OBJETIVOS: Comparar a expressão placentária de células natural killer deciduais (dNK), e a expressão sérica e placentária de suas citocinas reguladoras em gestações gemelares com pré-eclâmpsia (grupo pré-eclâmpsia, GPE) e sem comorbidades (grupo-controle, GC). MÉTODOS: Estudo transversal do tipo caso-controle, desenvolvido na Clínica Obstétrica do HC-FMUSP no período de julho de 2015 a junho de 2017. Foram obtidas amostras das regiões deciduais placentárias, para avaliação, por meio de técnica de imuno-histoquímica, da expressão de células dNK e suas interleucinas (IL) 10, 12 e 15, em pacientes que contemplaram os critérios de inclusão e concordaram em participar do estudo. Além disso, estas pacientes tiveram amostra sérica colhida no terceiro trimestre para dosagem de IL-10, IL-12 e IL-15 - por meio de kit comercial Milliplex®, que utiliza a tecnologia Luminex® xMAP®, da EMDMillipore (Merck Millipore Co., Alemanha) - e de fatores relacionados à angiogênese, como soluble fms-like tyrosine kinase-1 (sFlt-1) e placental growth factor (PlGF) - por meio de ensaio com imunoanalisador COBAS e411 (Roche Diagnostics, Alemanha). Os valores obtidos para as análises placentárias e séricas foram comparados entre o GPE e o GC, e a significância estatística estabelecida foi p < 0,05. RESULTADOS: Foram selecionadas 30 pacientes, sendo 20 no GC e 10 no GPE. Não se observaram diferenças significativas com relação às características maternas, gestacionais e de desfechos perinatais entre os dois grupos, exceto pela idade gestacional de início do pré-natal, menor no GPE (12,5 vs. 20,0 semanas, p = 0,015). Quanto à avaliação placentária, houve maior expressão de IL-15 no GPE (p = 0,001), e não houve diferença entre os grupos quanto à expressão placentária local de células dNK (p = 0,999), IL-10 (p = 0,063) e IL-12 (p = 0,135). Com relação às interleucinas séricas maternas, demonstrou-se aumento significativo nos níveis de IL-10 (22,7 vs. 11,9 pg/mL, p = 0,024) e IL-15 (15,9 vs. 7,4 pg/mL, p = 0,024) no GPE com relação ao GC, sem diferença entre os grupos para IL-12 (102,5 vs. 61,5 pg/mL, p = 0,373). A dosagem dos fatores relacionados à angiogênese demonstrou maiores níveis séricos maternos de sFlt-1 (15920 vs. 7978 pg/mL, p = 0,009) e da razão sFlt-1/PlGF (88,71 vs. 24,63, p = 0,002), e menores valores de PlGF (193,0 vs. 340,6 pg/mL, p = 0,036) no GPE. CONCLUSÃO: Observou-se maior concentração sérica materna tanto de fatores pró quanto anti-inflamatórios no GPE, quando comparado ao GC. Entretanto, não foram observadas diferenças entre os grupos quanto à expressão placentária de IL-10, importante fator anti-inflamatório. Estes achados podem sugerir que a tentativa sérica materna de equilibrar estas interleucinas não alcançou resposta localmente na placenta, contribuindo para o desenvolvimento da doença no GPE / OBJECTIVES: To compare the placental expression of decidual natural killer cells (dNK) and serum and placental expression of their regulatory cytokines in twin pregnancies with preeclampsia (preeclampsia group, PEG) and uncomplicated twin pregnancies (control group, CG). METHODS: This was a case-control study, developed in a tertiary referral center, from July 2015 to June 2017. Samples of the placental decidual region were obtained and analyzed by immunohistochemistry technique for the expression of dNK cells and interleukins (IL) 10, 12 and 15, in patients who met the inclusion criteria. In addition, maternal serum sample was collected in the third trimester for the dosage of IL-10, IL-12 and IL-15 - by means of a commercial Milliplex® kit using Luminex® xMAP® technology from EMDMillipore (Merck Millipore Co., Germany) - and angiogenesis factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) - by COBAS e411 immunoassay (Roche Diagnostics, Germany). The values obtained for the placental analyzes and maternal circulating factors were compared between PEG and CG and the statistical significance was set at p < 0.05. RESULTS: Thirty patients were selected, 20 in CG and 10 in PEG. There were no significant differences in maternal, gestational and perinatal outcomes between the two groups, except for the gestational age at the onset of prenatal care, which was lower in PEG (12.5 vs. 20.0 weeks, p = 0.015). PEG showed strong immunostaining for IL-15 (p = 0.001) when compared to CG, with no difference between the groups concerning the placental expression of dNK (p = 0.999), IL-10 (p = 0.063), and IL -12 (p = 0.135). Relating to maternal circulating interleukins, a significant increase in IL-10 (22.7 vs. 11.9 pg/mL, p = 0.024) and IL-15 (15.9 vs. 7.4 pg/mL, p = 0.024) was observed in PEG, with no difference between the groups for IL-12 (102.5 vs 61.5 pg/mL, p = 0.373). We also demonstrated higher maternal levels of sFlt-1 (15920 vs. 7978 pg/mL, p = 0.009) and sFlt-1/PlGF ratio (88.71 vs. 24.63, p = 0.002) and lower levels of PlGF (193 vs. 340.6 pg/mL, p = 0.036) in PEG. CONCLUSION: A higher maternal serum concentration of both pro- and anti-inflammatory factors was observed in the PEG. However, no difference was found between the groups regarding the placental expression of IL-10, an important anti-inflammatory factor. These findings may suggest that the maternal serum attempt to balance these interleukins did not reach local placental response, which contribute to the development of the disease in the PEG

