In vitro effects of fungi isolated from equine hooves on primary human keratinocytes.

This research article investigates how various fungi found on horse hooves affect human skin cells (keratinocytes) in a laboratory setting, gauging the fungi’s toxicity levels, how they cause cell death, and their consequences on cell structure.

Research Methodology

• The team examined the effects of two types of fungi typically associated with skin infections (Microsporum gypseum and Trichophyton mentagrophytes) and four kinds of mould (Scopulariopsis brevicaulis, Alternaria alternata, Geotrichum candidum and Penicillium spp.) on living human skin cell (keratinocyte) cultures in the lab.

Evaluation of Cell Death Rate and Toxicity Level

• The researchers evaluated the rate of programmed cell death (apoptosis) in the infected cells using a colorimetric TUNEL system. This system is designed to detect the fragmentation of nuclear DNA, which is a key indication of apoptosis.
• The toxicity of the individual fungi was measured by quantifying the amount of the enzyme lactate dehydrogenase present in the culture supernatants. This enzyme is released when cells undergo lysis (break down), another indication of cell damage or death.

Assessment of Cell Structures

• They then further examined the cell structures within the fungi-infected keratinocytes using scanning electron microscopy. This tool allowed them to view the structural changes at a very high level of detail.

Results of the Research

• All the fungi displayed high levels of toxicity, but increased apoptosis was observed only in the development of the two dermatophytes and the mould Scopulariopsis brevicaulis.
• The electron microscopy results revealed that all fungi investigated inflicted comparable structural alterations to the cells. Among the fungi studied, Microsporum gypseum was established as the most detrimental.
• The most observable changes were an increase in the loss of cell adhesion, which resulted from a decreased number of cell appendices, along with reduced cell plasticity.