Allogeneic hematopoietic chimerism leading to central tolerance has significant therapeutic potential. Establishment of hematopoietic chimerism created by stem cell transplantation has been shown to prevent and cure a number of autoimmune diseases and induce the most robust and long-lasting form of transplantation tolerance known. However, the realization of the vast clinical potential of hematopoietic chimerism for induction of transplantation tolerance has been impeded by the toxicity of the host conditioning regimen and the development of graft-versus-host disease (GVHD). This thesis describes the development of stem cell transplantation protocols that 1) reduce the host conditioning regimen; and 2) abrogate the development of GVHD. When applied to the treatment of autoimmune diabetic NOD mice, a model of type 1 diabetes, stem cell transplantation was able to 3) prevent autoimmune recurrence; and 4) permit curative pancreatic islet transplantation. I first describe a tolerance-based stem cell transplantation protocol that combines sub-lethal irradiation with transient blockade of the CD40-CD154 costimulatory pathway using an anti-CD154 antibody. With this protocol, I established hematopoietic chimerism in BALB/c mice transplanted with fully allogeneic C57BL/6 bone marrow. All chimeric mice treated with anti-CD154 antibody remained free of graft vs.host disease (GVHD) and accepted donor-origin but not third party skin allografts. It was similarly possible to create allogeneic hematopoietic chimerism in NOD/Lt mice with spontaneous autoimmune diabetes. Pancreatic islet allografts transplanted into chimeric NOD/Lt mice were resistant not only to allorejection but also to recurrence of autoimmunity. I conclude that it is possible to establish robust allogeneic hematopoietic chimerism in sub-lethally irradiated mice without subsequent GVHD by blocking the CD40-CD154 costimulatory pathway using as few as two injections of anti-CD154 antibody. I also conclude that chimerism created in this way generates donor-specific allograft tolerance and reverses the predisposition to recurrent autoimmune diabetes in NOD/Lt mice, enabling them to accept curative islet allografts. In order to further reduce the impediments associated with the implementation of allogeneic hematopoietic chimerism as a therapeutic modality, I adapted a costimulation blockade-based protocol developed for solid organ transplantation for use in stem cell transplantation. The protocol combines a donor-specific transfusion (DST) with anti-CD154 antibody to induce peripheral transplantation tolerance. When applied to stem cell transplantation, administration of DST, anti-CD154 antibody, and allogeneic bone marrow led to hematopoietic chimerism and central tolerance with no myeloablation (i.e. no radiation) and no GVHD in 3 different strains of mice. The development of donor-specific tolerance in this system was shown to involve deletion of both peripheral host alloreactive CD8+ T cells and nascent intrathymic alloreactive CD8+ T cells. In the absence of large numbers of host alloreactive CD8+ T cells, the cell transfusion that precedes transplantation need not be of donor-origin, suggesting that both allo-specific and non-allo-specific mechanisms regulate engraftment. Agents that interfere with peripheral transplantation tolerance partially impair establishment of chimerism. I conclude that robust allogeneic hematopoietic chimerism and central tolerance can be established in the absence of host myeloablative conditioning using a peripheral transplantation tolerance protocol.

The prevalence of diabetes is approaching epidemic proportions worldwide. There is currently no cure for type 1 diabetes, and successful treatment requires constant monitoring of blood sugars and use of exogenous insulin to prevent hyperglycemia. Diabetes will be curable when pancreatic β-islet cells can be transplanted into diabetes patients without requiring long-term immunosuppression. This will require learning more about the induction of functional tolerance, a state that maintains the competence of the immune system to most antigens but protects graft-specific antigens from immune rejection, permitting transplantation. One known mechanism of peripheral tolerance is T cell anergy, a phenotype of hypo-reponsiveness in CD4+ T cells. The focus of this thesis is a description of factors shown to be specific to the induction and maintenance of T cell anergy, whose loss reverses the anergic phenotype, restoring the ability of the cells to proliferate in response to antigen. The first of these is Egr-2, a zinc-finger transcription factor, whose presence is required for the induction of anergy induced in T cell clones by TCR stimulation in the absence of costimulation. Egr-2 is shown to be important to anergy induction but not anergy maintenance. In contrast, a negative costimulation receptor, PD-1, is shown to be necessary for the maintenance of anergy. It is possible that learning more about the genetic factors that orchestrate T cell anergy will prove useful in the development of tolerance-based protocols for organ and tissue transplantation without the use of long-term immunosuppression.

