Phorbol dibutyrate-induced megakaryocytic differentiation increases susceptibility of K562 cells to SA11 rotavirus infection.

The role of integrins previously implicated as rotavirus receptors in determining cellular susceptibility to SA11 rotavirus was studied, using phorbol dibutyrate (PDB) treatment of K562 cells to induce megakaryocytic differentiation. Expression of alpha2beta1 integrin was detected after 2 days in PDB, and peaked after PDB treatment for 4-7 days. SA11 titres were increased by 1.8- to 10.8-fold over untreated cells after PDB treatment for 2-7 days, and correlated with levels of alpha2beta1 integrin expression in PDB-treated K562 cells.

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells.

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.

Quantifying phagocytosis of Mycobacterium avium complex by human monocytes in whole blood.

Studies of phagocytic efficiency in cells of the macrophage lineage have assumed additional importance since the discovery that HIV infection of these cells impairs their immune function. A rapid method has been developed for measuring phagocytosis of the opportunistic pathogen Mycobacterium avium complex by human monocytes. Fluoresceinated M. avium complex (F-MAC) was incubated with whole blood at 37 degrees C and the fluorescence of extracellular F-MAC was quenched using a vital blue stain. Monocytes were then stained with a monoclonal antibody (mAb) to human CD14 conjugated to phycoerythrin (PE) red cells were lysed, and the percentage of monocytes which had phagocytosed F-MAC was measured by flow cytometry. The results were reproducible in samples of blood taken from individual donors over a period of 1 or 2 weeks, and optimum F-MAC concentrations and an optimum incubation time were determined by experiment. This method has the advantages of requiring only a small volume of blood, not necessitating manipulation of cells before testing, and using a phagocytic target relevant to the pathogenesis of HIV infection.

HIV infection of monocyte-derived macrophages in vitro reduces phagocytosis of Candida albicans.

HIV-1 infection of peripheral blood monocyte-derived macrophages (MDMs) is unrelated to the level of CD4 expression on the surface of the cell, is associated with considerable donor variability, causes minimal cytopathology, and results in peak viral antigen production after 2 weeks of infection. Phagocytosis of opsonized Candida albicans by MDMs infected in vitro with several strains of HIV was compared with that of uninfected cells from the same donors; the proportion of MDMs containing the fluorescein isothiocyanate-labeled yeast was determined by flow cytometry and phase contrast microscopy. The intracellular localization of C. albicans was confirmed by confocal microscopy. Using paired MDMs from nine donors, 81% of uninfected and 53% of HIV-infected MDMs phagocytosed C. albicans. In addition, the number of yeast per cell was significantly higher in uninfected MDMs than in HIV-infected cells (mean 6.1 versus 2.5). These findings may partially explain the high incidence of mucocutaneous candidiasis in HIV-infected patients with advanced disease.

Ureaplasma urealyticum serotypes in urinary tract disease.

Ureaplasma urealyticum cultures from 124 patients with urinary tract disease were serotyped by indirect immunofluorescence, using antisera to serotypes I to VIII. A similar range of serotypes was recovered from first-voided, midstream, and bladder-aspiration (SPA) urine, upper urinary tract samples, and vaginal swabs. Serotype VI was predominant (44/124) among the samples, whereas serotypes V (1/124 samples) and VII (0/124 samples) were uncommon. Twenty of 124 cultures contained more than one serotype, and three cultures were untypeable. Serotypes cultured from bladder urine were also present in vaginal and urethral samples, although these samples often carried additional serotypes. Consecutive SPA samples from the same patient invariably contained the same serotype, whereas some consecutive midstream urine samples showed a loss or gain of serotypes with time. One patient carried the same serotype in SPA urine over a period of 13 months. The pattern of serotypes recovered from the urinary tract was similar irrespective of the sampling site, the site of infection, the clinical diagnosis and renal function of the patient, and the presence or absence of other microorganisms. Colonization above the urethra and association with urinary tract disease appeared to be serotype independent.