In vitro Evaluation of Photodynamic Effects Against Biofilms of Dermatophytes Involved in Onychomycosis.

Dermatophytes are the most common cause of onychomycosis, counting for 90% fungal nail infection. Although dermatophyte pathogens are normally susceptible to antifungal agents, onychomycosis often results in refractory chronic disease, and the formation of biofilms frequently underlines the inadequate responses and resistance to standard antifungal treatment. Numerous in vitro and in vivo antimicrobial photodynamic therapy (aPDT) studies have shown biofilm eradication or substantial reduction, however, such investigation has not yet been expanded to the biofilms of dermatophytes involved in onychomycosis. To shed a light on the potential application of aPDT in the clinic management of onychomycosis, in particular with the manifestation of dermatophytoma, we investigated photodynamic effects on the viabilities and the drug susceptibilities of the biofilm of dermatophytes in vitro. Here, methylene blue at the concentration of 8, 16, and 32 µg/ml applied as photosensitizing agent and LED (635 ± 10 nm, 60 J/cm2) as light source were employed against six strains of Trichophyton rubrum, ten strains of Trichophyton mentagrophytes and three strains of Microsporum gypseum isolated from clinical specimens. Our results indicated highly efficient photodynamic inhibition, exhibiting CFU (colony forming unit) reduction up to 4.6 log10, 4.3 log10, and 4.7 log10 against the biofilms formed by T. rubrum, T. mentagrophytes, and M. gypseum, respectively. Subjected biofilms displayed considerable decreases in SMICs (sessile minimum inhibitory concentrations) to multiple antifungal agents when compared with untreated groups, indicating the biofilms of dermatophytes became more susceptible to conventional antifungal drugs after aPDT. Additionally, the obliteration of biofilm after aPDT could be observed as shattered and ruptured structures being evident in SEM (Scanning Electron Microscopy) images. These findings suggest that aPDT is an attractive alternative treatment holding great promise for combating recalcitrant onychomycosis associated with the biofilm formation.

[Therapeutic effects of photocatalytic nano-TiO2on human nasopharyngeal carcinoma xenografts in nude mice].

OBJECTIVE: To study the therapeutic effect of photocatalytic nano-TiO2 on nasopharyngeal carcinoma xenograft in nude mice and underlying mechanism. METHODS: Nude mice bearing human nasopharyngeal carcinoma xenograft were randomly divided into six groups: nano-TiO2 + UV irradiation (with gradient concentration of nano-TiO2); nano-TiO2 alone and UV irradiation alone and blank control. The nano-TiO2 suspension was injected into xenografts, and 24 h after UV light with the wave length of 330 - 400 nm, all the xenografts were removed and sectioned for HE staining. Ultrastructure and apoptosis of tumor cells in the xenografts were observed by transmission electron microscope (TEM). The expression of Caspase-3 was examined immunohistochemical staining and the apoptosis was detected with TUNEL. RESULTS: Pathological analysis showed significant inflammatory responses (grade II and III) with local necrosis occurred in tumor tissues after nano-TiO2 photodynamic therapy, but not in the negative control and blank control. TEM showed the nano-TiO2 particles entered into the cytoplasm and the nucleus of tumor cells and many tumor cells had morphological changes for apoptosis. Significant positive expression of Caspase-3 and TUNEL-positive cells were found in the the xenografts with the treatments of nano-TiO2 + UV irradiation compared to control (P < 0.01), which were enhanced with the increases in nano-TiO2 concentration (P < 0.01). CONCLUSION: Photocatalytic nano-TiO2 can inhibit the growth of nasopharyngeal carcinoma xenograft in nude mice by inducing Caspase-3 expression and apoptosis in the tumor cells.

[Effect of Bugu Mixture on all-trans retinoic acid-induced apoptosis of bone marrow stromal cells].

OBJECTIVE: We used the SD rat's bone marrow stromal cells (BMSCs) cultured in vitro to observe the effects of Bugu Mixture on the apoptosis and to explore the molecular biologic mechanism of the treatment of osteoporosis with Bugu Mixture. METHODS: BMSCs were separated from the bones of the extremities of SD rats in vitro. The morphologic changes, the apoptosis cell cycles, the mitochondrion membrane potential changes, and the Bcl-2 and Bax gene expression were observed, and the effects of Bugu Mixture on the course of cell apoptosis were evaluated. RESULTS: The earlier use of Bugu Mixture could decrease the cells blocked in G0/G1 phase, and promote their synthesis of DNA in S phase. The expression of Bcl-2 was higher in the Bugu Mixture group than that in the all-trans retinoic acid (ATRA) induced group, and the expression of Bax was lower in the Bugu Mixture group than that in the ATRA induced group. The mitochondrion membrane potential descended significantly in the Bugu Mixture group than that in the ATRA induced group. CONCLUSION: The mechanism of the treatment of osteoporosis with Bugu Mixture is that the earlier use of Bugu Mixture can decrease the amount of apoptostic cells induced by ATRA, thus promoting the cell mitosis and restraining the apoptosis. It can also act as a protector to Bcl-2 located on the mitochondrion membrane. By preventing the transferring of the Bax protein from cell-plasma to mitochondrion membrane, it takes the advantage of Bcl-2 in forming Bcl-2/Bax homodimer so as to prevent the opening of the permeability transition pore to avoid the changing of mitochondrion membrane potential and the destruction of biosynthesis caused by the mitochondrion release of apoptosis inducing factors and to reach the objective of restraining apoptosis.

