O melanoma é uma neoplasia de pele invasivo, com maior taxa de morte, sem tratamento efetivo. Nanocápsulas poliméricas de núcleo lipídico (LNC) tem sido empregadas com sucesso como carreadores de fármacos hidrofóbicos. Como o eugenol é um composto hidrofóbico com atividades antiproliferativas e pró-apoptóticas em células cancerosas, visamos avaliar os efeitos dos tratamentos com acetileugenol (AC), LNC ou LNC contendo acetileugenol (LNC-AC) em modelo de melanoma in vivo em camundongos C57B6, e a citotoxicidade dos mesmos em células endoteliais (HUVEC) e de melanoma (SK-Mel-28) in vitro. Os resultados obtidos mostraram que: 1) tratamentos i.p. com as LNC ou com LNC-AC (50 mg/kg, 3-10 dia de indução do tumor) induziram toxicidade sistêmica e, somente o tratamento com LNC inibiu o desenvolvimento do melanoma. O tratamento com LNC, mas não com a mistura de triglicerídeos de cadeia média, por via oral, inibiu o desenvolvimento tumoral, sem toxicidade. Adicionalmente, os tratamentos com AC, LNC ou LNC-AC não foram eficazes quando administrados em fase tardia de evolução tumoral (50 mg/kg, 7-17 dia de indução do tumor, via oral); 2) os tratamentos agudos com AC, LNC ou LNC-AC (20 mg/kg, 200 µL, e.v.) não alteraram o número de leucócitos circulantes, mas os tratamentos com LNC ou com LNC-AC reduziram o comportamento de rolling dos leucócitos em vênulas póscapilares do músculo cremaster e causaram hemólise, sendo que este último efeito também foi observado após tratamento in vitro em hemácias murinas; 3) Os estudos in vitro mostraram que as LNC e LNC-AC foram captadas pelas células HUVEC e SK-Mel-28 após 1 hora de incubação; que a incubação com LNC-AC induziu apoptose tardia e necrose com maior eficácia em SK-Mel-28 do que em HUVEC; que as incubações com LNC ou LNC-AC exerceram efeitos antiproliferativos, induzindo parada na fase G2/M do ciclo celular das duas linhagens de células avaliadas; que somente a incubação com AC ou LNC-AC inibiu a adesão ao Matrigel® com maior eficácia na linhagem SK-Mel-28 do que HUVEC; que somente a incubação com as LNC reduziram a expressão de VCAM-1 em HUVEC e que as incubações com LNC ou LNC-AC reduziram a expressão de β3 integrina em SK-Mel- 28; que nenhum dos tratamentos alterou a migração celular das HUVEC ou SK-Mel- 28; que somente a incubação com LNC-AC reduziu os níveis de espécies reativas de oxigênio em HUVEC e SK-Mel-28; que a incubação com LNC ou LNC-AC aumentou a produção de óxido nítrico (NO) pelas duas linhagens de células avaliadas; que o tratamento com L-NAME reverteu os níveis de NO e a inibição sobre a proliferação celular induzida pela incubação com LNC ou LNC-AC e; que o tratamento de células de melanoma murino com LNC ou LNC-AC parece alterar a polarizar os neutrófilos para o fenótipo N1. Associados, os resultados obtidos mostram o tratamento oral com LNC inibe o crescimento do melanoma sem induzir efeitos tóxicos, e que este efeito benéfico pode ser dependente, pelo menos em parte, da nanoencapsulação dos triglicerídios de cadeia média e da supraestrutura da formulação, com toxicidade direta sobre as células de melanoma e possível modulação do microambiente tumoral. / Melanoma is the most invasive skin cancer, with high rates of death without effective treatment. Polymeric lipid-core nanocapsules (LNC) has been successfully used as carriers of hydrophobic drugs. As eugenol is an hydrophobic compound with antiproliferative and pro-apoptotic activity in cancer cells, here we aimed to evaluate the effects of treatments with acetyleugenol (AC), LNC or LNC containing acetyleugenol (LNC-AC) in an in vivo melanoma model in C57BL6 mice and the cytotoxicity of the treatments in vitro, using endothelial (HUVEC) and melanoma (SK-Mel- 28) cells. The results obtained showed that: 1) i.p. treatments with LNC or LNCAC (50 mg/kg, 3-10 days of tumor injection) induced systemic toxicity and, only the treatment with LNC inhibited the melanoma development. Treatment with LNC, but not with mix of triglycerides of medium chain, by oral route, inhibited the tumor development, without toxicity. In addition, the treatments with AC, LNC or LNC-AC were not effective when administered in the late stage of tumor evolution (50 mg/kg, 10-20 days of tumor induction, oral route); 2) the acute treatments with AC, LNC or LNC-AC (20 mg/kg, 200 µL, intravenous route) did not altered the number of circulating leukocytes, but the treatments with LNC or LNC-AC reduced the rolling behavior of leukocytes in postcapillary venules of the cremaster muscle and induced hemolysis. The latter effect was also observed after in vitro treatment using murine erythrocytes; 3) In vitro studies showed that the LNC and LNC-AC suffered uptake by HUVEC and SK-Mel-28 cells after 1 hour of incubation; that the incubation with LNC-AC induced late apoptosis and necrosis more effectively in SK-Mel-28 than in HUVEC cells; that the incubation with LNC or LNC-AC presented antiproliferative effects, by inducing G2M arrest in cell cycle in both cells lines evaluated; that only the incubation with AC or LNC-AC inhibited the adhesion in Matrigel® with more efficaccy in SK-Mel-28 than in HUVEC cells; that only incubtion with LNC reduced the VCAM-1 expression in HUVEC and the incubation with LNC or LNC-AC reduced the β3 integrin expression in SK-Mel-28 cells; that any treatment affected the HUVEC or SK-Mel- 28 