Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

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

Fungicidal effect of photodynamic therapy against fluconazole-resistant Candida albicans and Candida glabrata

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

Experimental paracoccidioidomycosis of the Syrian hamster: fungicidal activity and production of inflammatory cytokines by macrophages

Phagocytic cells play an important role in nonspecific resistance to fungal infection by mediating an inflammatory response and by a direct fungicidal action. In this study, the functional activity of peritoneal macrophages obtained from hamsters experimentally infected with strain Pb18 of Paracoccidioides brasiliensis was evaluated during 16 weeks of infection. The results showed that macrophages had a higher spreading ability associated with increased production of tumor necrosis factor alpha (TNF-alpha) and enhanced fungicidal activity during the early periods of infection. TNF-alpha levels remained elevated during all periods studied, while low levels of interleukin-1 beta (IL-1 beta) were produced during the infection. A necrotic area with dead fungi was observed at the inoculation site and the infection disseminated only to liver and lymph nodes in a few animals. These results suggest that during the early stages of infection with P. brasiliensis, macrophage activation by the high levels of TNF-alpha limited fungal dissemination. In contrast, in the later stages of infection, high levels of TNF-alpha were observed while the fungicidal activity of macrophages was lower and the animals presented loss of vitality resulting in their death. These observations suggest a complex role of TNF-alpha in experimental paracoccidioidomycosis of Syrian hamsters, involving not only resistance but also pathogenesis.

Effects of propolis from Brazil and Bulgaria on fungicidal activity of macrophages against Paracoccidioides brasiliensis

Paracoccidioidomycosis is the most important systemic mycosis in Latin America. Its etiological agent, Paracoccidoides brasiliensis, affects individuals living in endemic areas through inhalation of airborne conidia or mycelial fragments. The disease may affect different organs and systems, with multiple clinical features, with cell-mediated immunity playing a significant role in host defence. Peritoneal macrophages from BALB/c mice were stimulated with Brazilian or Bulgarian propolis and subsequently challenged with P, brasiliensis. Data suggest an increase in the fungicidal activity of macrophages by propolis stimulation, independently from its geographic origin, (C) 2002 Elsevier B.V. Ireland Ltd. All rights reserved.

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

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

Comparison of the photodynamic fungicidal efficacy of methylene blue, toluidine blue, malachite green and low-power laser irradiation alone against Candida albicans

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

Role of Paracoccidioides brasiliensis cell wall fraction containing beta-glucan in tumor necrosis factor-alpha production by human monocytes: correlation with fungicidal activity

The polysaccharide fraction of Paracoccidioides brasiliensis mycelial cell wall (F1 fraction), the active component of which is composed of beta-glucan, was investigated in regard to the activation of human monocytes for fungal killing. The cells were primed with interferon-gamma (IFN-gamma) or F1 (100 and 200 mug ml(-1)) or F1 (100 and 200 mug ml(-1)) plus IFN-gamma for 24 h and then evaluated for H2O2 release. In other experiments, the cells were pretreated with the same stimuli, challenged with a virulent strain of P. brasiliensis and evaluated for fungicidal activity and levels of tumor necrosis factor (TNF-alpha) in the supernatants. F1 increased the levels of H2O2 in a similar manner to IFN-gamma. However, a synergistic effect between these two activators was not detected. on the contrary, a significant fungicidal activity was only obtained after priming with IFN-gamma plus F1. This higher activity was associated with high levels of TNF-alpha in the supernatants of the cocultures. Overall, P. brasiliensis F1 fraction induced human monocytes to release relatively high levels of TNF-alpha, which, in combination with IFN-gamma, is responsible for the activation of human monocytes for effective killing of P. brasiliensis.

Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient, monocytes activated with IFN-gamma (1 000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover,,patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H2O2 in vitro. Unlike the results obtained with Ph 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN-gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All fights reserved.

Fungicidal activity of human monocyte-derived multinucleated giant cells induced in vitro by Paracoccidioides brasiliensis antigen

Multinucleated giant cells (MGC) are characteristic cells in granulomatous disorders such as paracoccidioidomycosis (PCM) and also are formed in vitro from peripheral blood mononuclear cells by several stimuli. In this study, the authors investigated in vitro formation of MGC derived from monocytes of healthy individuals, stimulated with Paracoccidioides brasiliensis antigen (PbAg), compared with other stimuli such as IFN-gamma and supernatant of Con-A-stimulated peripheral blood mononuclear cells (CM-ConA). Besides, the fungicidal activity of monocytes and monocyte-derived MGC challenged with P. brasiliensis were compared, at a ratio of one fungus per 50 monocytes. Results demonstrated that PbAg, IFN-gamma, and CM-ConA stimuli were able to induce MGC generation, with fusion indices significantly higher than control cultures. Striking results were observed when MGC induced by PbAg and IFN-gamma presented higher fungicidal activity than monocytes, submitted to the same stimuli, showing a better capacity of these cells to kill P. brasiliensis. In summary, the results suggest that PbAg is able to induce MGC generation, and these cells presented higher fungicidal activity against P. brasiliensis than monocytes.

