Cytotoxiques en lysant différents types de cellules et régulent la réponse immunitaire. Leur rôle dans l’asthme et ses exacerbations reste encore à identifier même si des modifications phénotypiques ont été observées chez des patients asthmatiques et qu’il a été récemment montré dans un modèle murin qu’elles n’intervenaient pas dans le développement de l’asthme allergique. L’objectif de la thèse était de mieux comprendre la place des cellules NK dans la pathologie asthmatique en se focalisant sur deux aspects : l’exacerbation viro-induite et l’inhibition par des composants microbiens.L’hypothèse pour la 1ère partie de la thèse était que les cellules NK de patients asthmatiques pouvaient présenter une dysfonction dans leur réponse à des agents microbiens qui pourrait favoriser l’exacerbation de la réaction asthmatique. Pour cela, nous avons analysé l’activation, la cytotoxicité et la production de cytokines de cellules NK provenant de patients asthmatiques sévères stimulées avec des molécules mimant des microorganismes ou un rhinovirus vivant (HRV), en comparaison avec des donneurs sains. Nous avons montré que les cellules NK de patients sévères étaient moins cytotoxiques que les cellules NK de donneurs sains en réponse à la stimulation avec un agoniste de Toll-Like Receptor 3 ou du TLR7/8 et avec HRV. En outre, lorsqu’elles sont stimulées avec de l’IL-12 et de l’IL-15, des cytokines produites pendant l’infection virale, les cellules NK de patients asthmatiques sévères expriment moins d’IFN-γ que les cellules NK de donneurs sains. Nos résultats suggèrent que l’activation des cellules NK de patients asthmatiques pourrait être insuffisante pendant les infections respiratoires et pourraient participer à l’aggravation de l’asthme.L’hypothèse pour la 2ème partie de la thèse était que les cellules NK pourraient participer à l’inhibition de la réaction asthmatique allergique dans un modèle murin. Dans des souris C57BL/6 sensibilisées à l’ovalbumine, l’instillation de FSL1, un agoniste de TLR2/6 inhibe la réaction asthmatique allergique. Cette inhibition étant associée à des modifications de la population des cellules NK, nous avons analysé leur rôle grâce à des souris déficientes en cellules NK. En l’absence de cellules NK, les souris développent un asthme allergique, et l’inhibition par FSL1 est maintenue. Par conséquent, les cellules NK ne jouent pas de rôle dans le développement de l’asthme allergique expérimental, ni dans son inhibition induite par un agent microbien. Cependant, elles pourraient être modifiées par l’environnement allergique, et avoir ainsi un rôle dans les exacerbation viro-induites. Cette question cruciale rejoint le travail réalisé dans la première partie de la thèse.En conclusion, nos résultats suggèrent que les fonctions des cellules NK seraient modifiées dans la pathologie asthmatique, qu’elle soit allergique ou non. Notre hypothèse est que le défaut d’activation des cellules NK participerait aux exacerbations viro-induites de l’asthme. Les perspectives de ces travaux sont de poursuivre la caractérisation des cellules NK chez les patients asthmatiques sévères et d’évaluer le rôle des cellules NK dans un modèle murin d’exacerbation de la réaction asthmatique.L’hypothèse pour la première partie de la thèse était que les cellules NK de patients asthmatiques pouvaient présenter une dysfonction dans leur réponse à des agents microbiens qui pourrait favoriser l’exacerbation de la réaction asthmatique. Pour cela, nous avons analysé l’activation, la cytotoxicité et la production de cytokines de cellules NK provenant de patients asthmatiques sévères stimulées avec des molécules mimant des microorganismes ou un rhinovirus vivant (HRV), en comparaison avec des donneurs sains. Nous avons montré que les cellules NK de patients sévères étaient moins cytotoxiques que les cellules NK de donneurs sains en réponse à la stimulation avec un agoniste de Toll-Like Receptor 3 ou du TLR7/8 et avec HRV [...] / Asthma is a chronic inflammatory disease of the airways affecting 334 million of people worldwide. Among asthma patients, 5% suffers from severe asthma. Severe asthma represents a major unmet need because, despite heavy treatments, patients still suffer from uncontrolled asthma symptoms, frequent exacerbations and a dramatic decrease in their respiratory capacity. The role of microorganisms in asthma is complex. On one hand, a group of epidemiologic and experimental studies have shown