Une compréhension fine et détaillée ciblant les mécanismes d’action de nouvelles molécules adjuvantes sur notre système immunitaire vise de manière directe à l’élaboration de nouveaux vaccins plus ciblés et plus efficaces, mais aussi à élargir nos connaissances quant à l’induction d’une réponse immune protectrice.<p>Au cours de cette thèse nous avons voulu comprendre les modes d’action de deux molécules lipidiques distinctes.<p>La première est le lipide cationique diC14-amidine dont il avait été démontré une action sur les cellules dendritiques en culture par une voie qui restait à élucider. Ce lipide cationique s'organise sous forme de liposomes en milieu aqueux et peut s'associer à de nombreux antigènes. La seconde est un analogue synthétique de l'adjuvant monophosphoryl lipide A (MPL), un dérivé du LPS, nommé CRX-527. À l'instar de sa molécule parente, le CRX-527 active le récepteur TLR4 et est considéré comme un adjuvant potentiel de vaccin ou comme immunostimulant isolé.<p>Au cours de notre travail, nous avons démontré que la diC14-amidine active les cellules cibles via le récepteur TLR4. En effet, l'absence de ce récepteur abolit les réponses induites par le lipide cationique diC14-amidine et la transfection du gène codant pour TLR4 rend répondeuses des cellules qui n'exprimaient pas ce récepteur. De plus, la diC14-amidine active et mature des cellules dendritiques, aussi bien de provenance murine qu'humaine, suggérant qu'elle puisse être utilisée en tant qu’adjuvant. Il avait d’ailleurs été précédemment décrit que l'injection d'un complexe diC14-amidine / allergène chez la souris induisait une réponse immune suffisante pour conférer une protection contre cet allergène. Dans ce contexte, nous avons caractérisé au niveau cellulaire la réponse induite suite à l'injection du complexe diC14-amidine / ovalbumine chez la souris. Cette réponse se manifeste par une production d'IFNγ lors d'une re-stimulation ex vivo par l'antigène OVA. <p>En ce qui concerne la molécule CRX-527, nous nous sommes particulièrement focalisés sur le rôle du co-récepteur du TLR4, le CD14, dans les réponses innées induites par le CRX-527. Nous avons établi que, de manière inattendue et contrairement à la plupart des ligands TLR4, le CRX-527 induit la production de nombreuses cytokines et chimiokines en complète absence de CD14, même à faible dose. De plus, l'ajout de CD14 sous sa forme soluble ne modifie pas le niveau des réponses associées à la voie de signalisation MyD88 / NF-κB. Cependant, il semblerait que la stimulation de cellules par du CRX-527 en présence de CD14 soluble recombinant, favorise plutôt la voie TRIF / IRF3, comme le suggère l'augmentation du taux de production d'IFNβ et d'activation d'IRF3. La molécule CD14 (membranaire et/ou soluble) ne serait donc pas qu'un simple transporteur de ligands, comme il l'a été décrit par le passé, mais bien une protéine impliquée dans la modulation des réponses induites lors de l'activation du TLR4. Le CD14 jouerait donc un rôle, aussi bien au niveau de la discrimination des ligands, que celle des voies de signalisation activées.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Vaccinia virus (VACV) is the most intensively studied orthopoxvirus and acts as an excellent model to investigate host-pathogen interactions. VACV encodes about 200 proteins, many of which modulate the immune response. This study focusses on two of these: C16 and C4, that share 43.7 % amino acid identity. Given the sequence similarity, we explored whether C16 and C4 have any shared functions, whilst also searching for novel functions. To gain mechanistic insight, we sought to identify binding partners and determine the residues responsible. C16 has two reported functions. Firstly, it inhibits DNA-PK-mediated DNA sensing, and this study found that C4 can perform this function as well. Like C16, C4 associates with the Ku heterodimer to block its binding to DNA leading to reduced production of cytokines and chemokines. For both proteins, the function localised to the C termini and was abrogated by mutating three residues. Secondly, C16 induces a hypoxic response by binding to PHD2. This function was mapped to the N-terminal 156 residues and a full length C16 mutant (D70K,D82K) lost the ability to induce a hypoxic response. In contrast, C4 did not bind PHD2. C4 inhibits NF-κB signalling by an unknown mechanism. Reporter gene assays showed that C16 also suppresses NF-κB activity and, intriguingly, this was carried out by both the N and C termini. C16 acts at or downstream of p65 and the N terminus of C16 associated with p65 independently of PHD2-binding. Conversely, C4 acted upstream of p65, did not display an interaction with p65, and the function was restricted to its C-terminal region. Novel binding partners were identified by a screen utilising tandem mass tagging and mass spectrometry, and selected hits were validated. The C terminus of C16 associated with VACV protein K1, a known NF-κB inhibitor. Additionally, C16 bound to the transcriptional regulator ARID4B. C4 did not interact with these proteins, but the N-terminal region of C4 associated with filamins A and B. The functional consequences of these interactions remain to be determined. In vivo, C4 and C16 share some redundancy in that a double deletion virus exhibits an attenuated virulence phenotype that is not observed by single deletion viruses in the intradermal model of infection. However, non-redundant functions also contribute to virulence in that both single deletion viruses display attenuated virulence compared to a wild-type Western Reserve virus in the intranasal model of infection. Data presented also reveal that C4 inhibits the recruitment of immune cells to the site of infection, as was previously described for C16. Overall, this investigation highlights the complexity of host-pathogen interactions showing that VACV encodes two multifunctional proteins with both shared and unique functions. Moreover, their inhibition of DNA-PK emphasises the importance of this PRR as a DNA sensor in vivo.

