Maturation of mononuclear phagocytes in the lungs of young calves-In vitro study

The influences of age in calves' immune system are described in their first phase of life. We hypothesized that variations that occur in the main mechanisms of lung innate response can help to identify periods of greater susceptibility to the respiratory diseases that affect calves in the first stage of their life. This study aimed to evaluate the innate immune system. Nine healthy calves were monitored for 3 mo and 8 immunologic evaluations were performed. Bronchoalveolar lavage samples were recovered by bronchoscopy. The alveolar macrophages in samples were identified by protein expression of cluster of differentiation 14 (CD14) and underwent functional evaluation of phagocytosis (Staphylococcus aureus stained with propidium iodide and Escherichia coli). Data was assessed by one-way ANOVA (unstacked and parametric) and the Mann-Whitney test (nonparametric). Functional alterations in CD14-positive phagocytes were observed, with punctual higher intensity of phagocytosis in the third week and its decrease starting at 45 d of life. A gradual increase in phagocytosis rate was observed starting at this date. It is concluded that from 45 d of life on, alveolar macrophages have less phagocytic capacity but more cells perform this function. We suggest that this occurs because lung macrophages of calves start to maintain their immune response without passive immunity influence. Until 90 d of life, calves did not achieve the stability to conclude the maturation of local innate immune response.

BAY 41-2272, a soluble guanylate cyclase agonist, activates human mononuclear phagocytes

BACKGROUND AND PURPOSE Phagocyte function is critical for host defense against infections. Defects in phagocytic function lead to several primary immunodeficiencies characterized by early onset of recurrent and severe infections. In this work, we further investigated the effects of BAY 41-2272, a soluble guanylate cyclase (sGC) agonist, on the activation of human peripheral blood monocytes (PBM) and THP-1 cells. EXPERIMENTAL APPROACH THP-1 cells and PBM viability was evaluated by methylthiazoletetrazolium assay; reactive oxygen species production by lucigenin chemiluminescence; gene and protein expression of NAPDH oxidase components by qRT-PCR and Western blot analysis, respectively; phagocytosis and microbicidal activity by co-incubation, respectively, with zymosan and Escherichia coli; and cytokine release by elisa. KEY RESULTS BAY 41-2272, compared with the untreated group, increased spreading of monocytes by at least 35%, superoxide production by at least 50%, and gp91PHOX and p67PHOX gene expression 20 to 40 times, in both PBM and THP-1 cells. BAY 41-2272 also augmented phagocytosis of zymosan particles threefold compared with control, doubled microbicidal activity against E. coli and enhanced the release of TNF-a and IL-12p70 by both PBM and THP-1 cells. Finally, by inhibiting sGC with ODQ, we showed that BAY 41-2272-induced superoxide production and phagocytosis is not dependent exclusively on sGC activation. CONCLUSIONS AND IMPLICATIONS In addition to its ability to induce vasorelaxation and its potential application for therapy of vascular diseases, BAY 41-2272 was shown to activate human mononuclear phagocytes. Hence, it is a novel pro-inflammatory drug that may be useful for controlling infections in the immunocompromised host.

Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions

Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC50 of 11.26 mu g/ml against promastigotes and amastigotes of 0.27 mu g/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests. (C) 2012 Published by Elsevier B.V.

Viabilidade celular, fagocitose e espraiamento de fagócitos mononucleares, e liberação de peróxido de hidrogênio por leucócitos de glândulas mamárias bovinas sadias e infectadas

O presente estudo objetivou avaliar a viabilidade celular, a capacidade de fagocitose e espraiamento pelos fagócitos mononucleares, e a liberação de peróxido de hidrogênio (H2O2) por leucócitos oriundos de glândulas mamárias bovinas sadias e infectadas. Deste modo, 94 amostras foram divididas de acordo com os resultados da cultura bacteriológica e da contagem de células somáticas (CCS). O presente estudo não encontrou diferenças na viabilidade celular, e nos índices de fagocitose e espraiamento entre os diferentes grupos. No entanto, a liberação de H2O2 oriundos dos quartos mamários infectados, infectados por Streptococcus spp. ou Corynebacterium spp. foi menor do que nas amostras de leite provenientes dos quartos mamários sadios. Ao estimar a concentração de H2O2 mL-1 leite observou-se que as amostras de quartos mamários positivos no exame bacteriológico, infectados por Staphylococcus spp. e negativos no exame bacteriológico com alta celularidade foram maiores que aquelas provenientes de quartos mamários sadios. Observou-se também correlação positiva entre a CCS e a viabilidade celular e os índices de fagocitose e espraiamento; e correlação negativa entre a liberação de H2O2 e a CCS e a viabilidade celular. Conclui-se que a CCS, assim como a sua viabilidade e função, são conceitos intimamente relacionados com a saúde da glândula mamária.