that chronic exposure with bacteria or microbial compounds, particularly during early childhood, would provide protection against allergic asthma. On the other hand, respiratory viruses are responsible for 80% of exacerbations and are associated with an increasing risk of developing asthma in children, whether allergic or not. Natural Killer cells (NK) are lymphocytes involved in innate antiviral immunity. They have cytotoxic functions by lysing different types of cells but also regulatory functions by producing cytokines and activating other immune cells. Their role in asthma and its exacerbations has yet to be identified, although phenotypic changes have been observed in asthmatic patients and it has recently been shown in a mouse model that they are not involved in the development of allergic asthma. The objective of the thesis was to better understand the role of NK cells in asthmatic pathology by focusing on two aspects : virus-induced exacerbation and inhibition by microbial compounds.The hypothesis for the first part was that NK cells from asthma patients may present a dysfunction in their response to microbial agents that could promote the exacerbation of the asthmatic reaction. To do this, we analysed NK cell activation, cytotoxicity and production of cytokines from severe asthmatic patients stimulated with molecules mimicking microbes or a human rhinovirus (HRV), compared to healthy donors. We showed that NK cells from severe asthma patients were less cytotoxic than NK cells from healthy donors in response to stimulation with a Toll-like Receptor 3 or TLR7/8 agonist and HRV. Moreover, when stimulated with IL-12 and IL-15, cytokines produced during viral infections, NK cells from severe asthma patient express less IFN-γ than NK cells from healthy donors. Our results suggest that the activation of NK cells in asthma patients may be insufficient during respiratory infections and may contribute to the worsening of asthma.The hypothesis for the second part was that NK cells may participate to the inhibition of a mouse model of allergic asthma. In C57BL/6 mice sensitized with ovalbumin, instillation of FSL1, agonist of TLR2/6, inhibits the features of experimental asthma. Since this inhibition is associated with changes in the population of NK cells, we analysed their role using mice deficient in NK cells. In the absence of NK cells, mice develop allergic asthma, and inhibition by FSL1 is maintained. Therefore, NK cells do not play a role in the development of experimental allergic asthma or in its inhibition induced by a microbial agent. However, they may be modified by the allergic environment, and thus have a role in viro-induced exacerbations. This crucial question is in line with the work done in the first part of the thesis.In conclusion, our results suggest that the functions of NK cells may be modified in asthmatic pathology, whether allergic or not. Our hypothesis is that the defect in NK cell activation may participate to virus-induced asthma exacerbations. Perspectives of this work are to further characterize NK cells in severe asthma patients and to evaluate the role of NK cells in a mouse model of asthma exacerbation.

Department of Biochemistry, Faculty of Science, Charles University in Prague 2010 Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography Abstract of Ph.D. thesis Ondřej Vaněk Supervisor: Prof. RNDr. Karel Bezouška, DSc. Natural killer cells (NK cells) were found out for their ability to spontaneously kill certain allogeneic tumour cell lines, without any previous sensitization. NK cells are part of non- adaptive immune response with very short reaction time against pathogens such as viruses, intracellular bacteria, parasites, and they are responsible for elimination of certain tumour cells and thus they are able to fight against malignancy and formation of metastasis. Activity of NK cells is regulated by the balance between activation and inhibitory signals mediated by the NK cell surface receptors. From the structural point of view, the majority of NK cell surface receptors could be classified as the C-type lectin or immunoglobulin-like receptors. One of many C-type lectin subgroups are type II lymphocyte receptors that are expressed on the NK cell surface. This study had two main aims. The first one was to find suitable expression and purification systems for selected C-type lectin receptors of NK cells and the other one was to perform their...