OBJETIVOS: Comparar a expressão placentária de células natural killer deciduais (dNK), e a expressão sérica e placentária de suas citocinas reguladoras em gestações gemelares com pré-eclâmpsia (grupo pré-eclâmpsia, GPE) e sem comorbidades (grupo-controle, GC). MÉTODOS: Estudo transversal do tipo caso-controle, desenvolvido na Clínica Obstétrica do HC-FMUSP no período de julho de 2015 a junho de 2017. Foram obtidas amostras das regiões deciduais placentárias, para avaliação, por meio de técnica de imuno-histoquímica, da expressão de células dNK e suas interleucinas (IL) 10, 12 e 15, em pacientes que contemplaram os critérios de inclusão e concordaram em participar do estudo. Além disso, estas pacientes tiveram amostra sérica colhida no terceiro trimestre para dosagem de IL-10, IL-12 e IL-15 - por meio de kit comercial Milliplex®, que utiliza a tecnologia Luminex® xMAP®, da EMDMillipore (Merck Millipore Co., Alemanha) - e de fatores relacionados à angiogênese, como soluble fms-like tyrosine kinase-1 (sFlt-1) e placental growth factor (PlGF) - por meio de ensaio com imunoanalisador COBAS e411 (Roche Diagnostics, Alemanha). Os valores obtidos para as análises placentárias e séricas foram comparados entre o GPE e o GC, e a significância estatística estabelecida foi p < 0,05. RESULTADOS: Foram selecionadas 30 pacientes, sendo 20 no GC e 10 no GPE. Não se observaram diferenças significativas com relação às características maternas, gestacionais e de desfechos perinatais entre os dois grupos, exceto pela idade gestacional de início do pré-natal, menor no GPE (12,5 vs. 20,0 semanas, p = 0,015). Quanto à avaliação placentária, houve maior expressão de IL-15 no GPE (p = 0,001), e não houve diferença entre os grupos quanto à expressão placentária local de células dNK (p = 0,999), IL-10 (p = 0,063) e IL-12 (p = 0,135). Com relação às interleucinas séricas maternas, demonstrou-se aumento significativo nos níveis de IL-10 (22,7 vs. 11,9 pg/mL, p = 0,024) e IL-15 (15,9 vs. 7,4 pg/mL, p = 0,024) no GPE com relação ao GC, sem diferença entre os grupos para IL-12 (102,5 vs. 61,5 pg/mL, p = 0,373). A dosagem dos fatores relacionados à angiogênese demonstrou maiores níveis séricos maternos de sFlt-1 (15920 vs. 7978 pg/mL, p = 0,009) e da razão sFlt-1/PlGF (88,71 vs. 24,63, p = 0,002), e menores valores de PlGF (193,0 vs. 340,6 pg/mL, p = 0,036) no GPE. CONCLUSÃO: Observou-se maior concentração sérica materna tanto de fatores pró quanto anti-inflamatórios no GPE, quando comparado ao GC. Entretanto, não foram observadas diferenças entre os grupos quanto à expressão placentária de IL-10, importante fator anti-inflamatório. Estes achados podem sugerir que a tentativa sérica materna de equilibrar estas interleucinas não alcançou resposta localmente na placenta, contribuindo para o desenvolvimento da doença no GPE / OBJECTIVES: To compare the placental expression of decidual natural killer cells (dNK) and serum and placental expression of their regulatory cytokines in twin pregnancies with preeclampsia (preeclampsia group, PEG) and uncomplicated twin pregnancies (control group, CG). METHODS: This was a case-control study, developed in a tertiary referral center, from July 2015 to June 2017. Samples of the placental decidual region were obtained and analyzed by immunohistochemistry technique for the expression of dNK cells and interleukins (IL) 10, 12 and 15, in patients who met the inclusion criteria. In addition, maternal serum sample was collected in the third trimester for the dosage of IL-10, IL-12 and IL-15 - by means of a commercial Milliplex® kit using Luminex® xMAP® technology from EMDMillipore (Merck Millipore Co., Germany) - and angiogenesis factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) - by COBAS e411 immunoassay (Roche Diagnostics, Germany). The values obtained for the placental analyzes and maternal circulating factors were compared between PEG and CG and the statistical significance was set at p < 0.05. RESULTS: Thirty patients were selected, 20 in CG and 10 in PEG. There were no significant differences in maternal, gestational and perinatal outcomes between the two groups, except for the gestational age at the onset of prenatal care, which was lower in PEG (12.5 vs. 20.0 weeks, p = 0.015). PEG showed strong immunostaining for IL-15 (p = 0.001) when compared to CG, with no difference between the groups concerning the placental expression of dNK (p = 0.999), IL-10 (p = 0.063), and IL -12 (p = 0.135). Relating to maternal circulating interleukins, a significant increase in IL-10 (22.7 vs. 11.9 pg/mL, p = 0.024) and IL-15 (15.9 vs. 7.4 pg/mL, p = 0.024) was observed in PEG, with no difference between the groups for IL-12 (102.5 vs 61.5 pg/mL, p = 0.373). We also demonstrated higher maternal levels of sFlt-1 (15920 vs. 7978 pg/mL, p = 0.009) and sFlt-1/PlGF ratio (88.71 vs. 24.63, p = 0.002) and lower levels of PlGF (193 vs. 340.6 pg/mL, p = 0.036) in PEG. CONCLUSION: A higher maternal serum concentration of both pro- and anti-inflammatory factors was observed in the PEG. However, no difference was found between the groups regarding the placental expression of IL-10, an important anti-inflammatory factor. These findings may suggest that the maternal serum attempt to balance these interleukins did not reach local placental response, which contribute to the development of the disease in the PEG

