La maladie du greffon contre l’hôte (GvHD) est une complication majeure des greffes de cellules souches hématopoïétiques (HSCT) qui survient dans 30 à 70% des cas et peut causer la mort, malgré un traitement prophylactique bien conduit. Il existe donc une réelle demande clinique pour améliorer ces traitements prophylactiques. Parce que ces traitements prophylactiques reposent en général sur des agents immunosuppresseurs, ceux-ci contribuent à diminuer la reconstitution immunitaire du patient, ce qui a un impact défavorable sur les infections et les taux de rechute d’hémopathie maligne, et donc limite leur utilisation. Les immunoglobulines (IVIG) pourraient représenter une alternative intéressante puisqu’elles ont des propriétés immunomodulatrices et qu’elles sont de plus couramment utilisées en clinique pour traiter des patients ayant un déficit immunitaire. Leur capacité à réduire l’apparition et la sévérité de la GvHD, sans toutefois inhiber ou nuire à la reconstitution immunitaire chez le patient n’a néanmoins jamais été clairement démontrée. Les objectifs de ce projet sont donc d’évaluer l’efficacité des IVIG à réduire l’incidence et la sévérité de la GvHD dans un modèle murin humanisé de GvHD, ainsi que de déterminer le mécanisme d’action des IVIG. Ce modèle consiste à injecter des huPBMCs à des souris immunodéprimées ne pouvant les rejeter. Les résultats obtenus suggèrent que les IVIG possèdent un effet immunomodulateur permettant de réduire les signes cliniques et de retarder l’apparition de la GvHD, tout en permettant l’apparition de cellules NK. Les IVIG agiraient de façon indirecte sur les huPBMCs afin d’induire l’apparition des cellules NK. / Graft-versus-host disease (GvHD) is a major complication following hematopoietic stem cell transplantation (HSCT), occuring in 30 to 70% of HSCT despite proper prophylactic treatment, and can result in death. It is therefore primordial to improve the prophylactic treatments in order to reduce the frequency and severity of GvHD. Since these prophylactic treatments are based on the use of immunosuppressive agents, they inhibit the immune reconstitution and thus increase the risk of infections and relapse. Because of their immunomodulating properties, immunoglobulins (IVIG) represent a promissing alternative. Furthermore, IVIG are already widely used in patients with immune deficits. Since no study has yet clearly shown that IVIG can reduce GvHD without inhibiting the immune reconstitution in the patient, the scientific community is still debating their usefullness in GvHD prevention. Should their efficacy to reduce the incidence and severity of GvHD without impairment to the immune reconstitution be demonstrated, the use of IVIG could change the clinical outcome for HSCT patients, improving their remission and quality of life. The objectives of this project are to evaluate IVIG efficacy in GvHD prevention and to define the mechanism of action of IVIG. To fulfill these objectives, we will use a GvHD humanized mouse model, which consists of injecting huPBMCs in immunodeficient mice who can not reject them. Our results demonstrate an immunomodulatory effect of IVIG, allowing for reduced clinical signs and a slighlty increased survival in GvHD. A population of NK cells also appeared upon IVIG treatment and is believed to be indirectly induced by IVIG.

