EHS. No início do estudo, 1 e 3 meses após o último tratamento, o IIEF-5 no grupo TOCE foi de 10,9 ± 5,1, 15,6 ± 6,1 e 17,2 ± 5,7, respectivamente. O IIEF-5 no grupo placebo foi de 14,9 ± 3, 16,6 ± 5,4 e 16,5 ± 5, respectivamente. O comportamento entre os 2 grupos ao longo do tempo foi significativamente estatistico (p=0,0177). A melhora do escore IIEF-5 foi superior a 5 pontos em 70% (variou de 1-14) dos pacientes no grupo TOCE e em 10% (variou de 0-10) dos pacientes no grupo placebo, 3 meses após o tratamento. A análise após 12 meses foi realizada exclusivamente no grupo TOCE. A alteração média no IIEF-5 após 12 meses foi de 4,8 no grupo TOCE. A média do IIEF-5 após 12 meses foi de 15,7 ± 6,45, mostrando estabilidade da melhora inicial. No início do estudo, 1 e 3 meses após o último tratamento, o EHS no grupo TOCE foi de 2 ± 1,05, 2,5 ± 0,85 e 2,6 ± 0,84, respectivamente. O EHS no grupo placedo foi de 2 ± 0,67, 2,4 ± 0,7 e 2,4 ± 0,7, respectivamente. Percebemos um comportamento semelhante nos dois grupos (p=0,724). No grupo TOCE, notamos um EHS médio um pouco maior após 3 meses. Os parâmetros da ultra-sonografia doppler peniana não diferiram entre os grupos e não apresentaram melhoras significativas. CONCLUSÕES: A TOCE é um tratamento não farmacológico com eficácia clínica em homens transplantados renais com DE. Além do seu potencial de reabilitação, é viável e eficaz. Não verificamos impacto nos parâmetros do doppler peniano apesar do efeito da neoangiogênese do método / INTRODUCTION AND OBJECTIVE: Erectile dysfunction (ED) in kidney transplant patients is frequent. The ideal treatment modality should not interfere with the graft function. Low-intensity Extracorporeal Shock Wave Therapy Extracorporeal (Li-ESWT) has been of interest due to its angiogenic properties and has shown interesting results when used to treat patients with cardiovascular disease and non-transplanted men with ED. Our objective is to study the efficacy of Li-ESWT for the treatment of ED of probable vascular etiology in kidney transplanted men. METHODS: Twenty men (mean age = 53.7 years, range 46 to 61 years) that have been submitted to kidney transplant for at least 6 months and have been suffering from ED for at least 6 months were selected for the treatment. This was a double-blinded, single-center, prospective, randomized, placebo-controlled trial. Ten patients were randomized into the placebo therapy arm and 10 patients into the Li-ESWT arm. The Li-ESWT protocol consisted of a 2 treatment sessions per week for 3 weeks. The placebo treatment was performed using the same device replacing the effective probe for one that emits zero energy, but delivers a sound and pulse sensation during treatment. Initial and follow-up assessment was performed with International Index of Erectile Function Questionnaire-5 (IIEF-5) score and Erection Hardness Score (EHS) after 1, 3 and 12 months. Penile ultrasound Doppler with drug erection was performed before therapy and after treatment (3 months). RESULTS: There was no difference between these 2 groups in baseline IIEF-5 score and EHS. At baseline, 1 and 3 months after the last treatment, the IIEF-5 in the Li-ESWT group were 10.9 ± 5.1, 15.6 ± 6.1 and 17.2 ± 5.7, respectively. The IIEF-5 in the sham therapy group were 14.9 ± 3, 16.6 ± 5.4 and 16.5 ± 5, respectively. The interaction was significant (p = 0.0177), indicating significantly different behaviors between the groups over time. The IIEF-5 score improved to higher than 5 points in 70% (range, 1-14) of