L’hypertension artérielle pulmonaire (HTAP) est une maladie grave caractérisée par une obstruction progressive des artères pulmonaires de petit calibre, conduisant à une augmentation des résistances vasculaires pulmonaires et, à terme, à une défaillance cardiaque droite et au décès du patient. La vasoconstriction, le remodelage vasculaire et la dysfonction endothéliale pulmonaire sont autant de facteurs qui contribuent au développement et à la progression de la maladie. Plusieurs arguments sont également en faveur d’une hypothèse de désordres immunologiques, voire autoimmuns, dans la physiopathologie de l’HTAP. Malgré ces données, le lien entre endothélium pulmonaire et système immunitaire dans cette maladie restent peu connus. Ce travail de thèse a donc eu pour objectif d’étudier et mieux comprendre la nature et les conséquences d’une communication aberrante entre cellules endothéliales pulmonaires et système immunitaire dans la pathogénèse de l‘HTAP, afin d’identifier de nouvelles cibles thérapeutiques. Pour cela, nous avons analysé le rôle de la dysfonction endothéliale dans la régulation de deux processus de la dysfonction immunitaire : l’autoimmunité pour la réponse adaptative d’une part, et la sécrétion de cytokines en ce qui concerne la réponse innée d’autre part. A travers ce travail de thèse, nous avons mis en évidence l’existence d’une communication aberrante entre endothélium pulmonaire et système immunitaire dans l’HTAP et montré que l‘endothélium pulmonaire jouait un rôle primordial dans le contrôle des réponses adaptatives, en régulant la fonction des lymphocytes T régulateurs via la leptine, et dans la participation active à la réponse innée, en acquérant un phénotype pro-inflammatoire. Cette meilleure compréhension du rôle de la dysfonction endothéliale dans la dérégulation du système immunitaire présente dans l’HTAP pourrait aider au développement de nouvelles stratégies thérapeutiques dans cette maladie. / Pulmonary arterial hypertension (PAH) is a severe disease characterized by a progressive obstruction of small pulmonary arteries, leading to an increase in pulmonary vascular resistance and ultimately right heart failure and death. Vasoconstriction, vascular remodeling and pulmonary endothelial dysfunction contribute to the disease development and progression. Increasing evidence are also suggesting the importance of immune disorders, such as autoimmunity, in PAH pathophysiology. Despite these data, the link between pulmonary endothelium and immune system is still unclear. The objective of this work was to investigate and elucidate the nature and the consequences of an aberrant communication between pulmonary endothelial cells and immune system in PAH pathogenesis, in order to identify new therapeutical targets. Therefore, we analyzed the role of endothelial dysfunction in the control of two types of altered immune responses: autoimmunity for the adaptive response and cytokine secretion for the innate response. In this work, we highlighted the existence of an aberrant communication between pulmonary endothelium and immune system in PAH and showed that pulmonary endothelium played a key role in the control of adaptive responses, by regulating regulatory T lymphocyte function in a leptin-dependent manner, and by actively participating to the innate responses through a pro-inflammatory phenotype. A better understanding of the role of endothelial dysfunction in PAH immune system dysregulation may help to the development of new therapeutical strategies for this disease.

Os avanços relacionados à ciência dos biomateriais e engenharia tecidual buscam esclarecer os mecanismos envolvidos na resposta biológica associada ao uso desses dispositivos e sua interação com o sistema imune. Neste estudo, avaliou-se a resposta imune e inflamatória desenvolvida em camundongos frente à implantação de membrana de cortical óssea bovina no tecido subcutâneo, em implantação única e sequencial de duas membranas, assim como os possíveis mecanismos envolvidos no processo de reconhecimento e reabsorção desse biomaterial, de acordo com análise histomorfométrica, enzimática e molecular. Após a implantação da membrana, sinais de reabsorção que antes eram notados em pontos isolados, aos poucos se unem até sua completa degradação, observada somente após 15 dias. Todo o processo de reabsorção da membrana é acompanhado por uma reação inflamatória de magnitude moderada, seguida pelo declínio do número de leucócitos, surgimento de células gigantes multinucleadas e formação de uma cápsula de tecido conjuntivo fibroso. A cinética de TNF- e MMP-2, MMP-8, MMP-9 e MMP-13 apresentou um padrão de produção decrescente, entretanto os níveis dos inibidores de metaloproteinases (TIMPs) e TGF- parecem atuar de forma inversa. A velocidade de reabsorção após duas implantações consecutivas da membrana foi maior quando comparada ao grupo de animais que sofreu apenas uma implantação, porém os resultados do teste de hipersensibilidade do tipo tardia (DTH) demonstraram que a membrana é biocompatível, pois não elicitou resposta imunológica exacerbada após uma segunda implantação, confirmando então a natureza não imunogênica desse biomaterial. Finalmente, os animais CD14KO e MyD88KO apresentaram uma reabsorção mais lenta da membrana implantada, quando comparados aos animais C57Bl/6 (WT), demonstrando que as moléculas CD14 e MyD88 estão envolvidas no reconhecimento do biomaterial e desempenham importante papel no processo de reabsorção da membrana de cortical óssea bovina, indicando a participação de PAMPs e/ou DAMPs na resposta biológica gerada por esse biomaterial. / Advances related to the biomaterials science and tissue engineering seek to clarify the mechanisms involved in the biological response associated with the use of those devices and their interaction with the immune system. This study evaluated the inflammatory and immune response developed in mice after implantation bovine bone cortical membrane in subcutaneous tissue, in both, unique and sequential implantation of 2 membranes, as the mechanisms involved in this biomaterial recognition and resorption process, on regards to histomorphometric, enzymatic and molecular analysis. After membrane implantation, previously observed signs of resorption in isolated spots, gradually unite until their complete degradation after 15 days. The whole membrane resorption process is accompanied by a moderate inflammatory reaction, followed by a decline in the leukocytes number, appearance of multinucleated giant cells and