Embora a obesidade esteja associada à disfunção imune, com incidência aumentada de infecções e alguns tipos de cânceres, há poucos estudos que avaliaram parâmetros imunológicos em pacientes obesos graves. Além disso, há um número limitado de trabalhos analisando o efeito da perda de peso sobre parâmetros imunológicos na obesidade grave. Desta forma, o objetivo do presente trabalho foi avaliar a influência da perda de peso de pacientes com obesidade grau III submetidos à cirurgia de derivação gastrojejunal em Y de Roux (DGJYR) em parâmetros imunológicos. A produção de citocinas associadas com a resposta imune adquirida (IL-2, IL-4, IL-10 e IFN-) e inata (TNF- e IL-6) por células mononucleares de sangue periférico (PBMC), o perfil das populações de linfócitos e a atividade citotóxica de células natural killer (NK), além de citocinas associadas a sua função e desenvolvimento (IL-12 e IL-18), foram avaliados em vinte e oito pacientes não diabéticos, sedentários, com obesidade grau III (20 mulheres e 8 homens, com média de idade de 39,9 ± 10,9 anos e IMC de 49,5 ± 7,1kg/m2) no pré-operatório e 6 meses após a cirurgia. As PBMC foram estimuladas com o mitógeno fitohemaglutinina (PHA) e as citocinas produzidas foram quantificadas por ELISA. O perfil das populações de linfócitos foi avaliado por citometria de fluxo. A citotoxicidade mediada por células NK foi determinada pelo ensaio de liberação de LDH por células alvo K562. A perda de peso foi de 35,3 ± 4,5 kg, com uma significativa redução no IMC seis meses após a cirurgia (-12,9 ± 0,9 kg/m2, p< 0,001). Nenhuma das populações de linfócitos analisadas apresentou modificação no 6º mês após a cirurgia. Observou-se aumento significativo da proliferação de linfócitos seis meses após a cirurgia (p= 0,0026). Houve aumento pósoperatório nas concentrações de IFN-, IL-12 e IL-18 produzidas por PBMC após estímulo com PHA, enquanto a IL-2 apresentou uma tendência ao aumento (p= 0,07). As demais citocinas não apresentaram variação significativa. A atividade citotóxica das células NK aumentou seis meses após a cirurgia [17,1 ± 14,7% no pré vs 51,8 ± 11,3% 6 meses pósoperatório, na proporção 40:1 (célula NK:célula alvo); p< 0,001], mostrando recuperação quando se compara aos valores obtidos em indivíduos controle, pareados por idade e sexo, de peso normal [proporção 40:1 (célula NK:célula alvo) de 45,4 ± 7,8%]. Houve aumento de atividade citotóxica em todos os pontos da curva no pós-operatório em cerca de 79% da amostra (22 pacientes). Os resultados obtidos demonstram que a perda de peso induzida por DGJYR aumenta a produção de algumas citocinas relacionadas com a função das células NK e melhora a sua atividade citotóxica. As alterações na função de células NK e do nível de citocinas envolvidas com a atividade destas células podem explicar a propensão ao desenvolvimento de infecções e cânceres associados com a obesidade. Os dados obtidos neste estudo sugerem que a cirurgia bariátrica pode ter impacto positivo sobre estes fatores. / Although obesity is related to immune dysfunction, with a higher incidence of infections and some types of cancer, few studies have evaluated immunological parameters in severely obese patients. Moreover, a limited set of studies have analyzed the effect of weight loss in immunological parameters in severely obese patients. Thus, the objective of this thesis was to evaluate the influence of weight loss induced by Roux en-Y gastric bypass in patients with grade III obesity in immunological parameters. The production of cytokines associated with acquired (IL-2, IL-4, IL-10 and IFN-) and innate (TNF- e IL-6) immune responses from peripheral blood mononuclear cells (PBMCs), the profile of lymphocytes populations and the cytotoxic activity of natural killer cells (NK), besides cytokines related with NK cell cytotoxic function and development (IL-12 e IL-18), were analyzed in 28 non-diabetic and sedentary patients with grade III obesity (20 women and 8 men, 39,9 ± 10,9 years and BMI 49,5 ± 7,1 kg/m2) before and 6 months after RYGB. PBMCs were stimulated with the mitogen phytohemagglutinin (PHA) and cytokines were measure by ELISA. The profile of lymphocytes populations was evaluated by flow cytometry. NK cell cytotoxicity was determined by the lactate dehydrogenase release assay from K562 lysed target cells. The weight loss 6 months after surgery was 35.3±4.5 kg and there was a significant post-surgical decrease in BMI at this point (-12.9±0.9 kg/m2, p<0.001). No significant differences were found in the lymphocytes populations after surgery. It was observed a significant increase in the lymphocytes proliferation six months after surgery (p= 0.0026). There was also a post-surgical increase in the production of IFN-, IL-12 e IL-18 from PBMC stimulated with PHA, while there was a trend towards the increase of the IL-2 production (p=0.07). The other cytokines analyzed were not altered. Cytotoxic activity of NK cells was significantly enhanced 6 months after RYGB [17.1±14.7% before RYGB vs 51.8±11.3% at 6 months after, at effector to target cell (NK cell:K562 cell) ratio 40:1; p<0.001], and it was in the same range when compared to data obtained from controls with normal BMI matched for age and gender (45,4 ± 7,8% at NK cell:K562 cell ratio 40:1). There was a significant post-surgical improvement in all points of the cytotoxic activity curve in almost 79% of the sample (22 patients). In conclusion, the data obtained show that the weight loss induced by RYGB increases the production of cytokines related with NK cell cytotoxic function and improves its activity. The impairment in NK cells cytotoxic activity and cytokines observed in patients with severe obesity may explain their propensity to develop infections and cancer. Our data suggests that the weight loss induced by bariatric surgery can positively impact these factors.