Les cellules épithéliales qui produisent l’émail, les améloblastes, sont séparées de l'émail au niveau de la zone de maturation par une membrane basale spécialisée (MBS) enrichie en laminine 332 (LM-332). Cette protéine hétérotrimérique (composée des chaînes α3, ß3 et γ2) assure l'intégrité structurelle des membranes basales (MB) et influence divers processus cellulaires épithéliaux tels que l'adhésion et la différenciation cellulaire. Des modèles de souris « knockout » (KO), où les gènes codant pour LM-332 ont été supprimés, meurent peu après la naissance. Néanmoins, ce phénotype létal peut être contourné en substituant chez la souris le gène produisant la chaîne γ2 de la laminine (LAMC2) par sa forme humaine, sous le contrôle de l’expression du promoteur-rtTA, de la cytokératine 14, inductible par la prise de doxycycline (Dox) - (Tet-on). Le but de ce projet est d’examiner si l’utilisation de cette protéine humaine chez la souris a un effet sur la structuration de la MBS ainsi que sur la maturation de l'émail. La phase de maturation de l’organe de l’émail chez la souris transgénique a été sévèrement altérée par rapport à une souris normale (WT). La MBS n’est plus visible, une matrice dystrophique s’est formée dans la couche d'émail dans la phase de maturation, et la présence d’une matrice résiduelle de l'émail est observée durant la phase tardive de maturation. Des micro-analyses tomographiques ont révélé une usure excessive des surfaces occlusales des molaires, un écroulement de l'émail sur les pointes des incisives et une hypominéralisation de l'émail. Cependant, aucune altération structurale due à cette recombinaison transgénique n’a été observée dans d'autres sites épithéliaux, tels que la peau, le palais et la langue. Ces résultats indiquent que, bien que ce modèle de souris humanisée soit capable de rétablir ses fonctions dans divers tissus épithéliaux, il est incapable de soutenir la structuration d'une MBS à l'interface entre les améloblastes et l’émail en maturation. Cet échec peut être lié à la composition spécifique de la MBS dans la phase de maturation et supporte l’hypothèse que la MBS est essentielle pour la maturation adéquate de l'émail. / The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation.

O Vírus Sincicial respiratório (HRSV) é um patógeno de grande importância para crianças. É o maior causador de infecções respiratórias agudas e também um dos principais causadores de internações e mortes de crianças menores de 5 anos. Cepas de HRSV com mutações no sítio de ligação do Palivizumab vêm apresentando resistência ao medicamento. Pouco se sabe sobre a prevalência destas mutações em amostras clínicas, apesar de sua potencial importância na patogênese viral. Além disso, existem poucos dados sobre a evolução molecular da proteína F do HRSV, sobretudo no Brasil. O presente estudo tem como objetivo caracterizar fenotipicamente através de ensaios de microneutralização in vitro, cepas oriundas de crianças não tratadas com Palivizumab circulantes em 2013 apresentando mutações na proteína F, mais especificamente relacionadas a potencial resistência nos epítopos de ligação do Palivizumab. Como resultado deste trabalho todas as amostras testadas foram neutralizadas pelo Palivizumab, concluindo-se que as mutações encontradas não conferem resistência ao monoclonal. / Respiratory Syncytial Virus (HRSV) is a pathogen of great importance for children. It is the major cause of acute respiratory infections and also one of the main causes of hospitalizations and deaths of children under 5 years. Strains of HRSV with mutations in the binding site of Palivizumab have been showing resistance to the drug. Little is known about the prevalence of these mutations in clinical samples, despite their potential importance in viral pathology. In addition, there is little data on the molecular evolution of HRSV F protein, especially in Brazil. The present study aims to characterize phenotypically by in vitro microneutralization assays, strains from children not treated with Palivizumab circulating in 2013 showing mutations in F protein, more specifically related to potential resistance in the binding epitopes of Palivizumab. As result of this study all samples tested were neutralized by Palivizumab, concluding that the mutations found did not confer resistance to the monoclonal.

Este estudo, de natureza qualitativa, buscou apreender o sentido da arte de partejar para cinco parteiras tradicionais que atuaram, ou ainda atuam, no distrito de Regência, ES, como a única possibilidade de assistência ao parto na região. Ao longo deste trabalho, buscou-se uma fundamentação teórica sobre o tema e que permitiu reconhecer, na história da parturição, até meados do século XX, que foram as parteiras quem sempre fizeram o parto e auxiliaram as mulheres em doenças especificamente femininas. Mesmo nos dias atuais, em regiões afastadas dos grandes centros e em zonas rurais, as parteiras continuam constituindo a única fonte da população para resolver seus problemas de saúde, especialmente na assistência do parto, realidade também constatada no presente estudo.A entrevista semi-estruturada foi utilizada como instrumento para coleta de dados. Histórias de vida oral temática foram obtidas, mediante os relatos das entrevistadas, os quais foram gravados em fitas cassete e transcritos, para desenvolvimento da análise de conteúdo. Todas as parteiras entrevistadas são pessoas simples, com nenhum ou pouco estudo e que, apesar de viverem com poucos recursos, orgulham-se de nada cobrarem pelos serviços prestados, aceitando somente alguns presentes, muitas dependendo da ajuda dos filhos para sua sobrevivência. A análise da trajetória dos sujeitos da população de estudo revelou que o ofício de parteira tem o sentido de missão de vida, decorrente do dom de que são portadores, dom este descoberto diante do inesperado, de uma demanda concreta, e que se desenvolve pela própria experiência, a serviço da coletividade, muitas vezes, sem nenhuma estrutura de