Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.A01Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Antifungal and antitumor models of bioactive protective peptides

Peptides are remarkably reactive molecules produced by a great variety of species and able to display a number of functions in uni-and multicellular organisms as mediators, agonists and regulating substances. Some of them exert cytotoxic effects on cells other than those that produced them, and may have a role in controlling subpopulations and protecting certain species or cell types. Presently, we focus on antifungal and antitumor peptides and discuss a few models in which specific sequences and structures exerted direct inhibitory effects or stimulated a protective immune response. The killer peptide, deduced from an antiidiotypic antibody, with several antimicrobial activities and other Ig-derived peptides with cytotoxic activities including antitumor effects, are models studied in vitro and in vivo. Peptide 10 from gp43 of P. brasiliensis (P10) and the vaccine perspective against paracoccidioidomycosis is another topic illustrating the protective effect in vivo against a pathogenic fungus. The cationic antimicrobial peptides with antitumor activities are mostly reviewed here. Local treatment of murine melanoma by the peptide gomesin is another model studied at the Experimental Oncology Unit of UNIFESP.Peptídeos são moléculas particularmente reativas produzidas por uma grande variedade de espécies, aptos a exercer um número de funções em organismos uni-e multicelulares como mediadores, agonistas e substâncias regulatórias. Alguns deles exercem efeitos citotóxicos em células outras das que os produzem, e podem ter um papel controlando subpopulações e protegendo certas espécies ou tipos celulares. No presente, focalizamos peptídeos antifúngicos e antitumorais e discutimos alguns modelos nos quais seqüências específicas e estruturas exercem efeitos inibitórios diretos ou estimulam uma resposta imune protetora. O peptídeo letal (killer), deduzido de um anticorpo anti-idiotípico, com várias atividades antimicrobianas bem como outros peptídeos derivados de imunoglobulinas com atividades citotóxicas incluindo efeitos antitumorais são modelos estudados in vitro e in vivo. O peptídeo P10 da gp43 de P. brasiliensis e a perspectiva de vacina contra a paracoccidioidomicose é outro tópico ilustrando o efeito protetor in vivo contra um fungo patogênico. Peptídeos antimicrobianos catiônicos com atividades antitumorais são os principais revistos aqui. O tratamento local do melanoma murino com o peptídeo gomesina é outro modelo estudado na Unidade de Oncologia Experimental (UNONEX) da UNIFESP.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo (UNIFESP) Departamento de Microbiologia, Imunologia e Parasitologia Unidade de Oncologia ExperimentalUniversidade de São Paulo Instituto de Ciências Biomédicas Departamento de MicrobiologiaUNIFESP, Depto. de Microbiologia, Imunologia e Parasitologia Unidade de Oncologia ExperimentalSciEL

Characterization of an ecto-ATPase activity in Cryptococcus neoformans

Cryptococcus neoformans is the causative agent of pulmonary cryptococcosis and cryptococcal meningoencephalitis, which are major clinical manifestations in immunosuppressed patients. in the present study, a surface ATPase (ecto-ATPase) was identified in C. neoformans yeast cells. Intact yeasts hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 29.36 +/- 3.36 nmol Pi/h x 10(8) cells. in the presence of 5 mM MgCl2, this activity was enhanced around 70 times, and an apparent K-m for Mg-ATP corresponding to 0.61 mM was determined. Inhibitors of phosphatases, mitochondrial Mg2+-ATPases, V-ATPases, Na+-ATPases or P-ATPases had no effect on the cryptococcal ATPase, but extracellular impermeant compounds reduced enzyme activity in living cells. ATP was the best substrate for the cryptococcal ecto-enzyme, but it also efficiently hydrolyzed inosine 5'-triphosphate (ITP), cytidine 5'-triphosphate (CTP), guanosine 5'-triphosphate (GTP) and uridine-5'-triphosphate (UTP). in the presence of ATP, C. neoformans became less susceptible to the antifungal action of fluconazole. Our results are indicative of the occurrence of a C. neoformans ectoATPase that may have a role in fungal physiology. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.Univ Fed Rio de Janeiro, Inst Bioquim Med, CCS, BR-21541590 Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Microbiol Prof Paulo Goes, CCS, BR-21541590 Rio de Janeiro, BrazilUNIFESP, Disciplina Biol Celular, São Paulo, BrazilUNIFESP, Disciplina Biol Celular, São Paulo, BrazilWeb of Scienc

DNA vaccine encoding peptide P10 against experimental paracoccidioidomycosis induces long-term protection in presence of regulatory T cells

