OBJETIVO: avaliar a concordância entre duas titulações decrementais de PEEP guiadas por tomografia de impedância elétrica (TIE): uma rápida, com tempo total inferior a 7 minutos, e uma lenta, com 40 minutos, e comparar os efeitos hemodinâmicos causados pelas duas titulações; e como objetivo secundário, comparar os efeitos fisiológicos da PEEP ótima escolhida pela TIE com a PEEP escolhida pela tabela PEEP-FiO2 do ARDSNet, durante 4 horas de ventilação mecânica. MÉTODOS: Trata-se de um estudo clínico, randomizado, unicêntrico que incluiu pacientes hipoxêmicos no pós-operatório imediato de cirurgia cardíaca. Os pacientes foram randomizados em 3 grupos: Titulação Rápida, Titulação Lenta ou Controle (tabela PEEP-FiO2 do ARDSNet). Os grupos Titulação Rápida e Titulação Lenta foram submetidos a duas titulações de PEEP guiadas pela TIE (uma rápida e uma lenta) precedidas de manobra de recrutamento alveolar. A titulação da PEEP foi realizada da PEEP de 23 cmH2O até a PEEP de 5 cmH2O, em passos decrementais de 2 cmH2O, com duas durações: 40 segundos na Titulação Rápida ( < 7 minutos no total), ou 4 minutos na Titulação Lenta (40 minutos no total). A PEEP ótima (PEEPTIT) foi definida como a menor PEEP com menos de 5% de colapso estimado pela TIE. No grupo Controle, a PEEP foi ajustada de acordo com a oxigenação, baseada no protocolo ARDSNet. Todos os pacientes permaneceram em ventilação mecânica por 4 horas com a PEEP de acordo com seu grupo, e foram monitorados com TIE durante todo estudo. A comparação entre as manobras de Titulação Rápida e Lenta incluiu as seguintes variáveis: colapso recrutável e hiperdistensão estimados pela TIE, valor da PEEPTIT, menor pressão arterial média e dose de noradrenalina durante as titulações. A comparação com o grupo controle incluiu: nível de PEEP, complacência do sistema respiratório, driving pressure, colapso e hiperdistensão estimados pela TIE (associação entre o ZMIN - que representa a aeração - e a Complacência Z avaliados regionalmente), e oxigenação (PaO2/FiO2) ao longo das 4 horas de acompanhamento. RESULTADOS: Não houve diferença entre colapso e hiperdistensão estimados pela Titulação Rápida e Lenta, nem nos valores de PEEPTIT (13 ± 4 vs 14 ± 4 cmH2O, P=0,13). A pressão arterial média foi maior durante a titulação rápida (92mmHg [IQ25-75%: 81-111] vs 83mmHg [71-93], P=0,035) e não houve diferença no uso de noradrenalina. A PEEPTIT pela TIE foi significativamente mais alta do que a PEEP do Grupo Controle. Os grupos Titulação Rápida e Lenta apresentaram comportamentos semelhantes após ajuste da PEEPTIT, e houve aumento significativo na complacência do sistema respiratório no primeiro minuto, que permaneceu maior que a condição basal ao final das 4 horas (T. Lenta: 0,73 ± 0,2 vs 0,89 ± 0,1 mL/cmH2O/Kg do peso predito, P < 0,001; T. Rápida: 0,7 ± 0,1 vs 0,85 ± 0,2 mL/cmH2O/Kg do peso predito, P < 0,001); com a melhora da complacência houve redução da driving pressure e, ao final das 4 horas, esta permaneceu menor que no tempo basal. No grupo controle, a complacência não mudou durante as 4 horas de ventilação mecânica (0,63 ± 0,1 vs 0,58 ± 0,1 mL/cmH2O/Kg do peso predito, P=0,34) e a driving pressure aumentou significativamente. A oxigenação melhorou nos três grupos, mas foi mais alta nos grupos PEEPTIT guiados pela TIE. Após ajuste da PEEPTIT, em ambos os grupos (Titulação Rápida e Lenta) houve aumento da aeração nas regiões dependente e não dependente, recrutamento na região dependente e não houve hiperdistensão na região não dependente, mesmo com a PEEP mais alta. No grupo controle, de acordo com a tabela PEEP-FiO2, a necessidade de PEEP diminuiu ao longo do tempo (6,1 ± 1,4 cmH2O no tempo 4 horas), causando redução da aeração. CONCLUSÕES: Houve concordância entre titulações rápida e lenta guiadas pela TIE em relação à PEEPTIT, estimativa de colapso e hiperdistensão para cada passo de PEEP; a titulação rápida da PEEP pôde ser realizada com menor repercussão hemodinâmica quando comparada com a titulação convencional lenta. A PEEP individualizada pela TIE melhorou a complacência, reduziu a driving pressure e melhorou a oxigenação, sem causar hiperdistensão quando comparada com protocolo ARDSNet. Descritores: respiração com pressão positiva; mecânica respiratória; impedância elétrica; tomografia; procedimentos cirúrgicos cardíacos; respiração artificial; insuficiência respiratória; tomografia de impedância elétrica / OBJECTIVE: to assess the agreement of \"optimum-PEEP\" values selected by two decremental PEEP trials guided by electrical impedance tomography (EIT): a Fast one lasting less than 7 minutes, and a Slow one lasting 40 minutes, and to compare the hemodynamic effects caused by these two trials; as secondary objectives, we aimed at comparing the physiological effects of the optimum-PEEP chosen by EIT (Fast or Slow) with those chosen by ARDSNet PEEP-FiO2 table during the subsequent 4 hours of mechanical ventilation. METHODS: in this single center, randomized controlled trial, hypoxemic patients immediately after cardiac surgery were randomized into three groups: Fast Titration (FAST-EIT), Slow Titration (SLOW-EIT) and Control (ARDSNet PEEP-FiO2 table). After recruiting maneuvers, and starting from a PEEP of 23 cmH2O, the FAST-EIT and SLOW-EIT groups were submitted to decremental PEEP trials, in steps of 2 cmH2O, until reaching 5 cmH2O, with two different durations: 40 seconds (the entire maneuver lasted < 7 minutes) or 