Bancos de tumores foram criados para organizar a coleta, arm azenamento e distribuição de amostras biológicas de pacientes oncológicos, favorecendo seu uso nas pesquis as sobre o cân cer. Amostras ade quadas devem ter RNA, DNA e proteínas de boa qualidade. RNA de boa qualidade deve estar íntegro e puro e DNA deve ter boa c oncentração e pur eza. Basea do em norm as in ternacionais, f oi elaborado e implantado um abrangente sistema de controle de qualidade no banco de tumores do Hospital de Câncer de Barretos, que para fins de estudo foi dividido em banco pré-controle de qu alidade (den ominado b anco pré) e em ban co pós- controle de qualidade (denominado banco pós). Objetivando comparar a qualidade das amostras n os dois bancos, atra vés d a extração d e R NA total e d e DNA (utilizando-se homogeneizador de tecidos e Kits), selecionou-se de forma aleatória 200 a mostras tumorais, distribuídas ig ualitariamente entre mama, co lorreto, estômago, pulmão e tireóide, sendo 100 do banco pré e 100 do banco pós. Para se avaliar a influência do tempo de isquemia fria (tempo entre a excisão do e spécime cirúrgico e o congelamento rápido da amostra armazenada) na qualidade do RNA total de amostras tumorais do banco pós, foram coletadas 200 amostras tumorais, distribuídas igualitariamente entre mama, co lorreto, estômago, pulmão e ti reóide, de 100 doadores diferentes, metade com o tempo de isquemia fria (TIF) de até 30 minutos e a o utra metade do mesmo espécime com TIF de 45 minutos. Extraiu-se RNA total dessas amostras (com maceração manual e Trizol) e comparou-se a sua qualidade, através do núm ero de i ntegridade do RNA (RIN), dentr o dos d ois intervalos de tempo e nas diferentes top ografias. Ao c omparar-se amostras com RIN acima de 7 (consideradas ideais para experimentos de microarray), do banco pré e do b anco pó s, for am enc ontrados 73 (73%) no p rimeiro e 87 (87%) no segundo (p=0,013). Ao comparar-se o intervalo de TIF de até 30 minutos com o de 45 minutos, encontrou-se respectivamente 63 (64,3%) e 3 6 (36%) amostras com RNA total intacto, 11 (11,2%) e 17 (17 %) com RNA tot al parcialmente degradado e 24 (24, 5%) e 47 (47%) com RNA t otal degradado (p<0,001). Amostras tireoidianas e colorretais f oram mais sensíveis ao a umento d o T IF (p=0,006 e p=0,03, respectivamente), e as de estômago e pulmão menos sensíveis (p=0,919 e p=0,384, resp ectivamente). Ao comparar-se a s 200 amostras dos dois ban cos, constatou-se que a grande maioria apresentava boa qualidade, porém o banco pós se destacou ao avaliar-se o número de amostras ideais para estudos de microarray, por provável interferência d o TIF, ainda não controlado no banco pr é. Constatou-se também que algumas amostras do banco pré, armazenadas há mais de ci nco anos em freezer a -80ºC, apresentaram excelente qualidade. O presente estudo também mostrou que o TIF é muito importante para a preservação da qualidade do RNA total, por isso, deve-se sempre respeitar o tempo máximo de 30 minutos. Ainda se observou que a de gradação do RNA é tecido dependente e qu e amostras processadas com homogeneizador de tecidos e extraídas com RNeas y Mini Kit apresentaram melhor q ualidade do RNA, qu e as macer adas manualmente e extraídas com Trizol / Tumor banks were created to or ganize the collection, storage and d istribution of biological samples of cancer pa tients, favoring it\'s use in cancer rese arches. Appropriate samples should have good quality of RNA, DNA and p roteins. RNA of good quality should be intact and pure and DNA should have good concentration and pu rity. Ba sed on international sta ndards, we elabo rated and imp lanted an comprehensive s ystem of qu ality control in the tu mor bank of Ba rretos Cancer Hospital, w hich was divided for st udy purposes i n pre bank quality control (denominated pre bank) and post bank qu ality control (denominated post bank). Aiming to compare the quality of the samples in two banks, through the extraction of total RNA and DNA (b y tissue homogenizer and Kits), we se lected 200 tumor samples in a random way, distributed equally among breast, colorectal, stomach, lung and thyroid, being 100 of the pre-bank and 100 of the post bank. To evaluate the influence o f cold ischem ia time (time b etween t he ex cision o f the su rgical specimen and the fast freezing of the stored sample) in the quality of total of RNA tumor sa mples of th e po st bank , we collected 2 00 t umor s amples, distrib uted equally among breast, colorectal, stom ach, lung and th yroid, fro m 100 different donors, half with the cold ischemia time (CIT) up to 30 minutes and the other ha lf of the sam e specimen with CIT exact ly 45 minutes. We ex tracted total RNA of these samples (with manual maceration and T rizol) and c ompared their qu ality, through the RNA integri ty number (RIN), ins ide tw o intervals of time a nd in different topographies. Comparing samples with RIN above 7 (considered ideals for microarray experiments), of the pre bank and of the post bank, we found 73 (73%) in the first and 87 (87%) in the second (p=0,013). Comparing the interval of CIT up to 30 m inutes with the ex actly 45 minutes, we found respectively 63 (64,3%) and 36 (36%) samples with total RNA intact, 11 (11,2%) and 17 (17%) with total RNA partially degraded and 24 (2 4,5%) and 47 (47%) wit h total RNA de graded (p<0,001). Thyroid and colorectal samples were more sensitive to the increase of CIT (p =0,006 and p=0,03, respectively), a nd s tomach and lun g samples less sensitive (p=0,919 and p=0,384, respectively). C omparing the 200 samples from the two b anks, we v erified that the great ma jority had good qu ality; however the post bank stood out the evaluating number of the id eal samples for m icroarray studies, for probable interference of CIT, still n o controlled in the pre bank. We also verified that some samples of the pre bank, stored more than 5 years in freezer at -80 ºC presented e xcellent qu ality. T he stu dy still sho wed that CIT is ver y important to preserve the quality of total RNA, for that, we sh ould always respect the maximum time of 30 minutes. We still observed that the degradation of RNA is tissue dependent and that samples processed with tissue homogenizer and extracted using RNeasy Mini Kit showed better quality of RNA that macerated manually and extracted with Trizol

Bancos de tumores foram criados para organizar a coleta, arm azenamento e distribuição de amostras biológicas de pacientes oncológicos, favorecendo seu uso nas pesquis as sobre o cân cer. Amostras ade quadas devem ter RNA, DNA e proteínas de boa qualidade. RNA de boa qualidade deve estar íntegro e puro e DNA deve ter boa c oncentração e pur eza. Basea do em norm as in ternacionais, f oi elaborado e implantado um abrangente sistema de controle de qualidade no banco de tumores do Hospital de Câncer de Barretos, que para fins de estudo foi dividido em banco pré-controle de qu alidade (den ominado b anco pré) e em ban co pós- controle de qualidade (denominado banco pós). Objetivando comparar a qualidade das amostras n os dois bancos, atra vés d a extração d e R NA total e d e DNA (utilizando-se homogeneizador de tecidos e Kits), selecionou-se de forma aleatória 200 a mostras tumorais, distribuídas ig ualitariamente entre mama, co lorreto, estômago, pulmão e tireóide, sendo 100 do banco pré e 100 do banco pós. Para se avaliar a influência do tempo de isquemia fria (tempo entre a excisão do e spécime cirúrgico e o congelamento rápido da amostra armazenada) na qualidade do RNA total de amostras tumorais do banco pós, foram coletadas 200 amostras tumorais, distribuídas igualitariamente entre mama, co lorreto, estômago, pulmão e ti reóide, de 100 doadores diferentes, metade com o tempo de isquemia fria (TIF) de até 30 minutos e a o utra metade do mesmo espécime com TIF de 45 minutos. Extraiu-se RNA total dessas amostras (com maceração manual e Trizol) e comparou-se a sua qualidade, através do núm ero de i ntegridade do RNA (RIN), dentr o dos d ois intervalos de tempo e nas diferentes top ografias. Ao c omparar-se amostras com RIN acima de 7 (consideradas ideais para experimentos de microarray), do banco pré e do b anco pó s, for am enc ontrados 73 (73%) no p rimeiro e 87 (87%) no segundo (p=0,013). Ao comparar-se o intervalo de TIF de até 30 minutos com o de 45 minutos, encontrou-se respectivamente 63 (64,3%) e 3 6 (36%) amostras com RNA total intacto, 11 (11,2%) e 17 (17 %) com RNA tot al parcialmente degradado e 24 (24, 5%) e 47 (47%) com RNA t otal degradado (p<0,001). Amostras tireoidianas e colorretais f oram mais sensíveis ao a umento d o T IF (p=0,006 e p=0,03, respectivamente), e as de estômago e pulmão menos sensíveis (p=0,919 e p=0,384, resp ectivamente). Ao comparar-se a s 200 amostras dos dois ban cos, constatou-se que a grande maioria apresentava boa qualidade, porém o banco pós se destacou ao avaliar-se o número de amostras ideais para estudos de microarray, por provável interferência d o TIF, ainda não controlado no banco pr é. Constatou-se também que algumas amostras do banco pré, armazenadas há mais de ci nco anos em freezer a -80ºC, apresentaram excelente qualidade. O presente estudo também mostrou que o TIF é muito importante para a preservação da qualidade do RNA total, por isso, deve-se sempre respeitar o tempo máximo de 30 minutos. Ainda se observou que a de gradação do RNA é tecido dependente e qu e amostras processadas com homogeneizador de tecidos e extraídas com RNeas y Mini Kit apresentaram melhor q ualidade do RNA, qu e as macer adas manualmente e extraídas com Trizol / Tumor banks were created to or ganize the collection, storage and d istribution of biological samples of cancer pa tients, favoring it\'s use in cancer rese arches. Appropriate samples should have good quality of RNA, DNA and p roteins. RNA of good quality should be intact and pure and DNA should have good concentration and pu rity. Ba sed on international sta ndards, we elabo rated and imp lanted an comprehensive s ystem of qu ality control in the tu mor bank of Ba rretos Cancer Hospital, w hich was divided for st udy purposes i n pre bank quality control (denominated pre bank) and post bank qu ality control (denominated post bank). Aiming to compare the quality of the samples in two banks, through the extraction of total RNA and DNA (b y tissue homogenizer and Kits), we se lected 200 tumor samples in a random way, distributed equally among breast, colorectal, stomach, lung and thyroid, being 100 of the pre-bank and 100 of the post bank. To evaluate the influence o f cold ischem ia time (time b etween t he ex cision o f the su rgical specimen and the fast freezing of the stored sample) in the