Les fièvres hémorragiques à virus Lassa (LASV) et Ebola (EBOV) représentent un important problème de santé publique en Afrique. Les réponses immunes et la pathogenèse associées à ces maladies sont peu connues. Les cellules NK ont un rôle important dans la réponse immune innée par leurs propriétés cytotoxiques, mais également dans l'induction des réponses adaptatives par leur production de cytokines et leurs interactions avec les cellules dendritiques (DC) et les macrophages. Ce projet s'attache à comprendre le rôle des cellules NK dans le contrôle de la réplication virale et dans l'induction des réponses immunitaires au cours de ces infections. Un modèle in vitro de coculture de cellules NK humaines avec des DC et macrophages autologues a été développé. L'activation des cellules NK et leurs fonctions ont été analysées après l'infection par LASV et EBOV. Par ailleurs, les réponses des cellules NK en réponse à LASV ont été comparées avec celles induites lors de l'infection par le virus Mopeia (MOPV), très proche de LASV mais non pathogène pour l'homme. Les macrophages, mais pas les DC, infectés par LASV ou MOPV induisent l'activation et l'augmentation des capacités cytotoxiques des cellules NK. Toutefois, les cellules NK ne sont pas capables de lyser les cellules infectées et ne produisent pas d'IFN-γ. Les cellules NK s'activent et sont capables de lyser les cellules infectées en présence de macrophages mais également de DC infectés par des LASV mutants. Cependant, les IFN de type I sécrétés en grande quantité en réponse à ces virus ne sont pas impliqués dans l'activation des cellules NK. L'infection par EBOV n'induit qu'une très faible activation des cellules NK en présence de DC ou macrophages et ne conduit pas à la sécrétion de cytokines, ni à la modification du potentiel cytotoxique.Ces résultats permettent d'améliorer la compréhension des réponses immunes et des mécanismes de pathogenèse mis en place lors des fièvres hémorragiques Lassa et Ebola.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production, which can eventually lead to immune complex (IC)-mediated organ damage. Due to the stimulation of plasmacytoid dendritic cells (pDC) by nucleic acid-containing ICs (DNA- or RNA-IC), patients with SLE have an ongoing interferon (IFN)-α production. IFN-α induces a general activation of the immune system that may initiate or propagate an autoimmune process if not properly regulated. Previous studies have shown that natural killer (NK) cells potently enhance the IFN-α production by pDCs. In study I, the mechanisms behind the NK cell-mediated increased IFN-α production by RNA-IC-stimulated pDCs were investigated. ICs triggered CD56dim NK cells via FcγRIIIA to the secretion of cytokines (e.g. MIP-1β) that promoted IFN-α production. Additionally, an LFA-1-dependent cell-cell interaction between pDCs and NK cells strongly contributed to the increased production of IFN-α. In study II, the RNA-IC-induced regulation of surface molecules on pDCs and NK cells was investigated. The expression of CD319 and CD229, which are two SLAM family receptors genetically associated with SLE, was induced on pDCs and NK cells by RNA-IC. IFN-α-producing pDCs displayed an increased expression of CD319 and CD229, whereas pDCs from patients with SLE had a decreased expression of CD319. In study III, we serendipitously identified an SLE patient harboring autoantibodies to the NK cell receptor CD94/NKG2A. In study IV, sera from 203 patients with SLE were analyzed for autoantibodies to the CD94/NKG2A, CD94/NKG2C and NKG2D receptors. Seven patients harbored anti-CD94/NKG2A autoantibodies, and two of these patient’s autoantibodies also reacted with CD94/NKG2C. Anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies both interfered with the HLA-E-mediated regulation of NK cell cytotoxicity, and facilitated the elimination of target cells expressing these receptors. Furthermore, these autoantibodies were found in a group of severely diseased SLE patients and their titers closely followed disease activity. In conclusion, this thesis provides insights to molecular mechanisms whereby NK cells regulate the IFN-α production, it further links the SLAM receptors to SLE, and it describes novel autoantibodies to receptors regulating NK cell cytotoxicity. Together these findings strengthen the assumption that NK cells are involved in the pathogenesis of SLE.

