RESUMO
We evaluated the effects of glutamine on clastogenic and genotoxic damage prevention caused by the administration of cisplatin. Forty Swiss mice were divided into 8 experimental groups: G1 and G2, which were control groups; G3, G4, and G5, which were administered [2 doses of glutamine (orally)] separated by a 24-h period (150, 300, and 600 mg/kg, respectively), and a dose of phosphate-buffered saline by intraperitoneal injection; G6, G7, and G8, which were treated in the same manner as the previous groups, but received cisplatin rather than phosphate-buffered saline. The antimutagenicity groups showed damage reduction percentages of 79.05, 80.00, and 94.27% at the time point T1, 53.18, 67.05, and 64.74 at time point T2 for the 150, 300, and 600 mg/kg doses of glutamine, respectively. Antigenotoxic activity was evident for all 3 doses with damage reduction percentages of 115.05, 119.06, and 114.38 for the doses of glutamine of 150, 300, and 600 mg/ kg, respectively. These results suggest that further studies are needed to confirm the clastogenic activity of glutamine. However, our results may lead to rational strategies for supplementation of this antioxidant as an adjuvant in cancer treatment or for preventing genomic lesions.
Assuntos
Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Cisplatino/farmacologia , Glutamina/farmacologia , Mutagênicos/farmacologia , Animais , Antineoplásicos/farmacologia , Cisplatino/antagonistas & inibidores , Ensaio Cometa , Dano ao DNA , Injeções Intraperitoneais , Masculino , Camundongos , Testes para MicronúcleosRESUMO
The geographic distribution of paracoccidioidomycosis (PCM) in the Brazilian state of São Paulo was evaluated in a retrospective study using secondary data from serological analyses, carried out by double immunodiffusion assay of patients with PCM suspicion, from January 1999 to May 2010. Sixty percent of 10,176 patients, from 239 cities, were serologically reactive to P. brasiliensis. The cities that showed the most serological reactivity among patients were São João da Boa Vista (85%), Piracicaba (75%), Sorocaba (73%), Campinas (72%) and São Paulo (62%). São Paulo state has an area of 248,209.4 km²; the climate is tropical and sub-tropical with annual temperatures between 18 and 24ºC, high rainfall (900 to 1800 mm/year), rainy summers and mild winters. It also features large areas composed of acidic soils, and is one of the greatest contributors to Brazilian agricultural production and, separately, the largest producer of orange juice and, the ninth greatest producer of soy and sugar cane and the fourth largest coffee producer. We suggest that the climatic characteristics associated with soil type and development of primary activities can contribute to the endemic potential of PCM in São Paulo state.(AU)
Assuntos
Humanos , Animais , Paracoccidioides/patogenicidade , Paracoccidioidomicose/epidemiologia , Testes Sorológicos/estatística & dados numéricos , Testes Imunológicos/estatística & dados numéricosRESUMO
The geographic distribution of paracoccidioidomycosis (PCM) in the Brazilian state of São Paulo was evaluated in a retrospective study using secondary data from serological analyses, carried out by double immunodiffusion assay of patients with PCM suspicion, from January 1999 to May 2010. Sixty percent of 10,176 patients, from 239 cities, were serologically reactive to P. brasiliensis. The cities that showed the most serological reactivity among patients were São João da Boa Vista (85%), Piracicaba (75%), Sorocaba (73%), Campinas (72%) and São Paulo (62%). São Paulo state has an area of 248,209.4 km²; the climate is tropical and sub-tropical with annual temperatures between 18 and 24ºC, high rainfall (900 to 1800 mm/year), rainy summers and mild winters. It also features large areas composed of acidic soils, and is one of the greatest contributors to Brazilian agricultural production and, separately, the largest producer of orange juice and, the ninth greatest producer of soy and sugar cane and the fourth largest coffee producer. We suggest that the climatic characteristics associated with soil type and development of primary activities can contribute to the endemic potential of PCM in São Paulo state.