Gene expression profiles displayed by peripheral blood mononuclear cells from patients with type 2 diabetes mellitus focusing on biological processes implicated on the pathogenesis of the disease

Patients with type 2 diabetes mellitus (T2DM) exhibit insulin resistance associated with obesity and inflammatory response, besides an increased level of oxidative DNA damage as a consequence of the hyperglycemic condition and the generation of reactive oxygen species (ROS). In order to provide information on the mechanisms involved in the pathophysiology of T2DM, we analyzed the transcriptional expression patterns exhibited by peripheral blood mononuclear cells (PBMCs) from patients with T2DM compared to non-diabetic subjects, by investigating several biological processes: inflammatory and immune responses, responses to oxidative stress and hypoxia, fatty acid processing, and DNA repair. PBMCs were obtained from 20 T2DM patients and eight non-diabetic subjects. Total RNA was hybridized to Agilent whole human genome 4x44K one-color oligo-microarray. Microarray data were analyzed using the GeneSpring GX 11.0 software (Agilent). We used BRB-ArrayTools software (gene set analysis - GSA) to investigate significant gene sets and the Genomica tool to study a possible influence of clinical features on gene expression profiles. We showed that PBMCs from T2DM patients presented significant changes in gene expression, exhibiting 1320 differentially expressed genes compared to the control group. A great number of genes were involved in biological processes implicated in the pathogenesis of T2DM. Among the genes with high fold-change values, the up-regulated ones were associated with fatty acid metabolism and protection against lipid-induced oxidative stress, while the down-regulated ones were implicated in the suppression of pro-inflammatory cytokines production and DNA repair. Moreover, we identified two significant signaling pathways: adipocytokine, related to insulin resistance; and ceramide, related to oxidative stress and induction of apoptosis. In addition, expression profiles were not influenced by patient features, such as age, gender, obesity, pre/post-menopause age, neuropathy, glycemia, and HbA(1c) percentage. Hence, by studying expression profiles of PBMCs, we provided quantitative and qualitative differences and similarities between T2DM patients and non-diabetic individuals, contributing with new perspectives for a better understanding of the disease. (C) 2012 Elsevier B.V. All rights reserved.

Interferon-gamma and interleukin-10 production by mononuclear cells from patients with advanced head and neck cancer

OBJECTIVE: This study aims to evaluate the production of interferon-gamma and interleukin-10 by stimulated peripheral blood mononuclear cells isolated from patients with supraglottic laryngeal cancer before and after surgical treatment. METHODS: Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cultures of peripheral blood mononuclear cells isolated during the preoperative and late postoperative periods were stimulated with concanavalin A and Bacille Calmette-Guerin, and the supernatant concentrations of interferon-gamma and interleukin-10 were measured. RESULTS: For non-stimulated cultures, the interferon-gamma levels produced by the preoperative period and the late postoperative period cultures were lower than the levels produced by the control group cultures. The interferon-gamma levels after stimulation with concanavalin A were higher in the late postoperative period cultures than in the preoperative evaluation cultures. Stimulation with Bacille Calmette-Guerin led to the production of similar levels of interferon-gamma and interleukin-10 by all cultures; thus, stimulation increased the levels of interferon-gamma produced by both the preoperative and postoperative cultures relative to the levels produced by the corresponding unstimulated cultures. CONCLUSION: Patients with advanced supraglottic laryngeal cancer exhibit an in vitro deficiency in interferongamma secretion by mononuclear cells. Stimulated cells seem to recover this function during the postoperative period.