the patients in the Li-ESWT group and 10% (range, 0-10) of the patients in the placebo treatment group, 3 months after treatment. The analysis after 12 months was performed exclusively in the Li-ESWT group. The mean change in IIEF-5 after 12 months was 4.8 in the Li-ESWT group. The mean of the IIEF-5 score after 12 months was 15.7 ± 6.45, showing stability of the initial improvement. At baseline, 1 and 3 months after the last treatment, the EHSs in the Li-ESWT group were 2±1.05, 2.5±0.85, and 2.6±0.84, respectively. The EHSs in the sham treatment group were 2±0.67, 2.4±0.7, and 2.4±0.7, respectively. We noticed a similar behavior in both groups (P=0,724). In the Li-ESWT group, we noticed a slightly higher mean EHS after 3 months. Penile Doppler ultrasound parameters did not differ between groups and did not show significant improvements. CONCLUSIONS: Li-ESWT is a non pharmacological treatment with clinical efficacy in kidney transplant recipients with ED. Besides its restorative potential, this treatment is feasible and effective. Despite evidences suggesting neoagiogenesis, our protocol had no impact in penile Doppler parameters

Introdução: Os imunossupressores das classes inibidores da calcineurina (tacrolimo) e da rapamicina (sirolimo) requerem controle terapêutico por apresentarem grande variabilidade farmacocinética que tem sido atribuída a fatores genéticos, entre outros. Poucos estudos avaliaram a expressão de genes alvo de imunossupressores e sua relação com a resposta terapêutica. Objetivo: Estudar a relação entre o perfil de expressão de genes alvos de tacrolimo e sirolimo e de microRNAs reguladores e a resposta à imunossupressores utilizados na profilaxia de rejeição ao transplante renal. Métodos: Participaram deste estudo 37 indivíduos submetidos ao transplante renal, no Hospital do Rim e Hipertensão da UNIFESP, de ambos os sexos, com idade acima de 18 anos e de qualquer etnia. Os pacientes foram tratados com esquema imunossupressor contendo tacrolimo, micofenolato de sódio e prednisona até o 3º mês quando foram randomizados para manter a terapia inicial (grupo TAC) ou para a conversão para sirolimo (grupo SRL). Os pacientes com disfunção renal ou suspeita de rejeição, no 3º mês, seguiram o tratamento inicial e foram avaliados em separado (grupo TACex). Os parâmetros de função renal e concentração sanguínea dos fármacos foram utilizados para monitoramento da terapia. A expressão de mRNA de MTOR, PPP3CA, PPP3CB, FKBP1A, FKBP1B e FKBP5, em leucócitos do sangue periférico, foi analisada por PCR em tempo real; e a expressão de miR-99a, miR-100, miR-145, miR-30a, miR-10b e miR-103a foi avaliada por PCR Array. Resultados: No primeiro mês de tratamento, a expressão diferencial de mRNA de MTOR, PPP3CA e FKBP1B diminuiu em relação ao pré-transplante (pre-Tx) (p<0,05). Esse efeito se manteve, no 3º mês, para MTOR e PPP3CA (p<0,05). A expressão diferencial de PPP3CB, FKBP1A e FKBP5 não foi alterada pelos tratamentos (p>0,05). No 6º mês, os grupos TAC, TACex e SRL apresentaram perfil de expressão diferencial de mRNA similar à do pre-Tx (p>0,05). No 3º mês, foram encontradas correlações positivas entre a expressão relativa de PPP3CB e creatinina sérica (r=0,49, p=0,04), e entre a expressão de FKBP1A e ureia (r=0,49, p=0,04), HDL colesterol (r= 0,53; p=0,02) e triglicérides (r=0,68, p=0,003) no soro. O perfil de expressão relativa de mRNA não se correlacionou com a concentração sanguínea de tacrolimo (p>0,05). Houve redução na expressão diferencial de miR-99a, no 3º