L'hépatite chronique virale C est associée à une défaillance du système immunitaire. Nous nous sommes intéressés aux cellules NK et aux lymphocytes Treg, partenaires de la réponse immunitaire innée. Le nombre des NK, particulièrement les CD56dim, est significativement diminué chez les patients infectés, dans le foie plus que dans le sang, et s'accentue avec la fibrogenèse. Le nombre de CD3-CD56+brightNKG2A+ circulantes est corrélé à la sévérité de l'inflammation et de la fibrose et celui des CD3-CD56+dimNKG2A+ inversement corrélé à la charge virale. Les NK sont fonctionnelles, en capacité de produire de l'IFN-γ et d'engager un processus de cytolyse. L'expression de CD158 est significativement diminuée à la surface des NK hépatiques mais conservée dans les NK circulantes. L'expression de NKG2A,C,D dans les NK circulantes et hépatiques est identique à celles de patients non infectés. Les Treg intrahépatiques FoxP3+ sont quasi-exclusivement de phénotype CD4+. En analyse multivariée, le nombre de FoxP3+ est indépendamment associé à celui de CD8+, surtout dans les lésions nécrotico-inflammatoires et une corrélation forte est observée entre les transcrits CD8, FoxP3, IL-10 et TGF-β, suggérant que les Treg bloquent l'expansion et la cytotoxicité des TCD8 par contact cellulaire ou par le biais de cytokines immunosuppressives. L'équilibre entre FoxP3 et CD8 est rompu dans les grades et stades Métavir A>2 et F>3, avec un effondrement du rapport FoxP3/CD8. L'inflammation hépatique chronique s'accompagne de fibrose, aboutissant à la cirrhose, principale cause de CHC. Dans les cirrhoses virales C avec ou sans CHC, les lymphocytes CD3+, CD4+, CD8+, CD20+, CD56+, TCRγδ +, FoxP3+ sont plus nombreux dans la fibrose que dans le parenchyme. Le nombre de CD20+, CD3+, CD4+, CD8+ et l'expression d'IFN-γ et RANTES sont plus élevés dans les cirrhoses qui développent un CHC. En analyse multivariée, CD8 est le seul facteur indépendament associé à la récidive tumorale et à une diminution de la survie sans récidive à 5 ans. Les CD20+, CD3+, CD4+, CD8+, CD56+, TCRγδ+, FoxP3+ sont significativement moins nombreux dans le CHC que dans la cirrhose. Mais les FoxP3+ sont significativement plus nombreux et les CD56+ moins nombreux dans le CHC que dans le nodule parenchymateux, sans modification des LT, conduisant à une augmentation du rapport FoxP3/CD8 dans la tumeur. Les CD56+ diminuent de la cirrhose au CHC. Aucune corrélation n'est observée entre la densité intra-tumorale des lymphocytes étudiés et la récidive carcinomateuse. Conclusion. Un infiltrat inflammatoire dense au sein de la cirrhose C, particulièrement riche en CD8, favorise le développement et/ou la récidive du CHC.

S'ha establert un sistema experimental autòleg per a poder estudiar la resposta de les cèl.lules Natural Killer (NK) contra les cèl.lules dendrítiques derivades de monòcits (moDC), infectades pel Cytomegalovirus humà (HCMV). Els nostres resultats mostren que les cèl.lules NK responen contra les moDC infectades per HCMV, que presenten una expressió de les molècules MHC de classe I a superficie reduïda. Específicament, demostrem que la infecció per HCMV disminueix l'expressió en superficie d'HLA-E en les moDC, alliberant així la inhibició de les cèl.lules NK NKG2A+. Mostrem que els NKR anomenats NKp46 i DNAM-1 tenen un paper dominant en el reconeixement de les moDC infectades per HCMV i evidenciem la importància de la dinàmica dels mecanismes d'immunoevassió en la susceptibilitat a la resposta NK. Finalment, trobem que els interferons de tipus I i la IL-12 secretats en resposta a la infecció per HCMV, a més de participar en l'activació de la cèl.lula NK i en la secreció d'IFN-, inhibeixen l'expressió i la funció de NKG2D en les cèl.lules NK, com un mecanisme de regulació potencial per prevenir la reactivitat NK contra cèl.lules veïnes sanes. / Suitable experimental conditions have been established to dissect the role of NK cell receptors (NKR) and cytokines in the NK cell response against autologous human cytomegalovirus (HCMV) infected monocyte derived dendritic cells (moDC). Our results reveal that NK cells are capable of responding to HCMV infected moDC that have down-regulated surface MHC class I molecules. In particular, we prove that HCMV infection decreases surface HLA-E expression on moDC, thus releasing NKG2A+ NK cells from inhibition. We show that NKp46 and DNAM-1 NKR play a dominant role in the recognition of HCMV infected moDC and we provide evidences stressing the importance of the dynamics of viral immune evasion mechanisms in NK cell susceptibility. Finally, we find that type I interferons and IL-12 secreted in response to HCMV infection, beyond their participation in NK cell activation and IFN- secretion, transiently inhibit the expression and function of NKG2D in NK cells, thus providing a potential regulatory feedback mechanism to prevent NK cell reactivity against bystander healthy cells.

