An unexpected twist in alopecia areata pathogenesis: are NK cells protective and CD49b+ T cells pathogenic?

Natural killer (NK) cells have become a recent focus of interest in alopecia areata (AA) research. To further investigate their role in an established mouse model of AA, lesional skin from older C3H/HeJ mice with AA was grafted to young C3H/HeJ female mice, and NK cells were depleted by continuous administration of rabbit anti-asialo GM1. As expected, this significantly reduced the number of pure NK cells in murine skin, as assessed by NKp46 quantitative immunohistochemistry. Quite unexpectedly, however, the onset of hair loss in C3H/HeJ mice was accelerated, rather than retarded. NK cell depletion was accompanied by a significant increase in the number of perifollicular CD49b+T cells in the alopecic skin of anti-asialo GM1-treated mice. These findings underscore the need to carefully distinguish in future AA research between pure NK cells and defined subsets of CD49b+ lymphocytes, as they may exert diametrically opposed functions in hair follicle immunology and immunopathology.

Preliminary study of activity of the thioredoxin inhibitor pleurotin against Trichophyton mentagrophytes: a novel anti-dermatophyte possibility.

Dermatophyte infections, while not life-threatening, are very common, and there is great interest in developing new antifungal agents. Transcriptional profiling of Trichophyton on keratin has identified some antioxidant genes as induced on this host substrate, including a thioredoxin gene TmTRX1. If thioredoxin is a virulence factor, or necessary for the growth on keratin, thioredoxin inhibitors should act as antifungals. As a first evaluation of this hypothesis, we have tested the activity of a thioredoxin-inhibitory natural product, pleurotin, against a clinical isolate of each of two fungal pathogens: the dermatophyte T. mentagrophytes and Candida albicans. Pleurotin inhibited the growth of the dermatophyte in vitro and in an ex vivo skin model, but had no effect on Candida. It may be possible to develop and optimise thioredoxin inhibitors, some of which are already under study in cancer chemotherapy, as antifungals.

Targeting the calcineurin pathway enhances ergosterol biosynthesis inhibitors against Trichophyton mentagrophytes in vitro and in a human skin infection model.

Fluconazole-FK506 or fluconazole-cyclosporine drug combinations were tested in an ex vivo Trichophyton mentagrophytes human skin infection model. Conidia colonization was monitored by scanning electron microscopy over a 7-day treatment period. The fluconazole-FK506 combination demonstrated the most obvious advantage over single-drug therapy by clearing conidia and protecting skin from damage at low drug concentrations.

Infection stages of the dermatophyte pathogen Trichophyton: microscopic characterization and proteolytic enzymes.

Dermatophytes are pathogenic fungi that infect human skin, nails and hair and cause dermatophytosis. Trichophyton mentagrophytes is one of the most widespread species that belong to this group. Infection of the skin tissues include several stages, i.e., adhesion to the surface of the skin, invasion into the sublayers by the penetration of fungal elements and secretion of enzymes that degrade the skin components. In this study we have followed the morphology of the fungal elements, such as arthroconidia and hyphae, during the adhesion and invasion stages. Skin explants were inoculated with the dermatophyte and observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Skin explants were also inoculated with a transgenic isolate of T. mentagrophytes expressing the green fluorescent protein (GFP). The infected sublayers were investigated by confocal scanning laser microscopy (CSLM). As an adaptation to the tissue environment, the dermatophyte produced long fibrils when it is on the open surface of the stratum corneum, while short and thin fibrils are produced inside the dense sublayers. The short and long projections might have a role in adhesion. Invasion may be produced by mechanical and biochemical means. Invasion of the tissue showed hyphal branching and growth in multiple directions. The proteolytic profile was assayed by substrate gel and proteolytic activity. Two serine proteases of similar molecular weight were secreted during growth on the epidermal matrix components keratin and elastin. The dermatophyte may use the proteolytic enzymes to invade the surface and also the deep layer of the skin in immunocompromised patients. Dermatophytes, which are well adapted infectious agents, seem to use their mechanical and biochemical capabilities to invade the skin tissue effectively.

Markers for host-induced gene expression in Trichophyton dermatophytosis.

Dermatophytes are adapted to infect keratinized tissues by their ability to utilize keratin as a nutrient source. Although there have been numerous reports that dermatophytes like Trichophyton sp. secrete proteolytic enzymes, virtually nothing is known about the patterns of gene expression in the host or even when the organisms are cultured on protein substrates in the absence of a host. We characterized the expression of an aminopeptidase gene, the Trichophyton mentagrophytes homolog of the Trichophyton rubrum Tri r 4 gene. The T. rubrum gene was originally isolated based on the ability of the protein encoded by it to induce immediate and delayed-type hypersensitivity in skin tests. T. mentagrophytes Tri m 4 is closely related to Tri r 4 (almost 94% identity at the protein level). Tri m 4 resembles other protease-encoding genes thought to be virulence factors (for example, DPP V of Aspergillus fumigatus). The Tri m 4 protein was detected immunochemically both in fungal extracts and in the culture medium. Expression of the Tri m 4 gene was induced severalfold when T. mentagrophytes was grown on keratin and elastin. Ex vivo, strong induction was observed after culture on blood plasma, but the use of homogenized skin did not result in a significant increase in Tri m 4 transcript levels. In order to identify additional genes encoding putative virulence factors, differential cDNA screening was performed. By this method, a fungal thioredoxin and a cellulase homolog were identified, and both genes were found to be strongly induced by skin extracellular matrix proteins. Induction by superficial (keratin) and deep (elastin) skin elements suggests that the products of these genes may be important in both superficial and deep dermatophytosis, and models for their function are proposed. Upregulation of several newly identified T. mentagrophytes genes on protein substrates suggests that these genes encode proteins which are relevant to the dermatophyte-skin interaction.

Green fluorescent protein (GFP) as a vital marker for pathogenic development of the dermatophyte Trichophyton mentagrophytes.

Skin infections by dermatophytes of the genus Trichophyton are widespread, but methods to investigate the molecular basis of pathogenicity are only starting to be developed. The initial stages of growth on the host can only be studied by electron microscopy, which requires fixing the tissue. This paper shows that restriction-enzyme-mediated integration (REMI) provides stable expression of the green fluorescent protein (GFP) in a clinical isolate of Trichophyton mentagrophytes. Under control of a constitutively active fungal promoter, GFP renders the hyphae fluorescent both in culture and in a recently developed model using human skin explants. Stages of infection and penetration into the skin layers were visualized by confocal microscopy. The stages of infection can thus be followed using GFP as a vital marker, and this method will also provide, for the first time, a means to follow gene expression during infection of skin by dermatophyte fungi.