5 Abstract Tolerance to "self" is the fundamental property of the immune system and its breakdown can lead to autoimmune diseases. In order to eliminate self-reactive T- cells during their development in thymus (central tolerance), Aire promotes the expression of peripheral self-antigens in medullary thymic epithelial cells (mTECs). Recently, Aire was suggested to fulfil a similar function in rare lymph node and spleen cells (peripheral tolerance). However, the detection, characterization and function of these extrathymic Aire-expressing cells is still obscure. The main objective of presented thesis was to investigate if Aire positive cells are also present in other lymphoid as well as non-lymphoid tissues. Using two independent mouse transgenic models we identified the Aire-reporter expressing cells in several lymphoid tissues such as Peyer's patches, spleen and bone marrow as well as in one non-lymphoid organ, the lungs. We show here that based on the expression of B220, EpCAM and CD11c markers these heterogenic cells consist of at least five phenotypically distinct subpopulations, and with the exception of those from lungs, all of them are strictly of hematopoietic origin. This study also demonstrates that Aire on protein level is predominantly expressed by one of these subpopulations with CD45+ MHCII+...

O antígeno leucocitário humano (HLA)-G corresponde a uma molécula não clássica de classe I do complexo principal de histocompatibilidade. Segundo a literatura, tal molécula pode ser expressa em ambos os contextos patológico e fisiológico. Diversos autores têm apresentado evidências acerca do papel do HLA-G na tolerância imune do feto durante a gestação, bem como para o sucesso de alotransplantes. No entanto, até o momento, há poucas informações publicadas a respeito da expressão fisiológica dessa molécula nos diferentes órgãos humanos. Em acréscimo a isso, a participação do HLA-G em eventos fisiológicos é ainda um assunto controverso entre cientistas. Tendo em vista o exposto, o objetivo desse estudo foi investigar a expressão da proteína HLA-G em órgãos fetais durante o progredir da gestação, bem como em órgãos adultos. Trata-se de um estudo descritivo, comparativo, transversal e retrospectivo realizado com base na revisão de prontuários e análise de necropsias/biópsias de diferentes órgãos de fetos e adultos através do método de imunohistoquímica. Os resultados demonstraram a existência de diferença estatística significativa na imunomarcação da proteína HLA-G em glândulas adrenais (p= 0,0003), baço (p= 0,0276), coração (p= 0,0474), fígado (p= 0,0052), pulmões (p = 0,0367), rins (p = 0,0377) e timo (p= 0,0336) na comparação entre o primeiro e segundo trimestre gestacional; em glândulas adrenais (p= 0,0329), baço (p= 0,0095), pâncreas (p= 0,0009) e placenta (p= 0,0285) na comparação entre o segundo e terceiro trimestre gestacional; e no coração (p= 0,0304), fígado (p= 0,0055), pulmões (p= 0,0150) e rins (p= 0,0312) na comparação entre o terceiro trimestre gestacional e a fase adulta. Foi verificado um aumento na expressão do HLA-G fetal a partir do segundo trimestre gestacional em órgãos como glândulas adrenais, coração, fígado, rins, timo e pulmões. Entretanto, isso não foi uma constante, pois em outros, a exemplo do baço, pâncreas e placenta, não observouse essa tendência durante o mesmo período. Durante o terceiro trimestre gestacional e a fase adulta evidenciou-se valores mais elevados para a expressão do HLA-G nos rins, e valores bastante inferiores no fígado. A expressão fisiológica do HLA-G embora positiva em todos os órgãos avaliados, nos três trimestres gestacionais e/ou na fase adulta, apresentou diferenças na intensidade e localização nos diferentes órgãos e períodos. Os achados a partir dessa pesquisa certamente representam uma importante contribuição para um melhor entendimento do mecanismo gestacional, assim como da fisiologia do HLA-G em adultos, sobretudo no que concerne o estabelecimento da tolerância imunológica em transplante de órgãos / Human leukocyte antigen (HLA)-G is a nonclassical class I major histocompatibility complex molecule. According to the literature, this molecule can be expressed in both pathological and physiological contexts. Several authors have presented evidence about the role of HLA-G in the immune tolerance of the fetus during pregnancy, as well as for the success of allotransplants. However, until now, there are very few published data regarding the physiological expression of this molecule in different human organs. Moreover, the role of HLA-G in physiological events is still a controversial subject among scientists. In view of the above, the objective of this study was to investigate the expression of HLA-G protein in fetal organs during the progression of gestation, as well as in adult organs. This was a descriptive, comparative, cross-sectional and retrospective study based on the review of medical records and immunohistochemical analysis of different organs of fetuses and adult people. The results showed a statistically significant difference in the immunostaining of HLA-G protein in adrenal glands (p = 0.0003), spleen (p = 0.0276), heart (p = 0.0474), liver (p = 0.0367), kidneys (p = 0.0377) and thymus (p = 0.0336) in the comparison between the first and second gestational trimesters; in adrenal glands (p = 0.0329), spleen (p = 0.0095), pancreas (p = 0.0009) and placenta (p = 0.0285) in the comparison between the second and third gestational trimesters; and in the heart (p = 0.0304), liver (p = 0.0055), lungs (p = 0.0150) and kidneys (p = 0.0312) in the comparison between the third gestational trimester and the adult phase. An increase of fetal HLA-G expression was observed from the second gestational trimester in organs such as adrenal glands, heart, liver, kidneys, thymus and lungs. However, this was not a constant finding, since in other organs including spleen, pancreas and placenta, this trend was not observed during the same period. During the third gestational trimester and the adult phase, higher values for HLA-G expression in the kidneys and much lower values in the liver were observed. Although the physiological expression of HLA-G has been positive in all evaluated organs (in the three gestational trimesters and/or in adulthood), it showed differences in its intensity and location in the different organs and periods. The findings from this research certainly represent an important contribution to a better understanding of the gestational mechanism, as well as on the physiology of HLA-G in adults, especially regarding the establishment of immunological tolerance in organ transplantation

