RESUMO
A multiplex polymerase chain reaction (PCR) assay using oligonucleotide primers to detect mecA and 16S ribosomal RNA gene was developed to aid in identification of methicillin-resistant staphylococci. Validation included 99 isolates of staphylococcus grouped into one of five categories: methicillin-susceptible coagulase-negative staphylococcus (MSCNS), methicillin-resistant coagulase-negative staphylococcus (MRCNS), methicillin-susceptible Staphylococcus aureus (MSSA), high beta-lactamase producing S aureus (HiBSA), and methicillin-resistant S aureus (MRSA). mecA was detected in MRSA (21/21), and in MRCNS (20/20), but not in MSSA (0/20). mecA was occasionally detected in HiBSA (1/19) and MSCNS (3/19). This multiplex PCR assay was also used to test 30 clinical isolates of coagulase-negative staphylococci with discrepancies between results of in vitro tests for susceptibility to oxacillin and was found to be valuable when a more definitive determination of intrinsic methicillin-resistance was desired.
Assuntos
Resistência a Meticilina/genética , Reação em Cadeia da Polimerase , Staphylococcus aureus/genética , DNA Bacteriano/análiseRESUMO
Reactive oxygen species (ROS) are ubiquitous compounds produced by phagocytes with important roles in both host defense and pulmonary inflammation. Enhanced ROS metabolism by alveolar macrophages (AM) has been previously demonstrated in various interstitial lung diseases including sarcoidosis. We studied 17 healthy, nonsmoking volunteers and 10 patients with sarcoidosis by bronchoalveolar lavage, and separated AM on discontinuous Percoll gradients to determine patterns of airspace cell recovery and the corresponding ROS metabolism of density-defined AM subpopulations. AM subpopulations were largely purified from contaminating granulocytes, thereby allowing more accurate estimation of ROS metabolism by AM. In bronchoalveolar lavage material from sarcoidosis patients, increased recovery of cells of high (1.075 gm/ml) density was found, which contrasted with the pattern seen in the volunteers in whom cells of lowest density (1.045 gm/ml) predominated. In addition, dense cells from sarcoidosis patients exhibited enhanced stimulated ROS metabolism compared with cells of similar density obtained from the volunteers or with sarcoid cells of lower density. At least three mechanisms may contribute to increased lung oxidative burden in sarcoidosis. The combination of increased bronchoalveolar lavage cell counts, increased cell recovery at high density, and increased cell function produced substantial increases in the total oxidative burden imposed on the lungs of sarcoidosis patients by airspace cells. We conclude that AM metabolism of ROS is dependent on the density and, by implication, the maturity of the cells and that regulation of AM ROS metabolism differs markedly between sarcoidosis patients and healthy volunteers.
Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Macrófagos/metabolismo , Sarcoidose/metabolismo , Superóxidos/metabolismo , Análise de Variância , Líquido da Lavagem Broncoalveolar/metabolismo , Contagem de Células , Separação Celular , Sobrevivência Celular , Centrifugação com Gradiente de Concentração , Humanos , Macrófagos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Reactive oxygen species (ROS) have generated increasing interest for their possible role in a wide variety of diseases. Interferon-gamma (IFN-gamma), a potent immunoregulatory lymphokine, is likely involved in control of ROS metabolism. In this study, the superoxide release of cultured human peripheral blood monocytes (PBM) after exposure to IFN-gamma and lipopolysaccharide (LPS) was examined. Compared with controls, adherent monocytes cultured with 80 units of IFN-gamma for 48 hours demonstrated fourfold increased spontaneous and twofold increased PMA stimulated release of superoxide anion. In addition, the enhanced superoxide release was both dose and time dependent. Further experiments showed that bacterial LPS in concentrations as low as 4 ng/mL markedly reduced monocyte superoxide release and abrogated the enhancing effects of IFN-gamma.
Assuntos
Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo , Superóxidos/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Humanos , Interferon gama/administração & dosagem , Masculino , Espectrofotometria , Fatores de TempoRESUMO
Alveolar macrophages (AMs) and lymphocytes are activated in pulmonary sarcoidosis. Mediators from these cells are potentially important in the pathophysiology and pathogenesis of this disease. To determine whether the enhanced release of reactive oxygen species (ROS) participates in inflammatory events in sarcoidosis, and to explore the relationship between ROS release and clinical parameters, we studied ROS metabolism of AMs and other airspace cells by luminol-enhanced chemiluminescence and by direct biochemical measurement of superoxide anion production. Ten of 17 patients with sarcoidosis were prospectively found to have active disease by objective radiographic, functional, and laboratory criteria. In these subjects, ROS metabolism by AMs was significantly enhanced compared either with healthy control subjects or with patients with inactive sarcoidosis. Abnormalities in ROS metabolism were not seen in peripheral blood monocytes, suggesting that this increased metabolic activity is compartmentalized to the lung. Enhanced ROS metabolism by AMs was associated with recent adverse chest radiographic changes, recent decline in forced vital capacity, and more advanced radiographic type. These data support the hypothesis that ROS generated by airspace cells can promote parenchymal inflammation in sarcoidosis, are associated with physiologic and radiologic changes, and may thereby contribute to the pathogenesis of sarcoidosis.