migration; that only the incubation with LNC-AC reduced the levels of reactive species of oxygen in HUVEC and SK-Mel-28 cells; that the incubation with LNC or LNC-AC increased the nitric oxide (NO) production by both cell lines used; that the treatment with L-NAME reversed the NO levels and the inhibition on cell proliferation induced by incubation with LNC or LNC-AC and; that the in vitro treatment of murine with LNC or LNC-AC altered the neutrophil polarization to N1 phenotype. Together, results obtained show that the oral treatment with LNC inhibit the melanoma growth without any toxic effect, and that the beneficial effect could be dependent, at least in part, of nanoencapsulation of medium chain triglycerides and the supraestrucuture of the formulation, with direct toxicity on melanoma cells and possible modulation of tumor microenvironment.

Ventilator associated pneumonia (VAP) and acute respiratory distress syndrome (ARDS) are two respiratory conditions unique to mechanically ventilated patients. The diagnosis of these conditions, and therefore any subsequent treatment, are befuddled by uncertainty. VAP rates vary considerably according to the diagnostic or surveillance criteria used. The pathogenesis of ARDS is well understood but when the internationally agreed consensus criteria are employed, the histological hallmarks are absent about half the time, indicating a disconnection between the clinical diagnosis and what is known about the biology of this condition. It is argued that tests of biological function should be considered in addition to clinical characteristics in order to improve the utility of diagnosis. Given that the pathological sequelae of both VAP and ARDS are driven by an over exuberant host neutrophil response, the activated neutrophil was selected as a potential biological imaging target. Optical molecular imaging uses visible and near visible wavelengths from the electromagnetic spectrum to derive or visualize information based on the optical properties of the target tissue. Optical wavelengths are safe and cheap to work with, producing much higher resolution images than those relying on x-rays or gamma radiation. The imaging modality can be coupled with exogenously applied chemistry to identify specific biological targets or processes. The hypothesis that optical molecular imaging could be used to detect activated neutrophils in real time in the alveolar region of patients was tested. A bespoke optical molecular imaging agent called Neutrophil Activation Probe (NAP), designed in-house, was used to test the hypothesis. NAP is a dendrimeric compound delivered to the alveolar region of a patient in microdoses (≤100 micrograms), becoming fluorescent only on contact with activated neutrophils, and can be detected by optical endomicroscopy. Both the imaging agent and the endomicroscope are delivered to the distal lung via routine bronchoscopy. The agent was tested extensively in the laboratory to demonstrate function, specificity, and safety. Ex vivo testing took place using human and ovine lungs. A regulated dose escalation Phase I clinical trial of investigational medicinal product (CTIMP) in healthy volunteers, patients with bronchiectasis, and mechanically ventilated patients with a pulmonary infiltrate on chest radiography (NCT01532024) was designed and conducted. The aim of the Phase I study was to demonstrate the safety of the technique and to confirm proof of concept. In order to support the requirement for a technique that interrogates alveolar neutrophils two supplementary clinical studies were performed. Firstly, two VAP surveillance techniques (CDC surveillance and HELICS European VAP surveillance) were compared with clinically diagnosed VAP across consecutive admissions in two large tertiary centres for one year. Secondly, the utility of circulating neutrophils to permit discrimination between acute respiratory illnesses was examined. Blood samples from mechanically ventilated patients with and without ARDS underwent flow cytometric assessment using eight clusters of differentiation and internal markers of activation to determine neutrophil phenotype. All clinical studies received the appropriate regulatory, ethical, and/or Caldicott guardian approval prior to commencement. NAP became fluorescent only in the presence of three processes specific to neutrophil activation: active pinocytosis, progressive alkalinization of the phagolysosome, and the activity of human neutrophil elastase. High optical signal was detected following the application of NAP in the alveolar regions of explanted lungs from patients with cystic fibrosis, known to be rich in activated neutrophils. Using an ex vivo ovine lung ventilation and perfusion model optical signal was demonstrated following segmental lung injury. The safety and specificity of the technique in a small cohort of healthy volunteers and mechanically ventilated patients was demonstrated. The technique was tested on a small cohort of patients with bronchiectasis, which provided the first opportunity to obtain broncho-alveolar lavage samples for laboratory correlation. Fluorescent signal was shown in the lavaged neutrophils, labeling that could only have taken place in the alveolar region. The supportive clinical studies found the concordance between actual VAP events was virtually zero even though the reported VAP rates