Cytomegaloviruses (CMVs) are ubiquitous in nature, having evolved over many millenia with their hosts. While in healthy hosts most infections with CMV are asymptomatic, the virus can cause severe disease in immunocompromised hosts. Thus, the increase in organ transplantation and the HIV/AIDS pandemic have established human CMV (HCMV) as a clinically important pathogen. Indeed, HCMV infections are now the major cause of morbidity and mortality among immunocompromised patients, which has led to more research targeting CMV for effective anti-viral treatment. The discovery that cytomegaloviruses encode several genes which are involved in immune escape has prompted a new area of research, aimed at understanding immune escape mechanisms for exploitation as potential anti-viral therapeutics. By targeting the viral proteins directly, or their receptors in the host, it may be possible to treat CMV disease by agonistic/antagonistic therapy. The first part of this thesis describes the first demonstration of anti-NK1.1 staining in situ to identify NK cells using a modified in vivo perfusion/fixation method. Using this method, we have compared the acute NK1.1+ cellular response to wild-type MCMV infection in the visceral organs of genetically susceptible intra-NK complex recombinant BALB.B6-CT6 (Cmv1s, NK1.1+) mice with resistant C57B⁄J (Cmv1r, NK1.1+) and BALB.B6-Cmv1r mice (Cmv1r, NK1.1+). Expression of viral antigens and the consequences of infection on other cellular subsets, were also analyzed in this study. The data show that in susceptible mice (Cmv1s) MCMV infection is predominent in the marginal zone of splenic white pulp, resulting in local changes in various cellular constituents, including macrophages, NK cells and DC. In the liver, distinct foci of infection were comprised of large numbers of macrophages and NK1.1+ cells surrounding infected cytomegalic cells. In resistant mice (Cmv1r), 6 MCMV infection predominantly affected the red-pulp of the spleen and was associated with increased accumulation of NK1.1+ cells and macrophages at sites of viral infection

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

As células Natural Killer (NK) fazem parte da resposta imune inata, sendo a primeira linha de defesa do organismo contra vírus, bactérias, tumores e microorganismos. Estas células induzem a morte da célula-alvo quando não há o reconhecimento das moléculas de antígenos leucocitários humanos (HLA) de classe I, através de seus receptores, chamados Killer cell Immunoglobulin-like Receptor (KIR). Vários estudos demonstram o envolvimento dos genes KIR na patogênese das doenças auto-imunes. Acredita-se que combinações desses genes possam ser favoráveis para o desenvolvimento da esclerose sistêmica (ES). Portanto, o conhecimento destes genes relacionados às células NK poderiam ser úteis para o entendimento da patogênese da ES. O objetivo deste estudo é investigar o polimorfismo dos genes KIR em um grupo de pacientes com ES, incluindo a forma difusa e limitada da doença. A freqüência do receptor inibidor KIR2DL2 foi significantemente menor nos pacientes comparada com a do grupo controle (28,7% versus 65,2%; P<0,001; OR=0,21; IC95% 0,11–0,38). Quando analisamos a combinação do receptor inibidor 2DL2, com a presença do ativador 2DS2 (KI2DS2+/KIR2DL2-), encontramos uma maior freqüência nos pacientes (26,1% versus 1,7%; P<0,001; OR=19,94; IC95% 4,7–175,1). Por outro lado, a presença de ambos KIR2DL2 e KIR2DS2 foi mais freqüente no grupo controle (26,9% versus 57,3%; P<0,001; OR=0,27; 95%CI 0,1–0,4). Nenhuma diferença estatística no polimorfismo dos genes KIR foi encontrada entre a forma difusa e a forma limitada. A combinação KIR2DS2+/KIR2DL2– parece ser um fator de risco para o desenvolvimento da ES enquanto a alta freqüência do gene inibidor KIR2DL2 no grupo controle parece ter uma função protetora. Estes