Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity.

Monoclonal antibodies (mAbs) have been a valuable tool to elucidate several biological processes, such as stem cell differentiation and cancer, and contributed to virtually all areas of biomedical sciences. Yet, it remains a challenge to obtain mAbs specific to poorly expressed epitopes, or to epitopes that are actually involved in important biological phenomena, such as cell differentiation and metastasis. Drug-induced subtractive immunization, and recently the multiple tolerization subtractive immunization (MTSI) technique, reported by our group, have the potential to level up the field, as they direct the host´s immune response towards these epitopes. However, due to cyclophosphamide (CY) treatment, high mice mortality can be observed, and only a few data are available on how these techniques affect the immune system of mice. Tolerogen and immunogen cells, RWPE-1 and PC-3 cells, respectively, were individually seeded at 2 × 104 cells/cm2, and then adjusted to 2 × 106 cells per mouse before immunization, which was conducted in a subtractive approach (MTSI) with CY. Immunosuppression of mice was recorded via total white blood counting, as well the reactivity of circulating polyclonal antibodies (pAbs). General parameters, including weight, physical appearance, and behavior on mice subjected to three different concentrations of CY were recorded. mAbs were obtained using classical hybridoma techniques, using the spleen of immunized mice. After purification, antibodies were characterized by Western blotting, and Indirect immunofluorescence. In conclusion, all CY dosage were efficient in creating an immunosuppression state, but only the 100 mg/kg body weight was feasible, as the others resulted in extensive mice mortality. pAbs obtained in the peripheral blood of mice showed more reactivity towards tumor cells. MAbs 2-7A50 and 2-5C11 recognized antigens from tumor cells, but not from their non-tumor counterparts, as shown in western blotting and immunofluorescence assays. MTSI technique was successful in generating mAbs that recognize tumor-specific antigens.

Gentamicin encapsulated within a biopolymer for the treatment of Staphylococcus aureus and Escherichia coli infected skin ulcers.

Skin wound infection requires carefully long-term treatment with an immense financial burden to healthcare systems worldwide. Various strategies such as drug delivery systems using polymer matrix from natural source have been used to enhance wound healing. Natural rubber latex (NRL) from Hevea brasiliensis has shown angiogenic and tissue repair properties. Gentamicin sulfate (GS) is a broad-spectrum antibiotic which inhibits the growth of a wide variety of microorganisms and, because of this, it has also been applied topically for treatment of local infections. The aim of this study was to develop a GS release system using NRL as matrix for Staphylococcus aureus and Escherichia coli infected skin ulcers treatment, without changing drug antibiotic properties. The matrix did not change the GS antimicrobial activity against S. aureus and E. coli strains. Moreover, the NRL-GS biomembrane did not exhibit hemolytic activity, being non-toxic to red blood cells. The eluates of NRL-GS biomembranes and GS solutions did not significantly reduce the survival of Caenorhabditis elegans worms for 24 h at any of the tested concentrations. Thus, these results emphasize that the NRL-GS biomembrane proved to be a promising biomaterial for future studies on the development of dressings for topical uses, inexpensive and practicable, keeping drug antibiotic properties against pathogens and to reduce the side effects.

Selective photoinactivation of Histoplasma capsulatum by water-soluble derivatives chalcones.

Histoplasmosis is a respiratory and systemic disease caused by the dimorphic fungus Histoplasma capsulatum. The clinical features may vary from asymptomatic infections to disseminated severe form depending of patient immunity. The treatment of histoplasmosis can be performed with itraconazole, fluconazole, and in the disseminated forms is used amphotericin B. However, the critical side effects of amphotericin B, the cases of itraconazole therapy failure and the appearance of fluconozole-resistant strains makes necessary the search of new strategies to treat this disease. Antimicrobial photodynamic therapy (aPDT) seems to be a potential candidate once have been show efficacy to inhibit others dimorphic fungi. Although the photosensitizer (PS) chalcone aggregates in biological medium, it has antifungal activity and show a high quantum yield of ROS formation. So, the aim of this study was to obtain the experimental parameters to achieve an acceptable selective chalcone water-soluble derivatives photoinactivation of H. capsulatum comparing with fibroblastic and keratinocytes cells which are the constituents of some potential host tissues. Yeast and cells were incubated with the same chalchones concentrations and short incubation time followed by irradiation with equal dose of light. The best conditions to kill H. capsulatum selectively were very low photosensitizers concentration (1.95µgmL-1) incubated by 15min and irradiated with LED 450nm with 24Jcm-2. Key words: chalcone, Histoplasma capsulatum, aPDT, selectivity.

Alkaloids as inhibitors of malate synthase from Paracoccidioides spp.: receptor-ligand interaction-based virtual screening and molecular docking studies, antifungal activity, and the adhesion process.

Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify ß-carboline moiety as a standard key structure. The compounds with ß-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of ß-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four ß-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated.