Cyto and genotoxicity of gold nanoparticles in human hepatocellular carcinoma and peripheral blood mononuclear cells

Engineered nanomaterials have been extensively applied as active materials for technological applications. Since the impact of these nanomaterials on health and environment remains undefined, research on their possible toxic effects has attracted considerable attention. It is known that in humans, for example, the primary site of gold nanoparticles (AuNps) accumulation is the liver. The latter has motivated research regarding the use of AuNps for cancer therapy, since specific organs can be target upon appropriate functionalization of specific nanoparticles. In this study, we investigate the geno and cytotoxicity of two types of AuNps against human hepatocellular carcinoma cells (HepG2) and peripheral blood mononuclear cells (PBMC) from healthy human volunteers. The cells were incubated in the presence of different concentrations of AuNps capped with either sodium citrate or polyamidoamine dendrimers (PAMAM). Our results suggest that both types of AuNps interact with HepG2 cells and PBMC and may exhibit in vitro geno and cytotoxicity even at very low concentrations. In addition, the PBMC were less sensitive to DNA damage toxicity effects than cancer HepG2 cells upon exposure to AuNps. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model

We hypothesized that bone marrow-derived mononuclear cell (BMDMC) therapy protects the lung and consequently the heart in experimental elastase-induced emphysema. Twenty-four female C57BL/6 mice were intratracheally instilled with saline (C group) or porcine pancreatic elastase (E group) once a week during 4 weeks. C and E groups were randomized into subgroups receiving saline (SAL) or male BMDMCs (2 x 10(6), CELL) intravenously 3 h after the first saline or elastase instillation. Compared to E-SAL group, E-CELL mice showed, at 5 weeks: lower mean linear intercept, neutrophil infiltration, elastolysis, collagen fiber deposition in alveolar septa and pulmonary vessel wall, lung cell apoptosis, right ventricle wall thickness and area, higher endothelial growth factor and insulin-like growth factor mRNA expressions in lung tissue, and reduced platelet-derived growth factor, transforming growth factor-beta, and caspase-3 expressions. In conclusion, BMDMC therapy was effective at modulating the inflammatory and remodeling processes in the present model of elastase-induced emphysema. (c) 2012 Elsevier B.V. All rights reserved.

Detection of the Epstein-Barr virus in blood and bone marrow mononuclear cells of patients with aggressive B-cell non-Hodgkin's lymphoma is not associated with prognosis

The Epstein-Barr virus (EBV) is associated with a large spectrum of lymphoproliferative diseases. Traditional methods of EBV detection include the immunohistochemical identification of viral proteins and DNA probes to the viral genome in tumoral tissue. The present study explored the detection of the EBV genome, using the BALF5 gene, in the bone marrow or blood mononuclear cells of patients with diffuse large B-cell lymphomas (DLBCL) and related its presence to the clinical variables and risk factors. The results show that EBV detection in 21.5% of patients is not associated with age, gender, staging, B symptoms, international prognostic index scores or any analytical parameters, including lactate dehydrogenase (LDH) or beta-2 microglobulin (B2M). The majority of patients were treated with R-CHOP-like (rituximab. cyclophosphamide, doxorubicin, vincristine and prednisolone or an equivalent combination) and some with CHOP-like chemotherapy. Response rates [complete response (CR) + partial response (PR)] were not significantly different between EBV-negative and -positive cases, with 93.2 and 88.9%, respectively. The survival rate was also similar in the two groups, with 5-year overall survival (OS) rates of 64.3 and 76.7%, respectively. However, when analyzing the treatment groups separately there was a trend in EBV-positive patients for a worse prognosis in patients treated with CHOP-like regimens that was not identified in patients treated with R-CHOP-like regimens. We conclude that EBV detection in the bone marrow and blood mononuclear cells of DLBC patients has the same frequency of EBV detection on tumoral lymphoma tissue but is not associated with the risk factors, response rate and survival in patients treated mainly with immunochemotherapy plus rituximab. These results also suggest that the addition of rituximab to chemotherapy improves the prognosis associated with EBV detection in DLBCL.

Enhanced phagocytosis of Corynebacterium pseudotuberculosis by monocyte-macrophage cells from goats naturally infected with caprine arthritis encephalitis virus

