Biomedical subjects
L Polonelli
Publications and source records attributed to L Polonelli.
Cytological immunodetection of yeast glycoprotein secretion.
Expression of antigenic epitopes shared by secreted yeast glycoproteins was studied using specific immunological probes. Application of cytological and ultrastructural methods of immunodetection, employing monoclonal antibodies, permitted us to localize these glycoproteins in the cytoplasm, through the cell wall and at the yeast cell surface. Importance of glycosylation-secretion relationships were evaluated in the secretion process of these molecules. The cell wall crossing and the cell surface distribution of antigenic glycoproteins was described in immunoelectron microscopy and immunofluorescence. Some preferential secretion "ways" were suspected through the yeast cell wall leading to an heterogenous distribution of cell surface glycoproteins destined to be excreted into the medium. Antigenic variability of cell wall glycoproteins expression was discussed in relation with the glycoprotein secretion.
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.
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.
Nosocomial outbreak of systemic candidosis associated with parenteral nutrition.
Eight patients in two surgical units developed systemic candidosis during a 40-day period from June 5 to July 13, 1987 (in five cases Candida albicans was identified). Three of them died. All cases belonged to a group of 27 patients receiving parenteral nutrition (PN), while among the 108 patients who did not receive PN, no cases were observed (p = .000001). Candida was cultured from two PN bags administered to the cases. A specialized nutrition nurse was responsible for the PN compounding and for maintaining administration sets in the two wards involved. An epidemiological investigation, in which 19 uninfected patients who had had PN were used as controls, showed no significant difference between cases and controls except that lipids were more frequently added to bags administered to cases (p = .0005). Furthermore, the bags administered to cases contained a higher average number of multidose constituents (p = .0008) when the comparison was focused on the two days before the onset of symptoms. Given the favorable medium provided by lipids, even a low level contamination of PN solutions during compounding and/or administration could have been responsible for the exposure of cases to multidose vials suggests, although not conclusively, that an extrinsic contamination occurred during compounding. Six isolates of C albicans were available from four cases. C albicans was cultured from the pharyngeal swabs of two physicians and three nurses, including the specialized nutrition nurse.
Biotyping of bacterial isolates using the yeast killer system.
Forty-four presumptive killer yeasts were tested against bacterial isolates, including rapid-growing gram-positive and gram-negative bacteria, as well as slow-growing bacteria, such as the mycobacteria. A killer system, based on the patterns of bacterial susceptibility to the action of nine selected killer yeasts, was developed for epidemiological purposes. The killer system, previously standardized for yeasts and hyphomycetes, was adapted to the specific growth conditions of the bacterial isolates. The results obtained confirm that susceptibility to the yeast killer phenomenon is widespread among microorganisms unrelated to yeasts and that it could form the basis for a convenient and adaptable biotyping method in microbiological laboratories.
Studies on the epidemiology of Aspergillus fumigatus infections in a university hospital.
The impact of the airborne opportunistic fungus pathogen, Aspergillus fumigatus in patients hospitalized at the university hospital of Parma, Italy, and its outpatients was investigated during a period of six months. Sputum and bronchial washings were collected from 986 patients. The 2,437 specimens were culturally examined for the presence of A. fumigatus regardless of the patient's clinical diagnosis. This fungus was isolated from 32 patients (3.2%). Subsequently, immunological tests for aspergillosis were performed on 22 of these culture-positive patients. Eight of these patients (36.4%) were serologically proven to be affected by aspergillosis. The 32 A. fumigatus isolates were epidemiologically investigated by the killer system to determine the mode of spread of these infections. Among the patients, fifteen different biotypes were differentiated, and their value in studying the epidemiology of aspergillosis in the hospital environment was investigated.
Serological study of yeast killer toxins by monoclonal antibodies.
Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.
Biotyping of pathogenic fungi by the killer system and with monoclonal antibodies.
Biotyping of pathogenic yeasts and hyphomycetes based on their susceptibility to selected killer yeasts and their reactivity with monoclonal antibodies are described. Both methods were used to differentiate fungi isolated from patients providing valuable epidemiological information on mycotic infections. The functional biotyping obtained with the two systems and the conventional auxenographic biocoding approaches commercially available for opportunistic yeasts are comparatively evaluated. The potential for biotyping of industrial fungal isolates is also discussed.