mês, comparado com o pre-Tx (p<0,05) e a dos outros miRNAs não foi alterada. Não foi observada correlação entre o perfil de expressão relativo de miRNAs e mRNAs alvo, no 3º mês (p>0,05). Conclusão: A expressão diferencial de mRNA de MTOR, PPP3CA e FKBP1B e de miR-99a que tem como alvo o mRNA de MTOR, em leucócitos do sangue periférico, é modulada pelo tratamento imunossupressor a base de tacrolimo. A expressão diferencial de genes da via da calcineurina e do mTOR é sugestiva de sua potencial aplicação no monitoramento da terapia a base de tacrolimo. / Background: Immunosuppressive classes like calcineurin inhibitors (tacrolimus) and rapamycin (sirolimus) require therapeutic control because they have great pharmacokinetic variability that has been attributed to genetic factors, among others. Few studies have evaluated the expression of immunosuppressive target genes and their relation to therapeutic response. Objective: To study the relationship between the gene expression profile of tacrolimus and sirolimus targets and regulatory microRNAs with the response to immunosuppressive agents used in the prophylaxis of renal transplant rejection. Methods: This study included 37 patients undergoing kidney transplantation at the Hospital do Rim e Hipertensao/UNIFESP, age over 18 year old, both gender and any ethnicity. Patients were treated with an immunosuppressive regimen containing tacrolimus, mycophenolate sodium and prednisone during 3 months, when they were randomized to maintain the initial therapy (TAC group) or to convert to sirolimus (SRL group). Patients with renal dysfunction or signals of rejection in the 3rd month followed the initial treatment and were evaluated separately (TACex group). Parameters of renal function and blood concentration of immunosuppressive drugs were used to monitor therapy. The mRNA expression of MTOR, PPP3CA, PPP3CB, FKBP1A, FKBP1B and FKBP5 in peripheral blood leukocytes was analyzed by real-time PCR; and the expression of miR-99a, miR-100, miR-145, miR-30a, miR-10b and miR-103a was evaluated by PCR array. Results: In the first month of treatment, the differential mRNA expression of MTOR, PPP3CA and FKBP1B decreased relative to pre-transplant (pre-Tx) (p <0.05). This effect was maintained up to 3 month to MTOR and PPP3CA (p <0.05). The differential expression of PPP3CB, FKBP1A and FKBP5 was not affected by treatments (p> 0.05). On the 6th month, the TAC groups, TACex and SRL showed differential expression profile of mRNA similar to the pre-Tx (p> 0.05). On the 3rd month, positive correlations were found between the relative expression PPP3CB and serum creatinine (r =0.49, p =0.04) and between the expression of FKBP1A and urea (r=0.49, p=0.04), HDL cholesterol (r=0.53; p=0.02) and triglycerides (r = 0.68, p = 0.003) in serum. The relative mRNA expression profile was not correlated with tacrolimus blood concentrations (p> 0.05). There was a reduction in the differential expression of miR-99a, on the 3rd month, compared to the pre-Tx (p <0.05) and the other miRNAs has not changed. There was no correlation between the expression profile of miRNAs and mRNAs on target, on the 3rd month (p> 0.05). Conclusions: The differential expression of mRNA of MTOR, PPP3CA and FKBP1B and miR-99a which targets MTOR mRNA in peripheral blood leukocytes, is modulated by the immunosuppressant tacrolimus treatment base. The differential expression of genes of the calcineurin and mTOR is suggestive of their potential application in monitoring therapy tacrolimus base.

