No abstract

Around 90 % of chronic dermatophyte infections are caused by the fungi Trichophyton mentagrophytes and Trichophyton rubrum. One of the causes of the chronic infection resides in the immunosuppressive effects of the cell-wall components of these organisms. Therefore we have attempted to identify the chemical structure of galactomannan, one of the major cell-wall components. The cell-wall polysaccharides secreted by T. mentagrophytes and T. rubrum were isolated from the culture medium and fractionated into three subfractions by DEAE-Sephadex chromatography. Analysis of each subfraction by NMR indicated that there are two kinds of polysaccharides present, i.e. mannan and galactomannan. The mannan has a linear backbone consisting of α1,6-linked mannose units, with α1,2-linked mannose units as side chains. The core mannan moiety of the

Testing of pharmacological agents that affect growth of epidermal keratinocytes (EK) requires a standardized assay. We have developed an assay measuring net effects of stimulatory (e.g. growth factors), inhibitory (e.g. methotrexate) or toxic (e.g. Triton X-100) compounds. The amount of crystal violet staining viable EK attached to the wells of standard 96-well microplates is measured in situ using an ELISA plate reader. Optical density readings are directly converted into cell counts by computer software. Counts obtained by this method strongly correlate with the results obtained using the [³H]thymidine uptake assay and direct cell counts. The assay standardizes measurements of nonimmortalized EK lines with different innate proliferative properties and allows accurate quantitation of EK numbers in the range of 2,500–500,000 EK/well.

Resistance to dermatophyte infections has been shown to be mediated in part by T lymphocytes. The dermatophyte antigens recognized by human T lymphocytes and their degree of cross-reactivity were analyzed. Dermatophyte-responsive T-cell lines were generated by in vitro sensitization to crude fungal extracts obtained from Trichophyton rubrum, Trichophyton tonsurans, Microsporum canis and Epidermophyton floccosum. Proliferation was measured by incorporation of 3H-thymidine. The human T-cell lines responded to fungal extracts derived from these various dermatophyte species, demonstrating the recognition of cross-reactive antigens by human T cells. However, the T cells were dermatophyte-specific as they did not respond to herpes antigen, nor did herpes-specific T cells derived from the same donors respond to dermatophyte antigens. The mannose-rich glycoprotein fraction (mannan) isolated from T. rubrum was able to induce proliferation of T-cell lines generated by stimulation with various fungal extracts. Furthermore, a T-cell line generated by stimulation with mannan derived from T. rubrum proliferated in response to extracts from various fungal species, indicating that a major cross-reactive dermatophyte T-cell antigen was present in the mannose-rich glycoprotein fraction.