quality of total of RNA tumor sa mples of th e po st bank , we collected 2 00 t umor s amples, distrib uted equally among breast, colorectal, stom ach, lung and th yroid, fro m 100 different donors, half with the cold ischemia time (CIT) up to 30 minutes and the other ha lf of the sam e specimen with CIT exact ly 45 minutes. We ex tracted total RNA of these samples (with manual maceration and T rizol) and c ompared their qu ality, through the RNA integri ty number (RIN), ins ide tw o intervals of time a nd in different topographies. Comparing samples with RIN above 7 (considered ideals for microarray experiments), of the pre bank and of the post bank, we found 73 (73%) in the first and 87 (87%) in the second (p=0,013). Comparing the interval of CIT up to 30 m inutes with the ex actly 45 minutes, we found respectively 63 (64,3%) and 36 (36%) samples with total RNA intact, 11 (11,2%) and 17 (17%) with total RNA partially degraded and 24 (2 4,5%) and 47 (47%) wit h total RNA de graded (p<0,001). Thyroid and colorectal samples were more sensitive to the increase of CIT (p =0,006 and p=0,03, respectively), a nd s tomach and lun g samples less sensitive (p=0,919 and p=0,384, respectively). C omparing the 200 samples from the two b anks, we v erified that the great ma jority had good qu ality; however the post bank stood out the evaluating number of the id eal samples for m icroarray studies, for probable interference of CIT, still n o controlled in the pre bank. We also verified that some samples of the pre bank, stored more than 5 years in freezer at -80 ºC presented e xcellent qu ality. T he stu dy still sho wed that CIT is ver y important to preserve the quality of total RNA, for that, we sh ould always respect the maximum time of 30 minutes. We still observed that the degradation of RNA is tissue dependent and that samples processed with tissue homogenizer and extracted using RNeasy Mini Kit showed better quality of RNA that macerated manually and extracted with Trizol

Introduction : Partant du constat qu'il est possible de conserver plus longtemps les denrées alimentaires grâce à un conditionnement sous atmosphère modifiée enrichie en gaz nobles, nous avons développé une stratégie innovante de conservation de transplants rénaux. Nous avons évalué l'effet protecteur d'une solution de conservation saturée en gaz nobles pour la préservation des transplants rénaux.Matériels et méthodes : Dans un modèle d'autotransplantation rénale hétérotopique chez le porc, les transplants prélevés ont été rincés et mis en conservation 30h à 4°C dans du Celsior présaturée en gaz (air, azote, argon 100% ou xénon 100%, n=6 dans chaque groupe) avant transplantation. Les porcs ont été surveillés quotidiennement pendant 21 j.Résultats : L'argon a amélioré la survie (83,3% vs 33,3% avec l'Air, p=0,04) et la reprise de fonction du transplant. Une sortie de tubulopathie significativement plus précoce des transplants a été observé avec l'Argon. Tous les porcs xénon et azote sont décédés. A J21 les transplants argon avaient une meilleure préservation de leur intégrité structurelle cellulaire avec moins d'inflammation, de fibrose interstitielle et d'atrophie tubulaire. Les rapports RAA/TBARS, et d'Hsp 27, étaient significativement plus élevés avec l'argon. Les taux de TNF alpha, Il 6 et 8 ont montré une diminution de la réponse inflammatoire avec l'argon.Conclusion : Nous avons démontré l'effet bénéfique de l'argon sur la reprise précoce de fonction de transplant et en limitant les lésions d'ischémie-reperfusion. Bien que le mécanisme d'action de l'Argon ne soit pas élucidé, il semble que Hsp 27 soit un élément central de la renoprotection. / Introduction: Based on prolonged preservation of perishable food products under modified atmosphere, we developed an innovative method to preserve kidneys during transplantation using nobles gases. We evaluated the protective effect of argon and xenon on preserving kidney graft functionality and integrity in a clinically relevant pig model of transplantation. Methods: The left kidneys of pigs (n=6 per group) were removed, flushed and stored for 30 h in Celsior solution saturated with air, nitrogen, 100% argon, or 100% xenon. Next, autotransplantation and controlateral nephrectomy were performed. The survival rate, renal function, Hsp27, thiobarbituric acid (TBARS), reduced ascorbic acid (RAA), and TNF alpha were analyzed. A histological examination was completed.Results: Argon improved survival (83.3% for argon vs 33.3% for air, p=0.04) and transplant function recovery. All pigs in the nitrogen and the xenon group died. Diuresis recovery occurred earlier in the argon group (n= 5) when compared with the air group (n=2), p=0.05. On day 7 argon transplants had lower serum creatinine levels and a large reduction in primary non function than the air group. Argon-treated tissues showed better cell structural integrity with minor signs of inflammation, fibrosis, and tubular atrophy. The argon group showed significantly higher RAA/TBARS ratios and Hsp27 levels.Conclusion: We demonstrated that modified atmosphere preservation packaging with argon in cold-storage solution improved early transplant function recovery and long-term quality by minimizing IRI in a pig model of prolonged cold-ischemia. The renoprotective effect of argon may involve the Hsp27 pathway.