In dieser Arbeit sollen die Vermittelbarkeit der notwendigen methodischen Fähigkeiten zur Anwendung der EbM in der klinischen Entscheidungsfindung sowie die Qualität der dazu unverzichtbaren Publikationen klinischer Studien für das Gebiet der Nierentransplantation untersucht werden. Des Weiteren sollen angesichts der für viele Fragen nicht vorhandenen Studienergebnisse alternative Verfahren der Gewinnung von klinisch entscheidungsrelevanten Erkenntnissen in der Nierentransplantationsmedizin untersucht werden. Liegen in einer Entscheidungssituation relevante Ergebnisse qualitativ adäquater randomisierter Studien vor, sollte die Entscheidung auf diese Ergebnisse gestützt werden. Die klinische Entscheidungsfindung kann auf verschiedenen Wegen unterstützt werden. Der Königsweg ist die auf Erkenntnissen der Grundlagenforschung fußende randomisierte Studie. Deren Nutzung erfordert jedoch eine leitliniengemäße Publikation und auf Seiten des Lesers entsprechendes Methodenwissen zur Interpretation der Ergebnisse. Komplementäre Methoden, insbesondere Analysen klinischer Datenbanken können die aus randomisierten Studien stammenden Entscheidungsgrundlagen sinnvoll ergänzen und die Durchführung weiterer randomisierter Studien vorbereiten. Auf diesem Hintergrund erscheint es auch für den Bereich der Nierentransplantation sinnvoll, die bestehende Datenbasis konsequent weiter auszubauen. / This work aims to investigate the communicability of the necessary skills for the application of Evidence-based Medicine in everyday clinical decision making. In addition the publication quality and design of clinical trials in the field of kidney transplantation are observed. In this context and on the background of the lack of clinically relevant study data for many important aspects of transplantation medicine alternative methods of knowledge generation to support clinical decision making are assessed. Whenever results of adequate prospective randomized trials are available clinical decision making should take these into account. But clinical decision making can be supported in various ways. The best way is the conduction of randomized clinical trials based on the results of basic science investigations. Use of randomized trial results requires their guideline-conforming publication and on the reader’s side the corresponding methodological skills for the critical interpretation and assessment of the scientific medical literature. Complementary methods - especially analyses of clinical databases can achieve substantial enhancement of the foundation for clinical decision making. They can also help to prepare the conduct of further randomized studies. Thus, as in other fields it seems worthwile to expand the existing databases in the field of kidney transplantation.

O transplante renal é uma modalidade de tratamento para substituir os órgãos em falência. As manifestações orais mais freqüentes nos pacientes portadores de insuficiência renal crônica (IRC) e transplantados renais (TR) são as alterações do esmalte, as estomatites, gengivites, diminuição do fluxo salivar, hiperplasias gengivais e as infecções por vírus, bactérias e fungos. A prevalência de infecções ativas causadas pelos herpesvírus aumentam quando há imunossupressão dos indivíduos. Em transplantados renais, apesar de ser realizada a profilaxia antiviral, a infecção clínica pelos vírus desta família tem levado a muitas complicações póstransplante aumentando a mortalidade e a morbidade destes pacientes. O objetivo deste estudo foi avaliar comparativamente as condições bucais e a presença dos vírus HSV-1 e EBV na saliva de pacientes pediátricos com IRC, TR e em pacientes normorreativos, através da técnica de PCR. Aplicou-se questionário a todos os pais ou responsáveis para obtenção dos dados demográficos, histórias médica e odontológica. Realizou-se avaliação da saúde bucal e coleta de saliva de 100 pacientes pediátricos de 0 à 16 anos, divididos em 3 grupos. O grupo 1 foi composto por 25 crianças insuficientes renais crônicos, o grupo 2 por 25 crianças