INTRODUÇÃO: A Moléstia de Dupuytren (MD) é uma doença crônica progressiva fibroproliferativa caracterizada por contraturas em flexão dos dedos. A origem deste processo está na proliferação de miofibroblastos e na síntese de matriz extracelular. Diversas técnicas já foram descritas no tratamento da MD. A falta de uma técnica capaz de associar altas taxas de sucesso com baixos índices de complicações e recidivas estimulou a procura por novas técnicas. Acredita-se que a propriedade totipotente das células-tronco presentes no tecido adiposo seria capaz de atuar na proliferação e na diferenciação dos fibroblastos em miofibroblastos, interrompendo a formação da fibrose e consequentemente a progressão da deformidade dos dedos. OBJETIVO: O objetivo primário deste estudo foi avaliar o efeito do enxerto de gordura, rico em células-tronco, nos pacientes com MD, submetidos a fasciectomia parcial aberta, por meio de avaliação funcional e goniométrica, e comparar com a técnica convencional (sem adição de enxerto de gordura). MÉTODO: Dois grupos totalizando 45 pacientes (24 pacientes no Grupo Controle e 21 no Grupo com gordura) foram operados pela técnica da fasciectomia parcial aberta. No Grupo controle era realizada apenas a fasciectomia parcial aberta. No Grupo com gordura, era realizada a fasciectomia parcial aberta e injetado, no local da corda ressecada, o enxerto de gordura, após o processamento do lipoaspirado de adipócitos proposto por Coleman (2006). O estudo foi prospectivo, randomizado e terapêutico. Os desfechos foram avaliados pela medida goniométrica do Déficit de Extensão Passiva Total (DEPT) e pelo escore funcional Brief Michigan Hand Questionnaire (BMHQ). As avaliações eram feitas no pré-operatório, com 6 semanas, 6 meses, 1 ano e 2 anos de seguimento. RESULTADOS: Na comparação com 6 semanas de pós-operatório, houve um aumento significativo da dor no Grupo com Gordura [mediana 2 ± 2,82 versus 0 ± 1,86 no grupo Controle (p=0,045)]. Os resultados do DEPT, não mostraram diferença significativa entre os grupos. Observamos piores resultados do escore funcional BMHQ com 6 meses e 1 ano de pós-operatório no Grupo com gordura (p=0,040 e p=0,047, respectivamente). Observamos ainda 9 casos (43%) de complicações no Grupo com gordura e 2 (8%) no Grupo Controle (p=0,019). CONCLUSÃO: O uso do enxerto de gordura associado à fasciectomia parcial aberta promoveu piores resultados funcionais comparado com a fasciectomia parcial aberta convencional, no curto prazo (um ano de seguimento pós-operatório). No entanto, resta a dúvida de qual seriam os resultados a longo prazo e, se as células-tronco, presentes no enxerto de gordura, poderiam interferir na recidiva da doença futuramente / BACKGROUND: Dupuytren\'s disease (DD) is a progressive chronic fibroproliferative disease characterized by flexion contractures of the fingers. The origin of this process is the proliferation of myofibroblasts and extra-cellular matrix synthesis. Several techniques have been described to treat the DD. The lack of a technique capable to associate high success rates with low rates of complications and recurrence stimulated the search for new techniques. It is believed that the totipotent property of the adipose-derived stem cells present in the processed lipoaspirate tissue would be able to inhibit the proliferation and differentiation of fibroblasts in myofibroblasts, interrupting the formation of fibrosis and consequently the progression of finger deformity. The primary objective of this study was to evaluate the effect of adipose-derived stem cells in patients with DD who underwent to open limited fasciectomy and compare with the conventional technique of limited fasciectomy. METHODS: A total of 45 patients were assigned in two groups in a single blind, prospective, randomized, controlled trial. All the patients were treated by the limited fasciectomy technique. In the control group (24 patients), only limited fasciectomy was performed. In the study group (21 patients), after the limited fasciectomy procedure, autologous lipoaspirate was