Aim: Complex arterial reconstruction in kidney transplantation (KT) using kidneys from deceased donors (DD) warrants additional study since little is known about the effects on the mid- and long-term outcome and graft survival. Methods: A total of 451 patients receiving deceased donor KT in our department between 1993 and 2017 were included in our study. Patients were divided into three groups according to the number of arteries and anastomosis: (A) 1 renal artery, 1 arterial anastomosis (N = 369); (B) >1 renal artery, 1 arterial anastomosis (N = 47); and (C) >1 renal artery, >1 arterial anastomosis (N = 35). Furthermore, the influence of localization of the arterial anastomosis (common iliac artery (CIA), versus non-CIA) was analyzed. Clinicopathological characteristics, outcome, and graft and patient survival of all groups were compared retrospectively. Results: With growing vascular complexity, the time of warm ischemia increased significantly (groups A, B, and C: 40 ± 19 min, 45 ± 19 min, and 50 ± 17 min, respectively; p = 0.006). Furthermore, the duration of operation was prolonged, although this did not reach significance (groups A, B, and C: 175 ± 98 min, 180 ± 35 min, and 210 ± 43 min, respectively; p = 0.352). There were no significant differences regarding surgical complications, post-transplant kidney function (delayed graft function, initial non-function, episodes of acute rejection), or long-term graft survival. Regarding the localization of the arterial anastomosis, non-CIA was an independent prognostic factor for deep vein thrombosis in multivariate analysis (CIA versus non-CIA: OR 11.551; 95% CI, 1.218–109.554; p = 0.033). Conclusion: Multiple-donor renal arteries should not be considered a contraindication to deceased KT, as morbidity rates and long-term outcomes seem to be comparable with grafts with single arteries and less complex anastomoses.

INTRODUÇÃO: O transplante traqueal continua um desafio. Contudo, avanços nas técnicas de revascularização dos enxertos traqueais e no conhecimento da imunobiologia da traquéia, indicam que esta técnica pode ser utilizada com freqüência no futuro próximo. A depuração mucociliar (DM) é o mecanismo de defesa inato mais importante das vias aéreas. A traquéia age como um órgão de defesa devido à DM. A DM ocorre por ação do batimento ciliar do epitélio respiratório que impele o muco que atapeta as vias respiratórias, carreando substâncias nocivas. Idealmente, a DM deve ser preservada em enxertos traqueais passíveis de utilização para transplante traqueal. Nosso intuito foi: 1) avaliar os efeitos da isquemia fria sobre a DM; e 2) avaliar a ação de soluções de preservação administradas por via tópica na manutenção da DM após isquemia fria. MÉTODOS: De 109 ratos Wistar foram obtidos 217 segmentos traqueais. Os segmentos foram distribuídos entre três grupos experimentais e um grupo Controle. Cada segmento foi submergido em LPD-glicose (grupo LPD), histidina-triptofano-cetoglutarato (grupo HTK) ou solução salina (grupo Salina). Avaliou-se a DM após 6,10,16 ou 24 horas de isquemia fria. No grupo Controle os segmentos foram analisados imediatamente após a extração, não havendo isquemia fria, nem submersão em soluções. A velocidade de transporte mucociliar (VTM) foi medida através de microscópio de luz, pela observação do movimento das partículas de muco na superfície dos segmentos traqueais. A freqüência de batimento ciliar (FBC) foi obtida pela sincronização entre o movimento ciliar observado pelo microscópio de luz e um estroboscópio. Em seguida, os segmentos foram corados com hematoxilina-eosina para analisar a integridade epitelial (IE) e a inflamação subepitelial (IS). Foi realizada análise quantitativa do muco intracelular por um programa de computador após coloração com azul de alcião (MI-AA) e PAS (MI-PAS). As amostras mais significativas foram analisadas qualitativamente por microscopia eletrônica de transmissão (ME). Foram realizadas duas análises: 1) grupo Controle e tempos de isquemia; e 2) grupo Controle e soluções de preservação (agrupado pelo tempo de isquemia). RESULTADOS: 1) grupo Controle e tempos de isquemia: O grupo controle foi melhor que os grupos submetidos a isquemia fria quanto à VTM (p=0,0001) e FBC (p=0,012). Contudo, não houve diferença na IE, IS e MI entre o grupo Controle e os demais grupos. 2) grupo Controle e soluções de preservação: O grupo controle apresentou melhor VTM que os grupos LPD, HTK e Salina após isquemia de 6h (p=0,001), 16h (p=0,009) e 24h (p=0,001). O grupo controle apresentou melhor FBC que os grupos LPD, HTK e Salina após isquemia de 24h (p=0,001). Não houve diferença entre os grupos na IE e IS. O grupo Controle apresentou melhor MI-AA que os grupos LPD após 16h (p=0,02) e HTK após 24h de isquemia (p=0,04). Não houve diferença entre os grupos à MI-PAS. À ME, o grupo Salina apresentou maiores alterações secundárias à isquemia do que os demais grupos. CONCLUSÕES: 1) A isquemia fria piora a DM; e 2) O uso de soluções de preservação administradas por via tópica não contribui para manutenção da DM após isquemia fria / INTRODUCTION: Tracheal transplantation is a challenging problem. Recent