resultados indicam um potencial papel dos genes KIR na patogênese da ES. / Natural killer (NK) cells have an important role in the early responses to viral infections. They kill diverse target cells with decreased or absent expression of major histocompatibility complex (MHC) class I molecules through the Killer Cell Immunoglobulin-Like Receptors (KIR). Many studies have reported association of KIR genes with autoimmune diseases. The objective of this study is to investigate possible associations of KIR polymorphisms with systemic sclerosis (SSc), including the limited (lSSc) and diffuse (dSSc) forms of the disease. The frequency of inhibitory KIR2DL2 was significantly decreased among patients with SSc compared with healthy controls (28.7% versus 65.2; P<0.001, odds ratio [OR] 0.21, 95% confidence interval [95% CI] 0.11–0.38). When activatory and inhibitory KIR genes were analyzed in combination, the concomitant presence of KIR2DS2 and absence of KIR2DL2 (KI2DS2+/KIR2DL2-) phenotype was more frequent in SSc patients than in the control group (26.08% versus 1.75%; P<0.001, OR=19.94, 95%CI [4.78–175.10]). On the other hand, the presence of both KIR2DS2 and KIR2DL2 was more frequent in the control group (26.96% versus 57.39%; P=0.000005, OR=0.27, 95%CI [0.15–0.49]). No significant difference in KIR genes polymorphisms was found between lSSc and dSSc disease subsets. The combination of KIR2DS2+/KIR2DL2– may be a risk factor for development of SSc while the higher frequency of the inhibitory KIR2DL2 gene in the control group suggest to a protective effect. These results indicate a potential role of KIR genes in the SSc pathogenesis.

As células Natural Killer (NK) fazem parte da resposta imune inata, sendo a primeira linha de defesa do organismo contra vírus, bactérias, tumores e microorganismos. Estas células induzem a morte da célula-alvo quando não há o reconhecimento das moléculas de antígenos leucocitários humanos (HLA) de classe I, através de seus receptores, chamados Killer cell Immunoglobulin-like Receptor (KIR). Vários estudos demonstram o envolvimento dos genes KIR na patogênese das doenças auto-imunes. Acredita-se que combinações desses genes possam ser favoráveis para o desenvolvimento da esclerose sistêmica (ES). Portanto, o conhecimento destes genes relacionados às células NK poderiam ser úteis para o entendimento da patogênese da ES. O objetivo deste estudo é investigar o polimorfismo dos genes KIR em um grupo de pacientes com ES, incluindo a forma difusa e limitada da doença. A freqüência do receptor inibidor KIR2DL2 foi significantemente menor nos pacientes comparada com a do grupo controle (28,7% versus 65,2%; P<0,001; OR=0,21; IC95% 0,11–0,38). Quando analisamos a combinação do receptor inibidor 2DL2, com a presença do ativador 2DS2 (KI2DS2+/KIR2DL2-), encontramos uma maior freqüência nos pacientes (26,1% versus 1,7%; P<0,001; OR=19,94; IC95% 4,7–175,1). Por outro lado, a presença de ambos KIR2DL2 e KIR2DS2 foi mais freqüente no grupo controle (26,9% versus 57,3%; P<0,001; OR=0,27; 95%CI 0,1–0,4). Nenhuma diferença estatística no polimorfismo dos genes KIR foi encontrada entre a forma difusa e a forma limitada. A combinação KIR2DS2+/KIR2DL2– parece ser um fator de risco para o desenvolvimento da ES enquanto a alta freqüência do gene inibidor KIR2DL2 no grupo controle parece ter uma função protetora. Estes resultados indicam um potencial papel dos genes KIR na patogênese da ES. / Natural killer (NK) cells have an important role in the early responses to viral infections. They kill diverse target cells with decreased or absent expression of major histocompatibility complex (MHC) class I molecules through the Killer Cell Immunoglobulin-Like Receptors (KIR). Many studies have reported association of KIR genes with autoimmune diseases. The objective of this study is to investigate possible