A endometriose é uma doença inflamatória crônica, estrógeno-dependente e de etiologia multifatorial, caracterizada pela implantação e crescimento de tecido endometrial fora da cavidade uterina e associada à dor pélvica e infertilidade. A doença é classificada de acordo com os estádios e sítios de acometimento nos órgãos pélvicos. Variantes genéticas, endócrinas e ambientais podem contribuir para a geração de uma deficiência na resposta imune local permitindo a implantação das células ectópicas na cavidade pélvica. Alterações constatadas no padrão de citocinas presentes no microambiente pélvico poderiam promover um ambiente imunossupressor, justificando a diminuição da resposta imune efetora verificada na endometriose. Dentre os possíveis fatores imunomoduladores, está o antígeno leucocitário humano-G (HLA-G), cuja expressão se dá intensamente nas células trofoblásticas, sendo reconhecido por induzir a tolerância materno-fetal. A proteína HLA-G pode ser expressa na membrana celular ou ser secretada na forma solúvel. HLA-G encontra receptores inibitórios nas células do sistema imune inato e adaptativo e tem sua expressão induzida sob condições não fisiológicas, como em transplantes alogênicos, doenças inflamatórias ou neoplasias malignas. Assim, a hipótese deste estudo é a de que a proteína HLA-G seria produzida em níveis superiores nas mulheres com endometriose, o que poderia contribuir para a imunossupressão no microambiente da doença. Para testar esta hipótese, a proteína solúvel foi mensurada no soro e no fluido peritoneal de mulheres com e sem endometriose, por ensaio de imunoabsorção enzimática (ELISA). Além disto, a expressão gênica de HLA-G foi avaliada nos tecidos de endométrio, por qRT-PCR, bem como a expressão da proteína, avaliada por imunohistoquímica, nos tecidos de endométrio e de lesão de endometriose, em mulheres com e sem a doença. Como resultados, verificaram-se maiores níveis da proteína solúvel no soro de mulheres que apresentavam endometriose em estádios avançados, especialmente naquelas com endometriose ovariana. Entretanto, na comparação entre os fluidos peritoneais, não houve diferença significativa entre os grupos com e sem endometriose. A expressão do transcrito gênico (mRNA) se mostrou maior no endométrio de mulheres sem a doença, mas a presença da proteína foi semelhante entre os endométrios de mulheres com e sem endometriose. Por outro lado, a expressão da proteína HLA-G nos tecidos de lesão de endometriose avançada se mostrou superior à do endométrio de mulheres sem a doença, indicando que a expressão de HLA-G seria induzida ectopicamente, no microambiente pélvico da doença. Portanto, os resultados apontam para um aumento da expressão de HLA-G em endometriose avançada / Endometriosis is a chronic inflammatory, estrogen-dependent disease of multifactorial etiology characterized by implantation and growth of endometrial tissue outside the uterine cavity, and associated with pelvic pain and infertility. Endometriosis is classified according to the stages and sites of the disease. Genetic, endocrine and environmental factors may contribute to the deficit on local immune response, allowing ectopic implantation of endometrial cells into the pelvic cavity. Changes in cytokines pattern in the pelvic microenvironment might promote an immune suppressor environment and explain the decreased immune effector cells response verified in endometriosis. Among possible immunomodulatory factors is the human leucocytary antigen-G (HLA-G) which is intensively expressed in trophoblasts and recognized by inducing maternal-fetal tolerance. HLA-G protein is expressed in both membrane-bound and soluble forms. HLA-G binds inhibitory receptors on innate and adaptive immune cells surface and its expression is induced in non-physiological conditions, such as allogeneic transplants, inflammatory diseases or neoplastic malignancies. Thus, this study hypothesizes that the HLA-G protein would be overexpressed in women with endometriosis, and could contribute to the immunosuppression in the disease microenvironment. To test this hypothesis soluble HLA-G protein was measured in serum and peritoneal fluid of women with and without endometriosis. Moreover, HLA-G gene expression were evaluated on endometrial tissue using RT-qPCR, and HLA-G protein expression were evaluated in matched ectopic and eutopic endometrium of women with and without endometriosis. As results, higher levels of soluble HLA-G were found in serum of women with advanced endometriosis, especially in those with ovarian endometriosis. However, soluble HLA-G levels in peritoneal fluid did not show significant differences between women with and without endometriosis. HLA-G mRNA expression were higher in eutopic endometrium of women without endometriosis, but the HLA-G protein expression were similar in eutopic endometrium of women with and without endometriosis. On the other hand, HLA-G protein expression in ectopic endometrium of women with advanced endometriosis was higher than in eutopic endometrium of women without endometriosis, suggesting that HLA-G expression was induced ectopically, in the pelvic microenvironment of the disease. In conclusion, the results point to an upregulation of HLA-G expression in advanced endometriosis