advances in graft revascularization, and reepithelialization renewed the interest on airway transplantation. Mucociliary clearance (MC) is the most important innate defense mechanism of the respiratory system. MC works by mucous transport carried out by ciliary beating function of the airway epithelium. Trachea acts as a defense organ in the respiratory system through the MC. Ideally, MC should be preserved in tracheal grafts used for transplantation. Preservation solutions improve organ preservation by decreasing ischemic injury. The purpose of the study was: 1) to evaluate the effects of cold ischemia on MC; and 2) to evaluate the impact of topically-applied preservation solutions on MC after cold ischemia. METHODS: From 109 male Wistar rats we obtained 217 tracheal segments. The segments were allocated to one of four groups. Segments were submerged in LPD-glucose (LPD group), histidine-tryptophan-ketoglutarate (HTK group), or saline solution (Saline group), and stored at 4C. MC was analyzed after 6, 10, 16 or 24h of ischemia. Control group have only segments that were analyzed right after extraction, not submitted to cold ischemia or submersion in preservation solutions. The mucociliary transport velocity (MTV) was measured by observation of mucous particle under the surface of the segments, through a light microscope. Ciliary beating frequency (CBF) was achieved by synchronization between cilia movement and a stroboscope flashlight. Tracheas were stained with hematoxylin-eosin in order to analyze the epithelial integrity (EI) and the subepithelial inflammation (SI). A quantitative analysis of the intracellular mucus stained with alcian blue (IM -AB) and PAS (IM-PAS) was achieved by a software. The most significant samples of the tracheal segments were qualitatively analyzed by transmission electronic microscopy (TEM). Two analyses were made: 1) Control group and ischemic time; and 2) Control group and preservation solutions. RESULTS: 1) Control group and ischemic time: Control group had better VTM (p=0,0001) and CBF (p=0,012) than the groups submitted to cold ischemia. However, there was no difference among Control group and the other groups on EI, SI, IM-AB, and IM-PAS. 2) Control group and preservation solutions: Control group showed better MTV than the LPD, HTK, and Saline groups after 6h (p=0,001), 16h (p=0,009) and 24h (p=0,001) of cold ischemia. Control group showed better CBF than the LPD, HTK, and Saline groups after 24h of ischemia (p=0,001). There was no difference among groups on EI and SI. Control group showed better IM-AB than both the LPD group after 16h of cold ischemia (p=0,02), and the HTK group after 24h of cold ischemia (p=0,04). There was no difference among the groups on IM-PAS. TEM showed more findings of ischemic lesion on Saline group. CONCLUSIONS: 1) Cold ischemia impairs MC; and 2) Topically-applied preservation solutions do not ameliorate MC after cold ischemia

Die Schädigung des Organs durch Ischämie-Reperfusion (IR) im Rahmen der kadaverischen Organtransplantation hat bedeutenden Anteil an der Pathogenese verzögert einsetzender Organfunktion und Auswirkungen auf das Langzeitüberleben des Transplantats. In der vorliegenden Studie sollte der Einfluss unspezifischer Schädigung durch IR verglichen mit spezifischen Alloantigen-abhängigen Mechanismen während der Frühphase nach der Transplantation sowie die Auswirkungen prolongierter Aufbewahrung auf Schädigung und Immunogenität des Organs ermittelt werden. Nach vorausgegangener vierstündiger kalter Ischämiezeit wurden Organe aus syngen (Lew/Lew) und allogen (F344/Lew) transplantierten Ratten an 8 aufeinander folgenden Zeitpunkten innerhalb der ersten 10 Tage zu funktionellen, immunhistochemischen und morphologischen Veränderungen untersucht. In weiteren Gruppen wurden syngen transplantierte Organe 24 Stunden nach der Transplantation untersucht, die zuvor ansteigenden kalten Ischämiezeiten zwischen 2 und 48 Stunden ausgesetzt wurden. Im zeitlichen Verlauf zeigten sich bis 7 Tage nach der Transplantation keine wesentlichen Unterschiede zu Nierenfunktion, Morphologie, Zellinfiltration und Expression von Adhesionsmolekülen zwischen allogenen und isogenen Gruppen. Die zunächst eintretende Verschlechterung der Nierenfunktion war begleitet von einem Einstrom neutrophiler und monozytärer Zellen und morphologischen Veränderungen im Sinne von akuter Tubulusnekrose (ATN). Unter zunehmender Infiltration von Monozyten/Makrophagen kam es funktionell und morphologisch zur Regeneration. Neutrophile traten vornehmlich über Interaktion von ICAM-1/LFA-1 und Monozyten/Makrophagen über VCAM-1/VLA-4 aus dem Gefäßsystem aus. Gabe von Cyclosporin A führte zu signifikanter Reduktion ED-1-positiver Makrophagen nach 10 Tagen, ohne jedoch den Anteil des aktivierten Makrophagensubtyps ED-2 zu beeinflussen. Ansteigende kalte Aufbewahrung des Organs führte zu größerer vaskulärer Schädigung, die sich durch abnehmende Intensität und lückenhaftere Verteilung von PECAM-1 auf dem Endothel äußerte. Die Zunahme der Intensität von Tissue Factor auf Endothel und infiltrierenden Leukozyten deutete neben gesteigerter Thrombogenese auf