injected at the site of the resected cord. Outcomes were assessed by the Total Passive Extension Deficit (TPED) and by the Brief Michigan Hand Questionnaire (BMHQ) functional score. The evaluations were performed by occupational therapists in the preoperative and at 6 weeks, 6 months, 1 year and 2 years. RESULTS: The study group presented higher rates of pain at 6 weeks postoperative (median 2 ± 2,82 versus 0 ± 1,86 on control group, p=0,045). TPED showed no significant differences between groups. BMHQ score, at 6 months and 1 year after surgery, were significantly inferior in the study group (p=0,040 e p=0,047, respectively). Patients in the study group had higher incidence of complications (9 patients, 43% versus 2, 8% in the control group, p=0,019). CONCLUSIONS: The autologous lipoaspirate associated to limited fasciectomy demonstrates inferior results regarding to functional score and pain compared to conventional limited fasciectomy, in short-term. Further long-term analysis is required to observe the effect of adipose-derived stem cells in the recurrences rates

Os desfechos clínicos e oncológicos dos pacientes submetidos à ressecção endoscópica do câncer gástrico precoce (CGP), considerando os critérios de indicação, comparados à cirurgia, não foram relatados em revisões sistemáticas. A pesquisa foi desenvolvida para estabelecer os desfechos de curto e longo prazos da ressecção endoscópica comparada à cirurgia no tratamento do CGP, elevando as informações para o nível de evidência 2a, melhor respaldando a prática clínica. A revisão sistemática com metanálises foi procedida utilizando-se as bases Medline, Embase, Cochrane, LILACS, Scopus e CINAHL. Onze coortes retrospectivas foram selecionadas para análise qualitativa e quantitativa. Todos os estudos incluem pacientes com CGP e comparam os desfechos nos dois braços. Os dados envolveram 2654 pacientes que preenchiam os critérios absolutos ou expandidos para ressecção endoscópica. Diferentes modalidades de tratamento endoscópico foram avaliadas, principalmente os procedimentos de ressecção, como endoscopic mucosal resection (EMR) e endoscopic submucosal dissection (ESD). As informações basearam-se nas características dos participantes, critérios de inclusão e exclusão, tipos de intervenções e desfechos (diferentes taxas de sobrevida, eventos adversos, ressecção completa, recorrência e mortalidade). As análises dos riscos absolutos dos desfechos foram feitas com o software RevMan, computando-se as diferenças de risco (DR) das variáveis dicotômicas. Dados de DR e intervalo de confiança de 95% (IC) foram calculados utilizando-se o teste de Mantel-Haenszel e a inconsistência foi qualificada e reportada em ?2 e método Higgins (I2). A análise de sensibilidade foi feita quando a heterogeneidade era maior que 50%. Todas as análises basearam-se inicialmente no modelo de efeito fixo. Dados de sobrevida de 3 anos estavam disponíveis em seis estudos (n = 1197). Não houve DR após os dois tratamentos (DR = 0,01, IC 95% = -0,02 a 0,05). A sobrevida de 5 anos (n = 2310) não demonstrou diferença significativa entre os grupos analisados (DR = 0,01, IC 95% = -0,01 a 0,03). A avaliação de 551 pacientes não evidenciou desigualdade na sobrevida de 10 anos entre as diferentes abordagens (DR = -0,02, IC 95% = -0,15 a 0,10). Dados de complicação estavam presentes em oito estudos (n = 2439), e diferença significativa foi detectada (DR = -0,08, IC 95% = -0,10 a -0,05), demonstrando melhores resultados com a endoscopia. As taxas de ressecção completa foram analisadas em 536 pacientes. Evidenciou-se diferença significativa entre o tratamento endoscópico e cirúrgico (DR = -0,13, IC 95% = -0,17 a -0,09), validando melhores resultados no último grupo. A recorrência foi avaliada em cinco pesquisas (n = 1331) e não houve diferença entre as duas formas de terapêutica (DR = 0,01, IC 95% = -0,00 a 0,02). As taxas de mortalidade foram obtidas de quatro estudos (n = 1107), e não se evidenciou diferença entre os grupos envolvidos (DR = -0,01, IC 95% = -0,02 a 0,00). Conclui-se que as taxas de