4 minutes (entire maneuver lasted 40 minutes). The optimum-PEEP (PEEPTIT) was defined as the lowest PEEP with less than 5% of collapse estimated by EIT. In the control group, PEEP was adjusted according to oxygenation based on ARDSNet protocol. All patients were ventilated for 4 hours with PEEP according to their randomized groups, and all were monitored with EIT during the study. The comparison between Fast and Slow PEEP trials included: recruitable collapse and hyperdistension estimated by EIT, level of optimum PEEP, lowest mean arterial pressure and norepinephrine doses during the trials. The comparison with the control group included: level of PEEP, compliance and driving pressure, collapse (aeration) and hyperdistension estimated with EIT, and oxygenation (PaO2/FiO2) during 4 hours of mechanical ventilation. RESULTS: There was no difference between recruitable collapse and hyperdistension estimated by EIT between Fast and Slow maneuvers, as well as for the PEEPTIT (13 ± 4 vs 14 ± 4 cmH2O, P=0.13). Mean arterial pressure was higher during the Fast maneuver in comparison to the Slow maneuver (92mmHg [IQ25-75%: 81-111] vs 83mmHg [71-93], P=0.035), without differences in norepinephrine. FAST-EIT and SLOW-EIT groups presented similar changes during the time: after set PEEPTIT there was an immediate and significant improvement in respiratory-system compliance, which remained above baseline condition during the 4 hours of mechanical ventilation (SLOW-EIT: from 0.73 ± 0.2 to 0.89 ± 0.1 mL/cmH2O/Kg of PBW, P < 0.001; FAST-EIT: from 0.7 ± 0.1 to 0.85 ± 0.2 mL/cmH2O/Kg of PBW, P < 0.001); as respiratory compliance improved, driving pressure significantly reduced and remained lower than the baseline condition after 4 hours. In the control group, respiratory compliance did not change between baseline and 4 hours (from 0.63 ± 0.1 to 0.58 ± 0.1 mL/cmH2O/Kg of PBW, P=0.34) but driving pressure significantly increased as PEEP decreased. Oxygenation improved in all groups, but it was higher in the EIT groups. After setting PEEPTIT in both EIT groups (Fast or Slow), there was an increase in aeration in both, nondependent and dependent regions. In contrast, regional compliance increased in the dependent region and didn\'t change in nondependent region, suggesting that the strategy caused long-lasting recruitment of dependent regions and did not produced hyperdistension of non-dependent lung. In the control group, the required PEEP, adjusted by ARDSNet PEEP-FiO2 table, decreased along the time, causing evident collapse in EIT derived signals. CONCLUSION: There was no difference between recruitable collapse and hyperdistension estimated by EIT and PEEPTIT between Fast and Slow maneuvers; Fast PEEP trial guided by EIT could be performed in less than 7 minutes, with less hemodynamic consequences than the traditional Slow maneuver. Individualized PEEP guided by EIT improved respiratory compliance, reduced driving pressure and improved oxygenation without causing hyperdistension - when compared to a PEEP set according the ARDSNet protocol

Os shunts vasculares temporários (SVT) são utilizados como uma técnica eficaz para reestabelecer rapidamente o fluxo sanguíneo em casos de lesão vascular com isquemia do membro ou órgão acometido, no qual a revascularização deverá ser postergada. Habitualmente, o SVT é inserido dentro dos cotos proximal e distal do vaso lesado através de uma abertura na pele, visando restaurar a perfusão e interromper a isquemia. O objetivo deste estudo é comparar a pressão arterial média (PAM em mmHg) e o fluxo sanguíneo (em ml/min) em dois modelos de SVT, um habitual e outro implantado por punção, bem como o tempo para a inserção destes dispositivos e suas patências primária e secundária. Realizamos experimentos em 30 suínos, somando 60 intervenções de revascularização arterial temporária dos membros posteriores: trinta SVT habituais e trinta por punção. Analisamos a PAM durante os procedimentos nos membros posteriores e o fluxo através dos dois tipos de SVT. A análise de fluxo mostrou uma diferença significativa entre os SVT testados (p=0,001), sendo menor no grupo SVT por punção. No entanto, o tempo decorrido (min) para inserção do SVT habitual foi maior do que o tempo para inserção do SVT por punção (15,32 ± 3,08 vs. 10,37 ± 1,7, p=0,001). Além disso, observamos uma recuperação da PAM nos membros submetidos aos dois tipos de SVT próxima à PAM sistêmica em 100% dos experimentos. Os resultados revelaram patência primária, secundária, e taxa de complicações similares entre os dois tipos de SVT. Concluímos que o fluxo foi menor no SVT por punção, mas a recuperação da PAM foi semelhante e com menor tempo de inserção do SVT por punção / Temporary vascular shunts (TVS) are used as an effective technique to rapidly restore blood flow in cases of vascular injury with ischemia of the affected limb or organ, in which revascularization shall be postponed. Usually, TVS is positioned within the proximal and distal stumps of the injured vessel, through an opening of the skin, in order to restore perfusion and stop the ischemia. We sought to compare mean blood pressure (MBP in mmHg) and blood flow (ml/min) between two types of TVS, a standard one and a puncture one, as well as the time spent to insert these devices. We performed an experimental study on 30 pigs, including 60 vascular interventions in posterior limbs: thirty standard TVS and thirty puncture TVS. MBP was analyzed during the interventions in both posterior limbs and the flow through both types of TVS. Flow analysis revealed a significant difference between the two types of TVS (p=0,001), being lower in the puncture TVS. However, the time spent during standard TVS insertion was greater than that of the puncture shunt (15,32 ± 3,08 min vs.10,37 ± 1,7 min, p=0,001). In addition, we observed a limb MBP recovery close to systemic MBP in 100% of the experiments. The results show similar primary and secondary patency and complication rate in both TVS types. Therefore, we conclude that the flow was lower in the puncture TVS, but the MBP recovery was similar and it took less time to be inserted

