Investigation of conditions involved in the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment.

BACKGROUND: Photodynamic treatment (PDT) refers to a treatment with light-activated agents (photosensitizers) in combination with visible light and molecular oxygen. Recently, we have demonstrated that the porphyrins, 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester (DP mme) are excellent photosensitizers to be used against Trichophyton rubrum both in vitro and ex vivo. OBJECTIVES AND METHODS: The objective of this study was to investigate the key factors involved in PDT efficacy of both photosensitizers in an ex vivo situation during different fungal growth stages using a recently developed ex vivo model. The study focused on the influence of pH and ion strength of incubation media, photochemical properties of the photosensitizers (spectra and singlet oxygen production), and the effect of several scavengers of reactive oxygen species (sodium azide, histidine, mannitol) and phenylmethylsulphonylfluoride (keratinase inhibitor) on the PDT efficacy. RESULTS AND CONCLUSIONS: The results show that an optimal pH and low concentrations of calcium are crucial for a selective binding of Sylsens B to the fungus, leading to an increased PDT efficacy. This selective binding to T. rubrum cannot be accomplished for DP mme. It can be concluded that the prerequisite for successful treatment is a use of a low molarity solution of pH 5, supplemented with a chelating agent and a keratinase activity-repressing agent. Under these conditions, PDT with Sylsens B inactivates, initially via singlet oxygen, effectively the fungus in different fungal growth stages.

Ferrochelatase cDNA delivered by adenoviral vector corrects biochemical defect in protoporphyric cells.

Protoporphyria is generally an autosomal dominant disease characterized genetically by mutations in the ferrochelatase gene. The interaction between the wild-type and mutant ferrochelatase protein is unknown. The aim of this study was to evaluate the ability to correct the enzymatic and biochemical defects in cells from patients with protoporphyria, using a replication-defective human adenovirus for gene transfer. Overexpression of ferrochelatase was accomplished by construction of a vector in which expression of the wild-type ferrochelatase cDNA was driven by the constitutive cytomegalovirus (CMV) promoter, introduction and packaging of the cDNA into human adenovirus dl309, and transduction of normal and protoporphyric fibroblasts. Fibroblasts from controls and patients were infected with the ferrochelatase adenovirus or a control adenovirus and assayed for ferrochelatase activity and the accumulation of protoporphyrin upon challenge with the precursor delta-aminolevulinic acid (ALA). At a multiplicity of infection (moi) of 10, greater than 85% of both the wild-type and protoporphyric fibroblasts were infected. The recombinant adenovirus increased the ferrochelatase protein content and activity in the wild-type and protoporphyric fibroblasts with equal efficiency. Therefore, the presence of the mutant ferrochelatase protein did not inhibit the ferrochelatase activity expressed by the transgene.