Biomedical subjects
M Gerloni
Publications and source records attributed to M Gerloni.
Anaerobic yeast killer systems.
The influence of anaerobic conditions on the expression of the killer phenomenon of several yeast isolates belonging to recognized killer systems coded by different genetic determinants (Pichia spp., Kluyveromyces lactis, Saccharomyces cerevisiae) was studied. Anaerobiosis influenced the activity of killer toxins from some individual isolates of the genera Pichia and Saccharomyces on sensitive strains of P. anomala, K. lactis and Candida albicans. However, no influence was detectable on a S. cerevisiae sensitive isolate. Thus, anaerobic conditions seem to interfere more with the metabolic process of sensitive strains than with toxin production by killer yeasts. The selection of a panel of killer yeasts, able to display their activity against reference sensitive yeast isolates under anaerobic conditions in a medium that favored the growth of anaerobes, allowed the use of the killer system to type Bacteroides fragilis isolates for epidemiological purposes.
Genomic studies on killer yeasts belonging to the genus Pichia.
Twenty-four species belonging to the genus Pichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA. Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii and Candida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of the EcoRI and XhoI RFLP patterns with P. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis and C. albicans). However, one hybridized band, located at 3.9-4.1 Kb, seems to be peculiar to the Pichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.
Ultrastructural immunodetection of a Pichia anomala killer toxin: a preliminary study.
A monoclonal antibody (mAb KT4), produced against a Pichia anomala killer toxin, was used to study the secretion process of toxin producing cells. The indirect immunofluorescence assay, performed with large concentrations of mAb KT4, showed a homogeneous distribution of the epitope at the cell surface of the P anomala cells. When increasing dilutions of mAb KT4 were employed, a 'punctuated' labeling appeared on the yeast's cell wall which suggested a heterogeneous secretion of the killer toxin. Similar labeling was also observed by immunodetection on live yeast cells held in buffered suspension. These results confirmed that 'punctuated' labeling was not an artefact due to a distortion of the cell's shape by having been dried on glass slides. Indirect immunodetection was performed in electron microscopy on ultra-thin sections of cells embedded in Araldite resin. The labeling thus obtained showed both the presence of the epitope in the cytoplasm and its sensitivity to strong glutaraldehyde fixation. Indirect immunodetection, performed on ultra-thin frozen sections, showed a cytoplasmic and cell wall labelling. However, the amount of gold particles observed in the cell wall was too low to confirm the heterogeneous killer toxin secretion observed in immunofluorescence. In this case, killer cells were fixed with a low concentration of glutaraldehyde which preserved the structure of the epitope complementary with mAb KT4.
Improved immunoblotting for the detection and quantitative analysis of antibodies to human immunodeficiency virus.
Detection of antibodies to single HIV-1 proteins was performed by modified Western-blotting procedure. The use of [35S]-Streptavidin instead of Avidin-peroxidase conjugate greatly enhances the sensitivity of the method and enables useful quantitative analysis of results.
Diagnostic potential of IgA coated Candida cells in mucous membrane candidiasis.
The significance of in vivo IgA coated yeast cells for the diagnosis of candidiasis of the oral and vaginal mucosal membranes was evaluated by direct immunofluorescence in 70 patients with or without clinical symptoms, shown to be positive for yeast growth in the cultural test. Most of the patients with clinically suspected candidiasis of the mucosal membranes gave positive results by serologic assays in contrast to the majority of symptomless patients. The diagnostic approach proved to be essentially consistent with the clinical signs, persistence of infection, response to antifungal therapy and quantitative cultural data.
Differential toxinogenesis in the genus Pichia detected by an anti-yeast killer toxin monoclonal antibody.
