I conducted a four-month ethnographic fieldwork study to document the stories of thirteen post-kidney-transplant minority patients and three nephrologists at Boston Medical Center. My research explores how patients’ interactions with health professionals, medical regimens, dialysis treatments, and adaptation to living with transplanted kidneys constantly shape their identities and perceptual worlds. Patients’ narratives highlighted the emotional struggles they encountered along the path of End-Stage Renal Disease, which unfolded as distinct experiences influenced by their varied backgrounds. The majority of my patient-participants lived on the verge of poverty, and in some cases, their insurance status caused delays in their being registered on the transplant waiting list, making them endure a long wait. Some patients were afraid of wearing short sleeves because they thought the scars on their arms from dialysis treatment would lead others to think they were gangsters. Instantiations of various theories emerged from the saturated data and narrative analysis, from Bourdieu’s concept of habitus with regard to the process of how patients alter their consciousness through interactions with medicine to Foucault’s ideas of power relations and technologies of the self that address the issues of agency and power that influence the formation of patients’ identities. The intersection of these theoretical frameworks led me to develop the critical medical anthropological-oriented concept of biological citizenship. This paper examines 1) the ways in which “race” interacts with the theoretical concept of biological citizenship and 2) the ways in which socioeconomic status and race tailor a kidney transplant patient’s illness experience, and related discourse.

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

Background: Aim of this single center cross-sectional study was to investigate oral behavior, dental, periodontal and microbiological findings in patients undergoing hemodialysis (HD) and after kidney transplantation (KT). Methods: Patients undergoing HD for end-stage renal failure and after KT were investigated. Oral health behavior was recorded using a standardized questionnaire, e.g. dental behavior, tooth brushing, oral hygiene aids. Oral investigation included screening of oral mucosa, dental findings (DMF-T) and periodontal situation (Papilla bleeding index [PBI] periodontal probing depth [PPD] and clinical attachment loss [CAL]). Additionally, microbiological analysis of subgingival biofilm samples (PCR) was performed. Statistical analysis: Student’s t-test or Mann–Whitney-U-test, Fisher’s exact test (α = 5 %). Results: A total of 70 patients (HD: n = 35, KT: n = 35) with a mean age of 56.4 ± 11.1 (HD) and 55.8 ± 10.9 (KT) years were included. Lack in use of additional oral hygiene (dental floss, inter-dental brush) was found. KT group presented significantly more gingivial overgrowth (p = 0.01). DMF-T was 19.47 ± 5.84 (HD) and 17.61 ± 5.81 (KT; p = 0. 21). Majority of patients had clinically moderate and severe periodontitis; showing a need for periodontal treatment of 57 % (HD) and 71 % (KT; p = 0.30). Significantly higher prevalence of Parvimonas micra and Capnocytophaga species in the HD group were found (p < 0.01). Conclusion: Periodontal treatment need and lack in oral behavior for both groups indicate the necessity of an improved early treatment and prevention of dental and periodontal disease, e.g. in form of special care programs. Regarding microbiological findings, no major differences between KT and HD patients were found.