This study examines the psychology of fictional and real-life serial killers and the behavioral similarities between them. Three fictional murderers, mainly Macbeth (William Shakespeare’s Macbeth), Buffalo Bill (The Silence of the Lambs), and the Creature (Frankenstein),as well as real life killers such as Charles Manson, Ed Gein, and Edmund Kemper were researched in depth. The data for this study was gathered from a variety of sources such as biographies, television interviews, published novels, articles, and documentaries. This study also focuses on predispositional factors and personality traits that led these killers to a life of crime. While no single behavioral trait was found to be present in every murderer studied, some of the psychological factors that were found to have predictive value included: abusive upbringings, mother hate, adoption, pornography, and brain damage were also reliable predictors in the lives of fictional and nonfictional perpetrators.

O exercício aeróbio aumenta o fluxo sanguíneo para o músculo esquelético, diminuindo esse fluxo em outros órgãos como útero e placenta o que pode prejudicar a oxigenação e nutrição do embrião. O estímulo do exercício extenuante pode ser estressor, aumentando a secreção de glicocorticóides através do eixo Hipotálamo-Hipófise-adrenal e que podem causar complicações durante a gestação, como as observadas em outras situações como o estresse alimentar e após infecção. As células Natural Killer uterinas (uNK) correspondem a maior população de leucócitos deciduais de humanos e roedores. As uNK de camundongos têm sido muito estudas por meio da citoquímica de lectina DBA (Dolichos Biflorus Agglutinin), que tem afinidade para N-acetil-D-Galactosamia expressa seletivamente nos grânulos e membrana plasmática destas células. Esta seletividade propiciou a caracterização de quatro subtipos de uNK, relacionados aos estágios de diferenciação destas células. A função mais aceita das uNK é a secreção de INF-y levando a dilatação das artérias espiraladas uterinas para manutenção da decídua e nutrição do embrião. Este estudo teve o objetivo de investigar se o exercício extenuante exerceria efeitos na implantação e integridade embrionária, bem como, no número, diferenciação, distribuição e padrão de reatividade para lectina DBA das uNK. Dez camundongos Swiss fêmeas foram mantidos sedentários (grupo 1). Outros 10 (grupo 2) foram submetidos a natação, do 1º ao 10º dia de gestação (dg), durante 60 minutos diários, dos quais os últimos 20 minutos se procederam com halter com massa de 4% do peso corporal, preso a suas caudas, em dias alternados do 1º ao 5º dg, e de 10% dessa massa do 6º ao 10º dg. Os animais do grupo 3 após prenhez, foram submetidos a natação da mesma maneira que os do grupo 2, porém passaram por um programa de treinamento de 3 semanas anterior a prenhez durante 5 dias/semana, sendo realizado 15 minutos/dia na primeira semana, aumentando gradativamente em 10 minutos/dia até 60 minutos na segunda semana e 60 minutos/dia na terceira semana. Todos os camundongos foram anestesiados e perfundidos com PFA (paraformaldeído) 4% no 10º dg. As taxas de implantação, viabilidade e reabsorção embrionárias foram avaliadas e os sítios de implantação destes animais foram coletados e embebidos em parafina para obtenção de cortes histológicos que foram analisados por lectina DBA e HE (Hematoxilina e Eosina). Foram quantificados os 4 subtipos de células uNK DBA reativos em 3 áreas da região mesometrial dos sítios de implantação. Nossos resultados mostraram que o exercício físico extenuante é capaz de causar diminuição na implantação, viabilidade embrionária e aumento na perda fetal, bem como, aumento em número e na diferenciação das células uNK, sem aparente ativação de sua citotoxicidade. O exercício realizado previamente a prenhez diminuiu estes efeitos, pois restabeleceu as taxas normais de implantação e o número total de uNK, bem como, aumentou a viabilidade e diminuiu a perda embrionária, contudo sem impedir completamente os efeitos deletérios do exercício na gestação. Nossos resultados indicam que esse modelo de estresse poderá ser útil para estudos futuros da investigação dos mecanismos neuroimunoendócrinos, envolvidos com parto prematuro e aborto causado por fatores estressores muitas vezes desconhecidos. / Aerobic exercise increases blood flow to skeletal muscle, decreasing the flow in other organs such as uterus and placenta, which may impair embryo oxygenation and nutrition. Furthermore, the continuous exercise can be stressful, changing the hypothalamic-pituitary-adrenal axis leading to secretion of glucocorticoids that may cause complications during pregnancy, such as those observed in other situations such as dietary and infection-mediated stress . Uterine natural killer cells (uNK) are the largest population of decidual leukocytes from humans and rodents. The mouse uNK have been extensively investigated by the use of DBA lectin cytochemistry, which has affinity for N-acetyl-D-Galactosamia selectively expressed in the granules and plasma membrane of these cells. This selectivity allowed the characterization of four subtypes of uNK related to their stages of differentiation. The most accepted function of uNK is the secretion of IFN-y leading to dilation of the uterine spiral arteries, maintaining the decidua and embryo nutrition. the goal of this study was to investigate whether strenuous exercise has effect in the embryo implantation and integrity, as well as, in the number, differentiation, distribution and DBA lectin reactivity pattern of uNK cells. Ten female Swiss mice were sedentary (group 1). Other 10 (group 2) were