title: Application of a fluorochrome-lysostaphin assay to the detection of phagocytic and bactericidal disturbances in human neutrophils and monocytes.
abstract: The evaluation of phagocytic and microbicidal activities of the blood neutrophils has been recognized as one of the important tools for investigating phagocytic dysfunctions in patients with recurrent infections. In the present study, these activities were examined in neutrophils and monocytes from healthy adults and patients affected by primary phagocytic dysfunctions by using a modified fluorochromic microbicidal assay, discriminating simultaneously the extracellular adherence, ingestion and intracellular killing of Staphylococcus aureus Cowan I. The assay employs acridine orange staining, as described in Bellinati-Pires et al. (1989) (AO assay), but was modified by the addition of an alternative leukocyte treatment with 0.5 U/ml of lysostaphin (LS) for 5 min at 37 degrees C, after phagocytosis (AO-LS assay). The LS treatment was standardized to eliminate staphylococci adhered to the outer surface of the phagocytes without affecting the determination of intracellular live or dead bacteria, as demonstrated in normal neutrophils and monocytes. Our purpose in this study was to compare AO and AO-LS assays in order to evaluate the effect of LS on the determination of actually ingested staphylococci and to provide a means for improving the fluorochromic assay for detecting phagocytic defects, as well as bactericidal disturbances. By using the AO-LS assay, decreased ingestion of staphylococci by neutrophils in Chediak-Higashi Syndrome (CHS) was demonstrated. However, increased staphylococci adherence, as well as ingestion, was observed in neutrophils or monocytes from chronic granulomatous disease (CGD) patients, comparing AO and AO-LS assays. Bactericidal defect, which is a common feature in CHS and CGD, was detected in neutrophils or monocytes in both assays. We emphasize that such alterations were deduced by comparing the patients' results with those obtained from their respective normal controls and with the normal range of values previously established for 160 healthy adults. No alteration was observed in hyper IgE syndrome phagocytes. Despite the possible penetration of LS into the leukocytes, as stated in other studies, we concluded that a short period of phagocyte incubation with this enzyme increased the sensitivity of the fluorochromic assay to detect phagocytic defect without affecting the determination of the bactericidal activity. Moreover, comparations between AO and AO-LS assays may be important in the study of the initial pathways of staphylococci phagocyte interaction, including adherence by non-phagocytic receptors.
authors.affiliation: Seção de Imunologia, Instituto Adolfo Lutz, São Paulo, Brasil.
authors.forename: R
authors.initials: R
authors.lastname: Bellinati-Pires
authors.forename: M M
authors.initials: MM
authors.lastname: Salgado
authors.forename: I P
authors.initials: IP
authors.lastname: Hypolito
authors.forename: A S
authors.initials: AS
authors.lastname: Grumach
authors.forename: M M
authors.initials: MM
authors.lastname: Carneiro-Sampaio
mesh_terms: D000328:Adult; D001770:Blood Bactericidal Activity; D002648:Child; D002675:Child, Preschool; D005069:Evaluation Studies as Topic; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D008248:Lysostaphin; D008297:Male; D009000:Monocytes / Q000276:immunology* / Q000382:microbiology; D009504:Neutrophils / Q000276:immunology* / Q000382:microbiology; D010585:Phagocyte Bactericidal Dysfunction / Q000097:blood* / Q000175:diagnosis* / Q000209:etiology*; D013211:Staphylococcus aureus / Q000276:immunology
chemical_list: D005456:Fluorescent Dyes; D007073:Immunoglobulin E; D008248:Lysostaphin