apoio do sistema oficial de saúde. Diante dessa constatação, fica evidente a necessidade de uma articulação efetiva entre as parteiras e o sistema de saúde da região, visando a uma assistência de melhor qualidade à população local, respeitados os princípios do SUS. / This qualitative study tried to catch the art of midwife practice of five ladies who have been exercising it in Regência, ES, as the only possibility of helping pregnant women at baby delivering in that region. During this essay, a theorical embasement was searched, which allowed to recognize the role of midwives, until mid 20th Century, as the only assistance for delivery as well as specifically feminine diseases. Even nowadays, in the countryside or faraway counties, midwives still are the only resource to turn to in health cases or baby delivery assistance, as verified in the present study. The half-structured interview was used as a data collection tool. Verbal thematic stories of lives where obtained, by means of reported stories of the interviewed, which where recorded in cassette tapes and transcripts, for content analysis development. All the interviewed midwives are from humble background, with little or none education who, despite living short of money, feel proud of not charging anything for the job. Most depend on their own children’s help to survive financially. Sometimes, they accept gifts in return but never money. The analysis of the subjects of this study reveals that the craft of midwives is faced as a mission in life, due to the natural gift they were given, which was discovered in an unexpected situation, or a real demand and, eventually, was developed by experience itself, as a service to the community, most of the times, with no structural support on behalf of the official Healthy system. Aware of this, it becomes more and more evident that there is need for effective engagement among midwives and the Regional health system, having in mind a qualified and better assistance to the local population, respecting the principles of SUS (Unified Health System).

Made available in DSpace on 2016-04-04T18:29:44Z (GMT). No. of bitstreams: 1 Mara Aparecida Lissarassa Weber.pdf: 1216913 bytes, checksum: 2e85eb10324d7ee4690b83a985c7453f (MD5) Previous issue date: 2009-02-02 / Universidade Estadual Paulista J?lio de Mesquita Filho / Inequalities of our society and the difficulty of existing humanized relationship between adults and children based on the values of the Statute of Children and Adolescents has motivated this study. Thus, this research was aimed in knowing how the values of the triad of Entire Protection Doctrine- liberty, dignity and respect were understood by parents and teachers and its implication on the children?s daily life. This research was taken at a public preschool, having as participants its children?s parents and teachers. The data of this study was produced during the discussion groups as well as the researcher s field diaries records developed during her presence at the school. The results were analyzed according to the historical and dialectical materialism since we believe on the interpretation of the phenomenon from the existing contradictions of daily life. It was proved that the ideology of the capitalism system rules parents and teachers of which subdue to this system even believing that they are the ones who establish the rules and limits. For them the warranty of dignity value is associated with the family?s wealth, respect is related with children?s autonomy, even though adults and children subdue to the society?s oppressive relation and liberty has been understood as the permission for children to express themselves and do whatever they want to, thus it was not observed during our presence at the school. The Statute cannot guarantee a humanized life to this population once social inequalities restrain an equal treatment for those who live such distant reality. The school psychologist needs to assume a role of social transformation promoting the access, the argument and the criticism to what is imposed. / As desigualdades vividas em nossa sociedade e a dificuldade em existirem rela??es humanizadas entre adultos e crian?as tendo como base os valores atribu?dos no Estatuto da Crian?a e do Adolescente motivaram a realiza??o deste estudo. Assim, esta pesquisa teve como objetivo principal conhecer como os valores da trilogia da Doutrina da Prote??o Integral - liberdade, dignidade e respeito - eram compreendidos por pais e educadores e as suas implica??es no cotidiano da crian?a. Realizamos esta pesquisa em uma escola p?blica de educa??o infantil, tendo como participantes os pais e os educadores das crian?as que l? estudavam. Nossos dados foram produzidos nos grupos de discuss?o que realizamos com esses participantes, al?m dos registros dos di?rios de campo da pesquisadora, elaborados ao longo de sua inser??o na escola. Os resultados obtidos foram analisados ? luz do materialismo hist?rico e dial?tico, por acreditarmos na interpreta??o dos fen?menos a partir das contradi??es existentes na realidade cotidiana. Evidenciamos que a ideologia do sistema capitalista domina pais e educadores, os quais se submetem ao sistema, apesar de acreditarem que s?o eles que determinam suas regras e limites. Para eles a garantia do valor da dignidade est? associada ao poder aquisitivo da fam?lia, o respeito relaciona-se com a autonomia das crian?as, apesar de adultos e crian?as se submeterem ?s rela??es opressivas da sociedade e a liberdade foi compreendida como a permiss?o para a crian?a se expressar e realizar aquilo que desejasse, apesar de n?o ser isso que evidenci?vamos na nossa inser??o na escola. O Estatuto n?o ? capaz de garantir uma vida humanizada a essa popula??o uma vez que as desigualdades sociais impedem um tratamento igual para quem vive realidades t?o distantes. O psic?logo escolar, diante disso, precisa assumir um papel de transforma??o social oportunizando o acesso, o questionamento e as cr?ticas ?quilo que est? imposto.

