Combination of flow cytometry and qPCR to study the immune response of american cutaneous leishmaniasis patients.

American Cutaneous leishmaniasis (ACL) is a public health problem. The immunological response is mainly dependent on T cell cytokine responses and might influence disease presentation, susceptibility and development. The understanding of the host immune response role in the control and in the pathology of leishmaniasis is relevant and has implications on diagnosis, follow-up and vaccine development. In this study, the differences in the immune response and T cell profile of patients before treatment was investigated through flow cytometry and real time PCR in peripheral blood mononuclear cells after different antigenic stimulations. Among the main findings are the significant presence of TNF and IFN-γ gene expression after 24 h of in vitro stimulation, and 48 h later the presence of CD4+ T and CD8+ T cells producing IL-10 and IL-4. This may be due to the differences in cytokine release over time and the presence of cells other than lymphocytes influencing the mRNA transcript detection. Evaluation of the immune response of individuals with leishmaniasis or other diseases should associate different technologies and times points for a clear and more reliable assessment of the immune response. This would help in the design of vaccine strategies/immunotherapies.

Relation between neonatal malnutrition and gene expression: inflammasome function in infections caused by Candida Albicans.

PURPOSE: To investigate the effects of neonatal malnutrition followed by nutritional replacement on the signaling mechanisms developed by the inflammasome complex by analyzing the expression of the targeted TLR2, TLR4, NLRP3, caspase-1 and release of IL-1ß and IL-18 by alveolar macrophages infected in vitro with Candida albicans. METHODS: Male Wistar rats (n = 24), 90-120 days, were suckled by mothers whose diet during lactation contained 17 % protein in the nourish group and 8 % protein in the malnourished group. After weaning, both groups were fed a normal protein diet. Macrophages were obtained after tracheostomy, through the collection of bronchoalveolar lavage fluid. The quantification of the expression levels of targets (TLR2, TLR4, NLRP3 and caspase-1) was performed by real-time RT-PCR. Production of cytokines was performed by ELISA. RESULTS: The malnourished animals during lactation showed reduced body weight from the fifth day of life, remaining until adulthood. Further, the model applied malnutrition induced a lower expression of TLR4 and caspase-1. The quantification of the TLR2 and NLRP3, as well as the release of IL-1ß and IL-18, was not different between groups of animals nourished and malnourished. The system challenged with Candida albicans showed high expression levels of all targets in the study. CONCLUSIONS: The tests demonstrate nutritional restriction during critical periods of development, although nutritional supplementation may compromise defense patterns in adulthood in a timely manner, preserving distinct signaling mechanism, so that the individual does not become widely vulnerable to infections by opportunistic pathogens.

Cytokines and NO in American tegumentary leishmaniasis patients: profiles in active disease, after therapy and in self-healed individuals.

Studies suggest the influence of immune response on the successful treatment of American tegumentary leishmaniasis (ATL), and indicate the existence of protective immunity in self-healed patients. Thus, the aim of this work was to quantify interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL-) 10, IL-17, IL-22 and nitric oxide (NO) in culture supernatants of PBMC from patients with active disease (AD), after treatment (AT), and from self-healed (SH) and healthy subjects (CT), in response to Leishmania (Viannia) braziliensis insoluble antigen (AgIns). All groups of patients produced IFN-γ, indicating a predominant proinflammatory profile. AD and AT patients presented TNF-α levels, with a slight increase after therapy, whereas it was weakly quantified in SH. Interestingly, NO secretion was significant in these individuals, whereas IL-17 appeared in low levels and seems to be regulated by NO. Although IL-22 was detected in AD, its role is still questionable. The presence of IL-10 in all groups of patients suggests that the cytokine plays distinct roles in the disease. These results indicate that specific cellular immunity takes part against Leishmania, but with some similarities between the different clinical states herein described; these mediators seem to be necessary for the cure to occur.

[Factors influencing the bacterial translocation in mice with chronic schistosomiasis]. / Fatores que influenciam a translocação bacteriana em camundongos corn esquistossomose crônica.

INTRODUCTION: Schistosomiasis causes alterations of the intestinal mucosa and a low cellular immune response in its chronic phase. Gender may influence the inflammatory response against Schistosoma mansoni. We investigate the association between schistosomiasis and secondary infections by bacterial translocation. METHODS: Swiss Webster mice (Mus musculus) with 35 days were divided into two groups: control (10 male and 10 female) and schistosomiasis (10 male and 10 female infected with 50 cercariae percutaneously). Stools were examined by the Kato-Katz with 45 and 97 days of infection. Liver perfusion was performed for quantification of worms. The animals were weighed after 35, 80, 125 and 132 days old when they were euthanized for study of translocation, microbiota and duodenal mucosa. For microbiota, stools were collected from the middle of the small intestine. Segments of this region were sectioned for morphometric diagnosis. RESULTS: Females had higher schistosomotic number of adult worms and eggs in stools (P = 0. 0001). Both sexes had a higher number of eggs on the 45th day (P = 0.005), decreased weight gain with 80, 125 and 132 days old (P = 0.0001) and increased spleen weight (P = 0.0001). The animals with schistosomiasis had more bacterial species and colony-forming units. Morphometric analysis revealed a reduction in height and area of villus and of perimeter of the mucosal surface of both groups with chronic disease (P = 0.0001). Increased bacterial translocation occurred in schistosomiasis when compared to controls, being more prevalent in females. CONCLUSIONS: Chronic schistosomiasis modify weight gain and weight of spleen, duodenal mucosa and microbiota in mice and favors translocation, migration and sepsis, especially in females, probably due to the intensity of parasitism.

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.