The differential toxinogenesis of 25 isolates belonging to species of the potential yeast killer genus Pichia that were previously classified in the genus Hansenula was comparatively demonstrated by two serologic techniques (indirect immunofluorescence and double immunodiffusion) by using a monoclonal antibody against a yeast killer toxin produced by a selected strain of Pichia anomala (UCSC 25F). The killer phenotypes of the Pichia isolates were evaluated by their ability to kill each other. The results, although of insufficient taxonomic value for a reliable separation of either species or genera, attest to the genomic heterogeneity for the killer character in the genus Pichia as well as the presumptive dual killer/sensitive identity for each single isolate.
'Antibiobodies': antibiotic-like anti-idiotypic antibodies.
Pathogenic micro-organisms such as Candida albicans may be susceptible to the activity of antimicrobial products like yeast killer toxins due to the presence of specific cell wall receptors for these agents. Anti-idiotypic antibodies (anti-Ids) were produced that competed for these receptors with the yeast killer toxin of a strain of Pichia anomala. We report here that affinity chromatography purified anti-Ids may kill C. albicans cells in vitro which are susceptible to the activity of the yeast killer toxin, as well as P. anomala killer cells which are obviously immune to their own toxin despite possessing specific cell wall receptors which can be detected by indirect immunofluorescence with anti-Ids. We propose that these conceptually new antimicrobial immunoglobulins acting as antibiotics be called 'antibiobodies'.
Interfaces of the yeast killer phenomenon.
A new prophylactic and therapeutic antimicrobial strategy based on a specific physiological target that is effectively used by killer yeasts in their natural ecological competition is theorized. The natural system exploited is the yeast killer phenomenon previously adopted as an epidemiological marker for intraspecific differentiation of opportunistic yeasts, hyphomycetes, and bacteria. Pathogenic microorganisms (Candida albicans) may be susceptible to the activity of yeast killer toxins due to the presence of specific cell wall receptors. On the basis of the idiotypic network, we report that antiidiotypic antibodies, produced against a monoclonal antibody bearing the receptor-like idiotype, are in vivo protecting animals immunized through idiotypic vaccination and in vitro mimicking the antimicrobial activity of yeast killer toxins, thus acting as antibiotics.
[Diagnostic significance of secretory IgA coated yeast cells in oral candidiasis].
The significance of in vivo IgA coated yeast cells for the diagnosis of candidiasis of the oral mucosal membranes was evaluated by direct immunofluorescence in 42 patients with or without clinical symptoms, shown to be positive for yeast growth in the cultural test. Most of the patients with clinically suspected candidiasis of the mucosal membranes gave positive results by serologic assays in contrast to the majority of symptomless patients. The diagnostic approach proved to be essentially consistent with the clinical signs, persistance of infection, response to antifungal therapy and quantitative cultural data.
Botryomyces caespitosus as an agent of cutaneous phaeohyphomycosis.
The second known case of a skin infection caused by Botryomyces caespitosus is reported. This case has made it possible to describe the characteristics of this fungus in vivo and to establish it as another agent of phaeohyphomycosis.
Detection by immunofluorescent anti-idiotypic antibodies of yeast killer toxin cell wall receptors of Candida albicans.
Yeast killer toxin cell wall receptors of Candida albicans were observed by indirect immunofluorescence using an affinity purified rabbit anti-idiotypic antiserum. The antiserum had been raised against a monoclonal antibody neutralizing the in vitro activity of a killer toxin produced by a selected strain of Hansenula anomala UCSC 25F. This simple procedure permitted the location of killer toxin cell wall receptors in various morphological phases of the yeast cells. The use of the indirect immunofluorescence technique with anti-idiotypic antibodies may have potential value in determining the occurrence of killer toxin receptors in other microbial systems.
Production of yeast killer toxin in experimentally infected animals.
The ability of a killer yeast (Pichia anomala, UCSC 25F) to produce toxin in vivo was demonstrated, for the first time, in tissues of normal and immunosuppressed experimentally infected mice by means of a fluorescent antibody technique and a killer toxin specific monoclonal antibody. The possible significance of the findings is discussed.