Paracoccidioides brasiliensis interferes on dendritic cells maturation by inhibiting PGE(2) production

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE2, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE2 inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-alpha. Assays using exogenous PGE2 confirmed an association between PGE2 inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Effect of physical training on cytokine expression in CD4+ T lymphocytes in subjects with stable COPD

Introduction: Although evidence suggests that physical exercise reduces systemic inflammation, at the plasma level, there are still contradictions in chronic obstructive pulmonary disease (COPD). In this sense, analysis of intracellular cytokines could clear off the effect of physical exercise on the inflammatory profile of these subjects. Aim: The aim was to evaluate the effect of physical training on cytokine expression in CD4+ T lymphocytes from subjects with COPD. Methods: This is a randomized controlled trial. Subjects with stable COPD were grouped into two groups, exercise and control. In total, 23 subjects with stable COPD were evaluated, of which 15 underwent aerobic strength training [physical exercise group (PEG)] and 8 underwent breathing exercises [respiratory physiotherapy group (RPG)]. Intracellular cytokines [interleukin (IL)-8, IL-13, IL-17, IL-6, IL-2, IL-10, and tumor necrosis factor alpha (TNF-α)] from CD4+ T lymphocytes were analyzed from peripheral blood through flow cytometry, before and after 8 weeks of intervention. Results: The PEG and RPG groups had a mean age of 68 ± 5.96 and 72.25 ± 6.86 years and predicted forced expiratory volume in the first second (FEV 1 ) of 58.6 ± 15.99% and 39.75 ± 10.39%, respectively. It was possible to detect a significant reduction in IL-8 ( p  = 0.0125) and an increase in IL-13 ( p  = 0.0014) and an increase in TNF-α ( p  < 0.001) in both groups. Conclusion: Eight weeks of physical training, both peripheral and respiratory, were able to reduce concentrations of IL-8 and to increase IL-13, and TNF-α in CD4+ T lymphocytes in subjects with stable COPD. The findings reinforce the benefits of interventions in subjects with COPD, revealing data not previously investigated

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.O objetivo deste estudo foi isolar, cultivar e caracterizar as células mesenquimais multipotentes estromais derivadas do sangue periférico (SpCTMs) equino. O sangue periférico foi coletado, seguido do isolamento das células mononucleadas utilizando o reagente de gradiente de densidade e o cultivo das células aderentes. Os anticorpos monoclonais mouse anti-horse CD13, mouse anti-horse CD44 e mouse anti-rat CD90 foram utilizados para a caracterização imunofenotípica da superfície das SpCTMs. Estas células também foram cultivadas utilizando meio de cultura específico para a diferenciação adipogênica e condrogênica. Não houve expressão do marcador CD13, mas os marcadores CD44 e CD90 foram expressos em todas as passagens testadas. Após 14 dias da diferenciação das células em adipócitos, gotículas de lipídeos foram observados através da coloração com Oil Red O. Vinte e um dias após a diferenciação condrogênica, as células foram coradas com o Alcian Blue. Embora a técnica de isolamento destas células necessite ser otimizada, o presente estudo demonstra a caracterização parcial das SpCTMs, classificando-as como um tipo de células progenitoras promissoras para o uso na terapia celular em equinos.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

TNF-α production by DCs challenged with Pb18 or Pb265 or activated by LPS for 48 h, and measured by ELISA.

<p>The results are expressed as mean ± SD of independent experiments performed with cells obtained from 4 subjects. Statistically significant differences between groups are indicated: *p< 0.05.</p

Effect of exogenous PGE₂ on percentage of cells, mean of fluorescence intensity (MFI) relative to the expression of CD40, CD80 (A) CD83, CD86 (B), HLA-DR, CXCR4 (C) CCR5, and CCR7 (D) by DCs after challenge or not with Pb18 and Pb265.

<p>The results are expressed in mean ± SD of experiments performed with cells obtained from 4 subjects. Statistically significant differences between groups are indicated: *p< 0.05 versus without PGE₂.</p

Involvement of MR, TLR2, Dectin-1 and DC-SIGN on PGE₂ production inhibition induced by <i>P</i>. <i>brasiliensis</i> in human DCs.

<p>Cells were incubated with anti-MR, anti-TLR2, anti-Dectin-1 and/or anti-DC-SIGN monoclonal antibodies for 1 h, challenged with Pb18 or Pb265 (DCs/yeast ratio 5:1) for 4 and 24 h, and evaluated for PGE₂ production by ELISA. The results are expressed in mean ± SD of independent experiments performed with cells obtained from 4 subjects. Statistically significant differences between groups are indicated: *p< 0.05 versus control DCs and other available receptors.</p

PGE₂ production by DCs activated with LPS or challenged with Pb18 or Pb265 (DCs/Pb ratio 5:1) for different periods.

<p>The results are expressed in mean ± SD of experiments performed with cells from 5 subjects. Statistically significant differences between groups in the same period are indicated: *p< 0.05; ** p< 0.01; ***p<0.001 versus control DC; o p< 0.05 versus Pb18.</p

Effect of exogenous PGE₂ on TNF-α production by DCs challenged with Pb18 or Pb265 by 48 h and evaluated by ELISA.

<p>The results are expressed in mean ± SD of experiments performed with cells obtained from 4 subjects. Statistically significant differences between groups are indicated: *p< 0.05 versus without PGE₂.</p