Este estudo, de natureza qualitativa, buscou apreender o sentido da arte de partejar para cinco parteiras tradicionais que atuaram, ou ainda atuam, no distrito de Regência, ES, como a única possibilidade de assistência ao parto na região. Ao longo deste trabalho, buscou-se uma fundamentação teórica sobre o tema e que permitiu reconhecer, na história da parturição, até meados do século XX, que foram as parteiras quem sempre fizeram o parto e auxiliaram as mulheres em doenças especificamente femininas. Mesmo nos dias atuais, em regiões afastadas dos grandes centros e em zonas rurais, as parteiras continuam constituindo a única fonte da população para resolver seus problemas de saúde, especialmente na assistência do parto, realidade também constatada no presente estudo.A entrevista semi-estruturada foi utilizada como instrumento para coleta de dados. Histórias de vida oral temática foram obtidas, mediante os relatos das entrevistadas, os quais foram gravados em fitas cassete e transcritos, para desenvolvimento da análise de conteúdo. Todas as parteiras entrevistadas são pessoas simples, com nenhum ou pouco estudo e que, apesar de viverem com poucos recursos, orgulham-se de nada cobrarem pelos serviços prestados, aceitando somente alguns presentes, muitas dependendo da ajuda dos filhos para sua sobrevivência. A análise da trajetória dos sujeitos da população de estudo revelou que o ofício de parteira tem o sentido de missão de vida, decorrente do dom de que são portadores, dom este descoberto diante do inesperado, de uma demanda concreta, e que se desenvolve pela própria experiência, a serviço da coletividade, muitas vezes, sem nenhuma estrutura de apoio do sistema oficial de saúde. Diante dessa constatação, fica evidente a necessidade de uma articulação efetiva entre as parteiras e o sistema de saúde da região, visando a uma assistência de melhor qualidade à população local, respeitados os princípios do SUS. / This qualitative study tried to catch the art of midwife practice of five ladies who have been exercising it in Regência, ES, as the only possibility of helping pregnant women at baby delivering in that region. During this essay, a theorical embasement was searched, which allowed to recognize the role of midwives, until mid 20th Century, as the only assistance for delivery as well as specifically feminine diseases. Even nowadays, in the countryside or faraway counties, midwives still are the only resource to turn to in health cases or baby delivery assistance, as verified in the present study. The half-structured interview was used as a data collection tool. Verbal thematic stories of lives where obtained, by means of reported stories of the interviewed, which where recorded in cassette tapes and transcripts, for content analysis development. All the interviewed midwives are from humble background, with little or none education who, despite living short of money, feel proud of not charging anything for the job. Most depend on their own children’s help to survive financially. Sometimes, they accept gifts in return but never money. The analysis of the subjects of this study reveals that the craft of midwives is faced as a mission in life, due to the natural gift they were given, which was discovered in an unexpected situation, or a real demand and, eventually, was developed by experience itself, as a service to the community, most of the times, with no structural support on behalf of the official Healthy system. Aware of this, it becomes more and more evident that there is need for effective engagement among midwives and the Regional health system, having in mind a qualified and better assistance to the local population, respecting the principles of SUS (Unified Health System).