Assuntos
Humanos , Masculino , Feminino , Paracoccidioides/imunologia , Paracoccidioidomicose/epidemiologia , Estudos EpidemiológicosRESUMO
Yeast forms of Paracoccidioides brasiliensis produce polydispersed high molecular mass (h-MM) antigens. We investigated the antibodies to an h-MM antigen from P. brasiliensis by immunoblotting and ELISA in sera from paracoccidioidomycosis (PCM) patients. IgG from the sera of chronic PCM patients was able to recognize the h-MM antigen at a higher frequency in the cell-free antigen (CFA) (8/13) than in the somatic antigen (SA) (2/13), as assessed by immunoblotting. The CFA was fractionated by Sephadex G-200 chromatography, and fraction 17 (F17) with the h-MM antigen of approximately 366 kDa was used in ELISA to analyze specific levels of IgG and IgE. Patients with the chronic form showed significantly higher levels of IgG (P<0.05) but not IgE (P>0.05) to F17 by ELISA, compared to patients with the acute form or to healthy donors. In conclusion, CFA is better than SA as a source of the P. brasiliensis h-MM antigen. This study reveals a new characteristic to differentiate between the acute and chronic forms of PCM, by demonstrating a higher level of seric IgG to h-MM antigen in chronic compared to acute PCM patients.
Assuntos
Anticorpos Antifúngicos/imunologia , Antígenos de Fungos/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Doença Aguda , Reações Antígeno-Anticorpo , Antígenos de Fungos/isolamento & purificação , Sistema Livre de Células/imunologia , Cromatografia em Gel , Doença Crônica , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Imunoglobulina E/análise , Imunoglobulina G/análise , Peso MolecularRESUMO
Adhesion is regarded as an important step in the pathogenesis of several microorganisms. Thus, the ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. Studying the already characterized laminin-binding protein of Paracoccidioides brasiliensis, the 43 kDa glycoprotein (gp43), we evaluated whether MAb 1.H12, raised against the laminin-binding protein from Staphylococcus aureus, cross-reacts with that fungal protein. By immunoblot analysis we show that MAb 1.H12 recognizes gp43. This interaction is able to inhibit the laminin-mediated adhesion to epithelial cells as well as the P. brasiliensis infection in vivo. Moreover, through immunoenzymatic assays, we show that MAb 1.H12 recognizes gp43 in solid phase and that this interaction is partially inhibited by the addition of anti-gp43 MAbs. These results show that MAb 1.H12 recognizes the gp43, suggesting the presence of an epitope similar to those found in the other laminin-binding proteins from phylogenetically very distant cells. These findings reinforce the possibility of evolutionary conservation of such epitopes.
Assuntos
Anticorpos Monoclonais , Laminina/metabolismo , Paracoccidioides/fisiologia , Receptores de Laminina/fisiologia , Staphylococcus aureus/imunologia , Animais , Sítios de Ligação , Adesão Celular , Linhagem Celular , Cricetinae , Cães , Epitélio/microbiologia , Epitélio/ultraestrutura , Epitopos/análise , Proteínas da Matriz Extracelular/metabolismo , Granuloma/microbiologia , Granuloma/patologia , Humanos , Rim , Neoplasias Pulmonares , Masculino , Microscopia Eletrônica de Varredura , Paracoccidioides/imunologia , Paracoccidioides/ultraestrutura , Receptores de Laminina/análise , Receptores de Laminina/imunologia , Testículo/microbiologia , Testículo/patologiaRESUMO
The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin. Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction. Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells. Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity. Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.