alternative Adhäsionsmechanismen hin. Diese Ergebnisse zeigen, dass innerhalb der ersten 10 Tage nach der Transplantation wichtige Phasen der Gewebeschädigung und Regeneration ausgelöst durch die Schädigung nach IR und weitestgehend ohne Beteiligung Alloantigen-abhängiger Faktoren ablaufen. Eine bedeutende Rolle als Mediatoren während dieser Phasen kommt dabei den Monozyten/Makrophagen zu. / Organ damage due to long cold preservation is associated with delayed graft function and has important effects on graft survival. Aim of this study was to determine the impact of ischemia-reperfusion (IR) injury compared to antigen-specific mechanisms and the effect of prolonged cold ischemia on intragraft injury and antigenicity during the early phase post transplantation. Rat renal grafts were four-hours cold-preserved, transplanted to syngeneic (Lew/Lew) or allogeneic recipients (F344/Lew) and harvested at 8 different time points after transplantation for further investigation of functional, immunhistochemical and histologic changes. In five additional syngen groups organs were cold preserved from 2 hours to 48 hours and harvested after 24 hours post transplantation. No significant differences in renal function, morphologic changes, cellular infiltration and expression of adhesion molecules occurred between syngeneic and allogeneic groups within the first 7 days. Initial functional impairment was accompanied by the influx of neutrophils and monocytes/macrophages together with morphologic changes reflecting acute tubular necrosis (ATN). Increasing infiltration of monocytes/macrophages paralleled functional and morphologic regeneration. Extravasation of neutrophils was mediated mainly by interaction of ICAM-1/LFA-1 and infiltration of monocytes/macrophages by VCAM-1/VLA-4. Treatment with the standard dose of Cyclosporin A (CsA) lead to a significant decrease of ED1-positive macrophage infiltration 10 days post NTx but the portion of ED2-positive macrophage subtype was not affected. Prolonged cold organ preservation lead to more severe vascular damage indicated by decreased color intensity and continuity of PECAM-1 staining on endothelial cells. Higher staining intensity for Tissue Factor (TF) on endothelium and infiltrating leukocytes implicated enhanced intragraft procoagulant capacity and alternative adhesion mechanisms. These results show that within the first 10 days post transplantation phases of tissue injury and repair after ischemia-reperfusion are largely independent of the immunologic background and monocytes/macrophages play an important role as mediators during these processes.

INTRODUÇÃO: O transplante traqueal continua um desafio. Contudo, avanços nas técnicas de revascularização dos enxertos traqueais e no conhecimento da imunobiologia da traquéia, indicam que esta técnica pode ser utilizada com freqüência no futuro próximo. A depuração mucociliar (DM) é o mecanismo de defesa inato mais importante das vias aéreas. A traquéia age como um órgão de defesa devido à DM. A DM ocorre por ação do batimento ciliar do epitélio respiratório que impele o muco que atapeta as vias respiratórias, carreando substâncias nocivas. Idealmente, a DM deve ser preservada em enxertos traqueais passíveis de utilização para transplante traqueal. Nosso intuito foi: 1) avaliar os efeitos da isquemia fria sobre a DM; e 2) avaliar a ação de soluções de preservação administradas por via tópica na manutenção da DM após isquemia fria. MÉTODOS: De 109 ratos Wistar foram obtidos 217 segmentos traqueais. Os segmentos foram distribuídos entre três grupos experimentais e um grupo Controle. Cada segmento foi submergido em LPD-glicose (grupo LPD), histidina-triptofano-cetoglutarato (grupo HTK) ou solução salina (grupo Salina). Avaliou-se a DM após 6,10,16 ou 24 horas de isquemia fria. No grupo Controle os segmentos foram analisados imediatamente após a extração, não havendo isquemia fria, nem submersão em soluções. A velocidade de transporte mucociliar (VTM) foi medida através de microscópio de luz, pela observação do movimento das partículas de muco na superfície dos segmentos traqueais. A freqüência de batimento ciliar (FBC) foi obtida pela sincronização entre o movimento ciliar observado pelo microscópio de luz e um estroboscópio. Em seguida, os segmentos foram corados com hematoxilina-eosina para analisar a integridade epitelial (IE) e a inflamação subepitelial (IS). Foi realizada análise quantitativa do muco intracelular por um programa de computador após coloração com azul de alcião (MI-AA) e PAS (MI-PAS). As amostras mais significativas foram analisadas qualitativamente por microscopia eletrônica de transmissão (ME). Foram realizadas duas análises: 1) grupo Controle e tempos de isquemia; e 2) grupo Controle e soluções de preservação (agrupado pelo tempo de isquemia). RESULTADOS: 1) grupo Controle e tempos de isquemia: O grupo controle foi melhor que os grupos submetidos a isquemia fria quanto à VTM (p=0,0001) e FBC (p=0,012). Contudo, não houve diferença na IE, IS e MI entre o grupo Controle e os demais grupos. 