sobrevida de 3, 5 e 10 anos, recorrência e mortalidade são semelhantes em ambos os grupos. Considerando-se as taxas de complicação, a abordagem endoscópica confere resultados mais apropriados e, analisando-se as taxas de ressecção completa, ela é inferior à cirurgia / Clinical and oncological outcomes of endoscopic resection of early gastric cancer (EGC), considering the indication criteria, compared to surgery, have not been reported in systematic reviews. To address the short- and long-term outcomes of endoscopic resection compared to surgery in the treatment of EGC, a systematic review was performed, establishing the available data to an unpublished 2a strength of evidence, better handling clinical practice. A systematic review and meta-analysis using Medline, Embase, Cochrane, LILACS, Scopus and CINAHL databases were done. Eleven retrospective cohort studies were selected to quantitative and qualitative synthesis. All studies included patients diagnosed with EGC that compared outcomes considering endoscopic treatment and surgery. The included records involved 2654 patients with EGC that filled the standard or expanded indications for endoscopic resection. Different endoscopic treatment modalities were analyzed, mainly mucosal resection procedures such as endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD), compared to surgery. Information of the selected studies was extracted on characteristics of trial participants, inclusion and exclusion criteria, types of interventions and outcomes (different survival rates, adverse events, complete resection, recurrence and mortality rates). The analysis of the absolute risks of the outcomes was performed using the software RevMan, by computing risk differences (RD) of dichotomous variables. Data on RD and 95% confidence interval (CI) for each outcome were calculated using the Mantel-Haenszel test and inconsistency was qualified and reported in X2 and the Higgins method (I2). Sensitivity analysis was performed when heterogeneity was higher than 50%. All pooled analyses were initially based on fixed-effects model. Three-year survival data were available for six studies (n = 1197). There were no RD in 3-year survival data after endoscopic and surgical treatment of EGC (RD = 0.01, 95% CI = -0.02 to 0.05). Five-year survival data (n = 2310) showed no evidence of a difference between the two groups (RD = 0.01, 95% CI = -0.01 to 0.03). The data analysis, in 551 patients, showed no difference in 10-year survival rates between the approaches (RD = -0.02 and 95% CI = -0.15 to 0.10). Complication data were identified in eight studies (n = 2439). A significant difference was detected (RD = -0.08, 95% CI = -0.10 to -0.05), demonstrating better results with endoscopic approach. Complete resection data was analyzed in 536 patients. It showed significant difference in complete resection rates between endoscopic and surgical treatment of EGC (RD = -0.13, 95% CI = -0.17 to -0.09), exhibiting improved results in the surgical group. Recurrence data were analyzed in five studies (n = 1331) and there was no difference between the approaches (RD = 0.01, 95% CI = -0.00 to 0.02). Mortality data were obtained in four studies (n = 1107), and there was no difference between treatment modalities (RD = -0.01, 95% CI = -0.02 to 0.00). This systematic review concludes that 3-, 5- and 10-year survival, recurrence and mortality rates are similar for both groups. Considering procedure-related complication rates, endoscopic approach achieves significantly better results and, analyzing complete resection data, it is considered worse than surgery

