RESUMO
The Bacille Calmette-Guérin (BCG) vaccine comprises a family of genetically different strains derived by the loss of genomic regions (RDs) and other mutations. In BCG Moreau, loss of RD16 inactivates rv3405c * , encoding a transcriptional repressor that negatively regulates the expression of Rv3406, an alkyl sulfatase. To evaluate the impact of this loss on the BCG and host cell viability and the cytokine profile, THP-1 cells were infected with BCG Moreau (harbouring the empty vector) and a complemented strain carrying a functional copy of rv3405c. Viability of the host cells and bacteria as well as the pattern of cytokine secretion were evaluated. Our results show that the viability of BCG Moreau is higher than that of the complemented strain in an axenic medium, suggesting a possible functional gain associated with the constitutive expression of Rv3406. Viability of the host cells did not vary significantly between recombinant strains, but differences in the profiles of the cytokine secretion (IL-1ß and IL-6) were observed. Our results suggest an example of a functional gain due to gene loss contributing to the elucidation of the impact of RD16 on the physiology of BCG Moreau.
Assuntos
Vacina BCG/farmacologia , Sobrevivência Celular/genética , Citocinas/efeitos dos fármacos , Mutação com Ganho de Função/genética , Macrófagos/microbiologia , Mycobacterium bovis/genética , Transcrição Gênica/genética , Vacina BCG/genética , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Mutação com Ganho de Função/efeitos dos fármacos , Humanos , Mycobacterium bovis/fisiologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/microbiologiaRESUMO
The Bacille Calmette-Guérin (BCG) vaccine comprises a family of genetically different strains derived by the loss of genomic regions (RDs) and other mutations. In BCG Moreau, loss of RD16 inactivates rv3405c * , encoding a transcriptional repressor that negatively regulates the expression of Rv3406, an alkyl sulfatase. To evaluate the impact of this loss on the BCG and host cell viability and the cytokine profile, THP-1 cells were infected with BCG Moreau (harbouring the empty vector) and a complemented strain carrying a functional copy of rv3405c. Viability of the host cells and bacteria as well as the pattern of cytokine secretion were evaluated. Our results show that the viability of BCG Moreau is higher than that of the complemented strain in an axenic medium, suggesting a possible functional gain associated with the constitutive expression of Rv3406. Viability of the host cells did not vary significantly between recombinant strains, but differences in the profiles of the cytokine secretion (IL-1β and IL-6) were observed. Our results suggest an example of a functional gain due to gene loss contributing to the elucidation of the impact of RD16 on the physiology of BCG Moreau.
Assuntos
Humanos , Transcrição Gênica/genética , Vacina BCG/farmacologia , Sobrevivência Celular/genética , Citocinas/efeitos dos fármacos , Mutação com Ganho de Função/genética , Macrófagos/microbiologia , Mycobacterium bovis/genética , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/microbiologia , Vacina BCG/genética , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Mutação com Ganho de Função/efeitos dos fármacos , Mycobacterium bovis/fisiologiaRESUMO
Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized cause of diarrhea in children in developing and developed countries. EAEC is recognized by a characteristic aggregative pattern of adherence to human epithelial (HEp-2) cells cultured in vitro. This is the gold standard assay. The aggregative phenotype is associated with the presence of a 65 MDa plasmid (pAA) that also encodes several other putative virulence factors, such as the aggregative adherence fimbria I (AAF/I) and the enteroaggregative heat-stable enterotoxin (EAST1). The objective of this work was to evaluate the application of PCR (polymerase chain reaction) to identify EAEC strains in cases of acute diarrhea. A total of 87 E. coli strains, isolated from patients under 2 years of age with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by AAF/I- and EAST1-PCR. PCR sensitivity and specificity in comparison with the cell culture assay showed 94.4% sensitivity and 78.26% specificity. EAST1- and AAF/I-PCR could be recommended as a screening test, applicable to epidemiologic studies.
Assuntos
Aderência Bacteriana , Diarreia Infantil/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/isolamento & purificação , Argentina/epidemiologia , Toxinas Bacterianas/genética , Enterotoxinas/genética , Células Epiteliais/microbiologia , Escherichia coli/patogenicidade , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Humanos , Lactente , Programas de Rastreamento , Fenótipo , Plasmídeos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Células Tumorais Cultivadas/microbiologia , VirulênciaRESUMO
Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized cause of diarrhea in children in developing and developed countries. EAEC is recognized by a characteristic aggregative pattern of adherence to human epithelial (HEp-2) cells cultured in vitro. This is the gold standard assay. The aggregative phenotype is associated with the presence of a 65 MDa plasmid (pAA) that also encodes several other putative virulence factors, such as the aggregative adherence fimbria I (AAF/I) and the enteroaggregative heat-stable enterotoxin (EAST1). The objective of this work was to evaluate the application of PCR (polymerase chain reaction) to identify EAEC strains in cases of acute diarrhea. A total of 87 E. coli strains, isolated from patients under 2 years of age with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by AAF/I- and EAST1-PCR. PCR sensitivity and specificity in comparison with the cell culture assay showed 94.4 sensitivity and 78.26 specificity. EAST1- and AAF/I-PCR could be recommended as a screening test, applicable to epidemiologic studies.(AU)
Assuntos
Humanos , Lactente , Aderência Bacteriana , Diarreia Infantil/microbiologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Argentina/epidemiologia , Toxinas Bacterianas/genética , Enterotoxinas/genética , Células Epiteliais/microbiologia , Escherichia coli/patogenicidade , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Programas de Rastreamento , Fenótipo , Plasmídeos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Células Tumorais Cultivadas/microbiologia , VirulênciaRESUMO
Ecotropic recombinant virus (ERV), a relatively new class of murine retrovirus endogenous to mice, is expressed at significant levels by most murine myeloma and hybridoma cells examined. The routine XC, S+L-, mink cell focus-inducing (MCF), and reverse transcriptase (RT) tests are not suitable to detect and quantify the levels of ERV. A serological focus assay, based on specific anti-murine leukemia virus (MuLV) viral envelope (env) antibodies, is required to detect ERV. A more sensitive format of this serological focus assay includes co-cultivation of test article cells with the indicator (Mus dunni) cells. ERV isolated from murine hybridoma cells show a unique pattern of cross-reactivity with anti-MuLV env antibodies and this pattern is clearly distinct from that of ecotropic and xenotropic retroviruses.
Assuntos
Gammaretrovirus/isolamento & purificação , Hibridomas/microbiologia , Mieloma Múltiplo/microbiologia , Células Tumorais Cultivadas/microbiologia , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Imunofluorescência , Gammaretrovirus/imunologia , Hibridomas/citologia , Vírus da Leucemia Murina/imunologia , Camundongos , Vison , Muridae , Ensaio de Placa ViralRESUMO
S. saprophyticus has been frequently isolated from urinary tract infections in young women. In contrast with S. aureus, no defined virulence factors have been recognized for the coagulase negative Staphylococcus species. The objective this study was to analyze the adherence of S. saprophyticus to HEp-2 cells and sheep erythrocytes. The sample were isolated from urine of patients with urinary infection. Hemagglutination, adherence to HEp-2 cells tests and inhibition by specific carbohydrates of the interactions between these cells were analyzed. Most of the strains were hemagglutinating whose properties was inhibited by mannose (100mM). There was a high adherence level to HEp-2 cells. The differences in specificity and attachment level noted in this study suggest that multiple adhesins are involved in the mechanism of cellular interaction.