2) grupo Controle e soluções de preservação: O grupo controle apresentou melhor VTM que os grupos LPD, HTK e Salina após isquemia de 6h (p=0,001), 16h (p=0,009) e 24h (p=0,001). O grupo controle apresentou melhor FBC que os grupos LPD, HTK e Salina após isquemia de 24h (p=0,001). Não houve diferença entre os grupos na IE e IS. O grupo Controle apresentou melhor MI-AA que os grupos LPD após 16h (p=0,02) e HTK após 24h de isquemia (p=0,04). Não houve diferença entre os grupos à MI-PAS. À ME, o grupo Salina apresentou maiores alterações secundárias à isquemia do que os demais grupos. CONCLUSÕES: 1) A isquemia fria piora a DM; e 2) O uso de soluções de preservação administradas por via tópica não contribui para manutenção da DM após isquemia fria / INTRODUCTION: Tracheal transplantation is a challenging problem. Recent advances in graft revascularization, and reepithelialization renewed the interest on airway transplantation. Mucociliary clearance (MC) is the most important innate defense mechanism of the respiratory system. MC works by mucous transport carried out by ciliary beating function of the airway epithelium. Trachea acts as a defense organ in the respiratory system through the MC. Ideally, MC should be preserved in tracheal grafts used for transplantation. Preservation solutions improve organ preservation by decreasing ischemic injury. The purpose of the study was: 1) to evaluate the effects of cold ischemia on MC; and 2) to evaluate the impact of topically-applied preservation solutions on MC after cold ischemia. METHODS: From 109 male Wistar rats we obtained 217 tracheal segments. The segments were allocated to one of four groups. Segments were submerged in LPD-glucose (LPD group), histidine-tryptophan-ketoglutarate (HTK group), or saline solution (Saline group), and stored at 4C. MC was analyzed after 6, 10, 16 or 24h of ischemia. Control group have only segments that were analyzed right after extraction, not submitted to cold ischemia or submersion in preservation solutions. The mucociliary transport velocity (MTV) was measured by observation of mucous particle under the surface of the segments, through a light microscope. Ciliary beating frequency (CBF) was achieved by synchronization between cilia movement and a stroboscope flashlight. Tracheas were stained with hematoxylin-eosin in order to analyze the epithelial integrity (EI) and the subepithelial inflammation (SI). A quantitative analysis of the intracellular mucus stained with alcian blue (IM -AB) and PAS (IM-PAS) was achieved by a software. The most significant samples of the tracheal segments were qualitatively analyzed by transmission electronic microscopy (TEM). Two analyses were made: 1) Control group and ischemic time; and 2) Control group and preservation solutions. RESULTS: 1) Control group and ischemic time: Control group had better VTM (p=0,0001) and CBF (p=0,012) than the groups submitted to cold ischemia. However, there was no difference among Control group and the other groups on EI, SI, IM-AB, and IM-PAS. 2) Control group and preservation solutions: Control group showed better MTV than the LPD, HTK, and Saline groups after 6h (p=0,001), 16h (p=0,009) and 24h (p=0,001) of cold ischemia. Control group showed better CBF than the LPD, HTK, and Saline groups after 24h of ischemia (p=0,001). There was no difference among groups on EI and SI. Control group showed better IM-AB than both the LPD group after 16h of cold ischemia (p=0,02), and the HTK group after 24h of cold ischemia (p=0,04). There was no difference among the groups on IM-PAS. TEM showed more findings of ischemic lesion on Saline group. CONCLUSIONS: 1) Cold ischemia impairs MC; and 2) Topically-applied preservation solutions do not ameliorate MC after cold ischemia

Das zunehmende Durschnittsalter unserer Gesellschaft und der Mangel an Spenderorganen stellen bedeutende Herausforderungen für die Organtransplantation dar. Organe, die früher als "marginal" galten, werden heute routinemässig transplantiert. Gleichzeitig mit der Zunahme älterer Patienten auf der Warteliste steigt das Interesse an der Entwicklung von speziellen Allokations-Strategien. Basierend auf dem Konzept der Abstimmung des metabolischen Bedarfs des älteren Empfängers und der Kapazität der älteren Spenderniere entwickelte Eurotransplant daher das Eurotransplant Senior Program (ESP), welches im Januar 1999 gestartet wurde. Im Rahmen dieses Programms werden Nieren von über 65 Jahre alten Spendern lokal auf eine selektierte Gruppe über 65 Jahre alter, nicht immunisierter Empfänger übertragen. Das Ziel dieser 5-Jahres Analyse war es herauszufinden, ob das ESP erfolgreich seine Ziele erreicht hat, Organe von älteren Spendern optimal zu nutzen und die Zeit auf der Warteliste für ältere Empfänger zu verkürzen. Als Basis dienten Daten des Eurotransplant Information Systems (ENIS). Zusätzliche Informationen wurden für die ESP Patienten und zwei Kontrollgruppen mit entweder annähernd vergleichbarem Alter des Spenders (Kontrolle 1) oder des Empfängers (Kontrolle 2) erfasst. Insgesamt zeigt diese Auswertung, dass die Ziele des ESP erreicht wurden. Die Verfügbarkeit von älteren Spenderorganen wurde von 162 (10%) im Jahre 1998 auf 239 (fast 15%) im Jahre 2004 gesteigert. Die