[Identification of Malassezia species associated with seborrheic dermatitis using PCR-RFLP]

Malassezia is a lipophilic and dimorphic fungus which has different species. Some of them can be found as natural flora on the skin and in some conditions may cause seborrheic dermatitis. The aim of this study was to identify Malassezia species associated with seborrheic dermatitis in Iranian patients, using PCR-RFLP. In this study out of 79 patients with seborrheic dermatitis, isolates of 70 patients were positive for Malassezia species using PCR-RFLP. The Internal Transcribed Spacer 2 [ITS2] region was amplified by PCR employing the ITS3 and ITS4 primers and the restriction endonucleases AluI, BanI and MspAI were selected for producing distinct RFLP patterns. M. globosa [48.6%], M. furfur [40.0%], M. slooffiae [8.6%] and M. sympodialis [2.8%], were the microorganisms responsible for the infection among participants. M. pachydermatis, M. japonica, M. dermatis, M. restricta, M. obtuse, M. nana and M. yamatoensis were not isolated from any samples. Our findings suggest that the most common Malassezia species associated with seborrheic dermatitis was M. globosa, followed by M. furfur

[Identification of Malassezia species associated with pityriasis versicolor using PCR-RFLP]

Malassezia is a lipophilic and dimorphic fungus which has different species. Some of them can be found as natural flora on skin and in some conditions may cause pityriasis versicolor. The aim of this study was to identify Malassezia species associated with pityriasis versicolor in Iranian patients, using PCR-RFLP. In this study out of 65 patients with pityriasis versicolor to have pityriasis versicolor,isolates of 60 patients were positive. Malassezia species. using by PCR-RFLP. The Internal Transcribed Spacer 2 [ITS2] region was amplified by PCR employing the ITS3 and ITS4 primers and the restriction endonucleases AluI, BanI and MspAI were selected for producing distinct RFLP patterns. M. furfur [36.7%], M. globosa [30.0%], M. sympodialis [20.0%], M. slooffiae [8.3%], M. restricta [3.3%] and M. obtusa [1.7%] were the microorganisms responsible for the infection among participants. The M. sympodialis infection was strongly correlated with the female gender [P=0.02]. Our findings suggest that, the most common Malassezia species associated with pityriasis versicolor was M. furfur, followed by M. globosa

[Culturing of kerationocytes in serum-free medium]

To establish a simple method for preparation of pure epidermal cell in serum-free medium, without co culturing with lethally irradiated 3T3 cells. A piece of skin biopsy was taken from a rat. After trypsinazation to separate epidermal layer, the epidermis was cut into explants 1x1 mm and were laid at 50 mL on tissue cultured flasks. Then, the explants were covered with serum-free keratinocyte growth medium, while the culture medium was changed every two days. The keratinocyte colonies were expanded in flasks and proliferated to 70% confluency on day 21. After two subcultures, the cells were frozen. Pure epidermal cells are established by this technique in a serum-free keratinocyte growth medium, under feeder cell-free condition. Culturing of keratinocytes in serum-free medium is proved to be a useful and simple method for keratinocyte isolation. The successful culturing of keratinocytes from a small skin biopsy can be useful in the treatment of major burn wounds

Development of an allogeneic cultured dermal substitute using a standard human fibroblast bank

Fibroblasts are mesenchymal cells that can be readily cultured in the laboratory and play a significant role in epithelialmesenchymal interactions, secreting various growth factors and cytokines that have a direct effect on epidermal proliferation, differentiation and formation of extracellular matrix. They have been incorporated into various tissue-engineered and used for a variety of clinical applications, including the treatment of burns, chronic venous ulcers and several other clinical applications in dermatology and plastic surgery. Isolated fibroblasts by the enzymatic process from foreskin were cultivated successively in a culture medium to establish cell banking. Foreskin and the last subcultured cells were checked for HBV, HCV, HIV, HSV I, HSV II, HTLV I, HTLV II, EBV, CMV, Treponema Pallidum, Mycoplasma sp. and Clamydia. The 1[st], 5[th] and 10[th] subcultured cells were processed for immunocytochemistry studies using a panel of monoclonal antibodies including antibodies to MHC class I and II antigens for ensuring the elimination of superficial cell antigens during cultivation. Subcultured cells were karyotyped to find any chromosomal abnormalities. The best passages were chosen for culturing on silicone sheets provided by the Iran Polymer and Petrochemical Institute. Evaluation for bacteria and viruses by molecular methods was negative. Karyotyping of cultured fibroblasts after the 10[th] passage showed some abnormalities. HLA expression was imperceptible in the cells obtained from the 10[th] sub-culture. The best passages were from 5th to 10[th] for banking and culturing on silicone sheets. Expression of HLA on fibroblast surfaces was diminished during subculturing. To prevent chromosomal abnormalities in fibroblast passaging, we should select the best colony that is expected to be chromosomally stable with the least antigenicity. In our study, the 5[th] to 10[th] sub-cultures were the best cells for the purpose of grafting and acceleration of the wound healing

Establishment of a primary cell culture of human fibroblast in Iran

Human fibroblasts are the part of the dermis that secrete extracellular matrix for the purpose of tissue repair. Culturing fibroblasts, which leads to formation of a monolayer of these cells, is used for treating various conditions including thermal burns and other skin defects such as diabetic and varicose vein leg ulcers. Therefore, we aimed at developing a fibroblast bank to accomplish multiple goals including skin repair in defects such as burns and ulcers and also performing various research projects on these cells in order to further study of the mechanisms involved in wound healing, rejuvenation and medication effects. We initially developed primary cultures of skin fibroblasts in a DMEM medium. These primary cultures were formed by washing and trypsinizing foreskin specimens followed by separation of epidermis from dermis and cutting the dermis into small pieces. In about 10 days, a monolayer of fibroblasts was formed. We were able to develop the fibroblast bank successfully and to initiate other projects utilizing this bank. With these cultured cells, we would be able to perform different research projects including studying the mechanisms of wound healing, rejuvenation, drug affects, inflammatory mediators, growth factors, etc. Moreover, further progress in this field will result in our independence from requesting these cells from external sources