Estudos baseados em parâmetros fisiológicos tem mostrado que a fisioterapia tem papel imperativo na assistência de pacientes pré e pós-operatórios. Os efeitos da assistência fisioterapêutica na unidade de terapia intensiva (UTI) sobre o tempo de ventilação mecânica invasiva (VMI), tempo de internação e mortalidade do paciente crítico não foram elucidados. Tampouco, estudos sobre o impacto do turno diário da assistência fisioterapêutica nestes desfechos tem sido realizados. A despeito disto, e, possivelmente, baseadas na experiência clínica, as UTIs brasileiras adotarão turnos de 18 horas de assistência fisioterapêutica na UTI para atender a uma regulamentação governamental. O objetivo deste estudo foi comparar o efeito da assistência fisioterapêutica na UTI em turno diário de 24 horas (Fisio-24) ao turno diário de 12 horas (Fisio-12), sobre o tempo de VMI, tempo de internação na UTI, frequência de complicações respiratórias relacionadas a VMI e custos indiretos de pacientes pós-operatórios. Este estudo observacional, prospectivo, de coorte incluiu 114 pacientes de UTIs com Fisio-12 e 152 pacientes de UTIs com Fisio-24 em condição pós-operatória, idade 18 anos, submetidos a VMI por 24 horas e admitidos na UTI para rotina pós-operatória. Foram coletados dados demográficos e cirúrgicos. Os desfechos primários deste estudo foram tempo de VMI, tempo de internação na UTI, complicações respiratórias relacionadas a VMI e custos indiretos. O desfecho secundário foi o dia-livre de ventilação (VFD). Os custos foram avaliados através do Omega French Score que compreende três categorias (Omega 1, 2 e 3). Um modelo de regressão linear múltipla (MRL) foi construído para verificar a associação entre o turno diário de assistência fisioterapêutica na UTI e o tempo de VMI. A despeito dos pacientes Fisio-24 serem mais velhos (p=0,002), possuírem maior número de comorbidades (p=0,001), maior frequência de risco cirúrgico moderado a alto (p=0,003), maior frequência de complicações intra operatórias (p=0,012) e insuficiência renal aguda dialítica (p<0,001), comparados aos pacientes Fisio-12, apresentaram melhores desfechos clínicos, tais quais, menor mediana de tempo de VMI (4 dias versus 6 dias; p=0,002), maior mediana de VFD (24 dias versus 21 dias; p=0,004) e menor mediana de tempo de internação na UTI (10 dias versus 15 dias; p=0,015). Não foi encontrada diferença na frequência de complicações respiratórias relacionadas à VMI entre os dois grupos (p=0,704), embora pacientes Fisio-24 tenham recebido mais sessões de fisioterapia respiratória durante a internação na UTI (25 versus 20 sessões; p=0,014). Pacientes Fisio-24 apresentaram menor pontuação do Omega 2 (p=0,007). O MRL manteve como variáveis explicativas o número de sessões de fisioterapia respiratória, APACHE II, realização de Neurocirurgia e o turno diário de assistência fisioterapêutica na UTI. Mantidas constantes as outras variáveis explicativas, a presença de Fisio-24 na UTI reduziu o tempo de VMI em 2,80 unidades. Concluí-se que pacientes pós-operatórios admitidos em UTIs com Fisio-24 apresentaram menores tempo de VMI e tempo de internação na UTI, maior VFD, contudo, não foi encontrada diferença na frequência de complicações respiratórias relacionadas à VMI entre Fisio-12 e Fisio-24. A redução da pontuação de Omega 2 nos pacientes Fisio-24 não foi suficiente para promover diferenças no custo indireto entre os grupos / According to studies based on physiologic parameters, physiotherapy plays an imperative role on pre and postoperative patients. The effects of physiotherapy assistance (PTA) in the intensive care unit (ICU) on length of invasive mechanical ventilation (IMV), length of ICU stay, frequency of ventilator-associated pneumonia and mortality remain unclear. Moreover, studies about impact of PTA shifts have not been conducted. Despite this fact, and possibly based on clinical experiences, Brazilian ICUs are going to adopt 18 hours of PTA shifts in order to attend a governmental regulation. The objective of this study was to compare the effects of 24-hour PTA (Physio-24) to 12-hour PTA (Physio-12) daily shifts in the ICU on length of IMV, length of ICU stay, frequency of respiratory complications related to IMV and indirect costs of postoperative patients. This observational, prospective and cohort study included 114 patients from ICUs with Physio-12 and 152 patients from ICU with Physio-24. Patients presented postoperative conditions, were aged 18 years, who underwent IMV 24 hours and were admitted on