Orientador: Ricardo de Lima Zollner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T03:38:16Z (GMT). No. of bitstreams: 1 Pedrosa_Denise_M.pdf: 2680300 bytes, checksum: 4372cb3695e33fb5ec7321200d7d6035 (MD5) Previous issue date: 2013 / Resumo: Empregando a linhagem de camundongos NOD (non-obese diabetic) de desenvolvimento espontâneo do diabetes mellitus tipo 1 (DM-1) e a linhagem NOD.Scid como controle, foi realizada a análise da expressão dos genes de regeneração Reg1 e Reg2 e a sua relação com a expressão dos Receptores Toll-Like (TLRs), além da análise do estresse oxidativo em ilhotas pancreáticas. Foram avaliados animais durante o desenvolvimento do diabetes com 2, 8 e 24 semanas de vida, além de camundongos diabéticos e do controle NOD.Scid. Houve aumento progressivo da insulite e do estresse oxidativo em função do tempo, além da diminuição drástica do número de ilhotas verificado em camundongos diabéticos. Os genes Reg1 e Reg2 apresentaram aumento da expressão em ilhotas pancreáticas de camundongos com 2 semanas de vida e nos animais diabéticos, assim como no controle NOD.Scid. Os receptores TLR2, TLR4, TLR5 e TLR6 foram expressos, com aumento significativo em animais com 8 e 24 semanas de vida, exceto para o TLR2 que apresentou expressão gênica reduzida em camundongos de 24 semanas de vida, sugerindo envolvimento na indução da fase de insulite. O animal diabético apresentou expressão reduzida em todos os TLRs analisados e expressão elevada dos genes Reg1 e Reg2 em ilhotas pancreáticas. Além disso, foi possível verificar o contraste de expressão de Reg1 e Reg2 frente aos TLRs no período pré-diabético, em que Reg1 e Reg2 apresentaram expressão gênica reduzida, enquanto os TLRs apresentaram expressão elevada. Em conjunto, estes dados sugerem possível associação entre a ativação dos TLRs e a redução da expressão dos genes de regeneração Reg1 e Reg2 na fase de indução da insulite, tornando-se necessário avaliar se os fatores inflamatórios afetam o balanço dos sistemas biológicos com consequente controle da capacidade regenerativa celular ou se a falha no sistema biológico age como fator primário de indução ao dano tecidual / Abstract: Employing the lineage of NOD mice (non-obese diabetic) which presents spontaneous development of diabetes mellitus type 1 (T1D) and the lineage NOD.Scid as a control, gene expression of regenerating genes Reg1 and Reg2 and their relation with expression of Toll-Like Receptors (TLRs) was performed, in addition to oxidative stress analysis in pancreatic islets. Animals were assessed during the T1D development at 2, 8 and 24 weeks-old and also in diabetic animals and NOD.Scid control. The insulitis and the oxidative stress process increased progressively with age, beyond the drastically decreased of islets number in diabetic animals. Analysis of oxidative stress in islets has been shown progressive with age and may be related to the development of diabetes. Reg1 and Reg2 genes were high expressed in islets of NOD mice aged 2 weeks-old, diabetic animals and NOD.Scid control. Receptors TLR2, TLR4, TLR5 e TLR6 were expressed in all animals, however mice aged 8 and 24 weeks-old showed a significant increase expression, being TLR2 an exception where there was low expression in mice aged 24 weeks-old which suggests that their expression should be related to the induction phase of insulitis. Diabetic mice showed reduced expression in all TLRs analyzed and high expression of Reg1 and Reg2 in pancreatic islets. Furthermore, Reg1 and Reg2 showed a contrast of expression when related to TLRs at pre-diabetic stage, with low expression of Reg1 and Reg2 and high expression of TLRs. Taken together, these results suggest a possible correlation between the activation of TLRs and the reduced expression of regenerating genes Reg1 and Reg2 in the induction phase of insulitis. Thus, studies are needed to elucidate whether inflammatory factors affect the balance of biological systems with consequent regenerative cell control capacity or if the failure in the biological system acts as a primary factor inducing tissue damage / Mestrado / Clinica Medica / Mestra em Clínica Médica

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 1, 2010). Vita. Thesis advisor: Habib Zaghouani. "May 2008" Includes bibliographical references.

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.

Breast cancer, as the most prevalent cancer in women, is responsible for more than 15% of new cancer cases and about 6.9% of all cancer-related death in the US. A major cause of therapeutic failure in breast cancer is the development of resistance to chemotherapy, especially for triple-negative breast cancer (TNBC). Therefore, how to overcome chemoresistance is the major challenge to improve the life expectancy of breast cancer patients. Our studies demonstrate that TNBC cells surviving the chronic treatment of chemotherapeutic drugs show significantly higher expression of the dual serine/threonine and tyrosine protein kinase (DSTYK) than non-treated parental cells. In our in vitro cellular models, DSTYK knockout via the CRISPR/Cas9-mediated technique results in apoptotic cell death of chemoresistant cells upon drug treatment. Moreover, DSTYK knockout promotes chemotherapeutic drug-induced tumor cell death in an orthotopic mouse model. These findings suggest that DSTYK exerts an important and previously unknown role in promoting chemoresistance. Our studies provide fundamental insight into the role of DSTYK in chemoresistance in TNBC cells and lay the foundation for the development of new strategies targeting DSTYK for improving TNBC therapy.