Objetivos: Avaliar os desfechos da ressecção endoscópica em comparação à cirurgia no tratamento dos adenomas de papila. Métodos: Foi realizada uma revisão sistemática com metanálise de acordo com as recomendações do Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). As bases de dados Medline, Embase, Cochrane, Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS), Scopus and Cumulative Index to Nursing and Allied Health Literature (CINAHL) foram escaneadas. Os estudos incluíram pacientes com adenoma de papila e dados de comparação dos tratamentos endoscópico e cirúrgico para os seguintes desfechos: Ressecção primária completa; Sucesso primário; Recorrência; Sucesso final; e Complicações. A análise foi baseada em modelos de efeito randômico e fixo. Resultados: Cinco estudos de coorte retrospectivo foram selecionados, com um total de 465 pacientes. Todos os estudos tinham dados de ressecção primária completa disponível, mostrando uma diferença favorável ao tratamento cirúrgico (Diferença de riscos = -0,22; Intervalo de confiança de 95% = -0,41 a -0,04). Dados de Sucesso primário também foram identificados em todos os cinco estudos. A análise mostrou que a abordagem cirúrgica supera o tratamento endoscópico neste desfecho (DR = -0,13; IC 95% = -0,24 a -0,02). Dados de recorrência foram encontrados em todos os estudos (465 pacientes), com benefício para o tratamento cirúrgico (DR = 0,12; IC 95% = 0,01 a 0,22). Analisando o desfecho de Sucesso final, disponível em todos os estudos, não encontramos diferença entre as duas abordagens terapêuticas (DR = -0,06; IC 95% = -0,15 a 0,04). Três estudos (251 pacientes) apresentaram dados de complicação, e a análise não mostrou diferença entre os tratamentos endoscópico e cirúrgico (DR = -0,15; IC 95% = -0,53 a 0,23), sem a possibilidade de descartar o viés de seleção para este desfecho. Conclusões: Considerando os desfechos de ressecção primária completa, sucesso primário e recorrência na comparação do tratamento cirúrgico com o tratamento endoscópico para adenomas de papila, a abordagem cirúrgica tem resultados significativamente melhores. Com relação ao sucesso final, não houve diferença entre os dois tratamentos. No desfecho das taxas de complicação, esta revisão sistemática não permite uma conclusão confiável devido à presença de alta heterogeneidade e provável viés de publicação nesta comparação / Objectives: To address the outcomes of endoscopic resection compared to surgery in the treatment of ampullary adenomas. Methods: A systematic review and meta-analysis was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations. Medline, Embase, Cochrane, Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS), Scopus and Cumulative Index to Nursing and Allied Health Literature (CINAHL) databases were scanned. Studies included patients with ampullary adenomas and comparison data considering endoscopic treatment and surgery for the following outcomes: Complete primary resection; Primary success; Reccurence; Final success; and Complications. The analysis were based on both random and fixed effects model. Results: Five retrospective cohort studies were selected, with 465 patients. All five studies had complete primary resection data available, showing a difference that favours surgical treatment (Risk Difference = -0.22, 95% Confidence Interval = -0.41 to -0.04). Primary success data were identified in all five studies too. Analysis showed that surgical approach overcome endoscopic treatment in this outcome (RD = -0.13, 95% CI = -0.24 to -0.02). Recurrence data was found in all studies (465 patients), with benefit for the surgical treatment (RD = 0.12, 95% CI = -0.01 to 0.22). Analyzing the final success outcome, available in all studies, we found no difference between the two therapeutic approaches (RD = -0.06, 95% CI = -0.15 to 0.04). Three studies (251 patients) presented complication data and analysis shown no difference between the approaches (RD = -0.15, 95% CI = -0.53 to 0.23), not discarding the possibility of presence of selection bias for this outcome. Conclusions: Considering complete primary resection, primary success and recurrence outcomes, surgical approach achieves significantly better results. Regarding the final success, there was no difference between the two treatments. Addressing complication data, this systematic review does not allow for a reliable conclusion due to the presence of high heterogeneity and likely publication bias in this comparison

