The effects of fluconazole and cytokines on human mononuclear cells

Candida infections are common infections and fluconazole is one of the most frequently administered antifungal agents in their treatment. The resistance developed against antifungal agents has necessitated the improvement of new treatments. This study focuses on the investigation of the effect of fluconazole and cytokines such as interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF) on chemokine production and anticandidal activity of human monocytes. In the study it was observed that GM-CSF caused an increase in candidacidal activity of monocytes. Anticandidal activity of GM-CSF + IFN-gamma combination was not found to be more effective than GM-CSF or IFN-gamma alone. The presence of cytokine and fluconazole caused an increase in the levels of CCL3 and CCL4 chemokines. Accordingly, it was considered that chemokines could contribute to the efficacy of fluconazole in C. albicans infections. Besides, in order to strengthen the immune system some cytokines might be used in addition to antifungal agents for the treatment.

Efeitos da saliva de lutzomyia intermédia sobre monócitos humanos / Effects of the saliva of intermediate lutzomyia on human monocytes

A saliva dos flebotomíneos contém moléculas farmacológicas potentes e complexas que, quando inoculadas na pele do vertebrado, são capazes de modular os sistemas hemostático, inflamatório e imune do hospedeiro. No presente estudo, nós avaliamos os efeitos do conteúdo da glândula salivar de Lutzomyia intermedia, o vetor natural da Leishmania braziliensis, na produção de citocinas e na expressão de moléculas de superfície em monócitos estimulados com LPS, infectados ou não por L. braziliensis. Avaliamos também a carga parasitária em células infectadas e expressão de quimiocinas em monócitos pré-sensibilizados com a saliva de L. intermedia e com a saliva de L. longipalpis. Monócitos selecionados de células mononucleares do sangue periférico (CMSP) de doadores saudáveis foram pré-sensibilizados com SGS de L. intermedia, estimulados com LPS e, em seguida, infectados ou não com L. braziliensis. A produção de citocinas foi avaliada por ELlSA em sobrenadante de cultura; expressão de moléculas de superfície foi determinada por citometria de fluxo e a expressão de quimiocinas foi analisada por PCR em Tempo Real em ensaios de Quantificação Relativa (QR). Monócitos humanos pré-tratados com SGS de L. intermedia mostraram uma redução significante na produção de IL-10 e um aumento significante na expressão de CD86. A infecção com L. braziliensis aumentou significativamente a produção de TNF-a, porém não alterou a expressão de CD86, nem a taxa de infecção em monócitos pré-sensibilizados com SGS. A expressão de IL-8, MIP-1a, MIP-1f3 e Rantes aumentou após estímulo de monócitos humanos com SGS de L. intermedia e L. longipalpis. Nossos dados indicam que a saliva de L. intermedia é capaz de modificar a produção de citocinas, a expressão de moléculas de superfície e a expressão de quimiocinas em monócitos humanos e estas alterações podem afetar a evolução da leishmaniose em infecções naturais.

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: reversal by holotransferrin not by apotransferrin

Os mecanismos utilizados pelo Paracoccidioides brasiliensis para sobreviver em células fagocitárias ainda não estão elucidados. O metabolismo celular férrico é muito importante para o crescimento de inúmeros patógenos intracelulares cuja capacidade de se multiplicarem em fagócitos mononucleares é dependente da disponibilidade intracelular do íon ferro. Assim, o objetivo deste trabalho foi investigar o papel do ferro intracelular sobre a capacidade do P. brasiliensis sobreviver em monócitos humanos. O tratamento de monócitos com deferoxamina, uma droga quelante, diminuiu a sobrevivência de leveduras do fungo de forma dose-dependente. O efeito inibidor da deferoxamina sobre a sobrevivência do P. brasiliensis foi revertido por transferrina saturada com ferro (holotransferrina) mas não por transferrina insaturada (apotransferrina). Estes resultados sugerem que a sobrevivência do P. brasiliensis em monócitos humanos é dependente do íon ferro.

Leptospira interrogans activation of peripheral blood monocyte glycolipoprotein demonstrated in whole blood by the release of IL-6

Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80°C until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

Mycobacterium bovis BCG but not Mycobacterium leprae induces TNF-alpha secretion in human monocytic THP-1 cells

In this study, we compared the level of TNF-alpha secretion induced in monocytic THP-1 cells after phagocytosis of Mycobacterium leprae, the causative agent of leprosy, and M. bovis BCG, an attenuated strain used as a vaccine against leprosy and tuberculosis. The presence of M. leprae and BCG was observed in more than 80 percent of the cells after 24 h of exposure. However, BCG but not M. leprae was able to induce TNF-alpha secretion in these cells. Moreover, THP-1 cells treated simultaneously with BCG and M. leprae secreted lower levels of TNF-alpha compared to cells incubated with BCG alone. M. leprae was able, however, to induce TNF-alpha secretion both in blood-derived monocytes as well as in THP-1 cells pretreated with phorbol myristate acetate. The inclusion of streptomycin in our cultures, together with the fact that the use of both gamma-irradiated M. leprae and heat-killed BCG gave similar results, indicate that the differences observed were not due to differences in viability but in intrinsic properties between M. leprae and BCG. These data suggest that the capacity of M. leprae to induce TNF-alpha is dependent on the stage of cell maturation and emphasize the potential of this model to explore differences in the effects triggered by vaccine strain versus pathogenic species of mycobacteria on the host cell physiology and metabolism

Comparison of methods for HIV isolation from monocytes and lymphocytes of seropositive asymptomatic hemophiliacs and homosexuals

1. Since monocyte-macrophages have been recognized as HIV targets in addition to CD4+ T-lymphocytes, we have evaluated HIV infection of purified peripheral blood mononuclear cell fractions obtained from 10 seropositive asymptomatic hemophiliacs and compared with that of 10 assymptomatic homosexual patients. 2. HIV was isolated more frequently from the lymphocytes than the monocytes of both groups of patients. 3. HIV preferential replicated in phytohemagglutinin-stimulated lymphocytes compared with growth factor-treated monocytes. Monocytes did not preferentially harbour HIV in either group

Application of peroxidase-antiperoxidase (PAP) staining for detection and localization of dengue-2 viral antigen. II. Observations for the antibody enhancement activity in human monocytes.

Peroxidase-antiperoxidase (PAP) staining was applied to measure the antibody enhancement activity in human monocytes. Increasing in number of infected cells can be seen with increasing of staining intensity of the cells by ordinary light microscope. Shifting of the optimum enhancement activity was found in previously tritiated antiserum indicated that for titration of antibody enhancement activity several dilutions of antiserum should be included in each experiment. Validity of the PAP method was made by the comparison of the results with Infectious Center Assay (ICA). With this technique, titration for antibody enhancement for dengue virus infection can be done with non-expensive equipment and can be kept for comparison for months.

Early detection of Rickettsia tsutsugamushi in peripheral monocyte cultures derived from experimentally infected monkeys and dogs.

Rickettsia tsutsugamushi organisms were identified in Giemsa and fluorescent antibody stained monocyte cell cultures derived from experimentally infected monkeys and dogs. The identification of organisms in monocyte cell cultures compared favorably with the standard technique of mouse inoculation.