Ultrastructural and immunohistochemical studies on Trichomonas vaginalis adhering to and phagocytizing genitourinary epithelial cells.

BACKGROUND: Trichomonas vaginalis (T. vaginalis) belongs to a common sexually transmitted disease pathogen causing genitourinary trichomoniasis in both sexes. We investigated the pathogenetic mechanism of genitourinary trichomoniasis. METHODS: Cultured T. vaginalis bodies were injected into the vaginas of rats, or incubated with genitourinary epithelial cells of female subjects, male subjects, and sperm. The ultrastructural and microscopic changes were observed via transmission and scanning electron microscopy and through microscopic histochemistry. RESULTS: Groups of T. vaginalis adhered to PAS positive columnar cells at the surface of stratified epithelium in the middle and upper portions of the vaginas. They also traversed under these cells. The parasites were shown to be PAS, cathepsin D, and actin positive, and they could release hydrolase into the cytoplasm of adhered epithelial cells. In the amebiform T. vaginalis, microfilaments were arranged into reticular formation. Similar phenomena were found during the interaction of T. vaginalis with host cells, both in vitro and in vivo. Usually several protozoa adhered to an epithelial cell and formed polymorphic pseudopodia or surface invaginations to surround and phagocytize the microvilli or other parts of the epithelial cytoplasm. Adhesion and phagocytosis of sperm by the protozoa occurred at 15 - 30 minutes of incubation. Digestion of sperm was found at 45 - 75 minutes and was complete at 90 - 105 minutes. CONCLUSIONS: T. vaginalis tends to parasitize at the fornix of the vagina, because this is the site where columnar cells are rich in mucinogen granules and their microvilli are helpful for adhesion and nibbling. T. vaginalis possesses some invading and attacking abilities. Shape change, canalization, encystation, phagocytosis, digestion, the cell coat, cytoskeleton, and lysosome all play important roles in the process of adhesion. They have two methods of phagocytosis: nibbling and ingestion. Genitourinary epithelium may be injured directly by the digestive action of hydrolases, phagocytosis, and the mechanical action of pseudopodia.

[Electron microscopic study on Trichomonas vaginalis adhering to and phagocytizing male genitourinary epithelial cells].

OBJECTIVE: To observe Trichomonas vaginalis (T. vaginalis) adhering to and phagocytizing male genitourinary epithelial cells in order to study the pathogenetic mechanism of male trichomoniasis. METHODS: Cultured T. vaginalis bodies were incubated with male genitourinary epithelial cells, and then the ultrastructure was observed with transmission electron microscopy. RESULTS: T. vaginalis adhered to epithelial cells like amoeba, and formed pseudopodium or surface invagination surrounding or nibbling other parts of the epithelial cytoplasm. CONCLUSIONS: T. vaginalis has the speciality of adhering to and phagocytizing to male genitourinary epithelial cells. Genitourinary epithelial cells may be injured directly by the phagocytosis of T. vaginalis. Attention has to be paid to the correlation.

[The quantitative analysis of protein particles of erythrocyte membrane from Duchenne muscular dystrophy patients and the gene carriers].

OBJECTIVE: To study the changes of the intramembrane protein particles of erythrocyte from Duchenne muscular dystrophy (DMD) patients and the gene carriers and to explore the pathogenesis of DMD and the diagnostic value of erythrocyte freeze-fracture technology. METHODS: The fixed erythrocyte mass was treated to form replica membrane by means of the freeze-fracture technology. Then the replica membrane was observed and a picture was taken under electron microscope. The protein particles of extracellular face(EF) and protoplasmic face(PF) per square were counted. The statistical comparative analysis was performed. RESULTS: The protein particle counts of EF face and PF face of erythrocyte membrane from DMD patients and DMD carriers decreased obviously in comparison with the normal control group (P<0.001). CONCLUSION: The erythrocyte freeze-fracture electron microscopic technology may serve as a method for accessory examination of diagnosing DMD patients and a method for detecting DMD carriers. This investigation material supplies reliable evidence for the theory of the systemic membrane defect of DMD.