La non-adhésion (NA) est un enjeu majeur en transplantation rénale (TR). Nous avons réalisé une revue systématique dans laquelle les facteurs liés à la NA sont discutés. Et, sachant que la variabilité des taux sanguins d'anti-calcineurine (CNI) pose la question de NA, nous avons essayé d'identifier les facteurs qui lui sont associés. 37 articles sur l'adhésion ou NA en TR, publiés entre 2009 à 2014, ont été analysés. La NA fluctuait entre 2 et 96% et plusieurs facteurs lui étaient associés : a. jeune, homme, faible support social, sans emploi, faible éducation h. >3 mois post Tx, donneur vivant, >6 comorbidités c. >5 médicaments/j, >2 prises/j d. Croyances et/ou comportements négatifs e. Dépression et/ou anxiété. Puis, nous avons réalisé une étude transversale sur une cohorte de patients à 1 an post greffe de rein. . Les données cliniques, de l'entretien du pharmacien clinicien (PC) et de 6 questionnaires ont été collectées. 408 patients ont été inclus (61.2% d'hommes, âge médian 54 ans). Nous avons comparé 2 groupes selon le coefficient de variation (CV) des CNI : CV<30% (n=302) et >30% (n=106). En analyse univariée la distance hôpital-domicile, la ciclosporine, le délai post greffe et la présence de divergences à la conciliation médicamenteuse, étaient associés à un risque élevé de CV>30%. A l'inverse, le tacrolimus LP conférait un risque plus faible. En analyse multivariée, la présence de divergences était significative (OR=3.2 IC95% [1.21-9.01], p=0.02). Un CV>30% des CNI après 1 an de greffe semble refléter un phénomène de NA pouvant être confirmé par l'entretien avec le PC et constituer un outil simple pour la pratique clinique. / Non-adherence (NA) is a major issue after kidney transplantation (Tx). We realized a systematic review, in which criteria related to NA were discussed. And, considering that calcineurin inhibitors (CNI) blood levels variability raises the question of NA, we tried to identify factors associated to it. 37 studies on adherence and NA in TX, published between 2009 and 2014 were reviewed. NA fluctuated from 2 to 96% and sseveral factors were related to NA: a.Young age, male, low social support, unemployed, low education b. >3 months after Tx, living donor, >6 comorbidities c. >5 drugs/d, > 2 intakes/d d. Negative beliefs and/or behaviors e. Depression and anxiety. Then, we realised a cross sectional study on a cohort of kidney recipients grafted for more than 1 year. We recorded: clinical data, data from a clinical pharmacist (CP) interview and from 6 self-reports. 408 recipients were enrolled (61.2% male, median age 54 years old). We compared 2 groups according to a coefficient of variation (CV) for CNI blood levels: CV<30% (n=302) and >30% (n=106). In univariate analysis, the distance hospital-home, cyclosporine, time since Tx, discrepancies in the reconciliation process were associated with a greater risk of CV>30%. By contrast, tacrolimus once daily conferred a lower risk of CV >30%. In multivariate analysis discrepancies remained significant (OR=3.2 CI 95% [1.21-9.01], p=0.02). ACV >30% for CNI blood levels after lyear post Tx seems to reflect NA, and could easily be confirmed by the CP interview. This could be a simple method to detect NA in clinical routine.

O mecanismo de imunorregulação gerado pelo catabolismo do triptofano pela IDO protege o feto contra a resposta imunológica materna. Neste estudo, esse mecanismo foi em avaliado em gestantes imunossuprimidas e portadoras de transplante renal. Examinou-se a expressão da IDO e sua atividade nos compartimentos placentários de gestantes saudáveis e portadoras de transplante. Células produtoras de IDO e IFN-<font face=\"Symbol\">g foram imunolocalizadas na região vilosa e região decidual em ambos os grupos analisados, com mudanças no tipo celular envolvido nestas expressões nas gestantes transplantadas. Os níveis de IDO e sua atividade, assim como seus fatores de regulação NF-kB, IFN-<font face=\"Symbol\">g e IL-10 estavam diminuídos na região vilosa. No compartimento decidual a atividade enzimática da IDO estava aumentada nas gestantes transplantadas, mas não dos seus reguladores. Sendo assim, eixo de imunorregulação gerado por IDO-IFN-<font face=\"Symbol\">g na interface placentária de gestantes portadoras de transplante renal responde diferencialmente a insultos ocasionados pela utilização de imunossupressores durante a gestação. / The mechanism of immunoregulation generated by the catabolism of tryptophan by IDO protects the fetus against maternal immune response. In this study, this mechanism was evaluated in renal transplanted pregnant women and immunosuppressed. We examined the expression and activity of IDO in placental compartments of healthy pregnant women and patients with transplants. IDO and IFN-<font face=\"Symbol\">g producing cells were imunolocalizated in villous and decidual region in both groups analyzed, with changes in cell type involved in these expressions in pregnant patients transplanted. The levels and activity of IDO, as well as their regulatory factors NF-kB, IFN-<font face=\"Symbol\">g and IL-10 were decreased in the villous region. In the decidual compartment IDO activity was increased in pregnant women transplanted, but not their regulators. Thus, the axis of immunoregulation generated by IDO and IFN-<font face=\"Symbol\">g in placental interface of pregnant women with renal transplantation responds differently to insults caused by the use of immunosuppressive drugs during pregnancy.