Effect of prostaglandins on the production of H2O2 and cytokines that modulate the fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Human monocytes lack fungicidal activity against high virulent strains of Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, even after IFN-γ activation. However, monocytes treated with indomethacin (INDO) or INDO plus IFN-γ effectively killed this fungus, suggesting an inhibitory role of prostaglandins in this process. Thus, the purpose of this work was to test if this regulatory effect of prostaglandin was associated with alterations on H2O2 production and/or on modulatory cytokines levels, such as TNF-α, IL-10, and IL-6. Peripheral blood monocytes obtained from 10 healthy donors were incubated for 18 hours in the presence or absence of IFN-γ, INDO, or IFN-γ plus INDO, and further challenged with a high virulent strain of P. brasiliensis (Pb18) for 4 hours. Then, the monocytes cultures were evaluated for H2O2 release and fungicidal activity calculated by counting the colony forming units after plating. Moreover, on supernatants of the same cultures, TNF-α, IL-10, IL-6, and PGE2 concentrations were evaluated by ELISA. Monocytes treated with INDO or INDO plus IFN-γ presented higher fungicidal activity associated with the release of higher levels of H2O2 and TNF-α, but lesser levels of PGE2, when compared to nontreated cells. However, the levels of IL-10 and IL-6 were similar between treated and nontreated cells. The results suggest that human monocytes when challenged with high virulent strains of P. brasiliensis produce prostaglandins that inhibit the fungicidal activity of these cells by reducing H2O2 and TNF-α levels.

The immunomodulatory effect of propolis on receptors expression, cytokine production and fungicidal activity of human monocytes

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

Antimicrobial and Cytotoxic Activity of Fruit Extract from Syzygium cumini (L.) Skeels

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Application of an HPLC method for analysis of propolis extract

Propolis obtained from honeybee hives has been widely used in medicine, cosmetics, and industry due to its versatile biological activities (antioxidant, antimicrobial, fungicidal, antiviral, antiulcer, immunostimulating, and cytostatic). These activities are mainly attributed to the presence of flavonoids in propolis, which points out the interest in quantifying these constituents in propolis preparations, as well as validation of analytical methodologies. High-performance liquid chromatography (HPLC) methods have been reported to quantify isolated flavonoids or these compounds in complex biological matrices, such as herbal raw materials and extractive preparations. An efficient, precise, and reliable method was developed for quantification of propolis extractive solution using HPLC with UV detection. The chromatograms were obtained from various gradient elution systems (GES) tested in order to establish the ideal conditions for the analysis of propolis extractive solution, using methanol and water: acetonitrile (97.5 : 2.5, v/v) as mobile phase. Gradient reversed phase chromatography was performed using a stainless steel column (250 x 4.6 mm i.d., 5 mum) filled with Chromsep RP 18 (Varian), column temperature at 30.0 +/- 0.1degreesC and detection at 310 nm. The main validation parameters of the method were also determined. The method showed linearity for chrysin in the range 0.24-2.4 mug mL(-1) with good correlation coefficients (0.9975). Precision and accuracy were determined. The obtained results demonstrate the efficiency of the proposed method. The analytical procedure is reliable and offers advantages in terms of speed and cost of reagents.

Modulatory effect of prostaglandins on human monocyte activation for killing of high- and low-virulence strains of Paracoccidioides brasiliensis

The effect of indomethacin (Indo), a cyclo-oxygenase inhibitor, on the monocyte-mediated killing of a low-(Pb265) and a high-(Pb18) virulence strain of Paracoccidioides brasiliensis was examined. The Pb18 strain was not killed by either non-activated or interferon-gamma (IFN-gamma)-activated human monocytes but these cells did show fungicidal activity if pretreated with Indo. In contrast with IFN-gamma tumour necrosis factor-alpha (TNF-alpha) was very effective at stimulating the fungicidal activity of monocytes. While the low-virulence strain, Pb265, could not be killed by monocytes, cells preincubated with IFN-gamma demonstrated fungicidal activity. The killing of this strain was also induced by pretreatment of monocytes with Indo. The results suggest a negative role for prostaglandins, which are synthesized via the cyclo-oxygenase pathway, in the regulation of monocyte-mediated killing of virulent and avirulent strains of P. brasiliensis and that TNF-alpha generation during the fungus-monocyte interaction is more important in the killing of Pb265 than Pb18.