submitted to swimming, from 1st to 10th gestation day (gd) for 60 minutes daily, been the last 20 minutes conducted with a steel caudal dumbbell with 4% of their body masses, attached in alternately days from the 1st to 5th gd, and 10% of this mass from the 6th to 10th gd. The animals from group 3 after pregnancy were submitted to swimming in the same way as group 2, but went through a training program for 3 weeks, 5 days a week, being 15 minutes/day in the first week, increasing gradually by 10 minutes a day up to 60 minutes in the second week and 60 minutes a day in the third week. All mice were anesthetized and perfused with 4% PFA at 10°dg. The rates of embryo implantation, viability resorption were evaluated and the implantatio sites of these animals were collected and embedded in paraffin to obtain histological sections that were submitted to DBA lectin and H&E. We quantified the four DBA reactive uNK cells subtypes in three areas of the mesometrial region of the implantation sites. Our results showed that strenuous exercise can cause a decrease in implantation embryo viability and increased fetal loss, as well as increase in number and differentiation of uNK cells, without apparent activation of its cytotoxicity. The exercise performed prior to pregnancy decreased these effects, since restored the normal rates of implantation and the total number of UNK, as well as increased viability and decreased embryonic loss, without completely prevent the deleterious effects of exercise during pregnancy. Our results indicate that this stress model established here may be useful for future studies about the neuroimmunoendocrinology of preterm labor and abortion caused by often unknown stress factors. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

A sepse é caracterizada por ser um processo inflamatório sistêmico devido a uma infecção, levando a lesão pulmonar aguda, síndrome do desconforto respiratório agudo e a falência múltipla dos orgãos. São achados histopatológicos da sepse: edema intersticial e alveolar, acúmulo de neutrófilos e macrófagos, hemorragia, lesão endotelial e do epitélio alveolar, e colapso alveolar. Acredita-se que a prolactina, hormônio produzido nas células lactotróficas da hipófise interfira no processo inflamatório, ora estimulando-o, ora inibindo-o. Além da relação direta com a lactação, este hormônio tem sido considerado um importante modulador do sistema imune, estimulando a proliferação e a sobrevivência celular. No presente estudo, buscou-se determinar os aspectos morfofuncionais do pulmão comprometido pela sepse induzida pela ligação e perfuração do ceco (CLP) em ratos com hiperprolactinemia induzida por sulpiride, um antagonista da dopamina. O presente estudo mostrou que os ratos pré-tratados com salina e submetidos a CLP, desenvolveram o padrão morfológico da sepse como por exemplo, aumento nas células inflamatórias com predomínio dos polimorfonucleares (neutrófilos), edema intersticial e colapso alveolar. Além disso, o presente estudo mostrou que a sepse induzida por CLP produziu diminuição da pressão arterial média bem como queda em alguns parâmetros ventilatórios como por exemplo, volume corrente e volume pulmonar em ratos não anestesiados. Interessante observação foi que grupos de ratos pré-tratados com sulpiride e submetidos a CLP apresentaram um maior comprometimento nos achados morfológicos bem como uma potencialização na queda do volume corrente durante a análise ventilatória. O presente estudo sugere que a hiperprolactinemia deve ser atentamente observada, pela estreita relação com o agravamento do processo inflamatório provocado por uma infecção pré-existente, que resultaria no aumento do número de óbitos em pacientes sépticos. / Sepsis is characterized as a systemic inflammatory process due to an infection, leading to acute lung injury, acute respiratory distress syndrome and multiple organs failure. Histopathological findings of sepsis are: interstitial and alveolar edema, accumulation of neutrophils and macrophages, hemorrhage, endothelial and alveolar epithelial injury and alveolar collapse. It is believed that prolactin, a hormone produced by pituitary lactotroph cells, interferes on the inflammatory process, sometimes stimulating it and sometimes inhibiting it. In addition to the direct relationship on lactation, this hormone has been observed to play important role on the modulation of the immune system, stimulating the cell proliferation and cell survival. In the present study, we sought to determine the morphofunctional aspects of compromised lung during sepsis induced by cecal ligation and perforation (CLP) in rats with hyperprolactinemia induced by sulpiride, a dopamine antagonist. The present study showed that pretreated rats with saline and subjected to CLP developed the morphogical pattern of sepsis such as increase in inflammatory cells with predominance of polymorphonuclear cells, interstitial edema and alveolar collapse. In addition, this study showed that sepsis induced by CLP produced a decrease in mean arterial pressure and decrease in ventilatory parameters such as tidal and pulmonary volume in unanesthetized rats. An interesting observation was that pretreated rats with sulpiride and subjected to CLP had a greater impairment in the morphological findings as well as an increase in the fall of the tidal volume during the ventilation analysis. The present study suggests that the hyperprolactinemia should be closely observed by a close relationship with the worsening of the inflammatory process caused by a pre-existing infection, which would result in increasing the number of deaths in septic patients.