Introdução: A nefropatia crônica do enxerto permanece sendo a principal causa de perda tardia de enxertos renais. No momento, não existe uma estratégia terapêutica definida para minimizar ou reverter a perda da função renal. Diversas tentativas terapêuticas foram empregadas sem resultados definitivos. As estratégias de minimização de inibidores da calcineurina (CNI) com conversão para Micofenolato mofetil (MMF) e conversão para Sirolimo (SRL) são as mais promissoras. Este estudo avaliou a segurança e a eficácia dessas duas estratégias terapêuticas na progressão da nefropatia crônica do enxerto em pacientes transplantados renais. Métodos: Foram selecionados pacientes com filtração glomerular (RFG) medida por depuração de 51Cr-EDTA entre 25 e 60 ml/min/1,73 m2 que apresentaram alterações histológicas compatíveis com nefropatia crônica do enxerto e que não apresentaram proteinúria 24 h superior a 800 mg/24 h. Os pacientes foram randomizados para serem convertidos ao SRL ou manterem-se sob níveis baixos de CNI associados ao MMF e prednisona. O objetivo primário foi avaliar um objetivo composto pelos seguintes eventos: morte, perda do enxerto, rejeição aguda ou perda de RFG inicial superior a 20%. Os pacientes foram acompanhados por 12 meses e a uma análise por intenção de tratar foi realizada ao fim desse período. Resultados: Vinte e nove pacientes foram randomizados para os grupos SRL (n=14) e CNI (n=15). Não houve diferença entre os grupos quanto a os dados demográficos e imunológicos. Os valores de creatinina sérica e a TFG foram semelhantes no momento da randomização. A sobrevida dos pacientes e dos enxertos foi de 100%. Não foram observados episódios de rejeição aguda. Após 12 meses, não houve diferença significativa entre os grupos com relação à TFG. Houve maior número de eventos adversos não-graves no grupo SRL, destacandose, acne, edema, piora de dislipidemia e anemia. Entretanto, o número de eventos adversos graves não foi estatisticamente diferente entre os grupos. SRL foi descontinuado temporariamente em 1 paciente, mas não ocorreu descontinuação definitiva no estudo. Conclusão: Os dois esquemas terapêuticos apresentaram desempenhos rigorosamente semelhantes com relação à evolução da função renal e quanto à evolução histológica, mas houve um número maior de eventos adversos não-graves com o uso de sirolimo / Chronic allograft nephropathy is the main cause of late kidney graft loss. Several treatments have been proposed for this condition without conclusive results. Calcineurin inhibitors minimization and conversion to Sirolimus are the most promising alternatives. This study evaluated the safety and the efficacy of these therapeutic strategies on one-year progression of chronic allograft nephropathy in kidney transplant recipients. Patients with measured glomerular filtration rate (51Cr-EDTA plasmatic clearance) between 25 e 60 ml/min/1,73 m2 and histological findings of CAN, with proteinuria less than 800 mg/24 h were included. They were randomized either to Sirolimus or to low-level of CNI (both groups received MMF and prednisone). The primary end-point was a composite of first occurrence of death, graft loss, acute rejection or a 20% decrease of initial GFR. Patients were followed for 12 months and evaluated as intention-to-treat analysis. Twenty-nine patients were included in this study. Fourteen patients were randomized to SRL group and fifteen to CNI group. At baseline, no differences were detected in any of the demographic and immunologic group characteristics. Also, serum creatinine and GFR were not different at randomization. One year after conversion, patient and graft survival was 100%. At 12 months, there were no differences in GFR between two groups, in SRL group was 41,99 ± 13,48 ml/min/1,73 m2and in CNI group was 41,21 ± 9,10 ml/min/1,73 m2 (p=0,96). Non-serious adverse events, like anemia (p=0,006), acne (p=0,006), edema (p=0,005) and mouth ulcers (p=0,017) were more frequently found in the SRL group. No significant difference in serious adverse events was observed. SRL was temporarily interrupted in one patient. None of the patients dropped-out from the study and none required study drug discontinuation. In conclusion both regimens conferred equal beneficial in GFR preservation in CAN patients. However, SRL was associated with more adverse events