were similar. Furthermore, the rate at which clinicians initiate antibiotics for VAP was approximately five times higher than either surveillance VAP rate. The study of circulating neutrophils from the blood of healthy volunteers and mechanically ventilated patients with and without ARDS indicated circulating neutrophil activation phenotype was not capable of discriminating between clinically diagnosed ARDS and other acute respiratory illnesses. In summary, an ambitious programme of work was completed to develop and support an optical molecular imaging technique that meets the rigorous requirements for human application and can be applied at the bedside to yield immediate visual results. The spatiotemporal relationship of neutrophil activation in real time both in the laboratory and in volunteers and patients was visualized. The visualization of neutrophil activation at such a resolution has never been achieved before in humans, healthy or unhealthy. The Phase I study was not powered to determine utility but recruitment has begun to a Phase II CTIMP (NCT02804854) to investigate the utility, accuracy, and precision of the imaging technique in a large cohort of mechanically ventilated patients. Ultimately, it is proposed that the technique will facilitate diagnosis, stratify patients for treatment and monitor treatment response using this technique.

Através do presente trabalho investigou-se a expressão do comportamento doentio e a atividade de neutrófilos sanguíneos de camundongos adultos, de meia idade e idosos, após o tratamento com lipopolissacarídeo de bactéria gram negativa (Escherichia coli). O comportamento doentio foi avaliado através da ingestão de alimentos e do peso corpóreo. Nos aparatos comportamentais Campo Aberto e Labirinto em cruz elevado, o comportamento doentio foi avaliado através da atividade geral, da exploração e dos níveis de ansiedade. A atividade de neutrófilos sanguíneos foi estudada através de citometria de fluxo, analisando-se a intensidade e porcentagem de neutrófilos que realizaram fagocitose e da intensidade e porcentagem de neutrófilos que realizaram burst oxidativo, ambos induzidos pelo estímulo in vitro da bactéria Staphylococcus aureus. Os animais foram tratados com solução salina ou LPS (na dose de 200 µg/kg) avaliações foram realizadas 2 horas após o tratamento. No caso da ingestão de alimentos e do peso corpóreo, as avaliações foram realizadas durante 3 dias anteriores ao experimento, e durante 7 dias posteriores ao experimento. Os resultados demonstraram que animais idosos apresentaram diferenças na expressão do comportamento doentio, sendo que houve uma maior motivação em explorar os aparatos comportamentais. Apesar da manutenção da exploração no Campo Aberto e Labirinto em Cruz elevado, a redução da ingestão de alimentos e do peso corpóreo apresentou-se mais acentuada nos camundongos de meia idade e idosos, após tratamento com LPS. A atividade de neutrófilos sanguíneos, avaliadas pela fagocitose e burst oxidativo, apresentou-se aumentada em camundongos idosos após tratamento com LPS. A menor migração de neutrófilos sanguíneos à cavidade peritoneal parece ser responsável por esse aumento de atividade dos neutrófilos de camundongos idosos. Esses resultados sugerem que a expressão do comportamento doentio de camundongos idosos é diferente, com manutenção do comportamento de exploração, e que esses camundongos seriam mais susceptíveis aos efeitos do choque endotóxico, devido à presença de maior quantidade de neutrófilos ativados no sangue. / Through the present work, it has been investigated the expression of sickness behavior and blood neutrophils activity in adult, middle-aged and aged mice, after gram negative (Escherichia coli) lipopolysaccharide treatment. Sickness behavior was evaluated through food ingestion and body weight. On the behavioral apparatus Open Field and Elevated Plus Maze, sickness behavior was evaluated through general activity, exploration and anxiety levels. Blood neutrophils activity was studied through flow cytometric analysis, assessing neutrophil\'s intensity and percentage of phagocytosis and neutrophil\'s intensity and percentage of oxidative burst, both induced by in vitroM stimulus of Staphylococcus aureus bacteria. Animals were treated either with saline solution or LPS (in the 200 µg/kg dose) and analysis were done 2 hours after treatment. In the case of food ingestioin and body weight, analysis were done through 3 days before experiment, and during 7 days after experiment. Results demonstrated that aged animals showed differences in sickness behavior expression, with a greater motivation to explore behavioral apparatus. Despite maintenance of Open Field and Elevated Plus Maze exploration, reduction in food ingestion and body weight were more pronounced in middle-aged and aged mice, after LPS treatment. Blood neutrophils activity, analysed through phagocytosis and oxidative burst, were higher in aged mice after LPS treatment. Lesser blood neutrophils migration to peritoneal cavity seems to be responsible for this enhancement in neutrophil activity. These results suggest that the expression of sickness behavior in aged mice is different, with maintenance of exploratory behavior, and that these mice would be more susceptible to endotoxic effects, due to the presence of higher quantity of activated neutrophils in blood.