Lytic effector function of Natural Killer (NK) cells and CD8+ T cells occurs through discrete and regulated cell biological steps triggered by recognition of diseased cells. Recent studies of the NK cell synapse support the idea that dynamic nanoscale rearrangements in cortical filamentous (F)-actin are a critical cell biological checkpoint for lytic granule access to NK cell membrane. Loss of function mutations in the LYST gene, a well-characterised cause of Chediak- Hegashi syndrome (CHS), result in the formation of giant lysosomal organelles including lytic granules. Here, we report a mismatch between the extent of cortical F-actin remodelling and enlarged lytic granules that limits the functionality of LYST- deficient NK cells in a human model of CHS. Using super-resolution stimulated emission depletion (STED) microscopy we found that LYST-deficient NK cells had nanoscale rearrangements in the organisation of cortical actin filaments that were indistinguishable from control cells- despite a 2.5-fold increase in the size of polarised granules. Importantly, treatment of LYST-deficient NK cells with actin depolymerising drugs increased the formation of small secretory domains at the synapse and restored their ability to lyse target cells. These data establish that sub-synaptic F-actin is the major factor limiting the release of enlarged lytic granules from CHS NK cells, and reveal a novel target for therapeutic interventions. While the importance of cortical actin filaments in NK cell cytotoxicity have been established, its persistence at the early stages of T cell synapse formation is disputed. We studied the organisation of cortical actin filaments in synapses formed by primary human T cells using STED microscopy and detected intact cortical actin filaments in key T cell effector subsets including memory CD8+ T cells as early as 5-minutes post-activation. Quantitative analysis revealed that activation specific rearrangements in cortical actin filaments at both CD4+ and CD8+ T cell synapses serve to increase the space between filaments. Additionally, comparison of cytolytic T cells with freshly isolated and IL-2 activated primary NK cells revealed that rapid maturation of the cortical actin meshwork is a specific feature of CD8+ T cell lytic synapses. Using chemical inhibition of actin nucleators, we show that increased cortical relaxation is mediated primarily by the activity of actin related proteins (Arp) -2/3. Taken together, these data establish the critical requirement for dynamic rearrangements in cortical actin filaments at lytic synapses but underscore cell-specific differences in its regulation.

Base teórica: A sepse é uma síndrome heterogênea, definida como disfunção orgânica que ameaça à vida, causada por uma resposta desregulada do hospedeiro à infecção. É um problema de saúde mundial, graças à sua alta prevalência, morbimortalidade associada, além de custos para seu tratamento. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). A intensidade da resposta à infecção pode variar entre indivíduos, logo pode-se considerar que esta seja determinada por bases genéticas, e estas influenciem na ocorrência de sepse e variabilidade nos desfechos. Objetivos: Avaliar a associação entre os genes KIR e os ligantes HLA em pacientes críticos, comparando pacientes com sepse e controles não sépticos internados na mesma UTI. Métodos: Foi examinado o polimorfismo de 16 genes KIR e seus ligantes HLA em 271 pacientes críticos, caucasóides, sendo 211 pacientes com sepse e 60 controles, pela técnica de PCR-SSO e PCR-SSP, respectivamente. Resultados: Os genes ativadores KIR2DS1 e KIR3DS1 foram mais frequentes nos controles que nos pacientes com sepse (41,23% versus 55,00%, e 36,49% versus 51,67%; p = 0.041 e 0,025, respectivamente). Estes resultados fornecem informação inicial sobre o papel de polimorfismos de KIR na sepse, sugerindo que este possa ser um potencial marcador diagnóstico ou prognóstico da doença. / Background: Sepsis is a heterogeneous syndrome, defined a life-threatening organic dysfunction caused by a dysregulated host response to infection. Sepsis is a global health problem, due to its high prevalence, associated morbidity and mortality, and costs for its treatment. Cells Natural Killer (NK) cells are part of the innate immune system that recognize HLA class I molecules on target cells via membrane receptors called killer cell immunoglobulin-like receptors (KIR). The intensity of the response to an infection may vary among individuals and might be influenced genetic features affecting sepsis occurrence and variability in outcomes. Objectives: To evaluate the association between KIR genes and HLA ligands in critically ill patients, comparing patients with sepsis and without sepsis admitted to the same ICU. Methods: We examined the polymorphism of 16 KIR genes and their HLA ligands in 271 critically ill patients, Caucasians, and 211 patients with sepsis and 60 controls by PCR-SSO and PCR-SSP, respectively. Results: Activating KIR2DS1 and KIR3DS1 genes were more common in controls than in patients with sepsis (41.23% versus 55.00% and 36.49% versus 51.67%, p = 0.041 and 0.025, respectively). These results provide initial information on the role of polymorphism of KIR in sepsis, suggesting that this may be a potential diagnostic or prognostic marker of the disease.