formation of a capsule of fibrous connective tissue. The kinetics of TNF-, MMP-2, MMP-8, MMP-9 e MMP-13 showed a pattern of decreasing production, however, levels of metalloproteinases inhibitors (TIMPs) and TGF- seem to act in reverse way. The resorption rate after two successive membrane implantations was higher when compared to the group which suffered only one implantation, however, the results of delayed test hypersensity (DTH) demonstrated that the membrane is biocompatible, that is, it does not elicited too high immune response after a second position, confirming the non immunogenic nature of this biomaterial. Eventually, CD14KO and MyD88KO strains showed a slower membrane resorption when compared to animals C57Bl/6, demonstrating that the CD14 and MyD88 molecules are involved in biomaterial recognition and play an important role in bovine cortical bone membrane resorption process, indicating that PAMPs and/or DAMPs are involved in biological response generated by this biomaterial.

A esclerose múltipla (EM) é uma doença autoimune desmielinizante progressiva imunomediada por linfócitos T auto-reativos, que provocam uma cascata imunológica, amplificando a inflamação local. No Diabetes mellitus tipo 1 (DM1), existem linfócitos T auto reativos destroem as células beta do pâncreas, causando deficiência na produção de insulina. O desenvolvimento de terapêuticas específicas fica limitado pela etiologia indefinida destas doenças, apesar de avanços na terapêutica antiinflamatória e imunossupressora. Uma alternativa de tratamento atual para tais doenças é o transplante autólogo de células tronco hematopoéticas (TACTH). O presente estudo, observacional do tipo transversal, com a coleta de dados de caráter retrospectivo, tem como objetivo identificar as reações adversas manifestadas pelos pacientes diabéticos ou de esclerose múltipla, submetidos ao TACTH no período de 2004 a dezembro de 2010. Para a coleta de dados elaborou-se dois instrumentos que foram submetidos à validação aparente e de conteúdo por três juízes. A amostra final do estudo foi constituída pela obtenção dos dados de 72 prontuários, sendo 23 de pacientes diabéticos e 49 de pacientes com EM. Em relação aos pacientes diabéticos 16 pertenciam ao sexo masculino e a idade média foi 18,26 anos. Todos possuíam positividade para o anticorpo anti-carboxilase do ácido glutâmico (antiGAD65). Quanto aos pacientes com EM, trinta e três pertenciam ao sexo feminino e idade média foi de 37,2 anos. O subtipo da doença mais frequente foi o surto-remissivo em 21 (42,9%) pacientes. A escala expandida do estado de incapacidade (EDSS) variou entre 3,0 e 6,5. Em relação às reações adversas manifestadas pelos pacientes diabéticos foram mais frequentes os calafrios, febre, cefaléia, náusea e vômito e nos pacientes com esclerose múltipla foram retenção hídrica e cefaléia. As principais intervenções de enfermagem identificadas para os pacientes diabéticos e com EM foram monitorização dos sinais vitais, coleta de hemocultura, otimização da administração de medicamentos antieméticos, controle da infusão da globulina antitimocitária, orientações sobre alimentação e para reduzir o risco de queda. Os pacientes com DM1 apresentam reações mais agudas e necessitam de monitorização contínua. Já os pacientes com EM são mais dependentes dos cuidados de enfermagem, exigindo maior tempo de cuidados prestados pelo profissional. Embora o DM1 e a EM sejam doenças distintas, percebe-se que na prática clínica, exigem do enfermeiro uma excelência no cuidado, quer pelas particularidades do tratamento realizado ou pelas singularidades de cada uma delas. / Multiple sclerosis (MS) is a progressive demyelinating autoimmune disease, immune- mediated by auto-reactive T lymphocytes, which provoke an immunological cascade, enhancing the local inflammation. In type 1 diabetes mellitus (DM1), self-reactive T lymphocytes exist that destroy ? cells in the pancreas, causing insulin production deficiency. The development of specific therapeutics is limited by these diseases\' undefined etiology, despite advances in anti-inflammatory and immunosuppressive therapy. A current treatment alternative for these diseases is autologous hematopoietic stem cell transplantations (AHSCT). The aim of this observational and cross-sectional study with retrospective data collection is to identify the adverse reactions manifested by diabetic or MS patients who were submitted to AHSCT between 2004 and December 2010. For data collection, two instruments were elaborated, submitted to face and content validation with the help of three experts. The final study sample comprised data from 72 patient files, 23 from diabetic and 49 from MS patients. As for the diabetic patients, 16 were male and the mean age was 18.26 years. All were positive for the anti-glutamic acid decarboxylase (antiGAD65) antibody. Concerning MS patients, 33 were female and the mean age was 37.2 years. The most frequent disease subtype was relapsing-remitting in 21 (42.9%) patients. The expanded disability status scale (EDSS) score ranged between 3.0 and 6.5. As for the adverse reactions the diabetic patients manifested, shivers, fever, migraine, nausea and vomiting were the most frequent, while fluid retention and migraine were the most frequent among multiple sclerosis patients. The main nursing interventions identified for the diabetic and MS patients were vital sign monitoring, blood culture collection, optimization of anti-emetic drug administration, control of anti- thymocyte globulin infusion, dietary orientations and advice to reduce the risk of falls. DM1 patients present more acute reactions and need continuous monitoring. MS patients are more dependent on nursing care, demanding lower professional care time. Although DM1 and MS are distinct conditions, in clinical practice, they demand excellent care from nurses, whether due to the particularities of the treatment or the singularities of each disease.