Wartezeit für ältere Empfänger verkürzte sich signifikant im Vergleich zu vor der Einführung des ESP und weiter im Verlauf der ersten 5 Jahre auf deutlich unter 4 Jahre, während sich die Wartezeit für Patienten in den Kontrollen die über ETKAS transplantiert wurden um bis zu einem Jahr verlängerte. Die kalte Ischämiezeit für ESP Patienten war signifikant kürzer mit etwa 12 im Vergleich zu ca. 17 Stunden für beide Kontrollen.Das Patienten- und Transplantatüberleben der Empfänger von Organen von über 65-jähriger Spendern wurde durch die ESP-Allokation, trotz 5-10% höherer Abstossungraten, nich negativ beeinflusst. Die Analyse der unabhängigen Risikofaktoren für akute Abstoßungsreaktionen weist darauf hin, dass ein verbessertes HLA matching unter Beibehaltung kurzer Ischämiezeiten möglicherweise von Vorteil wäre. / The aging society and the shortage of organs impose significant challenges to organ transplantation. As a result, organs previously considered marginal are now routinely used. At the same time, an increase in the number of elderly patients on renal transplant waiting lists has heightened interest in the development of special allocation strategies for these patients. As a result, Eurotransplant started the Eurotransplant Senior Program (ESP) in January 1999, an allocation scheme based on the concept of matching the metabolic demand of the recipient and the excretory capacity of the donor. The program obtaines kidneys from donors over 65 years and locally allocates them to a selected group of non-immunized patients in the same age group. The main objective of this evaluation was to find out if the allocation scheme is effective in using kidneys from elderly donors and if it shortens the waiting time for elderly patients. The Eurotransplant database was used as a starting point, and data added to the database by collecting additional information on the ESP patients, and on two control groups. The controls were observed over the same time period as the ESP patients, and matched them for either donor age (Control 1) or recipient age (Control 2). Overall, this 5-year analysis of the ESP shows that the objectives of the program have been met. The availability of elderly donors increased from 169 (10%) in 1998 to 239 (almost 15%) in 2004. The waiting time for elderly recipients transplanted within the ESP was successfully reduced compared to the waiting time before introduction of ESP and is now below 4 years, while waiting time in both control groups has increased by up to one year. The cold ischemia time for ESP patients was significantly shorter, with a mean of approximately 12 hours compared with over 17 hours in both control groups. Graft and patient survival in recipients of organs from donors age over 65 were not negatively impacted by the ESP allocation despite 5-10% higher acute rejection rates.Based on an analysis of independent risk factors the use of HLA matching instead of waiting time should be considered as an allocation criterion while maintaining a short cold ischemia time.

Islet transplantation has found its niche in diabetes treatment. It has contributed to a better quality of life and better glycemic control of patients with diabetes suffering from severe hypoglycemia that are not eligible for vascularized pancreas transplantation. Islet isolation is a technically challenging procedure. The different studies within this doctoral thesis aim to improve and standardize different steps in the isolation procedure. They are in particular looking to improve human pancreas preservation during cold storage, to optimize islet release from the exocrine tissue and to assess whether the isolated islet yield can be predicted from a biopsy. We found that pancreas preservation with pre-oxygenated perfluorodecalin (two-layer method) did not improve the ischemic tolerance of the human pancreas as compared to cold storage with the University of Wisconsin (UW) solution. Furthermore, in pancreas with long cold ischemia time (CIT) (&gt;10 hours), Histidine-Tryptophan-Ketoglutarate (HTK) had a limited preservation capacity as compared with the UW solution with respect to isolation outcome. We also found that during enzymatic pancreas digestion, Vitacyte HA was able to provide a similar islet yield and quality as Serva NB1 with less collagenase activity and shorter digestion time. We further describe the first experience with a new GMP manufactured enzyme called Liberase MTF-S for successful human islet isolation. Finally, we found that the isolated islet yield could not be predicted from a biopsy taken from the head of the pancreas concerning solely morphological parameters of the islets tissue. The improvement of pancreas preservation will allow for marginal organs with prolonged cold ischemia time to expand the donor pool. Better knowledge of how the pancreatic extracellular matrix is digested by collagenase will lead to a fast and predictable islet release from the exocrine tissue. By standardizing the isolation procedure and improving organ selection we will increase the success rate in human islet isolation, thereby making islet transplantation available for more patients.