A qualidade do sono é um dos fatores que afetam a qualidade de vida após o transplante renal. O objetivo deste estudo foi investigar a prevalência de distúrbios do sono e avaliar as variáveis que afetam a qualidade do sono nos primeiros anos de transplante renal. Trata-se de um estudo prospectivo observacional, no qual os sujeitos foram abordados em dois momentos: fase 1: entre 3 e 6 meses e fase 2: 12 e 15 meses após o transplante renal.Os instrumentos utilizados foram: o Índice de Qualidade do Sono de Pittsburgh (PSQI); o Questionário de Qualidade de Vida: Short-form SF-36; a Escala Hospitalar de Ansiedade e Depressão (HAD); a Escala de Karnofsky. Também foi realizado o levantamento dos dados sócio-demográficos e o programa SPSS foi utilizado para obtenção dos gráficos e análise estatística. Não houve diferenças significativas nas características demográficas entre os pacientes com e sem distúrbio do sono nas fases 1 e 2. Não encontramos variações no padrão de sono no decorrer do estudo. Ocorreu uma piora dos escores de vitalidade e de aspectos emocionais da fase 1 para a fase 2. Nos demais escores não houve diferença significativa nas fases analisadas. Foi observado um aumento significativo do IMC destes pacientes ao longo dos dois primeiros anos de transplante renal. Entretanto, com relação à idade, sexo e tipo de doador não foram encontradas diferenças significativas. Conclusão: A qualidade do sono em pacientes transplantados renais não variou significativamente no período estudado (médias das Fases 1 e 2: 36,7% e 38,3%, respectivamente). Porém, houve diferença significativa entre qualidade do sono e a percepção da qualidade de vida nos domínios de vitalidade e aspectos emocionais. / The quality of sleep is one of the factors that affect quality of life after renal transplantation. The aim of this study was to investigate the prevalence of sleep disorders and evaluate the variables that affect the quality of sleep in the early years of renal transplantation. This is a prospective observational study in which subjects were addressed in two phases (phase 1: 3 to 6 months and phase 2: 12 and 15 months after renal transplantation). The instruments used were the Quality Score Pittsburgh Sleep (PSQI), the Quality of Life Questionnaire: Short-form SF-36, the Hospital Anxiety and Depression (HAD) Scale Karnofsky. Also was done a survey of the socio-demographic data and SPSS program was used to obtain the graphs and statistical analysis. There were no significant differences in demographic characteristics between patients with and without sleep disturbance in phases 1 and 2. We found no changes in sleep patterns during the study. There was a worsening of the scores for vitality and emotional aspects of phase 1 to phase 2. In other scores did not differ in the stages analyzed. We observed a significant increase in BMI of these patients over the first two years of kidney transplantation. However, with regard to age, sex and donor type were not significant differences. Conclusion: Sleep quality in renal transplant patients did not change during the study period (average of Phase 1 and Phase 2 36.7%, 38.3%). However, significant differences were found between sleep quality and perceived quality of life in the areas of vitality and emotional aspects

para diversos tipos de tratamento, como doenças auto-imunes, inflamatórias e até para pacientes submetidos a transplante de órgãos. Assim também, é capaz de causar efeitos adversos como toxicidade hepática e mais frequentemente a mielossupressão. Tiopurina Smetiltransferase (TPMT), uma enzima que catalisa a S-metilação dessas drogas, exibiu um polimorfismo genético em 10% de Caucasianos, sendo 1/300 indivíduos com completa deficiência. Pacientes intermediários ou completamente deficientes da atividade de TPMT estão em risco por excessiva toxicidade após receberem doses padrões de medicações tiopurínicas. O presente estudo visa a 1) pesquisar a frequência desses polimorfismos (TPMT*2, TPMT*3A, TPMT*3B e TPMT*3C) em populações de doadores de sangue em Hospital terciário Universitário e em pacientes que utilizem a Azatioprina para o tratamento da hepatite autoimune e transplantado renal, 2) padronizar os métodos para a identificação dos polimorfismos e associá-los com os níveis dos metabólitos da AZA. Esses achados podem explicar os efeitos benéficos da Azatioprina e ter importante implicação para o desenho de novas terapias específicas em doenças autoimunes e em transplante de orgãos / Thiopurine drugs such as azathioprine (AZA) are widely used for many types of treatment: autoimmune and inflammatory diseases and for patients who had undergone organ transplantation. AZA is capable of causing adverse effects such as hepatotoxicity and myelosuppression. Thiopurine S-methyltransferase (TPMT), which catalyzes the Smethylation of such drugs, exhibits a genetic polymorphism in 10% of Caucasians, and 1 / 300 individuals with complete deficiency. Patients who are intermediary or completely deficient in TPMT activity are at risk for excessive toxicity after receiving standard doses of thiopurine drugs. This recent study aims to 1) investigate the frequency of these polymorphisms (TPMT*2, TPMT*3A, TPMT * 3B and TPMT*3C) in blood donors from a terciary University Hospital and in patients using azathioprine for the treatment of autoimmune hepatitis and transplanted kidney; 2) standardize the methods for the identification of polymorphisms and associate with levels metabolites of AZA. These findings may explain the beneficial effects of azathioprine and have important implications to design new therapies for autoimmune diseases and organ transplant