Os estágios avançados da DPOC são longos e dolorosos processos onde há o aumento dos sintomas, fazendo com que o paciente entre em um ciclo vicioso de deterioração da capacidade física, dispneia, ansiedade e isolamento social. Deste modo, o exercício vem se mostrando um componente importante na DPOC, auxiliando no tratamento medicamentoso para a redução dos sintomas e melhora da qualidade de vida. Neste contexto, não há muitos relatos na literatura sobre o papel da atividade física no padrão de secreção de citocinas e na resposta proliferativa de pacientes com DPOC. Além disso, não há muita concordância sobre o comprimento do telômero e fenótipo celular quando a comparados tabagistas que não desenvolvem a doença e pacientes com DPOC. O objetivo deste trabalho foi comparar alguns parâmetros imunológicos entre pacientes com DPOC e indivíduos tabagistas sem DPOC, e em pacientes com DPOC antes e após o programa de reabilitação oferecido no Hospital das Clínicas da FMUSP. As coletas de sangue foram realizadas em dois momentos para o grupo DPOC (pré e pós-programa de reabilitação pulmonar), e em um único momento para o grupo tabagista. Estas amostras foram processadas para obtenção de células mononucleares do sangue periférico, onde foram analisados os seguintes parâmetros: proliferação celular e apoptose, fenotipagem de linfócitos, comprimento relativo do telômero e dosagem de citocinas. Verificamos que indivíduos tabagistas possuem menores quantidades de proteína C reativa que pacientes com DPOC, e uma tendência a maior número de linfócitos. Além disso, o comprimento relativo do telômero em tabagistas é maior do que em pacientes com DPOC, especialmente em linfócitos TCD8+, e em menor grau em linfócitos TCD4+. Linfócitos TCD8+ de portadores de DPOC apresentaram maiores porcentagens de células terminalmente diferenciadas, sugerindo exaustão celular destes linfócitos, e menores porcentagens de células de memória central e memória efetora. Pacientes com DPOC apresentam maiores quantidades de citocinas comparados aos tabagistas sem DPOC. Já na comparação pré e pós-reabilitação verificamos menores quantidades de leucócitos, menores pontuações nos questionários de sintomas, e maiores distâncias percorridas no teste de caminhada de 6 minutos. Na avaliação da linfoproliferação, para as células estimuladas com mitógeno (fitohemaglutinina) e antígenos (citomegalovirus e Haemophilus influenza) foi possível verificar melhora na resposta linfoproliferativa dos pacientes no período pós-reabilitação, assim como maiores níveis da citocina imunoreguladora IL-10. Deste modo concluímos que pacientes com DPOC possuem um perfil mais pró-inflamatório e de diferenciação terminal que tabagistas sem a doença e que exercício físico é capaz de modular o ambiente inflamatório melhorando alguns parâmetros da resposta imune celular / The advanced stages of COPD are long and painful with increase of symptoms making the patients enter a vicious cycle of deterioration of physical capacity, dyspnea, anxiety and social isolation. Therefore, the exercise shows an important component of COPD pathogenesis, assisting in pharmacological treatment, reducing symptoms and enhancing life quality. In this context, there are no concise reports in literature about the role of physical activity in the pattern of cytokine secretion and proliferative response in COPD patients. Besides this, there is no consensus in the data comparing smokers with COPD and smokers without COPD. Because of this, we compared some immune parameters of smokers with COPD and smokers without COPD, and evaluated the cellular immune response before and after a rehabilitation program offered at the Hospital das Clínicas FMUSP. Blood collection was performed in two moments in the COPD group (before and after the rehabilitation program), and once in smokers without COPD. After that the samples were processed to peripheral blood mononuclear cells isolation, in which were analyzed the cellular proliferation and apoptosis, the lymphocyte phenotypic characteristics, the telomere length and the cytokines levels in serum and culture supernatants. Smokers without COPD have lower levels of C reactive protein, and a trend to greater percentages of lymphocytes than in smoker with COPD. The telomere length of COPD patients was shorter than that of smokers without COPD, especially in TCD8+ lymphocytes, with a non-significant trend in the TCD4+ lymphocytes. The TCD8+ subpopulation of COPD patients comprised greater percentages of terminally differentiated cells, and lower percentages of central memory and effector memory cells, suggesting a bias to more terminally differentiated (and eventually exhausted) cells. Moreover, the levels of pro inflammatory cytokines were greater in COPD patients. Evaluation of the exercise effect, we found greater quantities of leucocytes, lower scores in the symptoms questionnaires and longer distances in the six minute walk test after the rehabilitation program. Besides this, the proliferative response to the mitogen phytohemaglutinin, and the antigens from cytomegalovirus and nontypeable Haemophilus influenza were all improved after the exercise program with greater levels of secretion of the anti-inflammatory cytokine IL-10. In conclusion, COPD patients have a pro inflammatory profile and a bias for more terminally differentiated (and eventually exhausted) cells when compared with smokers without COPD, and that the exercise program is capable of modulating the inflammatory microenvironment enhancing some parameters of the cellular immune response

