No abstract

The ability of recombinant gamma interferon (rIFN-gamma) to activate macrophages for Salmonella-killing activity was kinetically examined in relation to phagosome-lysosome fusion and H2O2 generation. Resident peritoneal macrophages of BALB/c mice incubated with 10(2) to 10(3) U of rIFN-gamma per ml for 12 h exhibited enhanced bactericidal activity against Salmonella typhimurium, although H2O2 generation was unaltered. In contrast, macrophages incubated with equal doses of rIFN-gamma for 48 h showed both an enhanced Salmonella-killing activity and an increased generation of H2O2. To evaluate Salmonella-killing activities of macrophages, intracellular bacteria were assayed at 0, 2, and 8 h after infection. During the initial 2 h of infection, 12-h-activated macrophages, as well as the unstimulated control macrophages, showed a decline in bacterial population at the same rate. Over the next 6 h of infection, however, the number of viable bacteria in activated macrophages remained unchanged, whereas the number of bacteria in control macrophages significantly (P less than 0.05) increased. Similar results were obtained in 48-h-activated macrophages. On the other hand, macrophages incubated with 10 to 10(3) U of rIFN-gamma exhibited enhanced fusion of lysosomes to Salmonella-containing phagosomes in both the 12-h- and 48-h-stimulated stages. Moreover, when 48-h-activated macrophages were incubated concomitantly with superoxide dismutase and catalase, Salmonella-killing activity was not affected. These results indicate that rIFN-gamma per se is able to activate peritoneal macrophages to induce Salmonella-killing activity and suggest that increased phagosome-lysosome fusion followed by an oxygen-independent killing mechanism is primarily responsible for the enhanced Salmonella-killing activity in rIFN-gamma-activated macrophages.

Candida albicans serotype A (C. albicans A) possesses a specific antigen, designated antigen 6, which resides in mannans on the cell surface. To determine the role of the mannan moiety of the C. albicans cell wall in adherence to buccal epithelial cells, we used antigen 6-deficient mutants which had been isolated by screening with an agglutinating monoclonal antibody against antigen 6 (MAb-6). 'H nuclear magnetic resonance spectral analysis of the purified mannans from the mutants showed a loss of the signals related to the 1-linkage of the side chains. Moreover, acetolyzed fragments of the mutant mannans showed a decreased amount of mannohexaose and mannopentaose. The mutant yeast cells exhibited significantly reduced ability to adhere both to exfoliated buccal epithelial cells and to a human buccal cell line. A number of strains of C. albicans A, C. tropicalis, and C. glabrata, all of which bear antigen 6, showed significantly higher adherence to the cell line than did those of C. albicans serotype B, which lack antigen 6. The whole mannan from the C. albicans A parent inhibited the adherence of C. albicans A to epithelial cells dose dependently, whereas mannan from a mutant strain did not. Moreover, C. albicans A treated with MAb-6 or polyclonal factor 6 serum showed reduced adherence. A close correlation was found between adhesive ability and agglutinability with MAb-6 in the C. albicans A parent, the antigenic mutants, and their spontaneous revertants. These results suggest that so far as mannan adhesin is concerned, serotype A-specific determinants are largely involved in the mechanisms of adherence of C. albicans A to human buccal epithelial cells.

A 2-kbp DNA fragment, E03, that was present in multiple copies in the Candida albicans genome was isolated for use in developing a detection method for C. albicans by polymerase chain reaction (PCR). Dot blot hybridization revealed that E03 was specific for the 40 isolates of C. albicans serotypes A and B used. Using a set of primers (20-mer each) derived from the nucleotide sequence of E03, we performed specific amplification of a 1.8-kbp DNA fragment within E03 by PCR. All 40 isolates belonging to C. albicans serotypes A and B contained amplifiable 1.8-kbp fragments, although the DNA of the amplified products exhibited small variations in size, yielding three different fragment groups. Southern blot hybridization probed with E03 showed that these 1.8-kbp fragments were derived from the E03 region. Conversely, the 1.8-kbp fragment was not amplified from 38 isolates belonging to seven other medically important Candida species or from isolates of Cryptococcus neoformans, Saccharomyces cerevisiae, various bacteria, and a human cell line. The detection limit of the PCR assay for C. albicans with the E03 fragment was shown to be approximately 2 to 10 cells and 100 cells in saline and human urine, respectively, by ethidium bromide staining and 2 and 10 cells, respectively, by Southern blot analysis. In addition, E03 was assumed to originate from mitochondrial DNA on the basis of the results of its characterizations. These results indicate that the PCR system using the 1.8-kbp fragment as a target is a reliable method for identifying C. albicans isolates, thereby suggesting its potentials for specific and sensitive detection of C. albicans in samples from patients with candidiasis.

The phagocytic and intracellular killing activities of normal mouse phagocytes against Candida albicans were studied to elucidate the role of these activities in nonspecific and specific defense mechanisms. In the presence of fresh normal mouse serum, viable C. albicans cells were ingested by mouse peripheral blood leukocytes (PBLs) and peritoneal macrophages (PMPs) at the same rate. Serum-chelation experiments indicated that the factors involved in the alternative complement pathway are opsonins for C. albicans. PBLs killed intracellular C. albicans more effectively than PlVIPs. Lymphokine-activated PMPs manifested marked intracellular killing activity and the occurrence of increased superoxide anion-and singlet oxygen production, in the absence of increased myeloperoxidase (MPO) production, suggests that the enhanced, MPO-independent, oxidative mechanism may play an important role in the candidacidal activity. Specific rabbit antibodies played no role in the phagocytosis and intracellular killing of C. albicans. These results suggest that PMNs and factors involved in the alternative complement pathway, and lymphokine-activated macrophages play major roles in the protection of mice against C. albicans infection.Our previous study (11) led us to suggest that cell-mediated immunity plays a major, and humoral immunity a side, role in the murine defense mechanism(s) against Candida albicans infection. However, at present, the role of phagocytosis and intracellular killing of C. albicans in nonspecific host defense and specific immunity are not fully understood, although their roles in the host defense mechanisms against several bacterial infections are well established (35). Therfore, we examined the in vitro phagocytic and intracellular killing activity of normal mouse phagocytes, and the effect of specific rabbit antibody and mouse lymphokines together with that of mouse serum chelation, on the in vitro phagocytosis and intracellular killing of C. albicans. We found that peripheral blood leukocytes (PBLs) killed these organisms more effectively than peritoneal macrophages (PMPs) and that while antibody did not play a role in the in vitro phagocytosis by PBLs and 807