RESUMO
C57BL/6 and BALB/c mice are prototype hosts for the study of resistance and susceptibility to several infectious diseases. In many cases, resistance of C57BL/6 is due to the microbicidal effect of nitric oxide (NO) produced by macrophages in response to interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), mainly secreted by Th1 cells and macrophages, respectively. BALB/c, usually unable to give rise to Th1 lymphocytes, does not control certain infections. However, we and others have previously observed that regardless of the adaptive immune response, C57BL/6 (M-1) macrophages are far more sensitive to the stimulus of IFN-gamma-plus lipopolysaccharide (LPS) for the production of NO than are BALB/c (M-2) cells, a feature that might also account for resistance. Here, we report that the differential production of NO by M-1 and M-2 macrophages correlates with the accumulation of inducible nitric oxide synthase (iNOS) mRNA and protein, which shows that expression of iNOS is differentially regulated in M-1 and M-2 cells. The higher accumulation of iNOS mRNA in M-1 cells is independent of its stability, and, thus, it is possible that transcription of the iNOS gene in these cells may be more efficient than in M-2 cells. A remarkable finding is that the level of iNOS protein is much higher in M-1 macrophages than in M-2 cells, as compared with the mRNA levels, which makes us speculate that differential translational or posttranslational controls of iNOS gene are operative.
Assuntos
Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Animais , Células Cultivadas , Interferon gama/imunologia , Ativação de Macrófagos , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Estabilidade de RNA , Fatores de TempoRESUMO
BACKGROUND: The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. RESULTS: Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. CONCLUSION: Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/genética , Galinhas/microbiologia , Lactobacillus/genética , Lactobacillus/metabolismo , Probióticos/administração & dosagem , Administração Oral , Animais , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Doenças das Aves/imunologia , Doenças das Aves/microbiologia , Doenças das Aves/prevenção & controle , Galinhas/imunologia , Melhoramento Genético/métodos , Lactobacillus/imunologia , Lactobacillus/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transformação Bacteriana/genéticaRESUMO
Plasmodium malariae is commonly confounded with Plasmodium vivax at the microscopic examination of thick blood smear. In the present study, we used a nested PCR assay to amplify a species-specific sequence of the 18S SSU rRNA gene of Plasmodium in blood samples of 497 individuals living in an endemic region of the Brazilian Amazon basin. We have found that, while the microscopic examination of thick blood smears showed a P. malariae prevalence of 1.2% (6 out of 497), the nested PCR revealed 11.9% (59 out of 497) of positive cases for this specie. These results point to the need of the development or use of a more accurate diagnosis method to distinguish between P. malariae and P. vivax, which is particularly important in view of the fact that the choice of drug for the antimalarial therapy depends on the parasite species.
Assuntos
Malária/epidemiologia , Plasmodium malariae/genética , Animais , Brasil/epidemiologia , Diagnóstico Diferencial , Humanos , Malária/sangue , Malária/diagnóstico , Reação em Cadeia da Polimerase , PrevalênciaRESUMO
Being an intracellular pathogen demands being able to invade a host cell, to circumvent the host immune response and to survive in the intracellular environment. Pore-forming proteins are among the innumerable tools used by intracellular microorganisms to achieve these goals. Remarkably, this seems to be a multipurpose group of proteins that can act in several ways. Making channels may signify entering into host cells, inhibiting phagocytosis, escaping phagosomes or promoting pathogen dissemination. In certain cases, pore-forming proteins are double-edged tools and may benefit the host by eliminating infected cells and/or inducing inflammation.
Assuntos
Porinas/metabolismo , Animais , Apoptose , Bactérias/imunologia , Bactérias/metabolismo , Bactérias/patogenicidade , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Eucariotos/imunologia , Eucariotos/metabolismo , Eucariotos/patogenicidade , Interações Hospedeiro-Parasita , Modelos Biológicos , Fagocitose , Porinas/biossíntese , Porinas/química , Infecções por Protozoários/imunologia , Infecções por Protozoários/metabolismo , Infecções por Protozoários/parasitologia , Infecções por Protozoários/patologiaRESUMO
Sera from patients with Chagas' disease were used to screen a Trypanosoma cruzi amastigote cDNA library. Characterization of 50 positive clones showed that 21 (42%) encode previously identified T. cruzi ribosomal and flagellar proteins, heat-shock proteins or proteins with repetitive motifs. Twenty-nine clones (58%) correspond to nine genes not previously described in T. cruzi. Three cDNAs, encoding novel repetitive antigens with homology to ribosomal proteins and to other RNA binding proteins, were further characterized. Patient humoral responses against the recombinant proteins encoded by these cDNAs were evaluated in anticipation that they may constitute potential new targets for serodiagnostic assays.