Anti-proliferative and anti-inflammatory effects of 3ß,6ß,16ß-Trihydroxylup-20(29)-ene on cutaneous inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: 3ß,6ß,16ß-Trihydroxylup-20(29)-ene (TTHL) is a triterpene isolated from the flowers of Combretum leprosum, a plant used in folk medicine in the north of Brazil for the treatment of skin disorders. AIM OF THE STUDY: In the present study, TTHL was evaluated as a potential topical anti-inflammatory and anti-proliferative agent through in vivo and in vitro models. MATERIAL AND METHODS: Anti-inflammmatory and anti-proliferative effects of TTHL were assessed using Swiss mice in acute and chronic models of skin inflammation induced by 12-O-tetradecanoylphorbol-acetate (TPA) application. Anti-proliferative activity was proved through in vitro experiments with the HaCaT human keratinocyte cell line. RESULTS: Treatment with TTHL inhibited inflammatory parameters such as oedema formation and cellular infiltration in acute and chronic models. In the chronic model, TTHL also inhibited epidermal hyperproliferation, as evidenced by reduction of epidermis thickness and proliferating cell nuclear antigen expression. The anti-proliferative effect was confirmed by the capability of TTHL in reducing the proliferation and inducing cell apoptosis of HaCaT cells. Suggesting a mechanism of action, TTHL showed activation of corticosteroid receptors, but without the induction of corticosteroid-related cutaneous side effects. CONCLUSION: Our results demonstrate consistent anti-inflammatory and anti-proliferative activity and assign TTHL as a valuable tool in the development of a new treatment for skin inflammatory and proliferative diseases, such as psoriasis.

Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes.

BACKGROUND: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. AIM: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. METHODS: The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed. RESULTS: Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model. CONCLUSION: We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.

Topical phenytoin for wound healing.

Oral phenytoin is used widely for the treatment of convulsive disorders and about half the patients treated develop gingival overgrowth as a side effect. The apparent stimulatory effect has prompted its assessment in wound healing. Studies have shown topical phenytoin to promote healing of decubitus ulcers, venous stasis ulcers, diabetic ulcers, traumatic wounds, burns, and leprosy trophic ulcers. The mechanism of action has been postulated to be multifactorial. The present literature indicates that topical phenytoin deserves further investigation as a wound-healing agent in controlled dose-finding clinical trials.

Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing.

Recombinant granulocyte/macrophage-colony-stimulating factor (rGM-CSF), prepared from Chinese hamster ovary (CHO) cells and Escherichia coli, was administered to 35 patients with the borderline and polar lepromatous forms of leprosy by the intradermal and subcutaneous routes at doses of 7.5-45.0 micrograms/d for 10 d. With each of these doses and routes, increases in the number of circulating eosinophils were noted. After the intradermal injection, the local skin sites demonstrated zones of roughening and micronodularity that appeared within 24-48 h and persisted for more than 6 d. Reinjection of sites led to enhanced areas of epidermal reaction. GM-CSF prepared from CHO cells was a more potent inducer of this effect. GM-CSF given by the subcutaneous route, at higher doses, failed to initiate these changes. At the microscopic level, the epidermis became thickened (+75%) with increased numbers and layers of enlarged keratinocytes. These contained increased numbers of ribosomes and prominent nucleoli, and were imbedded in a looser meshwork of the zona Pellucida. The modified keratinocytes remained MHC class II antigen negative throughout the course of the response. A major change in the dermis was the progressive accumulation of CD1+, Birbeck granule-positive cells. These Langerhans were recognizable at 48 h after intradermal injection and reached maximum numbers by 4 d. During this period the number of epidermal Langerhans cells remained relatively constant. No increment in dermal Langerhans cells occurred when GLM-CSF was injected by the subcutaneous route. No appreciable increase in the numbers of T cells and monocytes was noted, and granulocytes and eosinophils were largely present within the dermal microvasculature. 4-mm punch biopsies taken from injected sites and adjacent controls were compared in terms of the rapidity of wound healing. 22 of 26 sites demonstrated more rapid filling and hemostasis, whereas four were equivalent to controls. We conclude that rGM-CSF, when introduced into the skin, leads to enhanced keratinocyte growth, the selective recruitment of Langerhans cells into the dermis, and enhanced wound healing of the prepared site. There was no evidence of an enhanced cell-mediated response to Mycobacterium leprae, and bacillary numbers remained unchanged.