Introdução: Descrita em 1983 e de sólida aplicação clínica, o impacto da microcirurgia endoscópica transanal (TEM) sobre a função anorretal permanece pouco conhecido. Os objetivos do presente estudo foram avaliar o impacto da TEM na função anorretal conforme avaliações clínicas (Wexner score) e funcional (manometria anorretal) antes e após a cirurgia. Método: Prospectivamente, 23 pacientes consecutivos com lesões retais foram operados com o uso do equipamento TEO® (Karl Storz, Tuttlingen, Alemanha). Para todos os pacientes, o valor do escore de Wexner foi obtido antes e após a cirurgia (7, 30 e 90 dias), e a eletromanometria anorretal foi realizada antes da cirurgia e também no pós-operatório (30 e 90 dias). Resultados: Quatorze pacientes eram homens. A idade média foi 53,7 (24-81) anos. A distância média da lesão à linha pectínea foi de 7 (2-15) cm. A histopatologia revelou adenoma em 14 (61%), tumor neuroendócrino em 5 (21,7%), carcinoma invasivo em 3 (13%) e pólipo hiperplásico em 1 (4,3%) caso. A duração média do seguimento pós-operatório foi de 5 (3-7) meses. O escore de Wexner foi significativamente menor aos 30 dias em comparação com 7 dias (Wilcoxon, p = 0,03). A capacidade retal foi significativamente menor aos 30 dias após a cirurgia e recuperada aos 90 dias após a cirurgia (ANOVA, p = 0,04). Conclusões: Após TEM, um impacto modesto na função anorretal pode ser observado. O comprometimento transitório resulta de perda de capacidade retal e não por comprometimento dos esfíncteres anais cessando completamente 90 dias após a cirurgia. Em última análise, não conseguimos detectar um impacto na qualidade de vida após TEM / Background: The impact of transanal endoscopic microsurgery (TEM) on anorectal function remains poorly available, particularly when considering that the technique involves undertaking full- or partial-thickness excision of the rectal wall. Moreover, in spite of wide adoption of TEM, its impact on quality of life remains unknown since most evidence derives from retrospective studies. Objective: The objectives of the present study were to evaluate the impact of TEM on sphincter function determined by clinical (Wexner score), functional (anorectal manometry), and quality of life (FIQL) evaluations conducted before and after surgery. Design: prospective, observational, single-center, 23 consecutive patients with rectal lesions underwent were operated on using the TEO® equipment (Karl Storz, Tuttlingen, Germany). Wexner and FIQL scores were obtained before and after surgery (7 days, 30 days and 90 days postoperatively). Anorectal manometry was obtained before surgery, and postoperatively after 30 and 90 days. Main Outcome Measures: Wexner and FIQL scores; anorectal manometry results. Results: Fourteen patients were men. Mean age was 53.7 (24-81) yrs. Mean distance from the lesion to the dentate line was 7 (2-15) cm. A full- thickness resection was undertaken in 18 (78.3%) cases. Histopathology revealed adenoma in 14 (61%), neuroendocrine tumor in 5 (21.7%), invasive carcinoma in 3 (13%), and hyperplastic polyp in 1 (4.3%) case. Postoperative rectal wound separation occurred in 2 patients and 1 patient developed atrial fibrillation. The mean duration of postoperative follow-up was 5 (3-7) months. Overall, Wexner score significantly declined between postoperative days 7 and 30 (Wilcoxon, p = 0.03). Rectal compliance exhibited significant decline 30 days after surgery and recovery at 90 days after surgery (ANOVA, p = 0.04). It was not possible to measure any difference in the FIQL results before and after surgery. Limitations: small sample size; limited follow-up. Conclusions: Following TEM, a modest impact on anorectal function could be confirmed. Interestingly, anorectal function impairment after surgery was not due to sphincter dysfunction, but resulted from loss of rectal compliance. Ultimately, we could not detect a significant impact on quality of life after TEM

The transplantation of allogeneic cells and tissues for the treatment of human disease has been a life-saving procedure for many thousands of patients worldwide. However, to date, neither solid organ transplantation nor bone marrow transplantation have reached their full clinical potential. Significant limitations to the advancement of clinical transplantation stem from our current inability to prevent the rejection of allogeneic tissues by the immune system of the host. Similarly, in patients that receive allogeneic bone marrow transplants, we cannot permanently prevent the engrafted immune system from mounting a response against the patient. This problem, termed graft versus host disease is the most prevalent cause of morbidity and mortality in recipients of allogeneic bone marrow transplants. Clinically, we rely on lifelong immunosuppression to prolong survival of allogeneic tissues within the host. Our currently available therapeutics burden patients with side-effects that range from being unpleasant to life-threatening, while in most cases offering only a temporary solution to the problem of alloimmunity. Efforts are underway to develop protocols and therapeutics that more effectively prevent the pathology associated with alloimmunity. To minimize patient risk, extensive pre-clinical studies in laboratory animals are conducted to predict clinical responses. In the case of immunologic studies, many of these pre-clinical studies are carried out in murine models. Unfortunately, studies of murine immunity often do not predict outcomes in the clinic. One approach to overcome this limitation is the development of a small animal model of the human immune system. In this dissertation, we hypothesized that NOD-scid IL2rγnull mice engrafted with