Submitted by Maria Aparecida (cidazen@gmail.com) on 2017-04-19T12:20:43Z No. of bitstreams: 1 Alice Parentes da Silva.pdf: 2593569 bytes, checksum: 94296a54a64fb3bfbea374d603ced0cd (MD5) / Made available in DSpace on 2017-04-19T12:20:43Z (GMT). No. of bitstreams: 1 Alice Parentes da Silva.pdf: 2593569 bytes, checksum: 94296a54a64fb3bfbea374d603ced0cd (MD5) Previous issue date: 2017-02-01 / Introduction: Good practices towards parturition and birth are recomendations based on scientific evidence of researches performed in different countries, which are the foundation for the work of Rede Cegonha, which aims to promote humanized assistance with quality, in addition to give women the chance of having a safe and positive experience, during pregnancy, parturition and birth. Among the notable and proved practices, which must be stimulated during labor, parturition and post-parturition we shall highlight: the free will companion during labor, parturition and post-parturition; the skin-to-skin contact between mother and child and the breastfeeding as precocious practices developed, preferably, in the first hour of life of the newborn. Objectives: To analyze the expectations and experiences of women during parturition and birth. Method: Qualitative research divided in three phases: prenatal interview; the observation of parturition; interview during puerperium. For database analysis, it was used the Contest Analysis in the Theme modality. Results and discussion: Eighteen women in the prenatal phase were interviewed. Among them, twelve were interviewed during puerperium and four were observed during parturition. The categories that highlighted in the research were: Too many doctors, no bond; An individual of the family would bring us another perspective from the birth room; The kind of parturition expected and the kind of parturition experienced; Sometimes they already put on the chest. Among the practices studied, the free will companion was the nearest recommended by the good practices during parturition and birth. On the other hand, breastfeeding showed the worst results during the research. Final considerations: Despite the actions on national levels, that aim to encourage the implementation of good practices during parturition and birth, those practices still presents many challenges. / Introdução: As boas práticas de atenção ao parto e ao nascimento são recomendações baseadas em evidências científicas de pesquisas realizadas em diversos países e ancoram a formulação das ações da Rede Cegonha, que visa promover assistência humanizada e de qualidade, além de possibilitar às mulheres experiências seguras e positivas durante a gravidez, o parto e o nascimento. Dentre as práticas demonstradamente úteis, que devem ser estimuladas durante o trabalho de parto, parto e pós-parto destacamos: o acompanhante de livre escolha no trabalho de parto, parto e pós parto; e o contato pele a pele entre a díade mãe-bebê e a amamentação como práticas precoces, desenvolvidas, de preferência, na primeira hora de vida do recémnascido. Objetivos: Analisar as expectativas e experiências de mulheres em relação ao momento do parto e do nascimento. Método: Pesquisa qualitativa realizada em três fases: entrevista no pré-natal; observação do parto; e entrevista no puerpério. Para análise dos dados foi utilizada Análise de Conteúdo na modalidade Temática. Resultados e discussão: Foram entrevistadas 18 mulheres na fase do pré-natal. Dessas, 12 foram entrevistadas no puerpério e 4 foram observadas durante o parto. As categorias encontradas foram: Muitos médicos, pouco vínculo; Uma pessoa da família seria o outro olho da gente; Parto esperado e parto vivido; Às vezes eles já colocam no peito. Das práticas estudadas, o acompanhante de livre escolha foi a que se apresentou mais próxima do preconizado pela RC. Por sua vez, a amamentação apresentou os piores resultados no estudo. Considerações finais: Apesar das ações a nível nacional que estimulam as boas práticas ao parto e nascimento, a implementação das mesmas ainda apresenta muitos desafios.