transplantadas renais na Santa Casa de Misericórdia de São Paulo, e o grupo 3 por 50 crianças normorreativas na Clínica Odontológica da FOUSP. Independentemente do grupo, a maioria das crianças exibia pobre higiene oral, gengivite e moderada incidência de cárie. Entre as manifestações bucais avaliadas as que apresentaram diferença entre os grupos foram: No grupo 1 palidez da mucosa bucal (8/25, 32%), alteração de esmalte (5/25, 20%), manchas brancas (4/25, 16%), e sensação de boca seca (3/25, 12%). No grupo 2 foram: candidíase (1/25, 4%), queilite angular (1/25, 4%), e dentes conóides (1/25, 4%). A hiperplasia gengival medicamentosa esteve presente em 4 dos 25 (16%) dos pacientes desse grupo estudado, sendo em todos os casos grau 1 . No grupo 3 observou-se alteração de esmalte (6/50, 12%), manchas brancas (8/50, 16%) e fratura dental (1/50, 2%). Quanto a presença dos vírus observou-se que o HSV-1 foi encontrado na freqüência de: 4% no G1, 28% no G2 e 22% no G3 sendo essa diferença estatisticamente significante (p=0,039). O EBV foi encontrado em 24% dos pacientes do G1, 60% do G2 e em 44% dos pacientes do G3, sendo a diferença entre G1 e G3 estatisticamente significante (p=0,010). Frente as manifestações bucais e a presença do HSV-1 e do EBV encontradas nesta população evidencia-se a o importante papel do cirurgião dentista na equipe transplantadora, participando de novos métodos de diagnóstico, adequação bucal dos pacientes na fase pré transplante, como também nas intervenções curativas nas fases pré, trans e pós transplante. / Renal transplantation may be a treatment choice for patients with a failing kidney. Patients who present chronic renal failure and have undergone renal transplantation may show enamel abnormalities, periodontal disease, decrease of the salivary flow and gingival enlargement as common oral manifestations. Bacterial, fungal and viral infections can also develop, being the later a major cause of post-transplantation morbidity and mortality despite the antiviral prophylaxis. Thus, the purpose of this study was to observe the occurrence of oral manifestations and also to detect the presence of herpes virus 1 (HSV-1) and Epstein-Barr virus (EBV) in the saliva of pediatric renal transplant patients (RT), pediatric patients with chronic renal failure (CRF) and immunocompetent patients, comparing the results among the three experimental groups. All the analyses were performed through the polymerase chain reaction (PCR). A questionnaire was applied to the respective parents or legal responsible in order to obtain the demographic data, medical and dental histories. Oral manifestation records and saliva samples were obtained from 100 pediatric patients with an age that ranged from 0 to 16 years old. The experimental groups consisted of: Group 1 (G1): 25 children with chronic renal failure, group 2 (G2): 25 children after renal transplantation conducted at Santa Casa de Misericórdia of São Paulo city and group 3 (G3): 50 immunocompetent children clinically examined at the School of Dentistry, University of São Paulo. All of the analyzed children presented poor hygienic conditions, periodontal diseases and decreased rates of dental caries. G1 presented the following oral manifestations: paleness of oral mucosa (8/25, 32%), enamel abnormalities (5/25, 20%) and xerostomia (3/25, 12%). G2: oral candidiasis (1/25, 4%), angular cheilitis (1/25, 4%) and conic teeth (1/25, 4%). Four patients also presented gingival enlargement at degree one (16%). G3 presented enamel abnormalities (6/50, 12%) and dental fracture (1/50, 2%). HSV-1 was detected in G1 (4%), G2 (28%) and G3 (22%) and presenting statistically significant differences among the groups (p=0,039). EBV was detected in 24% of G1, 60% of G2 and 44% of G3, being this difference statistically significant (p=0,010). Oral manifestations as well as the presence of HSV-1 and EBV in CRF e RT patients highlight the importance of a dentist in the renal transplant team, providing optimal dental care of patients before and after the renal transplant.