human peripheral blood mononuclear cells (PBMC), termed the hu-PBMC-NOD-scid IL2rγnull model, would provide a model that more accurately reflects human immunity in vivo than other models currently available. To investigate this possibility, we first investigated whether NOD-scid IL2rγnull mice were able to support the engraftment of human PBMC. We found that NOD-scid IL2rγnull mice engraft with human PBMC at much higher levels then the previous gold standard model, the NOD-scid mouse. We then investigated the kinetics of human cell engraftment, determined the optimal cell dose, and defined the influence of injection route on engraftment levels. Even at low PBMC input, NOD-scid IL2rγnullmice reproducibly support high levels of human PBMC engraftment. In contrast to previous stocks of immunodeficient mice, we observed low intra- and interdonor variability of engraftment. We next hypothesized that the human PBMC engrafted in NOD-scid IL2rγnull mice were functional and would reject transplanted allogeneic human tissues. To test this, human islets were transplanted into the spleen of chemically diabetic NOD-scid IL2rγnull mice with or without intravenous injection of HLA-mismatched human PBMC. In the absence of allogeneic PBMC, the human islets were able to restore and maintain normoglycemia. In contrast, human islet grafts were completely rejected following injection of HLA-mismatched human PBMC as evidenced by return to hyperglycemia and loss of human C-peptide in the circulation. Thus, PBMC engrafted NOD-scid IL2rγnull mice are able to provide an in vivomodel of a functional human immune system and of human islet allograft rejection. The enhanced ability of NOD-scid IL2rγnull mice to support human cell engraftment gave rise to the possibility of creating a model of graft versus host disease mediated by a human immune system. To investigate this possibility, human PBMC were injected via the tail vein into lightly irradiated NOD-scid IL2rγnull mice. We found that in contrast to previous models of GVHD using human PBMC-injected immunodeficient mice, these mice consistently (100%) developed GVHD following injection of as few as 5x106PBMC, regardless of the PBMC donor used. We then tested the contribution of host MHC in the development of GVHD in this model. As in the human disease, the development of GVHD was highly dependent on host expression of MHC class I and class II molecules. To begin to evaluate the extent to which the PBMC-engrafted NOD-scid IL2rγnull humanized mouse model of GVHD represents the clinical disease, we tested the ability of a therapeutic in clinical trials to modulate GVHD in these mice. In agreement with the clinical experience, we found that interrupting the TNFα signaling cascade with etanercept delayed the onset and severity of disease in this model. In summary, we conclude that humanized NOD-scid IL2rγnull mice represent an important surrogate for investigating in vivo mechanisms of both human islet allograft rejection and graft versus host disease.

The NOD mouse is a widely studied model of type 1 diabetes. The loss of self-tolerance leading to autoimmune diabetes in NOD mice involves at least 27 genetic loci. Curing type I diabetes in mice and humans by islet transplantation requires overcoming both allorejection and recurrent autoimmunity. This has been achieved with systemic immunosuppression, but tolerance induction would be preferable. In addition to their genetic defects in self-tolerance, NOD mice resist peripheral transplantation tolerance induced by costimulation blockade using donor-specific transfusion and anti-CDl54 antibody. Failure has been attributed to the underlying autoimmunity, assuming that autoimmunity and resistance to transplantation tolerance have a common basis. Hypothesizing that these two abnormalities might be related, we investigated whether they had a common genetic basis. Diabetes-resistant NOD and C57BL/6 stocks congenic for various reciprocally introduced Idd loci were assessed for their ability to be tolerized. Surprisingly, in NOD congenic mice that are almost completely protected from diabetes, costimulation blockade failed to prolong skin allograft survival. In reciprocal C57BL/6 congenic mice with NOD-derived Idd loci, skin allograft survival was readily prolonged by costimulation blockade. Unexpectedly, we observed that (NOD x C57BL/6)F1 mice, which have no diabetes, nonetheless resist induction of tolerance to skin allografts. Further analyses revealed that the F1 mice shared the dendritic cell maturation defects and abnormal CD4+ T cell responses of the NOD but had lost its defects in macrophage maturation and NK cell activity. Finally, using a genome wide scan approach, we have identified four suggestive markers in the mouse genome that control the survival of skin allografts following DST and anti-CD154 mAb therapy. We suggest that mechanisms controlling autoimmunity and transplantation tolerance in NOD mice are not completely overlapping and are potentially distinct, or that the genetic threshold for normalizing the transplantation tolerance defect is higher than that for preventing autoimmune diabetes. We conclude that resistance to allograft tolerance induction in the NOD mouse is not a direct consequence of overt autoimmunity and that autoimmunity and resistance to costimulation blockade-induced transplantation tolerance phenotypes in NOD mice are not under identical genetic control.