Current immune suppressive strategies fail to induce donor-recipient immune tolerance after allogeneic hematopoietic cell transplantation. Accordingly, patients suffer morbidity and mortality from graft vs. host disease (GVHD) and prolonged immune suppressive therapy. Biologic insight into transplantation tolerance is needed, and translation of such insight to novel clinical strategies may improve clinical outcomes. We report original investigation at seminal phases of this process including initial prophylactic immune suppression, onset of acute graft vs. host disease, and ultimate immune suppression discontinuation: In a controlled randomized clinical trial, we demonstrate that sirolimus-based immune suppression reduces risk for acute GVHD, ameliorates the severity of subsequent chronic GVHD, and supports reconstitution of functional regulatory T cells. Study of tissue-infiltrating CD4+ T cell subsets in acute GVHD target organs supports a pathogenic role for Th17 cells. Finally, we demonstrate that peripheral blood transcriptional biomarkers provide mechanistic insight into human transplantation tolerance. These data signal progress, and suggest rational translational efforts to achieve transplantation tolerance.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph.D. in Cancer Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 136-169). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 136-160).

Thesis (Ph.D. in Immunology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 133-151). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 64-80).

Chronic immune insult and immunosuppressant-related toxicities have remained an enduring challenge in organ transplantation. Long-term survival of transplant patients has improved marginally in recent decades due to these challenges. To circumvent these issues, transplant investigators have researched immune tolerance mechanisms that demonstrate potential to induce immunosuppression and rejection-free survival in the clinic. One mechanism in particular, the liver tolerance effect, has already demonstrated this experimentally and clinically. Liver transplants in experimental models and human patients have exhibited the ability to become spontaneously accepted without being rejected by the recipient’s immune system. Research in recent decades has revealed that the liver parenchymal and non-parenchymal cell populations harbor potent immunomodulatory properties. In the context of liver transplantation, it has been found that two cell populations in particular, the mesenchyme-derived liver sinusoidal endothelial cells and hepatic stellate cells, mediate the induction of liver transplant tolerance through a mechanism known as mesenchyme-mediated immune control.