A transformação do mosto de uva em vinho envolve uma série de ações combinadas de diferentes gêneros e espécies de microrganismos. A espécie Saccharomyces cerevisiae domina a fase intermediária e a fase final da fermentação alcoólica. De modo geral, as leveduras enológicas podem ser caracterizadas pela capacidade fermentativa, produção de H2S (sulfeto de hidrogênio) e seu comportamento killer. A Embrapa uva e vinho possui em sua Coleção, diversas leveduras autóctones isoladas de bagas de uvas oriundas de diversas regiões do Brasil. Entretanto, a diversidade genética destes isolados não é conhecida. Neste estudo foram avaliados a capacidade fermentativa, formação de H2S, fator killer e sensibilidade ao fator killer de 150 leveduras provenientes das cultivares Malvasia Bianca (FMB14), Moscato Alexandria (FMA14) e Moscato Tradicional (MBTF14) todas oriundas da região de Farroupilha- RS. A capacidade fermentativa foi avaliada juntamente com a formação de H2S, inoculando as leveduras em meio mosto sulfito. Os testes ao fator killer e sensibilidade ao fator killer foram avaliados através do meio Lorena/ELNC (80:20). As linhagens com perfil para elaboração de vinhos e produtoras da toxina killer foram identificadas por amplificação da região ITS1- 5.S- ITS2 por PCR e por PCR-RFLP. Foi avaliada também a diversidade genética de 23 linhagens da espécie de Saccharomyces cerevisiae da Coleção da Embrapa Uva e Vinho, usando a técnica de PCR-RAPD. Foram empregados para detectar a variabilidade genética das leveduras os oligonucleotídeos iniciadores: (GTG)5, (GAC)5, (GACA)4 e M13. Os resultados mostraram que a maioria das linhagens apresentaram baixa velocidade fermentativa aliada à diferentes níveis de produção de H2S. Somente 3 linhagens apresentaram capacidade fermentativa adequada quando comparadas com as linhagens de referencia 1vvt/97 e K1, quais sejam, 29MBF14, 39MBTF14 e 50MBF14. Apenas a linhagem 29MBTF4 formou pequenas quantidades de H2S. Verificou-se que 64% das linhagens isoladas mostraram-se metabolicamente capazes de biossintetizar H2S. Somente 9,33% apresentaram comportamento killer e apenas 6,66% mostraram sensibilidade à proteína killer. Os resultados apresentados sugerem ter relação com as cultivares utilizadas no isolamento. Verificou-se a existência de diferenças genéticas entre as linhagens de Saccharomyces cerevisiae estudadas com todos os iniciadores utilizados. Os iniciadores que mais discriminaram linhagens de Saccharomyces cerevisiae foram (GTG)5 e (GAC)5. / Grape must conversion into wine involve combined actions of different genus and species of microorganism. The species Saccharomyces cerevisiae dominates intermediate and final stages of alcoholic fermentation. Generally, the oenological yeasts are characterized by their fermentative capacity, production of H2S (hydrogen sulfide) and killer behavior. Embrapa Grape and Wine has a Yeast Collection that encompasses many autochthonous strains isolated of grape berries from different regions of Brazil. However, the genetic diversity of these yeasts are stilling known. This study has evaluated the fermentative capacity, production of H2S, killer factor and killer factor sensibility of 150 yeasts isolated from the cultivars Malvasia Bianca (FMB14), Moscato Alexandria (FMA14) and Moscato Tradicional (MBTF14) all belonging from Farroupilha commune in Rio Grande do Sul State. The fermentative capacity has been tested along with the evaluation of H2S production by the inoculation of the yeasts in sulfite must medium. The production and detection of factor killer and the evaluation of sensitive characteristics have been measured in Lorena/ELNC (80:20) solid medium. Yeasts with optimal fermentative characteristics and the ones producing killer toxin have been identified by amplification of ITS1-5.8S-ITS2 region by PCR -RFPL. This study also has evaluated the genetic diversity of 23 yeasts strains of Saccharomyces cerevisiae, belonging to the Yeast Collection of Embrapa Grape and Wine, employing PCRRAPD technique. The primers (GTG)5, (GAC)5, (GACA)4 and M13 have been used to detect the yeasts genetic diversity. The results showed that the majority of the yeasts analyzed have demonstrated low