Introdução: Testes de histocompatibilidade são indispensáveis para viabilizar o transplante renal. A prova cruzada por citotoxicidade dependente de complemento (CDC) tem sido a técnica padrão para avaliar risco imunológico pré-transplante, no entanto, a prova cruzada por citometria de fluxo (FCXM) possui benefícios adicionais, como maior sensibilidade e análise semi-quantitativa através do Median Channel Shift (MCS). Objetivo: Definir pontos de corte de MCS baseado em correlação inter-técnicas e desfechos clínicos pós-transplante. Método: Estudo retrospectivo com pacientes candidatos a transplante renal no Hospital de Clínicas de Porto Alegre, entre janeiro/2016-agosto/2017. Foram avaliadas 1705 provas cruzadas e 221 pacientes submetidos ao transplante. Resultados: A FCXM, relacionada ao CDC, apresentou sensibilidade=87%(FCXM-T) e 90%(FCXM-B), e VPN=98% para ambos. FCXM-B apresentou especificidade=43%, relacionada aos casos CDC-/FCXMB+. FCXM-T e -B detectaram 53% e 76% dos casos de DSA≥5001 (Donor Specific Antibody). MCS apresentou desempenho satisfatório em detectar CDC+ (AUC/IC): MCST=0,909(0,886-0,933) e MCSB=0,775(0,724-0,826). Pontos de corte de MCST=245 e MCSB=282 apresentaram melhor predição de CDC+. Não houve diferença na função do enxerto de pacientes transplantados com FCXM+. Apenas 30% das FCXM+ estiveram diretamente relacionadas com DSA pré-tx. No entanto, episódios de rejeição foram mais frequentes no grupo FCXM+vs.FCXM- (95%vs.86%, p=0,04). Conclusão: É possível calibrar o MCS baseado no CDC+, no entanto, significa um risco em termos da não detecção de anticorpos de baixo título. A FCXM+, em curto prazo, não deve ser por si só um fator impeditivo para o transplante. A análise conjunta do MCS e DSA parece ser uma boa ferramenta de seleção dos receptores renais. / Introduction: Histocompatibility tests are indispensable for enable the renal transplantation. Crossmatching tests for complement dependent cytotoxicity (CDC) has been a standard technique for assess pre-transplant immunological risk, however, the flow cytometry crossmatching test (FCXM) has additional benefits, such as increased sensitivity and semi-quantitative analysis through the Median Channel Shift (MCS). Objective: Define MCS cutoff values based on inter-technical correlation and post-transplant clinical outcomes. Methods: A retrospective study with renal transplant candidates at the Hospital de Clínicas of Porto Alegre, between January/2016-August/2017. A total of 1705 crossmatching and 221 patients submitted to transplantation were evaluated. Results: The FCXM, related to CDC, resulted in sensitivity=87% (FCXM-T) and 90% (FCXM-B), and NPV=98%, for both. FCXM-B resulted in specificity=43%, related to cases CDC-/FCXMB+. FCXM-T and -B detected 53% and 76% of cases of DSA≥5001 (Donor Specific Antibody). The MCS showed satisfactory performance in detecting CDC + (AUC/IC): MCST=0.909(0.886-0.933) and MCSB=0.775(0.724-0.826). Cutoff values of MCST=245 and MCSB=282 showed better prediction of CDC+. There was no difference in the graft function of patients transplanted with FCXM+. Only 30% of FCXM + were directly related to pre-tx DSA. However, rejection episodes were more frequent in the group FCXM+vs.FCXM- (95%vs.86%, p=0,04). Conclusion: it is possible to calibrate MCS based on CDC +, however, that means a risk in terms as to the non-detection of low-titre antibodies. The FCXM+, in the short term, should not be by itself an impediment to transplantation. Joint analysis of MCS and DSA seems to be a good tool for selection of renal receptors.