Camundongos AIRmax e AIRmin exibem diferenças significativas no número médio de leucócitos migrantes e no conteúdo protéico do exsudato inflamatório produzido por partículas de poliacrilamida. Um dos fatores preponderantes para a maior capacidade inflamatória da linhagem AIRmax é a maior produção de neutrófilos maduros pela medula óssea. Assim, considerando a diferente capacidade de produção leucocitária, entre as linhagens AIRmax e AIRmin, nos propomos a estudar comparativamente nestas linhagens, o processo de mielopoese in vitro em resposta ao GM-CSF e ATRA. Verificamos que as células da medula óssea dos animais AIRmax apresentaram maior potencial proliferativo e maiores níveis de expressão de genes envolvidos nos estágios iniciais da mielopoese, do que os animais AIRmin. Além disso, o conteúdo protéico das células em cultura revelou diferenças quantitativas e qualitativas de proteínas provavelmente envolvidas no processo de mielopoese. / Mice AIRmax and AIRmin exhibit significant differences in the average number of migrating leukocytes and in the protein content of inflammatory exudate produced by polyacrylamide particles. This higher inflammatory capacity of the AIRmax mice is due to three convergent factors: higher local production of chemotactic factors, increased resistance of locally infiltrated neutrophils to spontaneous apoptosis and larger production of mature neutrophils by the bone marrow. Thus, considering the differential capacity of leukocyte production between AIRmax and AIRmin mice, we are studying comparatively the myelocytic differentiation process in vitro. We verified that the BM cells of AIRmax mice showed higher proliferation levels. In addition by qPCR technique it was verified a larger expression of genes involved in the initial stages of myelopoiesis in the AIRmax BM cultures. The analysis of BM cellular protein content by 2D gel electrophoresis revealed quantitative and qualitative differences between two mouse lines.

Com o objetivo de avaliar a evolução clínica, a persistência da infecção e a resposta imune humoral e celular, sete cães adultos desprovidos de anticorpos anti-Ehrlichia canis foram inoculados com E. canis e acompanhados durante vinte semanas. Três cães permaneceram como controle. Exame físico diário e avaliação laboratorial (hemograma, bioquímica sanguínea, fragilidade osmótica eritrocitária, teste de Coombs, teste de redução de tetrazólio nitroazul), reação em cadeia da polimerase (PCR), pesquisa de anticorpos anti-E. canis (IFI) e quantificação dos linfócitos T CD4+ e CD8+ por citometria de fluxo foram realizados, em diferentes momentos, durante o período de observação. A infecção foi comprovada pela presença de mórulas em linfócitos/monócitos no sangue circulante, soroconversão, com títulos de anticorpos entre 1.280 a 20.480 e a reação positiva ao PCR. Febre, esplenomegalia, linfadenomegalia, trombocitopenia, anemia não regenerativa, aumento das atividades séricas de ALT, AST e FA, foram as principais alterações clínicas e de patologia clínica observadas, com pico máximo entre 21 e 28 dias. Também nesses momentos foram observados o aumento de linfócitos T CD8+ e a diminuição dos linfócitos T CD4+. O aumento do metabolismo oxidativo dos neutrófilos, quando estimulados, entre o 28º e 35º dia p.i., o aumento de reticulócitos na circulação sanguínea nessa ocasião, a redução dos linfócitos T CD4+ e o aumento de linfócitos T CD8+ indicaram a resposta do hospedeiro, na tentativa de eliminar a infecção. No auge da infecção entre o 21º e 49º dia p.i., houve a inversão da relação CD4:CD8, principalmente o aumento dos linfócitos T CD8+. A fragilidade osmótica eritrocitária manteve-se inalterada, bem como o teste de Coombs, comprovando não ter ocorrido a hemólise imunomediada. Nos cães infectados não foram detectados a presença de anticorpos IgM anti-E. canis em nenhum momento de observação. O aumento de reticulócitos na circulação sanguínea a partir do 28º dia p.i. sugere ser temporária a supressão da atividade eritropoética. O baixo número de linfócitos T CD4+, coincidindo com a esplenomegalia e a PCR positiva até a vigésima semana p.i., sugere a relação entre a persistência da infecção, com a localização esplênica de E. canis e ativação de um mecanismo de escape pelo microrganismo, representado pelo baixo limiar de ativação de linfócitos T CD4+. Por outro lado, o aumento de linfócitos T CD8+ no auge da infecção coincidindo com a melhora clínica, sugere o envolvimento do mecanimo de citotoxicidade na patogenia da erliquiose canina. O bloqueio parcial da atividade dos linfócitos T CD4+, constitui-se provavelmente em um dos mecanismos de escape da E. canis, enquanto a contínua liberação de TNF-α pelas células citotóxicas, como parte do mecanismo do hospedeiro para a eliminação do parasita pode resultar na pancitopenia terminal observado freqüentemente nos cães cronicamente infectados por E. canis. / The clinical evolution, the persistence of infection and humoral and cellular immune response were evaluated on seven adult naïve dogs experimentally inoculated with an E. canis strain and followed during 20 weeks. Three dogs were remained as control. Daily clinical evaluation and laboratorial exams (hematological, biochemistry, erythrocyte osmotic fragility, Coombs´ test, neutrophils oxidative metabolism by nitroblue tetrazolium reduction test), polymerase chain reaction assay (PCR), anti-E. canis antibodies detection by indirect immunofluorescence assay (IFA) and quantification of CD4+ and CD8+ T lymphocytes by flow cytometry technique were evaluated, at different moments during all the experimental period. The presence of E. canis morulae, seroconversion with high antibodies titers (1,280 to 20,480) and positive PCR had confirmed the infection in all the inoculated animals. Fever, splenomegaly, lymphadenomegaly, thrombocytopenia, non-regenerative anemia and increase on liver´s enzymes serum activity (ALT, AST and ALP) were the main clinical and laboratorial changes observed, with the highest point between days 21 and 28 post-infection (p.i.). Also at these moments were observed increase of CD8+ T lymphocytes and reduction of the CD4+ T lymphocytes. The increase in the stimulated neutrophils oxidative metabolism between days 28 and 35 p.i., and the increase of reticulocytes in the same period, allied to the reduction of the CD4+ T lymphocytes and the increase of CD8+ T lymphocytes, indicated the host´s immune response in the attempt to eliminate the infection. On the peak of the infection, from the 21st to the 49th post-infection day, there was an inversion in the CD4:CD8 ratio of blood lymphocytes, mainly caused by increase of the CD8+ T lymphocytes. There was no variation on erythrocyte osmotic fragility and Coombs? test results, which suggested the absence of immune-mediated hemolysis. It wasn´t detected any anti-E. canis IgM antibodies in the infected group of dogs. The increase of reticulocytes only from the 28th post-infection day suggests a temporary erythropoietic suppression. The low number of CD4+ T lymphocytes, coinciding with the splenomegaly and the positive PCR until the twentieth week p.i., suggest that there is a relation between the persistent infection, the splenic localization of E. canis and an evasion mechanism, represented for the low threshold of activation of CD4+ T lymphocytes. On the other hand, the increase of CD8+ T lymphocytes in the peak of infection, coinciding with clinical improvement, suggests the involvement of the cytotoxicity mechanism on canine ehrlichiosis pathogenesis. The partial blockage of CD4+ T lymphocytes activity could probably be one of the E. canis escape mechanisms, while the continue TNF-α release by host´s cytotoxic cells, as a mechanism to eliminate the parasite, could result in terminal pancytopenia commonly observed in dogs chronically infected with E. canis.

Em trabalho anterior verificamos que neutrófilos oxidam o hormônio melatonina a N1-acetil-N2-formil-5-metoxiquinuramina (AFMK), numa reação catalisada por mieloperoxidase e dependente de ânion superóxido. Neste trabalho acompanhamos a cinética de formação de AFMK e de seu produto de deformilação N1-acetil-5-metoxiquinuramina (AMK) quando neutrófilos foram ativados por diferentes estímulos. No sentido de obtermos informações sobre a relevância biológica destas reações, avaliamos o efeito de melatonina, AFMK e AMK sobre a formação de HOCl, sobre a liberação de citocinas pró-inflamatórias (TNF-&#945 e IL-8) e sobre a apoptose de neutrófilos. Uma forte inibição da liberação de HOCl foi observada em concentrações de melatonina igual ou superior a 0,05 mM. Embora menos efetivo, AMK também inibiu a formação de HOCl, enquanto AFMK não teve efeito. Adicionalmente mostramos que todos estes compostos inibiram a produção de TNF-α e IL-8 por neutrófilos ativados e que AFMK foi o mais potente deles. Por exemplo, enquanto 1µM de AFMK inibiu eficientemente a liberação de TNF-α, efeito similar foi obtido com melatonina apenas a partir de 1mM. O mesmo padrão de resposta foi observado na morte celular. AFMK e AMK (1mM), mas não melatonina, inibiram significativamente (aproximadamente 25%) a morte celular de neutrófilos. Nossos dados sugerem que neutrófilos são um alvo importante para AFMK e que a rota de metabolização da melatonina pode ser útil no controle da intensidade do processo inflamatório através do consumo de ERO, controle da liberação de citocinas e da sobrevida dos neutrófilos. Este conjunto de dados associados com o fato de que células mononucleares ativadas sintetizam melatonina e que o foco inflamatório é uma fonte de espécies reativas de oxigênio (ERO), levou-nos a verificar se a oxidação de melatonina ocorreria in vivo em situações inflamatórias. Para isso, analisamos amostras de liquor de pacientes com meningite viral. AFMK foi detectado em 16 das 20 amostras de liquor de pacientes com meningite viral e em nenhuma das 8 amostras controle. Das 16 amostras nas quais AFMK foi detectado, ele pôde ser quantificado em 6 (concentrações variando de 50 a 500 nM). Adicionalmente analisamos a correlação entre a presença de AFMK com alguns parâmetros inflamatórios como: celularidade, concentração de proteínas totais, TNF-α, IL-8 e IL-1β. Verificamos que todos estes parâmetros diminuíram nas amostras de liquor onde a concentração de AFMK era maior. Em conjunto, nossos resultados mostram que pelo menos parte dos efeitos antiinflamatórios descritos para melatonina pode ser originado da ação de seus produtos de oxidação. Nossos resultados contribuem significativamente para a compreensão do papel biológico da melatonina e seus produtos de oxidação na imunomodulação durante a inflamação. / In a previous study we described that activated neutrophils are able to oxidize the pineal hormone melatonin to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) in a reaction dependent on myeloperoxidase and superoxide anion. Here we followed the formation of AFMK and its deformylated product N1-acetil-5-metoxiquinuramina (AMK) when neutrophils were activated by different stimuli. The biological significance of this reaction has been one of our research interests, therefore we study the effect of melatonin, AFMK and AMK on the HOCl formation, on the release of pro-inflammatory cytokines (TNFα- and IL-8) and on the apoptosis process. Melatonin caused an almost complete inhibition of HOCl formation at concentrations up to 0.05 mM. Although less effective, AMK also inhibited HOCl formation, while AFMK had no effect. Additionally all the compounds assayed efficiently inhibited the production of TNF-α and IL-8 by activated neutrophils. Moreover, the inhibitory activity of AFMK was stronger than that of melatonin and could be observed already at 1µM. A significant inhibition of neutrophil death (approximately 25 %) was triggered by AFMK and AMK (1 mM) but not by melatonin. Our data suggest that neutrophils are an important target for AFMK and that the route of melatonin metabolism may be useful in controlling the intensity of the inflammatory process by the consumption of reactive oxygen species, control of cytokine production and neutrophils life span. These findings, associated with the efficient synthesis of melatonin by activated-mononuclear cells and the presence of reactive oxygen species in the inflammatory focus, led us to verify if inflammation triggers the oxidation of melatonin in vivo. In this way we analyzed the cerebrospinal fluid (CSF) of patients with meningitis. AFMK was detected in 16 CSF from 20 samples of patients with viral meningitis and in none of 8 control samples. From the 16 samples in which AFMK was detected, it was quantified in 6 (at the concentration range of 50 500 nM). We also analyzed the correlation between the presences of AFMK with some inflammatory parameters like: cellularity and concentration of total proteins, TNF-α, IL-8 and IL-1β. We verify that all these parameters were decreased in samples in which AFMK was in higher concentrations. Our results show that, at least part of the antiinflammatory effects described for melatonin is indeed caused by its oxidation product. These findings add a new insight in the comprehension of the biological role of melatonin and its oxidation products in immunomodulation and during inflammation.

O retículo endoplasmático (RE) vem ganhando evidência quando se trata de morte celular. O acúmulo de proteínas mal formadas inicia a ativação da Unfolded Protein Response (UPR), com a finalidade de manter a homeostasia do RE. Porém, quando o estímulo do estresse perdura por muito tempo e não é resolvido, a UPR pode ativar genes que conduzem à morte celular. Hiperglicemia, presente no diabetes mellitus, induz estresse de RE em vários tipos celulares. Assim, este estudo teve como objetivo investigar o possível envolvimento do estresse do retículo no processo de morte celular em neutrófilos de ratos diabéticos. Nosso estudo revelou que os neutrófilos de ratos diabéticos quando estimulados com PMA apresentam uma maior suscetibilidade à morte devido à ativação de IRE1a e subsequente fosforilação de JNK, redução na interação mitocôndria-RE na MAM e aumento da atividade da caspase-3. Dentre os resultados apresentados, neutrófilos provenientes de animais controle parecem estar protegidos do estresse de RE por apresentar maior expressão de GRP78 e das proteínas da MAM. / Endoplasmic reticulum (ER) has been gaining evidence when it comes to cell death. The accumulation of unfolded proteins initiates the activation of the Unfolded Protein Response (UPR). The UPR may resolve the ER stress by upregulating genes responsible to maintain the ER homeostasis; or it can activate genes that lead to the cell death when the ER disbalance is not solved. Hyperglycemia, one of many symptoms observed is Diabetes, may cause ER stress in various types of cells. Thus, this study aims to investigate the possible involvement of the ER stress in the process of cell death in neutrophils from diabetic rats. In summary, our study found that neutrophils from diabetic rats when stimulated with PMA exhibit greater susceptibility to death due to activation of IRE1a and subsequent phosphorylation of JNK, reduced safety in mitochondria-ER interaction in the MAM compartment and increased caspase-3 activation. Control group seems to be protect against the ER stress by ROS production by higher expression of GRP78 and MAM proteins.

A proliferação acelerada de cianobactérias do gênero Microcystis em mananciais e reservatórios tem causado sérios danos ecológicos e à saúde pública, e é um problema que desafia as instituições responsáveis pelo fornecimento de água para a população. Essas cianobactérias produzem microcistinas (MC), heptapeptídeos cíclicos hepatotóxicos e que podem causar câncer. Neste trabalho, isolamos (CLAE preparativa) e identificamos (ESI-EM/EM) as microcistinas MC-LR e [ASp3]-MC-LR produzidas pela cianobactéria Microcystis panniformis. As concentrações dessas substâncias foram determinadas por CLAE e variaram de 0,25 a 2,75 (MC-LR) e 0,08 a 0,75 ([ASp3]-MC-LR) fmol. Célula-1. Analisamos as concentrações destes compostos em tempos diferentes durante o ciclo claro:escuro (C:E) e foi encontrado que a quantidade de MC por célula é pelo menos três vezes mais alta durante a fase clara do que a fase escura. Isto pode ser associado ao relógio biológico, pois as cianobactérias expressam um robusto ritmo circadiano no controle do mecanismo de tempo que é independente do ciclo de divisão celular. O mesmo ocorreu no ciclo claro:claro (C:C). Também estudamos os efeitos da MC-LR e [ASp3]-MC-LR sobre as funções de neutrófilos humanos in vitro. Essas substâncias têm capacidade quimiotáxica, uma vez que observamos um aumento de migração de neutrófilos, além de ativarem a formação de espécies reativas de oxigênio (ERO) e a fagocitose. Já a atividade microbicida é ativada somente pela MC-LR. Nossos resultados indicam que concentrações menores do que o limite máximo de exposição a MC-LR (1 µg.L-1), sugerido pela OMS, exercem efeito sobre funções de neutrófilos humanos in vitro, podendo contribuir com a toxicidade dessas MC. / Cyanobacterial blooms of the genus Microcystis in water reservoirs have caused serious ecological and public health concern due to their ability to produce toxins. Microcystis and some other cyanobacerial species biosynthesize microcystins (MC). These cyanotoxins are hepatotoxic cyclic heptapeptides which can induce tumor promotion. In this study, MC-LR and [Asp3]-MC-LR were isolated (by preparative HPLC) and identified (by ESI-MS/MS) in the strain Microcystis panniformis BCCUSP100. Their levels were determined by HPLC and ranged from 0.25-2.75 and 0.08-0.75 fmols. cell-1 , respectively. The levels of MC-LR and [Asp3]-MC-LR were analyzed at different times during the light:dark (L:D) and light:light (L:L) cycles. It was found that the levels of MC per cell were at least three-fold as high during the day-phase than during the night-phase (L:D experiment). This may be associated to the biological clock since prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a timing mechanism that is independent of the cell division cycle. Our findings also showed the same pattern under L:L cycle. The effects of MC-LR and [Asp3]-MC-LR in some human neutrophil functions were also studied by in vitro assays. We observed that MC have chemotactic capacity as well as can generate reactive oxygen species and increase phagocytosis activity. The killing activity was activated only by MC-LR. Our results indicated that lower concentrations of MC-LR than the one recommended by World Health Organization (1 µg.L-1) may affect human neutrophil functions in vitro. These findings can contribute to the elucidation of MC toxicity as well as its effects in human neutrophils.