associations of KIR polymorphisms with systemic sclerosis (SSc), including the limited (lSSc) and diffuse (dSSc) forms of the disease. The frequency of inhibitory KIR2DL2 was significantly decreased among patients with SSc compared with healthy controls (28.7% versus 65.2; P<0.001, odds ratio [OR] 0.21, 95% confidence interval [95% CI] 0.11–0.38). When activatory and inhibitory KIR genes were analyzed in combination, the concomitant presence of KIR2DS2 and absence of KIR2DL2 (KI2DS2+/KIR2DL2-) phenotype was more frequent in SSc patients than in the control group (26.08% versus 1.75%; P<0.001, OR=19.94, 95%CI [4.78–175.10]). On the other hand, the presence of both KIR2DS2 and KIR2DL2 was more frequent in the control group (26.96% versus 57.39%; P=0.000005, OR=0.27, 95%CI [0.15–0.49]). No significant difference in KIR genes polymorphisms was found between lSSc and dSSc disease subsets. The combination of KIR2DS2+/KIR2DL2– may be a risk factor for development of SSc while the higher frequency of the inhibitory KIR2DL2 gene in the control group suggest to a protective effect. These results indicate a potential role of KIR genes in the SSc pathogenesis.

O presente estudo tem como objetivo investigar a frequência dos diversos polimorfismos dos genes KIR (Killer Immunoglobulin-like Receptors) e HLA C1 e C2 em um grupo de pacientes com câncer de mama e comparar com um grupo controle de indivíduos sadios. As células natural killer (NK) são linfócitos que diferem das células T e B e que fazem parte da imunidade natural, reconhecendo as moléculas HLA (Antígenos Leucocitários Humano) de classe I em células infectadas por vírus ou em células tumorais, através de seus receptores de membrana. Os principais receptores das células NK são conhecidos como receptores KIR, sendo codificados por genes localizados no cromossomo 19q13.4 e classificados em grupos funcionais supressores e ativadores. Neste estudo, analisamos 15 genes KIR e alelos do sistema HLA de classe I em 230 pacientes caucasóides e em 278 controles, usando a técnica de PCR com primers específicos (PCR-SSO e PCR-SSP). Nossos resultados demonstraram uma frequência maior do genótipo supressor 2DL2 (P<0,001) em pacientes com câncer de mama, quando comparados ao grupo controle. Os genes HLA-C2 e HLA-BW4 não apresentaram diferenças significantes entre os grupos. Contudo, o gene HLA-C1 foi observado em maior frequência nos pacientes com câncer de mama. Considerando que estes achados sugerem uma potencial associação entre o sistema de genes KIR, HLA classe I e o câncer de mama, estudos adicionais sobre este tema são necessários. / We investigated the frequency of various KIR (Killer Immunoglobulin-like Receptors) and HLA C1 and C2 gene polymorphisms in a group of patients with breast cancer and healthy controls. Natural Killer (NK) cells are lymphocytes that differ from T and B cells and are part of the innate immune system, recognizing class I Human Leukocyte Antigens (HLA) molecules on target cells (virus-infected as well as cancer cells), through specific cell surface receptors. KIR comprises the main class of NK receptors, being encoded by genes located in chromosome 19q13.4. They possess both suppressor and activating functional groups. Fifteen KIR genes and class I HLA alleles obtained from 230 Caucasians patients, as well as 278 controls were studied, using PCR techniques with specific primers (PCR-SSO and PCR-SSP). Our results showed a higher frequency of suppressor genotype 2DL2 (P<0,001) in patients with breast cancer as compared to controls. No significant difference between HLA-C2 and HLA-BW4 alleles were observed between the study groups. Notably, a higher frequency of HLA-C1 gene was noted in patients with breast cancer. Our results suggest a potential association between KIR genes, HLA class I and breast cancer, deserving further investigation.