Um grupo especial de indivíduos transplantados renais mantém a função do enxerto estável após a total retirada da terapia imunossupressora, alcançando um estado imunológico chamado de Tolerância operacional. Ainda não há marcadores moleculares e celulares que discriminem a tolerância no transplante humano, e os seus mecanismos estão sendo investigados. O perfil de peptídeos presentes na urina pode trazer informações importantes sobre o estado fisiopatológico renal. Nós investigamos se a tolerância operacional apresenta um perfil diferencial de peptídeos urinários, potencialmente, relevante para diagnóstico deste estado imunológico, assim como para a compreensão de seus mecanismos. Realizamos análises qualitativas do extrato de peptídeos da urina de indivíduos dos diferentes grupos do estudo: saudáveis (SA, n=6), tolerantes operacionais (TO, n=5), rejeição crônica (RC, n=8) e estável sob terapia imunossupressora convencional (EST, n=5), utilizando a abordagem proteômica de Shotgun. Identificamos um total de 15283 diferentes peptídeos em todos os grupos, correspondentes a 646 proteínas distintas, distribuídas nos diferentes grupos: TO = 189, RC = 296, EST = 205 e no grupo SA 219 proteínas. Observamos proteínas exclusivas dos diferentes grupos: TO teve 87 proteínas exclusivas, RC 168, EST 106 e SA 108 proteínas. Apesar das proteínas exclusivas não terem sido compartilhadas por todos os indivíduos do mesmo grupo, a totalidade dos indivíduos de cada grupo apresentou várias dessas proteínas (cada indivíduo apresentou em média 15% das proteínas exclusivas de seu grupo). Das 646 proteínas identificadas, apenas 2,3% foram classificadas pelo Gene ontology como relacionadas ao sistema imune e os compartimentos celulares mais frequentes foram: núcleo 36% e citoplasma 23%. Destacamos algumas proteínas relacionadas à resposta imune, exclusivas de alguns grupos, como no grupo TO, a C-C motif chemokine 24 (CCL24) e Endothelin-1 e no grupo RC, a beta 2 microglobulina. Essas moléculas podem ter relevância nos mecanismos imunológicos desses estados clínicos. A abordagem proteômica aplicada neste trabalho permitiu a identificação de um perfil diferencial de peptídeos na urina de cada grupo diferente de estudo. Os peptídeos urinários diferenciais e suas respectivas proteínas podem ter relevância funcional ou como biomarcadores - em relação ao estado fisiológico e às diferentes evoluções clínicas no transplante renal / A special group of renal transplant recipients maintain stable graft function after the complete withdrawal of immunosuppression, achieving a state called operational tolerance. To date, there are no cellular or molecular biomarkers to discriminate human transplantation tolerance and the underlying mechanisms are being investigated. The profile of urinary peptides may provide important information about different renal physiopathological statuses. We investigated whether operational tolerance displays a differential urinary peptide profile, potentially relevant as biomarkers or for the understanding of mechanisms involved in tolerance. We performed qualitative analysis of peptide urinary extracts in individuals from different study groups: healthy (HI, n=6), operational tolerance (OT, n=5), chronic rejection (CR, n=8) e stable under conventional immunosuppression (Sta, n=5), using Shotgun proteomics. Altogether, we identified 15283 different peptides, corresponding to 646 distinct proteins, distributed in all groups: OT = 189, CR = 296, Sta = 205, and 219 proteins in HI. Several proteins were exclusively detected in specific groups: OT showed 87 exclusive proteins, CR 168, Sta 106 and HI 108 proteins. Although the exclusive proteins were not shared by all individuals from that specific group, all individuals from each group presented several of the group-exclusive proteins (each individual presented an average of 15% of the proteins exclusive to his group). Of the 646 proteins identified, only 2.3% were classified in Gene Ontology as related to the immune system and the most frequent cellular compartments were: 36% from nucleus and 23% cytoplasmic. Of notice, some proteins related to the immune response were also group-exclusive, such as, in OT, a C-C motif chemokine 24 (CCL24) and Endothelin-1, and beta 2 microglobulin in CR. These proteins may display relevant roles in the mechanisms involved in these clinical statuses. In conclusion, the proteomic approach used in this study allowed the identification of a differential urinary peptide profile in each different study groups. The differential urinary peptides and their corresponding proteins may display a relevant role functional or as biomarkers - in the state of homeostasis and in different clinical outcomes in renal transplantation