Simple method for culture of peripheral blood lymphocytes of Testudinidae.

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 - 10 pg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation.

T and B lymphocytes in the brains of dogs with concomitant seropositivity to three pathogenic protozoans: Leishmania chagasi, Toxoplasma gondii and Neospora caninum

Background: Visceral leishmaniasis is a disease with great variability regarding the clinical manifestations in humans and dogs. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterize the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis. Concomitant with Leishmania chagasi, dogs may be infected by opportunistic pathogens, such as Toxoplasma gondii and Neospora caninum, which may contribute to the occurrence of lesions in the central nervous system. Hence, we aimed to compare the T and B lymphocytes population in the brains of infected dogs with seropositivity to L. chagasi, T. gondii and N. caninum concurrently (n = 24), seropositivity only to L. chagasi (n = 31), and seropositivity to T. gondii and N. caninum (n = 16). Uninfected dogs were used as control (n = 10). Results: Inflammatory lesions, characterised by mononuclear cell accumulation, composed mainly of CD3+ T lymphocytes predominated in several encephalic regions of the dogs from all the three infected groups, with no difference among them (P = 0.0004), whereas CD79α+ B lymphocytes were detected in very small intensity and presented no difference among groups (P = 0.5313). Furthermore, no association among diseases was detected at the serological enquire. Conclusions: We demonstrate that the peripheral infection by L. chagasi per se can promote the influx of lymphocytes within the nervous milieu as occurs during Toxoplasma and Neospora infections, and the concomitant seropositivity against these pathogens does not exacerbate the inflammatory brain lesions. Therefore, these findings give additional support that the brain should be included in the list of organs affected by visceral leishmaniasis and that even asymptomatic infected dogs may develop brain lesions. © 2013 Sakamoto et al.; licensee BioMed Central Ltd.

Laser stimulated light reflection for TeO2-WO 3-Bi2O3 thin films with incorporated Si nanoparticles

A novel method of preparation of the Si nanoparticles (NPs) incorporated in tellurite TeO2-WO3-Bi2O3 (TWB) thin films is proposed. This mew method applies RF magnetron sputtering technique at room temperature. The incorporation of Si NP was confirmed by transmission electron microscopy (TEM); isolated Si NPs with diameters of around 6 nm are observed. Energy dispersive X-ray spectroscopy (EDS) was performed during TEM analysis in order to confirm the presence of Si NP and also the other elements of the thin film. The thin films are explored with respect to the photoinduced changes of the reflectivity within the 400-65 nm spectra range using a 10 ns pulsed Nd:YAG with power densities varying up to 400 MW/cm2 and beam diameter within the 3-5 mm range. The observed processes are analyzed within a framework of trapping level conceptions for the Si NP. The possible application of the discovered materials as optical sensitive sensors is proposed. © 2013 Elsevier B.V.

Comparison of Brazilian plants used to treat gastritis on the oxidative burst of helicobacter pylori -stimulated neutrophil

Ten Brazilian medicinal plants used to treat gastritis and ulcers were carefully selected on the basis of ethnopharmacological importance and antiulcerogenic activity previously described. The antioxidant activity of the methanolic extracts was determined in analysis conditions that simulate a real biological activity on inhibition of the oxidative burst induced in neutrophils using Helicobacter pylori as activator, by a luminol-amplified chemiluminescence assay. The extracts, at low concentration (5 g/mL), exhibited a large variation in inhibitory effects of H. pylori-induced oxidative burst ranging from 48% inhibition to inactive, but all extracts, excluding Byrsonima intermedia, had inhibitory activity over 80% at the concentration of 100 g/mL. The total suppressive antioxidant capacity measured as the effective concentration, which represents the extract concentration producing 50% inhibition of the chemiluminescence induced by H. pylori, varies from 27.2 to 56.8 g/mL and was in the following order: Qualea parviflora > Qualea multiflora > Alchornea triplinervia > Qualea grandiflora > Anacardium humile > Davilla elliptica > Mouriri pusa > Byrsonima basiloba > Alchornea glandulosa > Byrsonima intermedia. The main groups of compounds in tested extracts are presented. Differences in the phytochemical profile, quantitatively and qualitatively, of these plants can explain and justify their protective effect on the gastric mucosa caused by the neutrophil-generated ROS that occurs when H. pylori displays its evasion mechanisms. © 2013 Cibele Bonacorsi et al.

Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory response by blocking NF-κB and MAPK activation in macrophages

Ethnopharmacological relevance Propolis is a bee product with numerous biological and pharmacological properties, such as immunomodulatory and anti-inflammatory activities. It has been used in folk medicine as a healthy drink and in food to improve health and prevent inflammatory diseases. However, little is known about its mechanism of action. Thus, the goal of this study was to verify the antioxidant activity and to explore the anti-inflammatory properties of propolis by addressing its intracellular mechanism of action. Caffeic acid was investigated as a possible compound responsible for propolis action. Materials and methods The antioxidant properties of propolis and caffeic acid were evaluated by using the 2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging method. To analyze the anti-inflammatory activity, Raw 264.7 macrophages were treated with different concentrations of propolis or caffeic acid, and nitric oxide (NO) production, a strong pro-inflammatory mediator, was evaluated by the Griess reaction. The concentrations of propolis and caffeic acid that inhibited NO production were evaluated on intracellular signaling pathways triggered during inflammation, namely p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK1/2), the transcription nuclear factor (NF)-κB and extracellular signal-regulated kinase (ERK1/2), through Western blot using specific antibodies. A possible effect of propolis on the cytotoxicity of hepatocytes was also evaluated, since this product can be used in human diets. Results Caffeic acid showed a higher antioxidant activity than propolis extract. Propolis and caffeic acid inhibited NO production in macrophages, at concentrations without cytotoxicity. Furthermore, both propolis and caffeic acid suppressed LPS-induced signaling pathways, namely p38 MAPK, JNK1/2 and NF-κB. ERK1/2 was not affected by propolis extract and caffeic acid. In addition, propolis and caffeic acid did not induce hepatotoxicity at concentrations with strong anti-inflammatory potential. Conclusions Propolis exerted an antioxidant and anti-inflammatory action and caffeic acid may be involved in its inhibitory effects on NO production and intracellular signaling cascades, suggesting its use as a natural source of safe anti-inflammatory drugs. © 2013 Elsevier B.V.