Made available in DSpace on 2015-07-22T20:50:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-25 / Introdução: O tratamento da deformidade na parede abdominal resultante do emagrecimento após a cirurgia bariátrica é a abdominoplastia, a qual pode induzir anemia pós-operatória. Além disso, a cirurgia bariátrica promove uma tendência a deficiência de ferro. Baixas reservas de ferro comprometem a eritropoese. Não há estudo avaliando o grau de anemia e sua evolução após a abdominoplastia pós-bariátrica. Objetivo: Avaliar a anemia e sua evolução após a abdominoplastia pós-bariátrica. Métodos: Os valores de hemoglobina, reticulócitos, ferro, ferritina, e índice de saturação da transferrina foram mensurados na véspera da abdominoplastia e na primeira, quarta e oitava semana de pós-operatório. A hemoglobina também foi mensurada com 48h de pós-operatório. Vinte mulheres adultas foram operadas e tiveram seus dados comparados com 12 controles. Resultados: Os níveis de hemoglobina caíram, em média, de 12,98g/dL para 10,8g/dL com 48h. Houve um aumento significante da hemoglobina no sétimo dia com correção de um terço do déficit, sem aumentos significantes posteriores. Houve um aumento dos reticulócitos na primeira semana. O ferro sérico e índice de saturação de transferrina caíram na primeira semana e mantiveram-se baixos. Os níveis de ferritina apresentaram aumento não significante na primeira semana e posteriormente caíram. Nenhuma das pacientes foi transfundida. Conclusão: Os níveis de hemoglobina caíram após a abdominoplastia e demonstraram um aumento na primeira semana de pós-operatório, com correção de um terço do déficit de hemoglobina, porém, não recuperaram por completo na oitava semana. Ao término do seguimento, 45% das pacientes desenvolveram deficiência de ferro e apresentaram déficit de hemoglobina maior que as pacientes que mantiveram estoques de ferro normais / Background: The treatment of the abdominal wall deformity resulted from weight loss after bariatric surgery is a mixed type of abdominoplasty, which can induce post-operative anemia. In addition, bariatric surgery itself promotes a tendency to iron deficiency which could compromise erythropoiesis. To our knowledge, there is no study evaluating the degree of anemia and its recovery after post-bariatric abdominoplasty. Methods: The values of hemoglobin, reticulocytes, iron, ferritin and transferrin saturation index were measured the day prior to abdominoplasty and the first, fourth and eighth weeks after surgery. Hemoglobin was measured within 48h after surgery. Twenty adult women underwent surgery and had their data compared with 12 controls. Results: Hemoglobin levels dropped significantly from 12,98g/dL to 10,8g/dL within 48h. Hemoglobin increased significantly to 11,5g/dL by day seven, with correction of one third of the deficit, without significant increases thereafter. There was an increase in reticulocytes in the first week. Serum iron and transferrin saturation index fell in the first week and remained low. Ferritin levels showed no significant increase in the first week and subsequently fell. None of the patients received blood transfusion. Conclusion: Hemoglobin levels fell from 12,98 g/dL to 10,8 g/dL after abdominoplasty and showed an increase in the first week after surgery, with correction of one third of the deficit, but did not completely recover in the eighth week. At the end of the follow-up, 45% of the patients developed an iron deficiency and had a hemoglobin deficit higher than the patients that maintained normal iron stores. / TEDE / BV UNIFESP: Teses e dissertações

Hepatitis C Virus (HCV) is the primary cause of liver transplantation due to its chronic nature in up to eighty percent of infected cases. Around 3 percent of the world’s population is infected with HCV. Treatment for HCV is a combined Ribavirin and interferon-α (IFN-α) therapy effective in only fifty to eighty percent of patients depending on HCV genotype. The growing health concern with this disease is the lack of a cure despite liver transplantation. HCV targets hepatocytes, liver cells, but is not cytolytic. HCV has been shown to induce end stage liver disease through sustained inflammation from the host’s immune system in the liver. One of the key dilemmas in HCV research and the search for fully effective treatments or vaccines is the lack of animal models. HCV infectivity and disease is limited to primates, most specifically to humans, which cannot be fully replicated in any other living being. The mechanisms for HCV evasion or activation of the immune system are complex, many and discoveries within this field are crucial to overcoming this destructive hepatic infection. Toll-like receptors (TLR) are cellular activators of the innate immune system that have been a target of HCV. Activated TLRs trigger both the inflammatory and anti-viral pathways to produce inflammatory cytokines and interferons. HCV proteins have been reported to activate a number of TLRs in a variety of cell types. In order to identify possible targets of HCV within the TLR family, we first characterized TLR presence and function in both human hepatic carcinoma cell lines and purified primary human hepatocytes. RNA from TLRs 1-10 was observed to varying degrees in both the hepatoma cell lines and the primary hepatocytes. We show the extracellular and/or intracellular presence of TLR2, TLR1, TLR3 and TLR7 proteins in hepatoma cell lines. TLR3 and TLR7 are located within the endosome and recognize viral RNA products. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with either TLR1 or TLR6. We show NF-κB activation in hepatoma cells by TLR2/1, TLR2/6 ligand and HCV protein stimulation. In primary hepatocytes, HCV proteins induced both IL-8 and IL-6 production. We also show that primary hepatocytes initiate a Type 1 IFN response in addition to IL-8 and IL-6 production upon stimulation with a TLR7/8 ligand. Human hepatoma and primary hepatocytes are responsive to TLR2, TLR1, TLR6, TLR7/8 ligands and HCV proteins. Activation of these TLRs may contribute to the inflammatory mediated destruction caused by HCV or could be targets of HCV contributing to its immune evasion. We found previously that hepatoma cells and primary hepatocytes are responsive to TLR2 ligands and HCV proteins. We also reported that TLR2 is activated by HCV proteins. Here we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing siRNAs targeted to TLR2, TLR1 and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in Human Embryonic Kidney cells (HEK/TLR2) and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-α and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1 and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1 or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. TLR2 may utilize both TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins contributing to inflammatory disease. Two critical factors in chronic HCV infection are inflammatory disease and immune evasion. We have demonstrated that TLR2 and its co-receptors play a role in inflammatory-mediated induction via HCV NS3 and core administration. It has recently been shown that HCV targets the TLR3 pathway to aid in immune evasion. TLR3 is only one of four viral recognition receptors located within the endosome and it is plausible that HCV may target others. We hypothesized that HCV infection may interfere with the expression and function of TLR7, a sensor of single stranded RNA. Investigating any effect on TLR7 by HCV may reveal a new mechanism for HCV immune evasion. Low levels of both TLR7 mRNA and protein were measured in HCV replicating cells compared to control cells while reducing HCV infection with either IFNα or restrictive culture conditions restored the decreased TLR7 expression. Downstream of the TLR7 pathway, an increased baseline IRF7 nuclear translocation was observed in HCV replicating cells compared to controls. Stimulation with a TLR7 ligand, R837, resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV replicating cells showed impaired IRF7 activation. Use of RNA polymerase inhibitors on hepatoma cells, control and HCV replicating, revealed a shorter TLR7 half life in HCV replicating cells compared to control cells which was not seen in TLR5 mRNA. These data suggest that reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and results in impaired TLR7-induced IRF7-mediated cell activation. In conclusion, Hepatitis C Virus manipulates specific Toll-like receptors’ expression and their signaling pathways to induce cytokine production. HCV utilizes surface receptors TLR2 and its co-receptors which once activated could contribute to inflammatory disease by production of inflammatory cytokines and possibly immune evasion. HCV down-regulates TLR7, a viral recognition receptor, by decreasing mRNA stability which could facilitate evasion of host immune surveillance.

Tolerance-based stem cell transplantation using sub-lethal conditioning is being considered for the treatment of human disease, but safety and efficacy remain to be established. In order to study these two issues, we first established that mouse bone marrow recipients treated with sub-lethal irradiation plus transient blockade of the CD40-CD154 costimulatory pathway develop permanent hematopoietic chimerism across allogeneic barriers. Our conditioning regimen of 6 Gy irradiation, a short course of anti-CD154 mAb and 25 million fully allogeneic BALB/c bone marrow cells consistently produced long-term, stable, and multilineage chimerism in C57BL/6 recipients. Furthermore, chimeric mice displayed donor-specific transplantation tolerance, as BALB/c skin allografts were permanently accepted while third-party CBA/JCr skin allografts were promptly rejected. We next determined both the safety and efficacy of this protocol by infecting chimeric mice with lymphocytic choriomeningitis virus (LCMV) either at the time of transplantation or at several time points afterwards. Infection with LCMV at the time of transplantation prevented engraftment of allogeneic, but not syngeneic, bone marrow in similarly treated mice. Surprisingly, infected allograft recipients also failed to clear the virus and died. Post-mortem study revealed hypoplastic bone marrow and spleens. Hypoplasia and death in these mice required the combination of 6 Gy irradiation, LCMV infection on the day of transplantation, and an allogeneic bone marrow transplant but did not require the presence of anti-CDl54 mAb. Allochimeric mice infected with LCMV 15 days after transplantation were able to survive and maintain their bone marrow graft, indicating that the deleterious effects of LCMV infection on host and graft survival are confined to a narrow window of time during the tolerization and transplantation process. The final section of this thesis studied the mechanisms of graft rejection and death in sublethally irradiated recipients of allogeneic bone marrow and infection with LCMV at the time of bone marrow transplantation. Infection of interferon-α/β receptor knockout mice at the time of transplantation prevented the engraftment of allogeneic bone marrow, but the mice survived. Therefore, IFN-αβ is involved in the development of marrow hypoplasia and death, whereas a second mechanism is involved in blocking the development of chimerism in these mice. Through the use of depleting mAb's and knockout mice we demonstrate that three types of recipients survived and became chimeric after being given sublethal irradiation, anti-CD154 mAb, an allogeneic bone marrow transplant and a day 0 LCMV infection: mice depleted of CD8+ T cells, CD8 knockout mice, and TCR-αβ knockout mice. Our data indicate that the mediator of bone marrow allograft destruction in LCMV-infected mice treated with costimulatory blockade is a radioresistant CD8+ NK1.1- TCRαβ+ T cell. We conclude that a non-cytopathic viral infection at the time of transplantation can prevent engraftment of allogeneic bone marrow and result in the death of sub-lethally irradiated mice treated with costimulation blockade. The abrogation of allogeneic bone marrow engraftment is mediated by a population of CD8+ NK1.1- TCRαβ+ T cells and the mediator of hypoplasia and death is viral induction of IFN-αβ.