Assuntos
Anticorpos Antifúngicos/farmacologia , Anticorpos Antifúngicos/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos de Fungos/imunologia , Proteínas Fúngicas , Glicoproteínas/imunologia , Laminina/antagonistas & inibidores , Laminina/farmacologia , Oligossacarídeos/imunologia , Paracoccidioides/patogenicidade , Paracoccidioidomicose/etiologia , Animais , Anticorpos Antifúngicos/imunologia , Anticorpos Monoclonais/uso terapêutico , Ligação Competitiva/imunologia , Adesão Celular/efeitos dos fármacos , Cricetinae , Células Epiteliais , Epitélio/metabolismo , Humanos , Laminina/efeitos dos fármacos , Masculino , Paracoccidioides/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Four murine monoclonal antibodies (MAbs 17C, 21A, 21F, and 32B) raised against the 43-kDa glycoprotein of Paracoccidioides brasiliensis were tested in a capture enzyme immunoassay (EIA) for the detection of specific human anti-gp43 immunoglobulin G in patients with paracoccidioidomycosis (PCM). All MAbs reacted similarly in the assay. These MAbs, which detected anti-gp43 at levels of as low as 500 pg/ml, were demonstrated to specifically recognize at least two different epitopes in gp43 binding assays. Specific antibodies in the sera of patients with active PCM were detected at dilutions of as high as 1:819,200, and the reactivities of patient sera, as measured by optical densities, were found to be significantly higher than those of control sera. The comparison between classical ELISA and our capture enzyme immunoassay showed that both sensitivity and specificity were greatly improved by the latter. These MAbs represent the first specific reagents to P. brasiliensis described for use in serological tests for PCM.
Assuntos
Anticorpos Antifúngicos/análise , Anticorpos Monoclonais/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/diagnóstico , Animais , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/imunologia , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/análise , Camundongos , Camundongos Endogâmicos BALB C , CoelhosRESUMO
We have demonstrated that laminin mediates the adhesion of P. brasiliensis to monolayers of epithelial cells through specific binding to the surface glycoprotein gp43. This binding seems to be related to the fungal pathogenesis. We now report the confirmation of these findings by scanning electron microscopy and show that some isolates that do not secrete gp43 do express the protein as seen by studying whole cell extracts. These results confirm the ability of these strains to produce paracoccidioidomycosis but should not be used for serological purposes since the absence of gp43 in exoantigens may lead to false negative results.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Laminina/metabolismo , Glicoproteínas de Membrana/isolamento & purificação , Paracoccidioides/metabolismo , Animais , Bovinos , Adesão Celular , Cricetinae , Cães , Proteínas Fúngicas/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Paracoccidioides/patogenicidade , Paracoccidioides/ultraestruturaRESUMO
We have demonstrated that laminin mediates the adhesion of P. brasiliensis to monolayers of epithelial cells through specific binding to the surface glycoprotein gp43. This binding seems to be related to the fungal pathogenesis. We now report the confirmation of these findings by scanning electron microscopy and show that some isolates that do not secrete gp43 do express the protein as seen studying whole cell extracts. This results confirm the ability of these strains to produce paracoccidioidomycosis but should not be used for serological purposes since the absence of gp43 in exoantigens may lead to false negative results
Assuntos
Bovinos , Cães , Cricetinae , Camundongos , Animais , Laminina/metabolismo , Glicoproteínas de Membrana/isolamento & purificação , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidade , Paracoccidioides/ultraestrutura , Proteínas Fúngicas/isolamento & purificação , Adesividade , Anticorpos Monoclonais , Western Blotting , Células Cultivadas , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica de Varredura , Paracoccidioides/patogenicidade , Paracoccidioides/ultraestrutura , Proteínas Fúngicas/metabolismoRESUMO
Extracellular matrix protein laminin binds specifically to yeast forms of Paracoccidioides brasiliensis and enhances adhesion of the fungus to the surface of epithelial Madin-Darby canine kidney cells in vitro. Immunoblotting of fungal extracts showed that the gp43 glycoprotein is responsible for adhesion. This was confirmed by binding assays using purified gp43, with a Kd of 3.7 nM. The coating of P. brasiliensis yeast forms with laminin before injection into hamster testicles enhanced the fungus virulence, resulting in a faster and more severe granulomatous disease. These results indicate that interaction of fungi with extracellular matrix elements may constitute a basis for the evolution of fungal infection toward regional spreading and dissemination.