Introdução: O papel do sistema HLA na evolução do transplante é indiscutível. Dados da literatura internacional e do nosso laboratório têm mostrado que este não é o único sistema envolvido nos processos de RMA (rejeição mediada por anticorpo) Esse fato é comprovado a partir da constatação de que transplantes realizados entre irmãos com total identidade HLA também são alvos da RMA. Entretanto, os alvos antigênicos desses anticorpos permanecem desconhecidos, dificultando assim o diagnostico e tratamento da RMA não-HLA. No transplante renal a presença desses anticorpos têm sido associada com anticorpos anti células endoteliais, células epiteliais tubulares, podocitos, células mesangiais e monócitos. Nosso objetivo neste estudo foi avaliar a presença e a relevância clínica de anticorpos contra a megalina, membro da família de receptores de LDL, expressa na membrana apical dos túbulos proximais, com importante papel na reabsorção de proteínas no rim. Métodos: Soros pré-transplante de 105 pacientes submetidos a transplante renal, realizado no Serviço de Transplante Renal do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (UTR-HC-FMUSP) foram testados por ELISA contra 2 peptídeos da megalina estudados e sintetizados em nosso laboratório. (convencionalmente chamados 18 e 19) Resultado: Não foi detectada a presença de anticorpos do isotipo IgG nas amostras prétransplante dos pacientes estudados. Entretanto, foi detectada a presença de anticorpos do isotipo IgM anti-peptídeo 18 em 33 (31,4%) amostras de soro e anti-peptídeo 19 em 23 (21,9%). Para avaliar a significância clinica desses anticorpos dividimos os pacientes em 2 grupos: pacientes com pelo menos 1 episodio de rejeição aguda (Grupo I) e pacientes sem rejeição (Grupo II) e observamos a distribuição dos pacientes positivos nos 2 grupos. O numero de pacientes com anticorpos anti-peptídeo 18 foi igualmente distribuída nos dois grupos. (12/42, 28,5% no Grupo I e 21/63, 33,3% no Grupo II p=ns). A maioria dos pacientes com anticorpos anti-peptídeo 19 pertenciam ao grupo I (17/42, 40,5% ; 6/63, 9,5% p=0,0003). Esta analise demonstrou uma boa correlação entre presença de anticorpos anti-peptídeo 19 no soro pré-transplante e rejeição. Conclusão: Nossos dados sugerem que anticorpos IgM anti-megalina no pré-transplante podem ser um fator de risco na rejeição do aloenxerto renal. É importante salientar que a nosso conhecimento este é o primeiro estudo envolvendo megalina e rejeição no transplante clínico. / Background. Preformed donor-specific human leukocyte antigen (HLA)- antibodies are accountable for the majority of antibody-mediated rejections (AMR). However, recipients of HLA identical kidneys can develop AMR implicating putative pathogenic antibodies that are directed against non-HLA antigens. Unknown immune targets and consecutive lack of detection methods make non-HLA AMR particularly difficult to diagnose and treat. In renal transplant rejection, the presence of antibodies to non-HLA has been associated with antibodies against endothelial cells, tubular epithelial cells, podocytes, mesangial cells and monocytes. The aim of this study was to evaluate the presence and clinical relevance of preformed antibodies against megalin, a member of the LDL receptor family, expressed on the apical membrane of proximal tubules. Megalin performs a central role in renal protein reabsorption. Methods. Pré-transplant sera of 105 recipients of kidney allograft transplanted at Serviço de Transplante Renal do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (STRHCFMUSP) were tested by enzyme-linked immunosorbent assay (ELISA) against 2 megalin-peptides (conventionally named 18 and 19) studied and synthetized in our laboratory. Results. Antibodies were not detected in pretranplant sera when tested for IgG isotype. However, in 33 (31,4%) sera we detected the presence of IgM antibodies to megalin-peptide18 and in 23 (21,9%) to megalin-peptide 19. To evaluate the clinical significance of these antibodies we divided the patients in 2 groups: Group I - 42 patients with at least one rejection episode during the first post-transplant year and Group II - 63 patients without any rejection episode and observed the distribution of positive patients in each of the 2 groups. Patients with megalin-peptide18 antibodies had the same distribution in both groups. (12/42, 28, 5% Group I and 21/63, 33,3% no Group II p=ns). However, patients with megalinpeptide19 antibodies were more frequent in group I. (17/42, 40,5% group I and 6/63, 9,5% group II p=0,0003). This analysis demonstrated a good correlation between preformed anti-megalin-peptide19 antibodies and allograft rejection Conclusion. Our data suggest that presence of IgM megalin antibodies before transplantation might be a risk factor for kidney allograft rejection. To our knowledge, this is the first study involving megalin and rejection in clinical transplantation.