Objetivo: Novas tendências na avaliação de risco trazem evidências de que modelos externos recalibrados ou remodelados funcionam melhor localmente. O objetivo deste estudo foi validar dois modelos externos e formular um modelo local, comparando-os na predição de mortalidade nos pacientes operados de coronária e/ou valva no InCor-HCFMUSP. Método: Entre 2007 e 2009, 3.000 pacientes foram sequencialmente operados de coronária e/ou valva no InCor-HCFMUSP. No banco de dados, foi realizada a validação dos modelos 2000 Bernstein-Parsonnet (2000BP) e EuroSCORE (ES), mediante testes de calibração e discriminação. O InsCor de 2.000 pacientes foi elaborado mediante a utilização de técnicas de bootstrap. Nos próximos 1.000 pacientes foi realizada a validação interna do modelo, e seu desempenho medido frente ao 2000BP e ES. Resultados: Houve uma diferença significativa na prevalência dos fatores de risco entre as populações do estudo, ES e 2000BP. Na validação externa dos modelos, o ES apresentou uma boa calibração (P=0,596); no entanto, o 2000BP revelou-se inadequado (P=0,047). Na discriminação, a área abaixo da curva ROC revelou-se boa para ambos os modelos, ES (0,79) e 2000BP (0,80). Utilizando a técnica de bootstrap, 10 variáveis: idade >70 anos, sexo feminino, cirurgia associada, infarto do miocárdio <90 dias, reoperação, cirurgia da valva aórtica, cirurgia da valva tricúspide, creatinina <2mg/dl, fração de ejeção <30% e estado pré-operátorio crítico (eventos), foram selecionadas para formulacão do InsCor. Na validação interna do InsCor, a calibração foi adequada, com P=0,184. Na discriminação, a área abaixo da curva ROC foi boa (0,79). Neste grupo, a área abaixo da curva ROC foi de 0,81 e 0,82 para o ES e 2000BP, respectivamente, mostrando-se apropriada para ambos os modelos. Conclusões: O InsCor e o ES tiveram melhor desempenho que o 2000BP em todas as fases da validação; pórem o novo modelo, além de se identificar com os fatores de risco locais, é mais simples e objetivo para a predição de mortalidade nos pacientes operados de coronária e/ou valva no InCor-HCFMUSP / Background: New trends in risk assessment bring evidence that recalibrated or remodeled external models work best locally. The aim of this study was to validate two external models and formulate a local model, comparing them to predict mortality in patients who underwent coronary bypass surgery and/or heart valve surgery at InCor-HCFMUSP. Method Between 2007 and 2009, 3.000 patients were sequentially operated to coronary bypass surgery and/or heart valve surgery at InCor-HCFMUSP. The database was assessment to validate the models 2000 Bernstein-Parsonnet (2000BP) and EuroSCORE (ES) through calibration and discrimination tests. The InsCor of 2,000 patients (2/3 of database) was elaborated using bootstrap techniques. Over the next 1000 patients (1/3 of database) the internal validation of the InsCor was performed and its performance compared against the 2000BP and ES. Results: Significant difference in the prevalence of risk factors was found among the external and study populations (P<0,001). In the external validation of these models, the ES showed good calibration (P = 0.596); however, 2000BP was inadequate (P = 0.047). In discrimination, the area under the ROC curve was good for both models, ES (0.79) and 2000BP (0.80). With the bootstrap technique, 10 variables: age> 70 years, female, CABG + valve surgery, myocardial infarction <90 days, reoperation, aortic valve surgery, tricuspid valve surgery, creatinine <2mg/dl, ejection fraction <30% and critical preoperative state (events) were chosen to formulate the InsCor. In the validation of InsCor, the calibration was appropriate with P = 0.184. In discrimination, the area under the ROC curve was good (0.79). In this group, the area under the ROC curve was 0.81 and 0.82 for ES and 2000BP, respectively, being suitable for both models. Conclusions: The InsCor and ES outperformed the 2000BP at all stages of validation, but the new model, besides identifying itself with the local risk factors, is more simple and objective for the prediction of mortality in patients who underwent coronary bypass surgery and/or heart valve surgery at InCor-HCFMUSP.