Orientador: Aureo Tatsumi Yamada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T22:06:48Z (GMT). No. of bitstreams: 1 Lima_PatriciaDanieleAzevedo_M.pdf: 2003445 bytes, checksum: 597310400704b8df5b1e07544bd6caca (MD5) Previous issue date: 2008 / Resumo: Durante a gestação em animais que possuem placentação hemocorial, a hipóxia no primeiro terço da prenhez é um dos fatores cruciais para indução da angiogênese e o adequado desenvolvimento da placenta. Contudo, esta hipóxia se contrapõe à intensa atividade das células que requerem elevado metabolismo, gerando um estresse fisiológico para estas células presentes na interface materno-fetal. Presume-se que estas células necessitem de mecanismos apropriados de citoproteção para sua sobrevida enquanto comprometidos ativamente no suporte funcional do útero gestante. Neste sentido, o presente trabalho teve como objetivo investigar a expressão e a distribuição da proteína de choque térmico 70 (HSP70) na interface materno-fetal durante a gestação normal em camundongos e a sua possível variação em condição de estresse adicional induzido experimentalmente através da lesão embrionária. Sítios de desenvolvimento embrionário/fetal de camundongos prenhes do dia de gestação (dg) 6 ao 17 e, após 30 minutos, 1, 6 e 12 h dos animais submetidos à lesão cirúrgida do embrião (LCE) no dg 9 foram coletados para: - processamento histotécnico convencional de embebição em parafina destinados às análises citoquímicas (lectina DBA e reação de TUNEL) e imunocitoquímicas (anti-HSP72/73, anti-PCNA); - embebição em resina Lowilcryl- K4M para imunocitoquímica ultraestrutural (anti-HSP72/73); - obtenção de homogenados teciduais destinados à SDS-PAGE das frações protéicas e Westernblot (anti-HSP72/73) e, - extração de RNA de homogenados teciduais e de células uNK isoladas para análise de transcritos (HSP72 e 73) com amplificação pelo RTPCR. As análises imunocitoquímicas demonstraram que as células uNK eram as únicas células que expressavam de forma constante as isoformas HSP72/73 ao longo da gestação, sendo confirmada a expressão dos transcritos gênicos das isoformas HSP72/73 nas células uNK isoladas pelo RT-PCR. A imunomicroscopia eletrônica detectou marcação conspícua nas mitocôndrias das células uNK. A análise quantitativa demonstrou que a lesão do embrião reduz o número de células uNK positivas para HSP72/73 e, o SDS/PAGE/Western-blotting identificou as isoformas HSP72 e 73 presente nos homogenados teciduais do útero com uma perceptível redução na intensidade da banda correspondente ao HSP73 nas amostras de pós-lesão, sem afetar significativamente a isoforma HSP72. As análises realizadas com a dupla marcação de TUNEL e PCNA demonstrarm redução de células uNK PCNA positivas no útero submetido a lesão embrionária e aumento de núcleos marcadas positivamente pelo TUNEL. Estes resultados demonstram de forma inédita a expressão de HSP72/73 nas células uNK, sendo inédita também a constatação em leucócitos, sugerindo um papel citoprotetor para estas células importantes na manutenção da gestação. A redução de células uNK HSP72/73 positivas no útero gestante desencadeada pela lesão embrionária, consubstancia a hipótese da atuação da HSP 72/73 como chaperona citoprotetora nas células uNK sendo crítica a atuação da isoforma HSP73 presente na mitocôndria através da regulação negativa das vias de morte celular por apoptose nas células uNK / Resumo: Durante a gestação em animais que possuem placentação hemocorial, a hipóxia no primeiro terço da prenhez é um dos fatores cruciais para indução da angiogênese e o adequado desenvolvimento da placenta. Contudo, esta hipóxia se contrapõe à intensa atividade das células que requerem elevado metabolismo, gerando um estresse fisiológico para estas células presentes na interface materno-fetal. Presume-se que estas células necessitem de mecanismos apropriados de citoproteção para sua sobrevida enquanto comprometidos ativamente no suporte funcional do útero gestante. Neste sentido, o presente trabalho teve como objetivo investigar a expressão e a distribuição da proteína de choque térmico 70 (HSP70) na interface materno-fetal durante a gestação normal em camundongos