O aprimoramento das técnicas analíticas viabilizou a metabolômica, uma área da ciência que busca compreender, de forma comparativa, os metabólitos envolvidos nas vias bioquímicas. A metabolômica está inserida no contexto das \"ômicas\", que teve início na \"Era Genômica\", a qual permitiu a identificação de diversos genes. Em seguida, o interesse dos pesquisadores centrou no estudo dos metabólitos (metabolômica) mostrando ser uma ferramenta valiosa na pesquisa do transplante renal, que exige um tratamento medicamentoso por meio de imunossupressores. A combinação destes imunossupressores pode minimizar a rejeição do órgão transplantado, reforçando a necessidade de um estudo metabolômico, a fim de avaliar e comparar as mudanças ocorridas após o transplante em nível molecular, melhorando o conhecimento sobre a influência destes regimes e dando subsídios sobre prognósticos possíveis na área de transplante renal. Nesta tese foram avaliadas 2 terapias: Everolimo/ Prednisona/Tacrolimo (grupo 1) e Micofenolato mofetil/Prednisona/Tacrolimo (grupo 2) a partir de uma abordagem untargeted. No presente trabalho foram coletadas amostras de urina de pacientes ao longo de 6 meses. Foi necessário determinar a melhor condição para análise das amostras de urina dos pacientes. Desta forma, foram realizados estudos sobre alguns parâmetros que impactam no preparo de amostra abordando a influência da urease, tipos e proporção de solventes para precipitação de proteína, seleção do melhor agente derivatizante e tratamento de dados. A avaliação da medida de qualidade dos tratamentos com urease foi feita a partir do desvio padrão relativo (RSD) dos valores de intensidade de pico. A concentração de 10 mg mL-1 apresentou o melhor resultado. O estudo mostrou também que o teor de ureia na urina pode influenciar na identificação dos compostos. O número de compostos identificados foi menor quando a urina não foi tratada com urease, com aproximadamente 10 compostos a menos em relação à amostra tratada com a enzima, na mesma concentração de ureia adicionada. Dos solventes orgânicos testados para precipitação de proteínas nas amostras de urina, o isopropanol mostrou ser o solvente mais adequado na proporção 1:6 urina:solvente (v/v), utilizando-se 100 &#181;L de urina. Foram testados dois protocolos de derivatização para análises por GC-MS: metoximação e sililação utilizando BSTFA e cloroformiato de metila. A comparação mostrou que o procedimento por BSTFA, com 40 metabólitos identificados, foi superior ao cloroformiato de metila, com 13 compostos identificados. No tratamento de dados com o software XCMS, os seguintes parâmetros foram avaliados: largura a meia altura do pico (fwhm), largura da banda (bw) e threshold (sntresh). Para avaliar a melhor combinação destes parâmetros, foi feita uma variação univariada destes valores. A qualidade do resultado de cada combinação foi monitorada pelos valores gerados de número de missing values, quantidade de picos com RSD <15% e número de valores duplicados. Os valores ótimos foram obtidos para a combinação: fwhm=4, bw=2 e threshold=5. A abordagem do estudo dos dois grupos de pacientes baseou-se inicialmente na comparação entre o dia 7 da terapia com os demais períodos (dia 14, mês 1, mês 3 e mês 6) e posteriormente avaliou-se a evolução temporal. A partir do mês 3 os valores de correlação e predição dos modelos de PLS-DA são melhores e já é eficaz na diferenciação entre os dois grupos. Foram observadas perturbações no metabolismo de carboidratos em ambos os grupos, como açúcares, glicerol e N-acetil-D-manosamina. No grupo 1, foram observados metabólitos discriminantes da classe dos poliois e das vias do ciclo do ácido cítrico e degradação de xenobióticos, enquanto que, no grupo 2, foi observada alteração do hidroxibutirato, um corpo cetônico. Neste grupo, foi observado também um aumento do ácido hipúrico, ácido acetamido butírico, ácido benzoico, entre outros. Nesta tese foi possível demonstrar que a metabolômica é uma ferramenta importante para comparar metabólitos discriminantes entre dois regimes imunossupressores, sendo um estudo piloto que visa fornecer subsídios para avaliação da influência destas terapias no prognóstico