Sanches B.G.S., Souza F.N., Azedo M.R., Batista C.F., Bertagnon H.G., Blagitz M.G. & Della Libera A.M.M.P. 2012. [Enhanced phagocytosis of Corynebacterium pseudotuberculosis by monocyte-macrophage cells from goats naturally infected with caprine arthritis encephalitis virus.] Fagocitose intensificada de Corynebacterium pseudotuberculosis por celulas da serie monocito-macrofago de caprinos naturalmente infectados pelo virus da artrite encefalite. Pesquisa Veterinaria Brasileira 32(12):1225-1229. Departamento de Clinica Medica, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Avenida Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitaria, Sao Paulo, SP 05508-270, Brazil. E-mail: camilafb@usp.br Caprine arthritis encephalitis (CAE) and caseous lymphadenitis (CL) have high incidence and transmissibility in small ruminants. Since both virus have tropism for macrophages and monocytes and affect the innate immune response, it is believed that CAE can predispose the animal to infection by Corynebacteruim pseudotuberculosis, the etiological agent of CL. To confirm this hypothesis, we evaluated phagocytosis from the monocyte-macrophage cells from 30 Saanen goats. Goats were uniformly divided in two groups according to results of agar gel immunodiffusion test for CAE virus (CAEV). Peripheral blood mononuclear cells were isolated by density gradient centrifugation and the monocyte-macrophage cells were isolated from the mononuclear cells by their adhesion properties in plaques. Afterwards, phagocytosis of C. psudotuberculosis was performed for two hours at 37 degrees C, 5% of CO2, and assessed by microscopic visualization. There was no difference in the percentage of monocyte-macrophage cells that phagocytozed C. bovis between groups (P = 0.41). However, when phagocytosis rates were classified according to the number of C. pseudotuberculosis phagocyted, the percentage of monocyte-macrophage cells that internalized more than 12 bacteria were higher in serologically CAEV positive animals compared to the serologically negative ones (P < 0.001). Furthermore, a positive and significant correlation (r = 0.488; P = 0.006) between the percentage of monocyte-macrophage cells that internalized more than 12 bacteria and the percentage of monocyte that were carrying out phagocytosis was also encountered in serologically CAEV positive goats, however the same were not observed in serologically negative ones. These results demonstrated an alteration in the intensity of C. pseudotuberculosis phagocytosis by monocytes-macrophages from goats infected by CAEV. Thus, these results indicated that goats infected with CAEV may be more susceptible to CL.

Influence of Mid-Trimester Amniotic Fluid on Endogenous and Lipopolysaccharide-Mediated Responses of Mononuclear Lymphoid Cells

Problem We evaluated the influence of amniotic fluid (AF) on immune mediator production by mononuclear leukocytes. Method of study Thirty mid-gestation AFs were incubated with peripheral blood mononuclear cells (PBMCs) in the presence or absence of lipopolysaccharide (LPS). Supernatants were tested for interleukin (IL) -6, 10, 12, 23, tumor necrosis factor-alpha (TNF-alpha) and monocyte chemotactic protein (MCP)-1. Results Endogenous mediator production was minimal or non-detectable. AF stimulated endogenous MCP-1, IL-6 and TNF-alpha release. In the presence of LPS, production of MCP-1 and IL-10 by PBMCs was enhanced eightto ninefold by AF. Release of IL-6 and IL-23 was enhanced less than twofold by the addition of AF while TNF-alpha production was unchanged. AF-stimulated mediator production was similar irrespective of pregnancy outcome. Conclusion Selective AF stimulation of LPS-mediated MCP-1 and IL-10 release may be a mechanism to promote antibody production and the influx of phagocytic cells to engulf pathogens while downregulating the production of pro-inflammatory cytokines.

Mast cells act as phagocytes against the periodontopathogen Aggregatibacter Actinomycetemcomitans

Background: Evidence to date shows that mast cells play a critical role in immune defenses against infectious agents, but there have been no reports about involvement of these cells in eliminating periodontopathogens. In this study, the phagocytic ability of mast cells against Aggregatibacter actinomycetemcomitans compared with macrophages is evaluated. Methods: In vitro phagocytic assays were conducted using murine mast cells and macrophages, incubated with A. actinomycetemcomitans, either opsonized or not, with different bacterial load ratios. After 1 hour, cells were stained with acridine orange and assessed by confocal laser-scanning electronmicroscopy. Results: Phagocytic ability of murine mast cells against A. actinomycetemcomitans was confirmed. In addition, the percentage of mast cells with internalized bacteria was higher in the absence of opsonization than in the presence of opsonization. Both cell types showed significant phagocytic activity against A. actinomycetemcomitans. However, the percentage of mast cells with non-opsonized bacteria was higher than that of macrophages with opsonized bacteria in one of the ratios (1:10). Conclusions: This is the first report about the participation of murine mast cells as phagocytes against A. actinomycetemcomitans, mainly in the absence of opsonization with human serum. Our results may indicate that mast cells act as professional phagocytes in the pathogenesis of biofilmassociated periodontal disease

Apolipoprotein E mRNA expression in mononuclear cells from normolipidemic and hypercholesterolemic individuals treated with atorvastatin