Esta dissertação procura evidenciar a dimensão socioambiental do parto humanizado, com foco nos conceitos que permeiam a saúde da mulher e do seu protagonismo enquanto parturiente, revelando a importância do saber ambiental nas pesquisas sociais. Desde a caça às bruxas e a medicalização da saúde, culminando na retirada das parteiras no momento do parto, as mulheres vêm sofrendo controle e vigilância dos seus corpos e lidam frequentemente com autoritarismos médicos e violências obstétricas. Ao longo da história da humanidade, a mulher vem sendo inferiorizada nos mais diversos campos de atuação e do conhecimento, ocupando poucos cargos políticos, além disso sofrem feminicídios, estupros, violências, sendo as mais prejudicadas pela industrialização e pela deterioração do ambiental natural. Ou seja, é subjugada diante de diversos elementos socioambientais, demonstrando a necessidade de um olhar mais apurado, empático e científico para a importância de seu protagonismo para os diversos níveis da construção humana. Pensar a dimensão socioambiental que agrega a mulher na hora do parto é um importante olhar para integrar as problemáticas sociais e ambientais. Agregando textos de autores de diversos campos do conhecimento, como biologia, filosofia, geografia humana e ciências sociais, procura-se estabelecer um diálogo com tais estudos, culminando na análise do protagonismo da parturiente como um evento significativo do socioambientalismo, que, aliado a determinados movimentos sociais, é capaz de produzir efeitos para além de si, promovendo não apenas uma melhoria no bem-estar da parturiente e do bebê, mas modificando estruturalmente o funcionamento das instituições e da sociedade como um todo / This dissertation aims to evidence the socio-environmental dimension of humanized birth, with a focus on the concepts that permeate women\'s health and their autonomy as a parturient, highlighting the importance of environmental understanding in social researches. Ever since the witch hunting time and the medicalization of health, culminating withdrawal of midwives at the childbirth moment, women have been suffering control and surveillance over their bodies and frequently have to deal with the medical authoritarism and obstetric violence. Throughout the history of humanity, women have been diminished in the most diverse fields of activity and knowledge, occupying the minority of political positions, also suffering feminicides, rapes, violence, being the most harmed by industrialization and the deterioration of natural environment. Therefore, women are subjugated in the face of several socio-environmental elements, demonstrating the need for more accurate, empathic and scientific look at the importance of their protagonism on different levels human construction. To consider the socio-environmental dimension that women add at the moment of childbirth is an importance way to go to integrate social and environmental issues. This work seeks to demonstrate the need for a more accurate, empathetic and scientific look at the female protagonism and its importance in the most diverse levels of human construction, especially at the moment of childbirth, a context that involves different socioenvironmental problems. Aggregating texts from authors of different fields of knowledge as biology, philosophy, human geography and social studies, it seeks to establish a dialogue with such studies, culminating in the analysis of the autonomy of the parturient as a significant event of socio-environmentalism, which, together with certain social movements, is capable of producing effects beyond itself, promoting not only an improvement in the well- being of the parturient and the baby, but also structurally modifying the functioning of the institutions and society as a whole

La maladie du greffon contre l’hôte (GvHD) est une complication majeure des greffes de cellules souches hématopoïétiques (HSCT) qui survient dans 30 à 70% des cas et peut causer la mort, malgré un traitement prophylactique bien conduit. Il existe donc une réelle demande clinique pour améliorer ces traitements prophylactiques. Parce que ces traitements prophylactiques reposent en général sur des agents immunosuppresseurs, ceux-ci contribuent à diminuer la reconstitution immunitaire du patient, ce qui a un impact défavorable sur les infections et les taux de rechute d’hémopathie maligne, et donc limite leur utilisation. Les immunoglobulines (IVIG) pourraient représenter une alternative intéressante puisqu’elles ont des propriétés immunomodulatrices et qu’elles sont de plus couramment utilisées en clinique pour traiter des patients ayant un déficit immunitaire. Leur capacité à réduire l’apparition et la sévérité de la GvHD, sans toutefois