ICU for postoperative routine. We collected demographical and surgical data. Our primaries end-points were duration of IMV, length of ICU stay, frequency of respiratory complications related to IMV and indirect costs. The secondary end-point was ventilator-free days (VFD). Indirect costs were assessed by Omega French Score which comprises three categories (Omega 1, 2 and 3). In addition, a multiple linear regression model (MLR) was constructed to verify the association between daily shifts of PTA in ICU and length of IMV. Despite of the fact that Physio-24 patients were older (p=0.002), with more severe conditions such as higher number of co morbidities (p<0.001), higher presence of moderate to severe surgical risk (p=0.003), higher frequency of intraoperative complications (p=0.012) and dialytic acute renal failure in ICU (p<0.001), compared to Physio-12 patients, they presented better clinical outcomes such as fewer median days spent in IMV (4 versus 6 days; p=0.002), higher median of VFD (24 versus 21 days; p=0.004) and shorter median of ICU stay (10 versus 15 days; p=0.015). No differences were found concerning respiratory complications related to IMV between groups (p=0.704), although Physio-24 patients had received more sessions of chest physiotherapy during ICU stay (25 versus 20 sessions; p=0.014). Physio-24 patients presented lower scores of Omega 2 (p=0.007). The number of chest physiotherapy sessions, APACHE II, Neurosurgery, and daily shifts of PTA in ICU remained as independent variables to length of IMV in the MLR model. According to this model, Physio-24 may reduce 2.80 units from length of IMV if the other independent variables are constant. We concluded that postoperative patients admitted in ICUs with daily shifts of 24-hour PTA showed shorter length of IMV and length of ICU stay and increased VFD; however, no reduction in frequency of respiratory complications related to IMV was found between groups. Despite the fact that Physio-24 patients had lower score of Omega 2, it was not enough to provoke a difference on indirect costs between Physio-12 and Physio-24 patients

Magnesium alloys have been widely explored as potential biomaterials, but several limitations to using these materials have prevented their widespread use, such as uncontrollable degradation kinetics which alter their mechanical properties. In an attempt to further the applicability of magnesium and its alloys for biomedical purposes, two novel magnesium alloys Mg-Zn-Cu and Mg-Zn-Se were developed with the expectation of improving upon the unfavorable qualities shown by similar magnesium based materials that have previously been explored. The overall performance of these novel magnesium alloys has been assessesed in three distinct phases of research: 1) analysing the mechanical properties of the as-cast magnesium alloys, 2) evaluating the biocompatibility of the as-cast magnesium alloys through the use of in-vitro cellular studies, and 3) profiling the degradation kinetics of the as-cast magnesium alloys through the use of electrochemical potentiodynamic polarization techqnique as well as gravimetric weight-loss methods. As compared to currently available shape memory alloys and degradable as-cast alloys, these experimental alloys possess superior as-cast mechanical properties with elongation at failure values of 12% and 13% for the Mg-Zn-Se and Mg-Zn-Se alloys, respectively. This is substantially higher than other as-cast magnesium alloys that have elongation at failure values that range from 7-10%. Biocompatibility tests revealed that both the Mg-Zn-Se and Mg-Zn-Cu alloys exhibit low cytotoxicity levels which are suitable for biomaterial applications. Gravimetric and electrochemical testing was indicative of the weight loss and initial corrosion behavior of the alloys once immersed within a simulated body fluid. The development of these novel as-cast magnesium alloys provide an advancement to the field of degradable metallic materials, while experimental results indicate their potential as cost-effective medical devices.