fermentative velocity combined with different levels of H2S production. From the three cultivars analyzed, only Moscato Tradicional showed yeasts with a suitable fermentative capacity when compared to the reference yeasts EMBRAPA 1vvt97 e K1, and they were named as 29MBF14, 39MBTF14 and 50MBF14. It was verified that 84%, 76% and 36% of the isolated strains from Malvasia Bianca, Moscato Tradicional and Moscato Alexandria, respectively, were capable to biosynthesize H2S. Concerning to killer behavior, 14%, 12% and 2% of the isolated strains from Moscato Tradicional, Moscato Alexandria and Malvasia Bianca, respectively, were capable of producing killer factor. These outcomes suggest the influence of the cultivar into the microflora biodiversity. Genetic differences were also demonstrated between the strains of Saccharomyces cerevisiae for all the primers tested. Primers GTG5 and GAC5 were the most discriminative.

OBJETIVOS: Avaliar o perfil e o número absoluto e percentual de células NK verdadeiras (CD56+CD16+CD3-) e de células NK e NKT (CD56+) no sangue periférico de uma coorte de pacientes com artrite reumatóide (AR) antes e durante o tratamento com rituximabe (RTX). MÉTODOS: Foram analisados dez pacientes do grupo controle (doadores de sangue) e dez pacientes com AR que receberam duas infusões de RTX 1g separadas por intervalo de 14 dias. As análises imunofenotípicas para avaliação do perfil e quantificação de células NK foram realizadas pré e após a infusão ou até a recaída clínica. Pacientes respondedores e não respondedores foram classificados de acordo com os critérios do Colégio Americano de Reumatologia (ACR) em 6 meses. RESULTADOS: A quantidade de células NK verdadeiras não demonstrou variação significativa pré e após o tratamento com RTX. Contudo, houve aumento percentual de células CD56+ entre o primeiro e o segundo mês após a infusão com RTX. Além disso, os pacientes respondedores apresentaram uma tendência de aumento do número absoluto de células NK verdadeiras após dois meses de tratamento. Já em relação ao grupo controle, observou-se um aumento significativo do número de células NK basais nos pacientes com AR (p<0,05). CONCLUSÕES: Foi identificada uma tendência de aumento nos valores absolutos de células NK verdadeiras entre os pacientes respondedores no segundo mês após a infusão com RTX. Não foi identificada uma variação significativa no perfil e quantidade de células NK nos pacientes com AR pré e após o tratamento com RTX. Contudo, foi observado que os pacientes com AR possuem uma quantidade maior de células NK do que os controles, sugerindo um possível envolvimento destas células na AR. / OBJECTIVES: To assess the profile as well as the absolute number and percentage of true NK cells (CD56+CD16+CD3-), and NK and NKT cells (CD56+) in the peripheral blood of a cohort of patients with rheumatoid arthritis (RA) before and during rituximab (RTX) therapy. METHODS: Ten control patients (blood donors) and ten patients with RA were assessed. The latter group received two intravenous infusions of 1g RTX, separated by a 14 day interval. Immunophenotypic analyses of NK cells were conducted before and after infusion, or until clinical relapse. After six months, respondents and nonrespondents were reassessed according to American Rheumatology Criteria (ARC). RESULTS: The number of true NK cells did not significantly change after treatment with RTX. However, an increase in the percentage of CD56+ cells was observed between the first and second month after RTX infusion. Respondents also displayed a tendency toward an increased number of true NK cells after two months of treatment. At baseline, the number of NK cells was also found to be significantly higher in patients with RA than in control individuals (p<0.05). CONCLUSIONS: Respondents displayed a tendency toward an increase in the absolute number of true NK cells in the second month after RTX infusion. No significant changes in the profile and frequency of NK cells were found between preand post-RTX treatment assessments of patients with RA. However, it was found that patients with RA have a higher number of NK cells than control partcipants, suggesting a possible role of these cells in RA.