de transplante renal. / The improvement of analytical techniques enabled the emergence of metabolomics, which aims to compare the metabolites involved in biochemical pathways, in certain biological conditions. Metabolomics is inserted in the \"omics\" context, which began in the \"Genomic Age\", and allowed the identification of several genes. After that, the researchers focused on the study of metabolites. Among several applications, metabolomics can be a valuable tool in renal transplant research, which requires a drug treatment through immunosuppressants. The combination of these immunosuppressants can minimize toxicity and rejection of the transplanted organ, reinforcing the need for a metabolomic study, in order to evaluate and compare changes after transplantation at the molecular level, improving knowledge about the influence of these regimens and giving subsidies on prognosis in the area of renal transplantation. In this thesis two immunosuppressive therapies were evaluated by an untargeted approach: Everolimus/Prednisone/Tacrolimus (group 1) and Mycophenolate mofetil/Prednisone/Tacrolimus (group 2). In this study, urine samples were collected from patients over 6 months. It was necessary to determine the best condition for analysis of patients\' urine samples. Thus, studies were carried out on some parameters that impact on sample preparation, evaluating the influence of urease, types and proportion of solvents for protein precipitation, selection of the best derivatizing agent, and data treatment. The evaluation of the quality measure of the urease treatments was made from the relative standard deviation (RSD) of the peak intensity values. The concentration of 10 mg mL-1 presented the best result. The study also showed that urine urea content may influence the identification of the compounds. The number of identified compounds was lower when urine was not treated with urease, with approximately 10 compounds less than the enzyme-treated sample, at the same concentration of urea added. In the evaluation of the organic solvents tested for protein precipitation in the urine samples, isopropanol was the most suitable solvent in the ratio 1: 6 urine:solvent (v/v), using 100 &#181;L of urine. Two derivatization protocols were tested for GC-MS analysis: metoximation and silylation with BSTFA and methyl chloroformate. The comparison between the two derivatization protocols showed that the BSTFA procedure, with 40 identified metabolites, was superior to methyl chloroformate with 13 compounds identified. In data processing with the XCMS software, the following parameters were evaluated: full width at half maximum of the peak (fwhm), bandwidth (bw) and threshold (sntresh). To evaluate the best combination of these parameters, a univariate variation of these values was made. The quality of the result of each combination was monitored by the number of missing values, number of peaks with RSD <15%, and number of duplicate values. The optimal values were obtained for the combination: fwhm=4, bw=2 and threshold =5. The study of the two groups of patients was initially based on the comparison between day 7 of the therapy with the other periods (day 14, month 1, month 3 and month 6) and later the temporal evolution was evaluated. From month 3 the values of correlation and prediction of the PLS-DA models are better and already effective in the differentiation between the two groups. Disorders in carbohydrate metabolism were observed in both groups with sugars and glycerol and N-acetyl-D-mannosamine as discriminant metabolites. In group 1, discriminant metabolites of the class of polyols and citric acid cycle pathways and degradation of xenobiotics were observed, and in group 2 alteration of hydroxybutyrate, a ketone body, was observed. In this group an increase of hippuric acid, acetamido butyric acid, benzoic acid, among others, was also observed. In this thesis it was possible to demonstrate that metabolomics is an important tool to compare discriminant metabolites between two immunosuppressive regimens, being a pilot study that aims to provide future subsidies to evaluate the influence of these therapies on the renal transplant prognosis