Francisella tularensis, l'agent de la tularémie, est une bactérie intracellulaire capable d'infecter un grand nombre de cellules dont les macrophages. Le système immunitaire inné cytosolique est capable de détecter la bactérie à différents stades de son cycle d'infection. Dans un premier temps, le macrophage détecte la bactérie cytosolique et produit de l'interféron de type I. Cet interféron induit l'expression de milliers de gènes. Le macrophage est ensuite capable de détecter l'ADN cytosolique de la bactérie via un récepteur spécifique AIM2. La liaison AIM2-ADN entraine la formation d'un complexe multi-protéique appelé inflammasome et se composant de AIM2-ASC-caspase-1. L'activation de ce complexe conduit à la maturation de la caspase-1. Caspase-1 permet la sécrétion de deux cytokines majeures antimicrobiennes : l'IL-1beta et l'IL-18. De plus, caspase-1 induit une mort programmée des cellules infectées appelée pyroptose. La sécrétion de cytokines et la pyroptose sont deux évènements majeurs pour lutter contre les pathogènes. Ma thèse a consisté à identifier le lien entre l'interféron et l'activation de l inflammasome AIM2 dans des macrophages infectés par la bactérie Francisella. En réalisant un crible a l'aide d'ARNs interférents, j'ai découvert que 2 protéines sont impliquées dans l'activation de cet inflammasome, les guanylate binding proteins 2 et 5 (GBP2 et GBP5). En collaboration avec l'équipe du Dr. Broz en Suisse, nous avons démontré que les GBPs étaient impliquées dans le contrôle de la réplication intracellulaire de Francisella et également dans la lyse de la bactérie permettant le relargage d'ADN et l'activation de l'inflammasome AIM2. Les GBPs sont induites par l'interféron de type I mais très majoritairement par l'interféron de type II (IFN- gamma). Nous avons mis en évidence que le contrôle de la réplication bactérienne est GB dépendant et inflammasome-dépendant en absence d'IFN- gamma mais qu'il devient totalement GB dépendant et inflammasome-indépendant dans des macrophages pré-stimulés avec de l'IFN- gamma. De plus, la mort des macrophages pré-stimulés avec de l'IFN- gamma et infectés par Francisella est également GBP-dépendante et inflammasome-indépendante. En prenant en compte tous ces résultats, nous concluons que les GBPs sont des protéines impliquées dans l'immunité des macrophages infectés par Francisella mais qu'elles ont un double rôle : d'une part celui d'induire l'activation de l'inflammasome (la pyroptose) sous le contrôle de l'interféron de type I et d'autre part, d'induire une mort cellulaire et la lyse des bactéries cytosoliques de manière indépendante de l'inflammasome sous le contrôle d'IFN- gamma. Nos résultats placent donc les GBPs comme les effecteurs majeurs de l'immunité cytosolique antibactérienne suite au traitement par l'IFN-gamma / Francisella tularensis is an intracellular bacterium, and the causative agent of tularemia, capable of infecting a large number of cells including macrophages. The innate cytosolic immune system is capable of detecting the bacterium at different stages of its infection cycle. Macrophages first detect the DNA of the cytosolic bacterium and produce type I interferon. Type I interferon subsequently induces the expression of thousands of genes. The macrophages then detect the cytosolic DNA of the bacterium via a cytosolic DNA sensor called AIM2. The AIM2-DNA binding results in the formation of a multi-protein complex called the AIM2 inflammasome composed of AIM2-ASC-caspase-1. Activation of this complex leads to the maturation of caspase-1. Caspase-1 activation leads to the secretion of two major antimicrobial cytokines, IL-1ß and IL-18. In addition, caspase-1 induces a programmed cell death termed pyroptosis. Cytokine secretion and pyroptosis are two major events in the control of pathogens. My PhD focused in identifying the link between interferon and activation of the AIM2 inflammasome in macrophages infected with the pathogenic bacterium Francisella. I performed a RNA interference screening and identified two proteins involved in the activation of the AIM2 inflammasome: guanylate binding proteins 2 and 5 (GBP2 and GBP5). In collaboration with Dr. Broz’s team in Switzerland, we demonstrated that GBPs are involved in the control of intracellular replication of Francisella and also in the lysis of the bacterium allowing the release of bacterial DNA and the activation of inflammasome AIM2. GBPs are induced by type I interferon but to a much greater extent by type II interferon (IFN-gamma). In the second part of my work, we demonstrate that the control of bacterial replication is GBP-dependent and inflammasome-dependent in the absence of IFN-gamma but that it becomes fully GBP-dependent and inflammasome-independent in macrophages primed with IFN-gamma. Cell-death of macrophages primed with IFN-? and infected with Francisella is also GBP-dependent and inflammasome-independent. Taken together, these results demonstrate that GBPs are innate immunity proteins involved in the death of macrophages and the bacterial growth restriction through two differents pathways : one induces the activation of inflammasome (induction of Pyroptosis) controlled with type I interferon signaling and, another induces cell-death and bacterial killing in an inflammasome-independent manner under the control of IFN-gamma. Our results thus discriminates the antimicrobial action of the inflammasome and of GBPs and position GBPs as the master antibacterial effectors of IFN-gamma, a key cytokine to fight cytosolic bacteria

