Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to alpha 3 and alpha 5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, alpha 3 and alpha 5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-beta-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with alpha 3 and alpha 5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.Brazilian agencies: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Univ Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilFAPESP: 2011/22773-6FAPESP: 2012/11792-2Web of Scienc

Evaluation of Oxidative-Stress Pathway and Recovery of Sudden Sensorineural Hearing Loss

Introduction Although the pathogenesis of sudden sensorineural hearing loss (SSNHL) has been discussed in the literature, many unclear points remain. Several authors have hypothesized that oxidative stress plays a role in the pathogenesis of noise-related hearing loss, as well as in drug- and aging-related hearing loss. Reactive oxygen species (ROS) may contribute to the pathogenesis of SSNHL in a similar way as in cases of ototoxicity, noise-induced hearing loss and presbyacusis. Objective The aim of the present study was to find potential peripheral biomarkers to show the levels of oxidative stress in samples of peripheral blood collected from SSNHL patients with and withouth metabolic disease. Methods In total, 80 consecutive patients with SSNHL were evaluated in the otolaryngology emergency room and outpatient clinic of a tertiary hospital between May 2017 and May 2019. All patients underwent detailed anamnesis, physical examination, audiometry, magnetic resonance imaging (MRI) of the inner ears, and blood tests for serum lipids and plasma activity of thiobarbituric acid reactive species (TBARS). Results No significant effect of malondialdehyde (MDA) activity was observed regarding the hearing recovery of patients who developed SSNHL. Conclusion We did not observe a significant correlation between the concentration of TBARs in the peripheral blood or the presence of arterial hypertension and the severity of the initial hearing loss or the prognosis of hearing recovery in patients with SSNHL. The concentration of TBARs in the peripheral blood may not adequately represent the abnormalities that occur in the intracoclear environment

Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner

Paracoccidioides brasiliensis is one of the etiological agents of the human systemic mycosis paracoccidioidomycosis. Protease-activated receptors (PARs) are expressed in many cell types and comprise a family of G protein-coupled receptors (PAR-1, PAR-2, and PAR-4), which may be activated by proteases secreted by several pathogens. In the present study, we showed that the pathogenic fungus P. brasiliensis secretes components that promote interleukin (IL)-6 and IL-8 secretion by the lung epithelial cell line A549. Cytokine secretion was reduced by antagonistic peptides for PAR-1 and PAR-2, but not for PAR-4. P. brasiliensis proteases were isolated from fungal culture supernatants in a p-aminomethylbenzamidine-Sepharose column. The obtained fractions were tested for enzymatic activity against fluorescence resonance energy transfer (FRET) peptides derived from sequences that spanned the activation sites of human PARs. The eluted fraction, termed PbP, contained protease activities that were able to hydrolyze the FRET peptides. PbP also induced IL-6 and IL-8 secretion in A549 epithelial cells, which was reduced upon heat inactivation of PbP, incubation with antagonistic peptides for PAR-1 and PAR-2, and the protease inhibitors aprotinin, leupeptin, and E-64. Together, these results show for the first time that P. brasiliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion.Sao Paulo Research Foundation (FAPESP)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES AUX PE PNPD Grant)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Univ Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, Rua Botucatu,862 Ed Antonio CM Paiva 6 Andar, BR-04023062 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biophys, Escola Paulista Med, Rua Tres Maio,100 INFAR 1 Andar, BR-04044020 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, Rua Tres Maio,100 INFAR 5 Andar, BR-04044020 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biophys, Escola Paulista Med, Rua Pedro Toledo,699 7 Andar, BR-04039032 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, Rua Botucatu,862 Ed Antonio CM Paiva 8 Andar, BR-04023062 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, Rua Botucatu,862 Ed Antonio CM Paiva 6 Andar, BR-04023062 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biophys, Escola Paulista Med, Rua Tres Maio,100 INFAR 1 Andar, BR-04044020 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, Rua Tres Maio,100 INFAR 5 Andar, BR-04044020 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biophys, Escola Paulista Med, Rua Pedro Toledo,699 7 Andar, BR-04039032 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, Rua Botucatu,862 Ed Antonio CM Paiva 8 Andar, BR-04023062 Sao Paulo, SP, BrazilFAPESP: 2011/23096-8FAPESP: 2011/22773-6FAPESP: 2015/25652-6CAPES AUX PE PNPD Grant: 2386/2011CNPq: 303357/2012-6CNPq: 306163/2015-2Web of Scienc

Enterolobium contortisiliquum Trypsin Inhibitor (EcTI), a Plant Proteinase Inhibitor, Decreases in Vitro Cell Adhesion and Invasion by Inhibition of Src Protein-Focal Adhesion Kinase (FAK) Signaling Pathways

Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. the effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin beta 1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Dept Biochem & Mol Biol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem & Mol Biol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04044020 São Paulo, BrazilFAPESP: 2009/53766-5Web of Scienc