Anti dermatophytic therapy: prospects for the discovery of new drugs from natural products

Millions of people and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which only infect keratinized structures. With the appearance of AIDS, the incidence of dermatophytosis has increased. Current drug therapy used for these infections is often toxic, long-term, and expensive and has limited effectiveness; therefore, the discovery of new anti dermatophytic compounds is a necessity. Natural products have been the most productive source for new drug development. This paper provides a brief review of the current literature regarding the presence of dermatophytes in immunocompromised patients, drug resistance to conventional treatments and new anti dermatophytic treatments.

Anti dermatophytic therapy--prospects for the discovery of new drugs from natural products.

Millions of people and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which only infect keratinized structures. With the appearance of AIDS, the incidence of dermatophytosis has increased. Current drug therapy used for these infections is often toxic, long-term, and expensive and has limited effectiveness; therefore, the discovery of new anti dermatophytic compounds is a necessity. Natural products have been the most productive source for new drug development. This paper provides a brief review of the current literature regarding the presence of dermatophytes in immunocompromised patients, drug resistance to conventional treatments and new anti dermatophytic treatments.

Antimicrobial activity of Pterogyne nitens Tul., Fabaceae, against opportunistic fungi / Atividade antimicrobiana de Pterogyne nitens Tul., Fabaceae, contra fungos oportunistas

As part of our ongoing research on antifungal agents from Brazilian flora, eight extracts and twelve fractions from Pterogyne nitens Tul., Fabaceae, were screened for antimicrobial activity against four opportunistic fungi species (Candida albicans, Candida krusei, Candida parapsilosis and Cryptococcus neoformans) using a broth microdilution method. The present investigation reveals that P. nitens extracts and fractions were more effective against C. krusei and C. parapsilosis than against C. neoformans. The growth of C. albicans was moderately affected by all tested extracts and fractions. The strongest effects were observed for n-butanol fractions from branches (MIC = 15.6 μg/mL) and roots (MIC = 31.2 μg/mL) against C. krusei. Additionally, the chromatographic fractionation of the n-butanol fraction from branches afforded four guanidine alkaloids; N-1,N-2,N-3-triisopentenylguanidine (1), described for the first time in the Fabaceae family, and nitensidines A-C (2-4), which showed moderate activity towards C. krusei (MIC = 62.5 μg/mL) and C. parapsilosis (MIC = 31.2 μg/mL).

No contexto de nossas pesquisas por novos agentes antifúngicos obtidos da flora brasileira, oito extratos e doze frações de Pterogyne nitens Tul., Fabaceae, foram submetidos ao ensaio antifúngico pelo método de microdiluição, contra quatro espécies de fungos oportunistas, Candida albicans, Candida krusei, Candida parapsilosis e Cryptococcus neoformans. Este trabalho revelou que os extratos e frações de P. nitens foram mais ativos contra C. krusei e C. parapsilosis quando comparados a C. neoformans, sendo que o crescimento de C. albicans foi moderadamente afetado por todos os extratos e frações. As atividades mais potentes foram observadas para as frações n-butanólica dos galhos (CIM = 15,6 μg/mL) e raízes (CIM = 31,2 μg/mL) contra C. krusei. Adicionalmente, a fração n-butanólica dos galhos foi submetida ao fracionamento cromatográfico, resultando no isolamento de quatro alcaloides guanidínicos, sendo N-1,N-2,N-3-tri-isopentenilguanidina (1), descrito pela primeira vez em espécies da família Fabaceae e nitensidinas A-C (2-4), os quais apresentaram atividade antifúngica moderada contra C. krusei (CIM = 62,5 μg/mL) e C. parapsilosis (CIM = 31,2 μg/mL).

Induction of apoptosis in A549 pulmonary cells by two Paracoccidioides brasiliensis samples.

Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.

Induction of apoptosis in A549 pulmonary cells by two Paracoccidioides brasiliensis samples

Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.

Estudo de colonização por Candida sp. na cavidade oral de indivíduos soropositivos e soronegativos para HIV-1 no Noroeste Paulista, Brasil / Candida sp. in the oral cavities of HIV-1 serum positive and serum negative individuals from the Northeastern São Paulo State region, Brazil

Este estudo avaliou Candida sp. isolada da mucosa orofaríngea e sua sensibilidade antifúngica em 52 portadores do HIV/Aids, residentes na Região Noroeste Paulista e respectivos controles. Para tanto, foram utilizados testes clássicos, o “kit” API20IDAUX e a disco difusão para: anfotericina B, fluconazol, itraconazol e cetoconazol. O isolamento de Candida sp. foi maior (p < 0,05) em pacientes e controles, sendo o índice de colonização/infecção superior entre usuários de próteses dentárias, em ambos os grupos (p < 0,05). A freqüência de espécies “não-albicans” não diferiu (21%) nesses grupos. Todas as cepas obtidas foram sensíveis a anfotericina B, enquanto uma cepa de C. albicans foi resistente a todos os azóis. Treze por cento das cepas foram resistentes ao fluconazol, em ambos os grupos. Nossos resultados mostram que o grupo HIV/Aids é mais colonizado por Candida sp. que os soronegativos para este vírus, sem mudança na proporção Candida albicans/“não-albicans” ou no perfil de resistência aos azóis.