e a sua possível variação em condição de estresse adicional induzido experimentalmente através da lesão embrionária. Sítios de desenvolvimento embrionário/fetal de camundongos prenhes do dia de gestação (dg) 6 ao 17 e, após 30 minutos, 1, 6 e 12 h dos animais submetidos à lesão cirúrgida do embrião (LCE) no dg 9 foram coletados para: - processamento histotécnico convencional de embebição em parafina destinados às análises citoquímicas (lectina DBA e reação de TUNEL) e imunocitoquímicas (anti-HSP72/73, anti-PCNA); - embebição em resina Lowilcryl- K4M para imunocitoquímica ultraestrutural (anti-HSP72/73); - obtenção de homogenados teciduais destinados à SDS-PAGE das frações protéicas e Westernblot (anti-HSP72/73) e, - extração de RNA de homogenados teciduais e de células uNK isoladas para análise de transcritos (HSP72 e 73) com amplificação pelo RTPCR. As análises imunocitoquímicas demonstraram que as células uNK eram as únicas células que expressavam de forma constante as isoformas HSP72/73 ao longo da gestação, sendo confirmada a expressão dos transcritos gênicos das isoformas HSP72/73 nas células uNK isoladas pelo RT-PCR. A imunomicroscopia eletrônica detectou marcação conspícua nas mitocôndrias das células uNK. A análise quantitativa demonstrou que a lesão do embrião reduz o número de células uNK positivas para HSP72/73 e, o SDS/PAGE/Western-blotting identificou as isoformas HSP72 e 73 presente nos homogenados teciduais do útero com uma perceptível redução na intensidade da banda correspondente ao HSP73 nas amostras de pós-lesão, sem afetar significativamente a isoforma HSP72. As análises realizadas com a dupla marcação de TUNEL e PCNA demonstrarm redução de células uNK PCNA positivas no útero submetido a lesão embrionária e aumento de núcleos marcadas positivamente pelo TUNEL. Estes resultados demonstram de forma inédita a expressão de HSP72/73 nas células uNK, sendo inédita também a constatação em leucócitos, sugerindo um papel citoprotetor para estas células importantes na manutenção da gestação. A redução de células uNK HSP72/73 positivas no útero gestante desencadeada pela lesão embrionária, consubstancia a hipótese da atuação da HSP 72/73 como chaperona citoprotetora nas células uNK sendo crítica a atuação da isoforma HSP73 presente na mitocôndria através da regulação negativa das vias de morte celular por apoptose nas células uNK / Abstract: During the pregnancy of animals developing hemochorial placenta, the hypoxia in the first third of pregnancy is one of the crucial factor for induction of angiogenesis and adequate placental development. However, this hypoxia is contradictory to the great dynamism and metabolism of cells required in the pregnant uterus, conditioning a physiological stress for the cells present at the maternal-fetal interface. It is presumed these cells demand appropriate cytoprotective mechanism for their survival while are committed to actively support the pregnancy. In this way, the present work aimed to investigate the expression and distribution of the chapelone isoforms heat shock protein 72 and 73 (HSP72/73) at the maternal fetal-interface through the pregnancy in mice and its possible variations under additional stressing condition induced experimentally by embryo lesion. Embryo/fetus developing sites of pregnant mice from gestational days (gd) 6 to 17 and, after 30min, 1h and 6h of surgical embryo lesion (SEL) on gd 9 mice, were collected for: - conventional paraffin embedding for cytochemical (DBA lectin and TUNEL reaction) and immunocytochemical (anti-HSP72/73, anti-PCNA) analysis; - LR-white resin embedding for ultrastructural immunocytochemistry (anti- HSP72/73); - uterine tissue homogenates for SDS-PAGE of proteins fractions and Western-blot (anti-HSP7273) and; - RNA extratction form uterine tissue homogenates and isolated uNK cells for transcripts (HSP72 and 73) amplification by RT-PCR. The immunocytcchemical analysis showed the uNK cells as the only cell expressing constantly the HSP72/73 isoforms throughout the gestation, being confirmed the expression of both gene isoforms by RT-PCR in uNK cells. The immunoelectron microscopy detected conspicuous labeling in the