Improvement in clinical signs and cellular immunity of dogs with visceral leishmaniasis using the immunomodulator P-MAPA

This study investigated the immunotherapeutic potential of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-γ in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4+ and CD8+ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-γ. An increase in CD8+ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs. © 2013 Elsevier B.V.

Desempenho produtivo e resposta inflamatória de Pacu (Piaractus mesopotamicus) alimentado com ração suplementada com cromo trivalente

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Papel de RAGE e TLR4 na modulação da resposta imune inflamatoria em PBMC de pacientes diabéticos e não diabéticos: (estudo in vitro)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Modulação da proliferação, morte celular e expressão gênica de mediadores inflamatórios pela ativação de RAGE e TLR4 em células da resposta imune inata e adaptativa: papel das vias de sinalização p38 MAPK e NF-kB

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise citogenética clássica e molecular em Chelonoidis carbonaria e Phrynops geoffroanus (Testudines)

Pós-graduação em Genética - IBILCE

Mutagenicity of hydroxyurea in lymphocytes from patients with sickle cell disease

Hydroxyurea is commonly used in the treatment of myeloproliferative diseases and in patients with sickle cell disease (SCD). The use of this antineoplastic agent in patients with SCD is justified because of the drug's ability to increase fetal hemoglobin levels, thereby decreasing the severity of SCD. However, high doses or prolonged treatment with hydroxyurea can be cytotoxic or genotoxic for these patients, with an increased risk of developing acute leukemia. This danger can be avoided by monitoring the lymphocytes of patients treated with hydroxyurea. Cytogenetic tests are important endpoints for monitoring the physiological effects of physical and chemical agents, including drugs. In this work, we assessed the genotoxicity of hydroxyurea in short-term cultures of lymphocytes from SCD patients. Hydroxyurea was not cytotoxic or genotoxic at the concentrations tested in the G2 phase of the cell cycle. These results support the use of hydroxyurea in the treatment of SCD, although further work is necessary to understand the effects of this drug in vivo

Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 µg/l) of HgCl2 and CH3HgCl and incubated at 37ºC for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 µg/l alone, and at 1, 10, 100, and 1000 µg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.

Establishing the reference range for t lymphocytes subpopulations in adults and children from Brazil

Os valores de referências de linfócitos T existentes no Brasil são baseados em dados originados de outros países. Não existem dados locais da variação normal para estes parâmetros em adultos e crianças brasileiras. Avaliamos a variação normal encontrada em doadores de sangue de cinco grandes cidades brasileiras em diferentes regiões e em crianças residentes em Salvador e Rio de Janeiro. Todas as amostras foram processadas por citometria de fluxo. Os resultados foram analisados de acordo com região, gênero e estilo de vida dos doadores. Um total de 641 adultos (63% homens) e 280 crianças (58% meninos) participaram do estudo. Valores absolutos de CD3+ e CD4+ foram significantemente maiores no gênero feminino (adultos e crianças). Maiores valores de CD4+ em adultos foram associados com tabagismo, enquanto que maiores valores de CD8+ foram encontrados entre crianças do sexo feminino. Adultos das regiões sul e sudeste apresentaram maiores valores absolutos para todas as células T enquanto que adultos da região norte, apresentaram menores valores. Indivíduos residentes no nordeste e centro-oeste obtiveram contagens intermediárias para todas as populações de células T. Entretanto, estas diferenças entre as regiões, não demonstraram diferença estatística. No Brasil, gênero e tabagismo foram os principais determinantes para diferenças em valores de referências de linfócitos T.

Reference range for T lymphocytes populations in blood donors from two different regions in Brazil

ABSTRACT: This study defined the normal variation range for different subsets of T-lymphocyte cells count in two different Brazilian regions. We analysed the T-lymphocytes subpopulations (CD3+, CD4+, CD8+) in blood donors of two Brazilian cities, located in North (Belem, capital state of Para, indian background) and Northeast (Salvador, capital state od Bahia, African background) regions of Brazil. Results were compared according to gender, stress level (sleep time lower than 8 hours/day), smoking, and alcohol intake. Lymphocytes subpopulations were measured by flow cytometry. Five hundred twenty-six blood donors from two Brazilians cities participated in the study: 450 samples from Bahia and 76 samples from Pará. Most (60%) were men, 59% reported alcohol intake, 12% were smokers, and 80% slept at least 8 h/day. Donors from Bahia presented with significantly higher counts for all parameters, compared with Para. Women had higher lymphocytes levels, in both states, but only CD4+ cells count was significantly higher than men's values. Smokers had higher CD4+ counts, but sleep time had effect on lymphocytes levels only for Para's donors (higher CD3+ and CD4+ counts). That state had also, a higher proportion of donors reporting sleep time <8 h/day. The values for CD3, CD4 and CD8+ cells count were significantly higher in blood donors from Bahia than among those from Pará. Female gender, alcohol intake, stress level, and smoking were associated with higher lymphocyte counts. The use of a single reference range for normal lymphocytes count is not appropriate for a country with such diversity, like Brazil is.