Os estágios avançados da DPOC são longos e dolorosos processos onde há o aumento dos sintomas, fazendo com que o paciente entre em um ciclo vicioso de deterioração da capacidade física, dispneia, ansiedade e isolamento social. Deste modo, o exercício vem se mostrando um componente importante na DPOC, auxiliando no tratamento medicamentoso para a redução dos sintomas e melhora da qualidade de vida. Neste contexto, não há muitos relatos na literatura sobre o papel da atividade física no padrão de secreção de citocinas e na resposta proliferativa de pacientes com DPOC. Além disso, não há muita concordância sobre o comprimento do telômero e fenótipo celular quando a comparados tabagistas que não desenvolvem a doença e pacientes com DPOC. O objetivo deste trabalho foi comparar alguns parâmetros imunológicos entre pacientes com DPOC e indivíduos tabagistas sem DPOC, e em pacientes com DPOC antes e após o programa de reabilitação oferecido no Hospital das Clínicas da FMUSP. As coletas de sangue foram realizadas em dois momentos para o grupo DPOC (pré e pós-programa de reabilitação pulmonar), e em um único momento para o grupo tabagista. Estas amostras foram processadas para obtenção de células mononucleares do sangue periférico, onde foram analisados os seguintes parâmetros: proliferação celular e apoptose, fenotipagem de linfócitos, comprimento relativo do telômero e dosagem de citocinas. Verificamos que indivíduos tabagistas possuem menores quantidades de proteína C reativa que pacientes com DPOC, e uma tendência a maior número de linfócitos. Além disso, o comprimento relativo do telômero em tabagistas é maior do que em pacientes com DPOC, especialmente em linfócitos TCD8+, e em menor grau em linfócitos TCD4+. Linfócitos TCD8+ de portadores de DPOC apresentaram maiores porcentagens de células terminalmente diferenciadas, sugerindo exaustão celular destes linfócitos, e menores porcentagens de células de memória central e memória efetora. Pacientes com DPOC apresentam maiores quantidades de citocinas comparados aos tabagistas sem DPOC. Já na comparação pré e pós-reabilitação verificamos menores quantidades de leucócitos, menores pontuações nos questionários de sintomas, e maiores distâncias percorridas no teste de caminhada de 6 minutos. Na avaliação da linfoproliferação, para as células estimuladas com mitógeno (fitohemaglutinina) e antígenos (citomegalovirus e Haemophilus influenza) foi possível verificar melhora na resposta linfoproliferativa dos pacientes no período pós-reabilitação, assim como maiores níveis da citocina imunoreguladora IL-10. Deste modo concluímos que pacientes com DPOC possuem um perfil mais pró-inflamatório e de diferenciação terminal que tabagistas sem a doença e que exercício físico é capaz de modular o ambiente inflamatório melhorando alguns parâmetros da resposta imune celular / The advanced stages of COPD are long and painful with increase of symptoms making the patients enter a vicious cycle of deterioration of physical capacity, dyspnea, anxiety and social isolation. Therefore, the exercise shows an important component of COPD pathogenesis, assisting in pharmacological treatment, reducing symptoms and enhancing life quality. In this context, there are no concise reports in literature about the role of physical activity in the pattern of cytokine secretion and proliferative response in COPD patients. Besides this, there is no consensus in the data comparing smokers with COPD and smokers without COPD. Because of this, we compared some immune parameters of smokers with COPD and smokers without COPD, and evaluated the cellular immune response before and after a rehabilitation program offered at the Hospital das Clínicas FMUSP. Blood collection was performed in two moments in the COPD group (before and after the rehabilitation program), and once in smokers without COPD. After that the samples were processed to peripheral blood mononuclear cells isolation, in which were analyzed the cellular proliferation and apoptosis, the lymphocyte phenotypic characteristics, the telomere length and the cytokines levels in serum and culture supernatants. Smokers without COPD have lower levels of C reactive protein, and a trend to greater percentages of lymphocytes than in smoker with COPD. The telomere length of COPD patients was shorter than that of smokers without COPD, especially in TCD8+ lymphocytes, with a non-significant trend in the TCD4+ lymphocytes. The TCD8+ subpopulation of COPD patients comprised greater percentages of terminally differentiated cells, and lower percentages of central memory and effector memory cells, suggesting a bias to more terminally differentiated (and eventually exhausted) cells. Moreover, the levels of pro inflammatory cytokines were greater in COPD patients. Evaluation of the exercise effect, we found greater quantities of leucocytes, lower scores in the symptoms questionnaires and longer distances in the six minute walk test after the rehabilitation program. Besides this, the proliferative response to the mitogen phytohemaglutinin, and the antigens from cytomegalovirus and nontypeable Haemophilus influenza were all improved after the exercise program with greater levels of secretion of the anti-inflammatory cytokine IL-10. In conclusion, COPD patients have a pro inflammatory profile and a bias for more terminally differentiated (and eventually exhausted) cells when compared with smokers without COPD, and that the exercise program is capable of modulating the inflammatory microenvironment enhancing some parameters of the cellular immune response