Abstract Background Apolipoprotein E (apoE) is a key component of the lipid metabolism. Polymorphisms at the apoE gene (APOE) have been associated with cardiovascular disease, lipid levels and lipid-lowering response to statins. We evaluated the effects on APOE expression of hypercholesterolemia, APOE ε2/ε3/ε4 genotypes and atorvastatin treatment in Brazilian individuals. The relationship of APOE genotypes and plasma lipids and atorvastatin response was also tested in this population. Methods APOE ε2/ε3/ε4 and plasma lipids were evaluated in 181 normolipidemic (NL) and 181 hypercholesterolemic (HC) subjects. HC individuals with indication for lowering-cholesterol treatment (n = 141) were treated with atorvastatin (10 mg/day/4-weeks). APOE genotypes and APOE mRNA in peripheral blood mononuclear cells (PBMC) were analyzed by TaqMan real time PCR. Results HC had lower APOE expression than NL group (p < 0.05) and individuals with low APOE expression showed higher plasma total and LDL cholesterol and apoB, as well as higher apoAI (p < 0.05). Individuals carrying ε2 allele have reduced risk for hypercholesterolemia (OR: 0.27, 95% I.C.: 0.08-0.85, p < 0.05) and NL ε2 carriers had lower total and LDL cholesterol and apoB levels, and higher HDL cholesterol than non-carriers (p < 0.05). APOE genotypes did not affect APOE expression and atorvastatin response. Atorvastatin treatment do not modify APOE expression, however those individuals without LDL cholesterol goal achievement after atorvastatin treatment according to the IV Brazilian Guidelines for Dyslipidemia and Atherosclerosis Prevention had lower APOE expression than patients with desirable response after the treatment (p < 0.05). Conclusions APOE expression in PBMC is modulated by hypercholesterolemia and the APOE mRNA level regulates the plasma lipid profile. Moreover the expression profile is not modulated neither by atorvastatin nor APOE genotypes. In our population, APOE ε2 allele confers protection against hypercholesterolemia and a less atherogenic lipid profile. Moreover, low APOE expression after treatment of patients with poor response suggests a possible role of APOE level in atorvastatin response.

Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia

Background We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoKATP protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoKATP activity and evaluate the influence of this trait on cell redox state and viability. Methods Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. Results HTG mice mononuclear cells displayed increased mitoKATP activity, as evidenced by higher resting respiration rates that were sensitive to mitoKATP antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoKATP further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. Conclusions These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoKATP channels. Thus, mitoKATP opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

Fagocitose intensificada de Corynebacterium pseudotuberculosis por células da série monócito-macrófago de caprinos naturalmente infectados pelo vírus da artrite encefalite

A Artrite Encefalite Caprina (AEC) e a Linfadenite Caseosa (LC) possuem alta incidência e transmissibilidade em pequenos ruminantes. Como ambas possuem tropismo por monócitos-macrófagos e afetam mecanismos da resposta inata do hospedeiro, acredita-se que a AEC predispõe o animal a infecções por Corynebacteruim pseudotuberculosis, agente etiológico da LC. Para confirmar esta hipótese, avaliou-se a fagocitose de células da série monócito-macrófago de cabras naturalmente infectadas pelo vírus da AEC (VAEC). Para tanto, foram utilizadas 30 cabras da raça Saanen, alocadas em dois grupos distintos, com 15 animais cada, conforme a sororreatividade de anticorpos séricos antivírus da AEC. Células mononucleares de sangue periférico foram isoladas por gradiente de densidade e plaqueadas para isolamento de células da série monócito-macrófago. Posteriormente, o ensaio de fagocitose de C. pseudotuberculosis foi realizado, após incubação por duas horas a 37ºC a 5% de CO2, e a visualização da fagocitose foi identificada por microscopia óptica. O presente estudo não encontrou diferença na porcentagem de monócito-macrófagos que realizaram fagocitose entre os diferentes grupos (P = 0,41). Todavia, a análise quantitativa de bactérias fagocitadas, demonstrou maior capacidade fagocítica pelos macrófagos-monócitos do grupo sororreagente ao vírus da AEC. Correlação entre monócitos fagocitando e macrófagos que fagocitaram mais de 12 bactérias foi observado neste grupo (r = 0,488; P = 0,006), não sendo o mesmo encontrado no grupo de animais sorroreagentes negativos. Os dados demonstram aumento na intensidade da fagocitose de macrófagos de animais infectados com o vírus da AEC.