inhiber ou nuire à la reconstitution immunitaire chez le patient n’a néanmoins jamais été clairement démontrée. Les objectifs de ce projet sont donc d’évaluer l’efficacité des IVIG à réduire l’incidence et la sévérité de la GvHD dans un modèle murin humanisé de GvHD, ainsi que de déterminer le mécanisme d’action des IVIG. Ce modèle consiste à injecter des huPBMCs à des souris immunodéprimées ne pouvant les rejeter. Les résultats obtenus suggèrent que les IVIG possèdent un effet immunomodulateur permettant de réduire les signes cliniques et de retarder l’apparition de la GvHD, tout en permettant l’apparition de cellules NK. Les IVIG agiraient de façon indirecte sur les huPBMCs afin d’induire l’apparition des cellules NK. / Graft-versus-host disease (GvHD) is a major complication following hematopoietic stem cell transplantation (HSCT), occuring in 30 to 70% of HSCT despite proper prophylactic treatment, and can result in death. It is therefore primordial to improve the prophylactic treatments in order to reduce the frequency and severity of GvHD. Since these prophylactic treatments are based on the use of immunosuppressive agents, they inhibit the immune reconstitution and thus increase the risk of infections and relapse. Because of their immunomodulating properties, immunoglobulins (IVIG) represent a promissing alternative. Furthermore, IVIG are already widely used in patients with immune deficits. Since no study has yet clearly shown that IVIG can reduce GvHD without inhibiting the immune reconstitution in the patient, the scientific community is still debating their usefullness in GvHD prevention. Should their efficacy to reduce the incidence and severity of GvHD without impairment to the immune reconstitution be demonstrated, the use of IVIG could change the clinical outcome for HSCT patients, improving their remission and quality of life. The objectives of this project are to evaluate IVIG efficacy in GvHD prevention and to define the mechanism of action of IVIG. To fulfill these objectives, we will use a GvHD humanized mouse model, which consists of injecting huPBMCs in immunodeficient mice who can not reject them. Our results demonstrate an immunomodulatory effect of IVIG, allowing for reduced clinical signs and a slighlty increased survival in GvHD. A population of NK cells also appeared upon IVIG treatment and is believed to be indirectly induced by IVIG.

La Granulomatose Septique Chronique (CGD) est une maladie génétique rare de l’immunodéficience innée affectant les cellules phagocytaires (neutrophiles, macrophages). Elle est causée par des mutations dans les sous-unités du complexe NADPH oxydase formé du cytochrome b558 membranaire (NOX2 associé à p22phox) et de facteurs cytosoliques (p47phox, p67phox et p40phox). La déficience de ce complexe enzymatique va conduire à l’absence de formation de formes réactives de l’oxygène (FRO) microbicides et donc à l’apparition d’infections graves et récurrentes très tôt dans l’enfance. La chimioprophylaxie à vie permet de protéger ces patients mais peut être responsable d’effets indésirables. La seule thérapie curative est la transplantation de moelle osseuse mais tous les patients ne peuvent en bénéficier, et la thérapie génique n’est pas encore envisageable. Il y a donc un manque réel de nouvelles thérapies pour cette maladie. Cependant pour développer de nouveaux traitements, il faut disposer de modèles physiopathologiques pertinents. Or, les modèles existants sont imparfaits ou manquants. Le but de notre travail est donc de produire des modèles cellulaires et animaux de la CGD pour développer dans un second temps, une nouvelle approche thérapeutique basée sur l’utilisation de protéoliposomes.Grâce à leurs propriétés de pluripotence et d’auto-renouvellement à l’infini, les cellules souches pluripotentes induites (iPS) sont un outil puissant pour la modélisation physiopathologique. Ainsi, à partir de fibroblastes de patients atteints de CGD reprogrammés en cellules iPS, nous avons mis au point un protocole efficace de différenciation hématopoïétique in vitro en neutrophiles et macrophages. Nous avons montré que ces cellules phagocytaires sont matures et reproduisent parfaitement le phénotype déficient en FRO des patients CGD. Nous avons donc obtenu des modèles cellulaires pertinent modélisant trois formes génétiques de CGD, la CGD liée à l’X et deux formes autosomiques récessives, CGDAR22 et CGDAR47.Nous avons ensuite réalisé la preuve du concept de l’efficacité de protéoliposomes thérapeutiques sur les macrophages modélisés de la forme CGDX, la forme génétique la plus fréquente (70 % des cas) due à l’absence du cytochrome b558 membranaire (NOX2/p22phox). Grâce à une collaboration avec la start-up Synthelis SAS, des liposomes contenant le cytochrome b558 au niveau de la membrane lipidique ont été produits dans un système d’expression acellulaire basé sur l’utilisation d’extraits d’Escherichia coli. Ces liposomes NOX2/p22phox sont capables de reconstituer une enzyme NADPH oxydase fonctionnelle in vitro et de délivrer le cytochrome b558 à la membrane plasmique des macrophages CGDX qui présentent alors une restauration de l’activité NADPH oxydase avec la production de FRO.Enfin, nous nous sommes proposés de générer des souris dites « humanisées » par transplantation de cellules souches hématopoïétiques CD34+ capables de prise de greffe et de reconstitution hématopoïétique dans des souris immunodéficientes. A partir de cellules iPS saines, nous avons réussi à produire des cellules hématopoïétiques CD34+ possédant un potentiel hématopoïétique in vitro. Cependant, malgré des résultats encourageants, aucune prise de greffe in vivo n’a pu être réellement confirmée à ce jour.Pour conclure, nous avons donc montré au cours de ce projet, la production de modèles cellulaires de trois formes génétiques de CGD à partir de cellules iPS. Puis le modèle de macrophages CGDX nous a permis de faire la preuve de l’efficacité d’une nouvelle thérapie in vitro, une « enzymothérapie substitutive liposomale », qui pourrait à terme, offrir une alternative thérapeutique pour le traitement des infections aigües pulmonaires des patients CGD réfractaires aux traitements antibiotiques et antifongiques conventionnels. / Chronic Granulomatous Disease (CGD) is a rare inherited pathology of the innate immune system that affects the phagocytic cells (neutrophils, macrophages). This disease is caused by mutations in the subunits of the NADPH oxidase complex composed of the membrane cytochrome b558 (NOX2 associated with p22phox) and the cytosolic components (p47phox, p67phox et p40phox). Dysfunction in this enzymatic complex leads to the absence of microbicidal reactive oxygen species (ROS) and therefore to the development of recurrent and life-threatening infections in early childhood. Life-long prophylaxis is used to protect these patients but it may be responsible for side effects. Bone marrow transplantation is the only curative treatment but it can not be proposed to all the patients. In addition, gene therapy is not possible up to now. So there is a real lack of new therapies for this disease. However, to develop new therapeutic approaches, relevant physiopathological models must be available. Actually, existing models are imperfect or missing. Thus, the goal of our work is to produce cellular and animal models of CGD to develop a new proteoliposome-based therapy.Induced pluripotent stem cells (iPSCs) are a powerful tool for physiopathologic modeling due to their pluripotency and self-renewal properties. Using CGD patient-specific iPSCs regrogrammed from fibroblasts, we developped an efficient protocol for in vitro hematopoietic differentiation into neutrophils and macrophages. We showed that the phagocytic cells produced are mature and reproduce the ROS-deficient phenotype found in CGD patients. Thus, we obtained relevant cellular models for three genetic forms of CGD: X-linked CGD and the two autosomal recessive forms AR22CGD and AR47CGD.Then, we demonstrated the proof-of-concept of the efficacy of therapeutic proteoliposomes on X-CGD iPS-derived macrophages. Indeed, X-CGD is the main form of the disease (70% of cases) and is caused by the absence of the membrane cytochrome b558 (NOX2/p22phox). Thanks to a collaboration with the start-up Synthelis SAS, liposomes integrating the cytochrome b558 into lipid bilayers were produced in an E. coli-based cell-free protein expression system. These NOX2/p22phox liposomes were able to reconstitute a functional NADPH oxidase enzyme in vitro and to deliver the cytochrome b558 at the plasma membrane of X-CGD macrophages, leading to restore the NADPH oxidase activity with a ROS production.Finally, we proposed to generate « humanized » mice models with a human immune system after transplantation of CD34+ hematopoietic stem cells able to engraft and reconstitute long-term hematopoiesis in immunodeficient mice. Using healthy iPSCs, we successfuly produced CD34+ hematopoietic cells with in vitro hematopoietic potential. However, no in vivo engraftment was really confirmed yet.In conclusion, during this project, we produced cellular models of three genetic forms of CGD using patient-specific iPSCs. Then, X-CGD macrophages were used to demonstrate in vitro the efficacy of a new therapy. This « liposomal replacement enzymotherapy » could, in the future, represents a curative alternative against life-threatening lung infections refractory to conventional antibiotic and antifungal therapy.