mitochondria of uNK cells. The quantitative analysis demonstrated that embryo-lesion reduced the number of HSP72/73 positive uNK cells in the uterus and, SDS/PAGE and Westernblot identified the HSP72 and 73 isoforms present in the tissue homogenates with low reactive intensity of the band corresponding to HSP73 in the after-lesion samples, without affecting significantly the HSP72 isoform. The analysis of TUNEL and PCNA double labelling showed decreasing of PCNA positive-uNK cells in the uterus after embryo-lesion and increasing of TUNEL positive nuclei. These results confirms the expression of HSP72 and HSP73 isoforms in the uNK cells through the gestation and to date, this is also the first report showing HSP70 in leukocytes, suggesting a cytoptotective function to this cell while working actively as important cells supporting the pregnancy. The decreasing of HSP72/73 positive uNK cells in the pregnant uterus triggered by embryo lesion consubstantiate the hypothesis of HSP72/73 working as cytoprotective chaperone in the uNK cells, and the HSP73 isoform in the mitochondria seems to be critical on down-regulation of apoptotic cell depth pathway / Abstract: During the pregnancy of animals developing hemochorial placenta, the hypoxia in the first third of pregnancy is one of the crucial factor for induction of angiogenesis and adequate placental development. However, this hypoxia is contradictory to the great dynamism and metabolism of cells required in the pregnant uterus, conditioning a physiological stress for the cells present at the maternal-fetal interface. It is presumed these cells demand appropriate cytoprotective mechanism for their survival while are committed to actively support the pregnancy. In this way, the present work aimed to investigate the expression and distribution of the chapelone isoforms heat shock protein 72 and 73 (HSP72/73) at the maternal fetal-interface through the pregnancy in mice and its possible variations under additional stressing condition induced experimentally by embryo lesion. Embryo/fetus developing sites of pregnant mice from gestational days (gd) 6 to 17 and, after 30min, 1h and 6h of surgical embryo lesion (SEL) on gd 9 mice, were collected for: - conventional paraffin embedding for cytochemical (DBA lectin and TUNEL reaction) and immunocytochemical (anti-HSP72/73, anti-PCNA) analysis; - LR-white resin embedding for ultrastructural immunocytochemistry (anti- HSP72/73); - uterine tissue homogenates for SDS-PAGE of proteins fractions and Western-blot (anti-HSP7273) and; - RNA extratction form uterine tissue homogenates and isolated uNK cells for transcripts (HSP72 and 73) amplification by RT-PCR. The immunocytcchemical analysis showed the uNK cells as the only cell expressing constantly the HSP72/73 isoforms throughout the gestation, being confirmed the expression of both gene isoforms by RT-PCR in uNK cells. The immunoelectron microscopy detected conspicuous labeling in the mitochondria of uNK cells. The quantitative analysis demonstrated that embryo-lesion reduced the number of HSP72/73 positive uNK cells in the uterus and, SDS/PAGE and Westernblot identified the HSP72 and 73 isoforms present in the tissue homogenates with low reactive intensity of the band corresponding to HSP73 in the after-lesion samples, without affecting significantly the HSP72 isoform. The analysis of TUNEL and PCNA double labelling showed decreasing of PCNA positive-uNK cells in the uterus after embryo-lesion and increasing of TUNEL positive nuclei. These results confirms the expression of HSP72 and HSP73 isoforms in the uNK cells through the gestation and to date, this is also the first report showing HSP70 in leukocytes, suggesting a cytoptotective function to this cell while working actively as important cells supporting the pregnancy. The decreasing of HSP72/73 positive uNK cells in the pregnant uterus triggered by embryo lesion consubstantiate the hypothesis of HSP72/73 working as cytoprotective chaperone in the uNK cells, and the HSP73 isoform in the mitochondria seems to be critical on down-regulation of apoptotic cell depth pathway / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural