Impact of neonatal malnutrition on expression TLR-9, NF-kB and cytokines of macrophages infected in vitro with methicillin resistant Staphylococcus aureus.

Early nutritional aggressions promote epigenetic adjustments that culminate in the loss of phenotype plasticity (with permanent long-term modifications). Maternal diet and inadequate neonatal nutrition can result in fetal programming that presents susceptibility to infections in adult life. Thus, it becomes essential to verify the impacts of neonatal malnutrition (even following nutritional replacement) on the immunological response to methicillin resistant Staphylococcus aureus (MRSA) infections. Male rats were divided into two distinct groups: Nourished and Malnourished. After isolation of mononuclear cells, four systems were established: negative control, positive control and two testing systems, (MSSA and MRSA). Tests were performed to analyze expression of TLR-9, NF-kB, IL-1ß, IL-18 and IL-33. For statistical analysis, we used the Student t and ANOVA tests p < 0.05. Even after nutritional replacement, malnutrition in the neonatal period compromised the animals' weight gains p < 0.05. There was a reduction in the expression of the immunological response in the positive control, however deregulation was observed in the gene expression of MRSA-infected macrophages, with a reduction in TLR-9 expression, and overexpression in NF-kB and cytokines p < 0.05. Puppies inflicted with protein-calorie malnutrition were compromised; (long-term) body growth and immune response. In the infectious scenario, immune collapse is reflected in inflammatory response exacerbation with a likely histolytic character. Immune disabling (resulting from gene expression deregulation) causes susceptibility to infections due to ineffective recognition, intense pro-inflammatory mediation, and cell death. It is suggested that neonatal malnutrition can program susceptibility to multiresistant bacterial infections, and generally favors a triggering of more intense confrontations with fatal outcomes.

Neonatal malnutrition programs the oxidant function of macrophages in response to Candida albicans.

Experimental maternal nutrition restriction models are used to investigate short or long-term consequences of nutritional deficiency on puppies' growth. By assuming that the immune function is directly related to host's nutritional status, the current study aims to investigate the effects of neonatal malnutrition on oxidative stress and on the cell death of the alveolar macrophage after in vitro infection by Candida albicans. Wistar rats were suckled by mothers fed on diets containing 17% protein (Nourished group) or 8% protein (Malnourished group) in the current assay. Both groups received the standard diet used in the vivarium until adulthood, after weaning. The results showed that the offspring from mothers fed on low-protein diet presented lower body weight from 5 days of life on. Their low weight remained until adulthood when it was compared to that of rats in the nourished group. Superoxide and nitric oxide production was lower in malnourished animals and it was accompanied by low inducible nitric oxide synthase gene expression levels in systems in which the alveolar macrophages were challenged by immunogenic stimulus. No significant differences were observed in comparisons performed between the nourished and malnourished groups in any of the analyzed cell viability (apoptosis/necrosis) parameters. The fungal inoculum-stimulated system induced higher oxidative stress and cell death by necrosis. The current study demonstrated that dietary restriction during lactation alters the oxidant function of alveolar macrophages in puppies; It happens from the gene transcription step to the release of mediators, thus compromising the host's defenses against Candida albicans. It raises the possibility that Candida albicans may cease to be a commensal fungus to become a pathogen in offspring that have suffered nutritional deficiency during critical developmental periods, due to impaired immune responses.

Antioxidant supplementation for the treatment of acute lung injury: a meta-analysis. / Suplementação de antioxidantes no tratamento da lesão pulmonar aguda: meta-análise.

OBJECTIVE: This meta-analysis was performed to evaluate the evidence supporting antioxidant supplementation as an adjunct therapy to prevent oxidative damage and improve the clinical outcomes (mortality, length of hospital stay and duration of mechanical ventilation). METHODS: The search strategy for randomized controlled trials (RCTs) involved the participation of two researchers who independently assessed the methodological quality of each full-text article that was available in the PubMed, ISI WEB of Knowledge and ScienceDirect databases. RESULTS: We extracted 110 studies from the past 10 years, but only 30 articles met the methodological criteria (RCT, blinded and statistically significant results), for a total of 241 animals and 256 patients. This study found an odds ratio (OR) of 0.45 [95% confidence interval (CI): 0.26 to 0.79] for death in the experimental group compared with placebo (six trials, n = 256), an OR of 0.46 [95% CI: 0.26 to 0.87] for hospitalization time and an OR of 0.63 [95% CI: 0.35 to 1.12] for mechanical ventilation time between groups. CONCLUSION: Conflicting evidence makes it impossible to recommend the routine use of antioxidant supplementation in critically ill patients.

Long-term effects of neonatal malnutrition on microbicide response, production of cytokines, and survival of macrophages infected by Staphylococcus aureus sensitive/resistant to methicillin / Efeitos tardios da desnutrição neonatal na resposta microbicida, produção de citocinas e viabilidade de macrófagos na infecção por Staphylococcus aureus sensível/resistente a meticilina

OBJECTIVE: To assess microbicide function and macrophage viability after in vitro cellular infection by methicillin-sensitive/resistant Staphylococcus aureus in nourished rats and rats subjected to neonatal malnutrition. METHODS: Male Wistar rats (n=40) were divided in two groups: Nourished (rats suckled by dams consuming a 17% casein diet) and Malnourished (rats suckled by dams consuming an 8% casein diet). Macrophages were recovered after tracheotomy, by bronchoalveolar lavage. After mononuclear cell isolation, four systems were established: negative control composed exclusively of phagocytes; positive control composed of macrophages plus lipopolysaccharide; and two testing systems, macrophages plus methicillin-sensitive Staphylococcus aureus and macrophages plus methicillin-resistant Staphylococcus aureus. The plates were incubated in a humid atmosphere at 37 degrees Celsius containing 5% CO2 for 24 hours. After this period tests the microbicidal response, cytokine production, and cell viability were analyzed. The statistical analysis consisted of analysis of variance (p<0.05). RESULTS: Malnutrition reduced weight gain, rate of phagocytosis, production of superoxide anion and nitric oxide, and macrophage viability. Production of nitrite and interleukin 18, and viability of macrophages infected with methicillin-resistant Staphylococcus aureus were lower. CONCLUSION: The neonatal malnutrition model compromised phagocyte function and reduced microbicidal response and cell viability. Interaction between malnutrition and the methicillin-resistant strain decreased the production of inflammatory mediators by effector cells of the immune response, which may compromise the immune system's defense ability. .

OBJETIVO: Avaliar a função microbicida e a viabilidade de macrófagos, após infecção celular in vitro, com Staphylococcus aureus sensível/resistente a meticilina, em ratos nutridos ou submetidos a desnutrição neonatal. MÉTODOS: Ratos machos Wistar (n=40) foram divididos em dois grupos distintos: Nutrido (ratos amamentados por mães submetidas a dieta com 17% de caseína) e Desnutrido (ratos amamentados por mães submetidas a dieta com 8% de caseína). Os macrófagos foram recuperados após procedimento cirúrgico de traqueostomia, através da coleta do lavado broncoalveolar. Após o isolamento dos mononucleares, foram estabelecidos quatro sistemas: controle negativo, composto apenas pelos fagócitos; controle positivo, macrófagos mais lipopolissacarídeo; e dois sistemas teste, macrófagos mais Staphylococcus aureus sensível e resistente a meticilina. As placas foram incubadas por 24 horas, à temperatura de 37ºC, com atmosfera úmida e 5% de dióxido de carbono. Transcorrido esse período, foram realizados ensaios para análise da resposta microbicida, produção de citocinas e viabilidade celular. Na análise estatística, utilizou-se analysis of variance, admitindo-se p<0,05. RESULTADOS: A desnutrição acarretou redução do crescimento ponderal dos animais, da taxa de fagocitose, da produção de óxido nítrico, do ânion superóxido e da viabilidade de macrófagos. Houve menor produção de nitrito, de interleucina 18 e da viabilidade dos macrófagos infectados com Staphylococcus aureus meticilina-resistente. CONCLUSÃO: O modelo de desnutrição neonatal adotado comprometeu a função dos fagócitos, com redução da resposta microbicida e da viabilidade celular. A interação ...

Immunological parameters of macrophages infected by methicillin resistant/sensitive Staphylococcus aureus / Parâmetros imunológicos de macrófagos frente à infecção por Staphylococcus aureus meticilina sensível/resistente

INTRODUCTION: Could changes in Staphylococcus aureus cellular walls, which are commonly associated with multidrug resistance phenotype, influence their immune evasion mechanisms? OBJECTIVE: To evaluate the microbicide response and survival of alveolar macrophages after in vitro infection with methicillin-sensitive and methicillin-resistant Staphylococcus aureus. MATERIAL AND METHODS: We used 20 adult, male, albino, Wistar rats. The alveolar macrophage samples were obtained after tracheostomy and bronchoalveolar lavage. The alveolar macrophages were cultured in the proportion of 1:1 cells/ml Roswell Park Memorial Institute (RPMI) medium/well and isolated by plate adhesion. For the assessment of immunological parameters, four systems were established: negative control, positive control, methicillin sensitive S. aureus (MSSA) and methicillin resistant S. aureus (MRSA). RESULTS: When comparing MRSA and MSSA systems, there was no significant difference as to adhesion and phagocytosis rates, superoxide anion production and macrophage viability. By analyzing the kinetics of nitric oxide production, after 4 to 10 hours of cellular culture incubation, there was lower average production of this radical in the MRSA system when compared to MSSA. However, after 12 hours, no differences were detected between both systems. CONCLUSION: It is claimed that methicillin resistance may be a factor that influences the bacteria's ability to escape from macrophage microbicide response. Although the results of some immunological parameters were similar in the surveyed systems, the oscillations occurred during the production of nitric oxide may contribute significantly to the survival of Staphylococcus aureus.

INTRODUÇÃO: Modificações nas paredes celulares das cepas de Staphylococcus aureus relacionadas com o fenótipo de multirresistência poderiam influenciar seus mecanismos de evasão frente à resposta imune? OBJETIVO: Avaliar a resposta microbicida e a sobrevivência de macrófagos alveolares após infecção in vitro com Staphylococcus aureus meticilina sensível/resistente. MATERIAL E MÉTODOS: Utilizaram-se 20 ratos adultos, machos, albinos e da linhagem Wistar. Os macrófagos alveolares foram obtidos após procedimento cirúrgico de traqueostomia, por meios da coleta do lavado broncoalveolar. Os macrófagos alveolares foram cultivados na proporção de 1:1 células/ml de Roswell Park Memorial Institute (RPMI)/poço e isolados pela capacidade de adesão à placa. Para avaliação de parâmetros imunológicos, foram estabelecidos quatro sistemas: controle negativo, controle positivo, S. aureus sensível a meticilina (MSSA) e S. aureus resistente à meticilina (MRSA). RESULTADOS: Ao comparar os sistemas de MSSA e MRSA, não foi observada diferença no índice de aderência, na taxa de fagocitose, na produção do ânion superóxido e na viabilidade dos macrófagos. Ao analisar a cinética de óxido nítrico, houve menor produção média desse radical para o MRSA quando comparado com o MSSA, no período de 4 a 10 horas de incubação das culturas celulares. Entretanto, após 12 horas, não foi detectada divergências entre esses sistemas. CONCLUSÃO: Sugere-se que a resistência à meticilina poderá ser um fator que influenciará a capacidade de evasão da bactéria à resposta microbicida dos macrófagos. Apesar dos resultados de alguns parâmetros imunológicos terem sido similares entre os sistemas analisados, as oscilações ocorridas durante a produção do óxido nítrico poderão contribuir de forma importante para a sobrevivência da bactéria Staphylococcus aureus.

Desnutrição neonatal e produção de IFN-γ IL-12 e IL-10 por macrófagos/linfócitos: estudo da infecção celular, in vitro, por Staphylococcus aureus meticilina sensível e meticilina resistente / Early malnutrition and production of IFN, IL-12 and IL-10 by macrophages/lymphocytes: in vitro study of cell infection by methicillin-sensitive and methicillin-resistant Staphylococcus aureus

OBJETIVO: Avaliar a influência da desnutrição neonatal sobre a produção de Interferon gama, Interleucina-12 e Interleucina-10 em cultura de macrófagos alveolares e linfócitos infectados, in vitro, com Staphylococcus aureus sensível/resistente à meticilina. MÉTODOS: Ratos machos Wistar foram amamentados por mães cuja dieta, durante a lactação, continha 17% de proteína no grupo nutrido e 8% no grupo desnutrido. Após desmame, ambos os grupos receberam a dieta normoproteica. Os macrófagos foram obtidos após traqueostomia, através da coleta do lavado broncoalveolar. Para obtenção dos linfócitos, foi realizado o procedimento cirúrgico de punção cardíaca. Após o isolamento dos diferentes tipos celulares, procedeuse à realização dos estímulos com as cepas de estudo. A dosagem das citocinas foi realizada pelo método de Enzyme-Linked Immunosorbent Assay, a partir de amostras coletadas do sobrenadante das culturas após 24 horas de incubação. RESULTADOS: A desnutrição acarretou diminuição do crescimento ponderal, redução na produção de Interferon gama em cultura de macrófagos alveolares e linfócitos e diminuição na produção de Interleucina-12 em cultura de macrófagos alveolares. Apenas a produção de Interferon gama e Interleucina-10 em cultura de macrófagos alveolares apresentou diferença entre as cepas analisadas, em ambos os grupos estudados. CONCLUSÃO: O modelo de desnutrição neonatal produziu sequela no peso corporal e reduziu a produção de citocinas próinflamatórias (Interleucina-12 e Interferon gama), indicando que esse modelo de desnutrição pode comprometer a resolução de um processo infeccioso. A cepa de Staphylococcus aureus resistente à meticilina estimulou uma maior produção de Interferon gama e Interleucina-10 por macrófagos alveolares, o que sugeriu estimulação imunológica mais intensa, por essa cepa, nesse tipo celular especificamente.

OBJECTIVE: The present study assessed the influence of neonatal malnutrition on the production of interferon gamma, interleukin-12 and interleukin-10 in cultured macrophages and lymphocytes infected in vitro with methicillin-sensitive or methicillin-resistant Staphylococcus aureus. METHODS: Male Wistar rats were suckled by mothers fed during lactation a chow containing 17% protein for the nourished group and 8% for the undernourished group. After weaning, both groups received a normal diet in terms of protein content. The macrophages were obtained by bronchoalveolar lavage after tracheostomy. Cardiac puncture was used for the collection of lymphocytes. After isolation of different cell types, the challenge with the different strains was performed. Cytokines were measured by enzymelinked immunosorbent assay (ELISA), using samples collected from the culture supernatant after an incubation period of 24 hours. RESULTS: Malnutrition let to slow weight gain, low interferon gamma in cultured alveolar macrophages and lymphocytes and low production of interleukin-12 in cultured alveolar macrophages. Only interferon gamma and interleukin-10 in cultured alveolar macrophages differed between the two groups and study strains. CONCLUSION: The neonatal malnutrition model used impaired weight gain and reduced production of proinflammatory cytokines (interleukin-12 and interferon gamma), indicating that protein malnutrition may result in an inability to fight infections. The methicillin-resistant Staphylococcus aureus strain stimulated macrophages to produce interferon gamma and interleukin-10, suggesting that this strain better provokes the immune system, specifically for this cell type.

Suplementação de antioxidantes no tratamento da lesão pulmonar aguda: meta-análise / Antioxidant supplementation for the treatment of acute lung injury: a meta-analysis

OBJETIVO: A pesquisa foi conduzida de maneira a se esclarecer, através de uma meta-análise, as evidências da suplementação de antioxidantes como terapia adjuvante na prevenção dos danos oxidativos e melhora do desfecho clínico, tais como mortalidade, tempo de hospitalização e ventilação mecânica. MÉTODOS: A estratégia de busca de ensaios clínicos randomizados (ECRs) envolveu a participação de dois pesquisadores que avaliaram, de forma independente, a qualidade metodológica de cada artigo, disponível full text, nas bases de dados PubMed, ISI of Knowledge e ScienceDirect. RESULTADOS: Foram extraídos 110 estudos dos últimos 10 anos, porém somente 30 artigos preencheram os critérios metodológicos (ensaios controlados, randomizados, cego e estatisticamente significativo), totalizando 241 animais e 256 pacientes. Este trabalho encontrou um OR de 0,45 [intervalo de confiança (IC) 95 por cento: 0,26 - 0,79] para a mortalidade na comparação do grupo experimental com placebo (6 estudos, n = 256), um OR de de 0,46 [intervalo de confiança (IC) 95 por cento: 0,26 - 0,87] para tempo de hospitalização e um OR de 0,63 [intervalo de confiança (IC) 95 por cento: 0,35 - 1,12] para o tempo de ventilação mecânica assistida entre os grupos. CONCLUSÃO: As evidências são conflitantes e, desta forma, ainda não é possível recomendar o uso rotineiro da suplementação com antioxidantes em pacientes criticamente enfermos.

OBJECTIVE: This meta-analysis was performed to evaluate the evidence supporting antioxidant supplementation as an adjunct therapy to prevent oxidative damage and improve the clinical outcomes (mortality, length of hospital stay and duration of mechanical ventilation). METHODS: The search strategy for randomized controlled trials (RCTs) involved the participation of two researchers who independently assessed the methodological quality of each full-text article that was available in the PubMed, ISI WEB of Knowledge and ScienceDirect databases. RESULTS: We extracted 110 studies from the past 10 years, but only 30 articles met the methodological criteria (RCT, blinded and statistically significant results), for a total of 241 animals and 256 patients. This study found an odds ratio (OR) of 0.45 [95 percent confidence interval (CI): 0.26 to 0.79] for death in the experimental group compared with placebo (six trials, n = 256), an OR of 0.46 [95 percent CI: 0.26 to 0.87] for hospitalization time and an OR of 0.63 [95 percent CI: 0.35 to 1.12] for mechanical ventilation time between groups. CONCLUSION: Conflicting evidence makes it impossible to recommend the routine use of antioxidant supplementation in critically ill patients.