β-Adrenergic receptor (β-AR) stimulation increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases β-AR-stimulated myocyte apoptosis and myocardial fibrosis. Here, we hypothesized that exogenous UB modulates the inflammatory response, thereby playing a protective role in cardiac remodeling after ischemia-reperfusion (I/R) injury. C57BL/6 mice infused with vehicle or UB (1 μg·g−1·h−1) were subjected to myocardial I/R injury. Functional and biochemical parameters of the heart were examined 3 days post-I/R. Heart weight-to-body weight ratios were similarly increased in I/R and UB + I/R groups. The area at risk and infarct size were significantly lower in UB + I/R versus I/R groups. Measurement of heart function using echocardiography revealed that I/R decreases percent fractional shortening and percent ejection fraction. However, the decrease in fractional shortening and ejection fraction was significantly lower in the UB + I/R group. The UB + I/R group displayed a significant decrease in inflammatory infiltrates, neutrophils, and macrophages versus the I/R group. Neutrophil activity was significantly lower in the UB + I/R group. Analysis of the concentration of a panel of 23 cytokines/chemokines in the serum using a Bio-Plex assay revealed a significantly lower concentration of IL-12 subunit p40 in the UB + I/R versus I/R group. The concentration of monocyte chemotactic protein-1 was lower, whereas the concentration of macrophage inflammatory protein-1α was significantly higher, in the UB+I/R group versus the sham group. Expression of matrix metalloproteinase (MMP)-2 and activity of MMP-9 were higher in the UB + I/R group versus the I/R group. Levels of ubiquitinated proteins and tissue inhibitor of metalloproteinase 2 expression were increased to a similar extent in both I/R groups. Thus, exogenous UB plays a protective role in myocardial remodeling post-I/R with effects on cardiac function, area at risk/infarct size, the inflammatory response, levels of serum cytokines/chemokines, and MMP expression and activity.

Estudos recentes demonstraram que as armadilhas extracelulares de neutrófilos (NETs) formam uma estrutura capaz de anular fatores de virulência e destruir microrganismos. Pacientes portadores da síndrome de Hiper-IgM ligada ao X (X-HIGM), imunodeficiência causada por mutações no gene do CD40 ligante (CD40L), são altamente suscetíveis a infecções oportunistas causadas por fungos. Uma vez que a resposta de neutrófilos apresenta grande importância para o controle de infecções fúngicas e estudos que apontam influencia da interação CD40-CD40L na granulopoiese, propusemos investigar se a suscetibilidade a infecções em pacientes com X-HIGM estariam relacionadas à falha na formação de NETs. Os resultados obtidos indicam que pacientes X-HIGM apresentam falha na geração de NETs de maneira estímulo-dependente, possivelmente relacionada a maturação incompleta dos neutrófilos. O estudo contribui para o avanço no conhecimento sobre a imunopatologia da deficiência de CD40L e para elucidação dos mecanismos envolvidos na suscetibilidade a infecções em pacientes com X-HIGM. / Recent studies have shown that neutrophil extracellular traps (NETs) release an structure able to annul virulence factors and destroying microorganisms. Patients with X-linked Hyper-IgM syndrome (X-HIGM), a rare immunodeficiency caused by mutations in the CD40 ligand (CD40L) gene, are highly susceptible to opportunistic infections caused by fungi. Since neutrophil responses are important to control fungal infection and studies that indicate the influence of CD40-CD40L interaction on granulopoiesis, we propose to investigate whether the susceptibility to infections in X-HIGM patients could be related to the failure in NET formation. The results indicate that X-HIGM patients present a failure in NET generation in a stimulus-dependent manner, likely related to incomplete neutrophil maturation. The study contributes to the advancement of knowledge on the immunopathology of CD40L deficiency and contributes to elucidate the mechanisms involved in the susceptibility to infections in X-HIGM patients.