Paracoccidioidomycosis is a granulomatous systemic mycosis endemic in Brazil and other Latin America countries. A DNA vaccine encoding the inununoprotective peptide 10 (P10) significantly reduced the fungal burden in mice when given prior to or after intratracheal challenge with Paracoccidioides brasiliensis. Presently, the generation/expansion of CD4(+) CD44(hi) memory T cells as well as Foxp3(+) Treg cells in mice immunized with the DNA vaccine (pcDNA3-P10) before and after infection with P. brasiliensis was investigated. Memory CD4(+) CD44(hi) T cells simultaneously with Foxp3(+) Treg cells increased in the spleens and lungs of pcDNA3-P10 immunized mice on day 0, 30, 60 and 120 postinfection. Histopathology of the lung tissue showed minimal inflammation in immunized mice compared with the unimmunized group, suggesting a role for regulatory T cells in controlling the immunopathology. the DNA vaccine shows that the repeated immunization generates memory cells and regulatory T cells that replace the initially protective pro-inflammatory T cells conferring a long term protection while preserving the integrity of the infected tissue. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ São Paulo, Dept Microbiol, Inst Biomed Sci, BR-05508900 São Paulo, BrazilAlbert Einstein Coll Med, Dept Med, Bronx, NY 10467 USAAlbert Einstein Coll Med, Dept Microbiol & Immunol, Bronx, NY 10467 USAUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniv São Paulo, Lab Med Mycol IMT SP LIM53, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFAPESP: 11/17267-4FAPESP: 09/15823-7FAPESP: 10/51423-0Web of Scienc

A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems

The Wilms tumor protein 1 (WT1) transcription factor has been associated in malignant melanoma with cell survival and metastasis, thus emerging as a candidate for targeted therapy. A lysine-arginine rich peptide, WT1-pTj, derived from the ZF domain of WT1 was evaluated as an antitumor agent against A2058 human melanoma cells and B16F10-Nex2 syngeneic murine melanoma. Peptide WT1-pTj quickly penetrated human melanoma cells and induced senescence, recognized by increased SA-beta-galactosidase activity, enhanced transcriptional activity of p53, and induction of the cell cycle inhibitors p21 and p27. Moreover, the peptide bound to p53 and competed with WT1 protein for binding to p53. WT1-pTj treatment led to sustained cell growth suppression, abrogation of clonogenicity and G2/M cell cycle arrest. Notably, in vivo studies showed that WT1-pTj inhibited both the metastases and subcutaneous growth of murine melanoma in syngeneic mice, and prolonged the survival of nude mice challenged with human melanoma cells. the 27-amino acid cell-penetrating WT1-derived peptide, depends on C-3 and H-16 for effective antimelanoma activity, inhibits proliferation of WT1-expressing human tumor cell lines, and may have an effective role in the treatment of WT1-expressing malignancies. (C) 2014 the Authors. Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, UNIFESP, Expt Oncol Unit UNONEX, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, UNIFESP, Expt Oncol Unit UNONEX, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilFAPESP: 2010/51423-0FAPESP: 2012/19476-2Web of Scienc

Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.Universidade de São Paulo Faculdade de Medicina Laboratório de Micologia Médica - LIM53Albert Einstein College of Medicine Departments of Medicine and Microbiology and ImmunologyUniversidade Federal de São Paulo (UNIFESP) Departamento de Microbiologia, Imunologia e ParasitologiaUNIFESP, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

Paracoccidioidomycosis vaccine

Paracoccidioidomycosis is a granulomatous pulmonary infection that is generally controlled by chemotherapy. the efficacy of treatment, however, is limited by the status of the host immune response. the inhibition of a Th-2 immunity or the stimulation of Th-1 cytokines generally increases the efficacy of antifungal drugs.(1) This has been achieved by immunization with an internal peptide of the major diagnostic antigen gp43 of Paracoccidioides brasiliensis. Peptide 10 (QTLIAIHTLAIRYAN) elicits an IFN-gamma rich Th-1 immune response that protects against experimental intratracheal infection by this fungus. the combination of chemotherapy with P10 immunization showed additive protective effect even after 30 d of infection or in anergic mice, rendering in general, increased production of IL-12 and IFN-gamma and reduction of IL-4 and IL-10. Immunotherapy with P10 even in the absence of simultaneous chemotherapy has been effective using various protocols, adjuvants, nanoparticles, P10-primed dendritic cells, and especially a combination of plasmids encoding the P10 minigene and IL-12. Gene therapy, in a long-term infection protocol succeeded in the virtual elimination of the fungus, preserving the lung structure, free from immunopathological side effects.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Div Cell Biol, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Dept Microbiol, São Paulo, BrazilUniv São Paulo, Lab Med Mycol IMT SP LIM53, São Paulo, BrazilUniversidade Federal de São Paulo, Div Cell Biol, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFAPESP: 2010/51423-0Web of Scienc

Identification of antigenic polypeptides of Paracoccidioides brasiliensis by immunoblotting

ESCOLA PAULISTA MED SCH,DISCIPLINA MICOL,RUA BOTUCATU 862,8 ANDAR,BR-04023 SAO PAULO,SP,BRAZILESCOLA PAULISTA MED SCH,DISCIPLINA MICOL,RUA BOTUCATU 862,8 ANDAR,BR-04023 SAO PAULO,SP,BRAZILWeb of Scienc