Species diversity of yeast in oral colonization of insulin-treated diabetes mellitus patients.

The aim of this study was to investigate oral yeast colonization, antifungal susceptibility and strain diversity in insulin-dependent diabetes mellitus patients (175), as well as to evaluate the influence of dental prostheses. Oral rinse samples were cultured on selective media, in order to isolate, count and identify the yeasts recovered. More than half of the diabetic subjects (53%) carried significant amounts of Candida cells in the buccal cavity and these organisms were recovered at higher densities in diabetics wearing dentures. A total of 93 yeast strains were isolated from these patients, including: Candida spp. (n = 89); Pichia (n = 02); Trichosporon (n = 1), and Geotrichum (n = 1). C. albicans represented 56% of these strains, non-albicans Candida 39.8%, and other genera of yeast 4.3%. C. albicans was prevalent, followed by C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, C. rugosa and C. guilliermondii. Agar disk-diffusion tests of the susceptibility of non-albicans Candida and other genera of yeast to fluconazole showed resistance in 21.9%, mainly in C. rugosa (100%), C. glabrata (57%) and C. krusei (50%). Local oral factors, such as the presence of dentures, in association with diabetes, seemed to have the effect of increasing the amount and variety of Candida species in the oral cavities, mainly those with lower drug susceptibilities.

Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD)

Trichophyton rubrum é um importante agente causal de dermatomicose. Os métodos de tipagem molecular têm sido recentemente desenvolvidos para responder questões sobre epidemiologia e auxiliar no esclarecimento de recidivas, após o tratamento. As seqüências aleatórias 1- (5'-d[GGTGCGGGAA]-3') e 6- (5'-d[CCCGTCAGCA]-3') foram usadas para tipagem molecular deste fungo por RAPD produzindo variabilidade intraespecífica. Cinco padrões foram observados entre os 10 isolados de T. rubrum, com ambas as seqüências. Foi concluído que a análise por RAPD pode ser utilizada para estudos epidemiológicos.

Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD).

Trichophyton rubrum is an important cause of dermatomycoses. Molecular strain typing methods have recently been developed to address questions about epidemiology and source of relapse following treatment. This report describes the application of RAPD for molecular strain differentiation of this fungus utilizing the primers 1- (5'-d[GGTGCGGGAA]-3') and 6- (5'-d[CCCGTCAGCA]-3'). A total of five RAPD patterns were observed among 10 strains of T. rubrum, with each of the primers used. We conclude that RAPD analysis using primers 1 and 6 can be used in epidemiological studies.

Estudo comparativo (in vitro) da açäo de diluiçöes e dinamizaçöes de Kalium iodatum sobre culturas de Aspergillus fumigatus / Comparative in-vitro study of the effect of dilution and dinamizations of Kalium iodatum on Aspergillus fumigatus culture

Entre os inúmeros medicamentos homeopáticos de origem mineral estäo os sais de potássio e entre estes últimos, o iodeto de potássio (Kalium expectorante, como fonte de iodo e como antifúngico

Presence of Mycobacterium marinum and other opportunist Mycobacteria in swimming pool waters in Araraquara, SP

O isolamento de Mycobacterium marinum é relatado pela primeira vez no Brasil. Analisaram-se amostras de águas de piscinas quanto à presença de micobactérias, para avaliar a participaçäo dessas águas como possível fonte de micobactérias patogênicas. Isolaram-se 24 cepas de micobactérias a partir de 72 amostras analisadas. A freqüência de isolamento foi de 37,5% para M. gordonae, de 20,8% para M. chelonae, de 4,2% para M. marinum, de 4,2% para M. scrofulaceum, de 4,2% para M. avium-intracellulare e de 29,2% para cepas näo pertencentes a qualquer espécie de micobactérias conhecida. As cepas näo identificadas constituem um novo grupo de micobactérias com características morfológicas e bioquímicas semelhantes às de M. gordonae mas com um perfil de ácidos micólicos distinto. Salienta-se, em conseqüência, a importância do estudo dos ácidos micólicos no rastreio de identificaçäo de micobactérias do ambiente. Concluiu-se que as águas das piscinas de Araraquara contêm micobactérias oportunistas potencialmente patogênicas para o homem

Indicadores de contaminaçäo fecal e enteropatógenos em águas de recreaçäo / Indicators of fecal contamination and enteropathogens in recreational waters

As águas de recreaçäo desempenham importante papel na disseminaçäo de infecçöes entre seus usuários. En 108 amostras de água de recreaçäo (72 de piscinas e 36 de lagos com praias) pesquisaram-se Salmonella, Shigella, Yersinia, E. coli enteropatogênica clássica e E. coli enteroinvasora; determinou-se o NMP de coliformes fecais e de E. coli; realizaram-se contagens de microrganismos heterotróficos e de estreptococos fecais. Näo foram isoladas Shigella, Yersinia e E. colli enteroinvasora. Observaram-se reaçöes positivas para todos os outros microrganismos ou grupos de microrganismos, em proporçöes variáveis. Conclui-se pelo perigo potencial que estas águas representam para os banhistas que as utilizam