Does the presence of unwanted dermal fibroblasts limit the usefulness of autologous epidermal keratinocyte grafts?

OBJECTIVES: Fibroblasts sometimes occur after enzymatic isolation of epidermis. They proliferate quickly, overgrowing the culture. A pure epithelial culture is essential for therapy using a keratinocyte graft. The aim of this study was to determine the possibility of fibroblast elimination from culture to prevent fibroblast overgrowth and obtain a pure monolayer of keratinocytes. MATERIAL AND METHODS: We analyzed three epidermal-derived cultures. Cells were cultured in medium contained Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12 at a 3:1 ratio with 5% autologous serum and additives. The epithelial culture was confirmed using pancytokeratin MMF. If fibroblast like cells were present, they were removed using 0.01% edetate disodium dihydrate (Na2EDDA). This procedure was repeated until we obtained pure primary keratinocyte cultures. RESULTS: Fibroblast detachment was observed after Na2EDDA treatment. The procedure was performed twice and pure primary cultures of keratinocyte were achieved in two cases. These two cultures maintained their epithelial-like morphology and cytokeratin expression. One culture was treated four times with Na2EDDA with no effect; the morphology of the cultures became fibroblast-like with no observed cytokeratin expression. CONCLUSIONS: Unwanted dermal fibroblasts can be separated from primary keratinocyte cultures during the first few days after the isolation. Cocultures of unwanted dermal fibroblasts and epidermal keratinocytes can be reverted to pure keratinocyte monolayers suitable as grafts for transplantation.

Activity of cathepsin D and alpha(1)-antitrypsin in the blood serum of patients with mammary carcinoma.

UNLABELLED: THE AIM of this study was to determine the activity of cathepsin D and alpha(1)-antitrypsin in the blood serum of patients with mammary carcinoma. PATIENTS AND METHODS: The study was conducted on 52 women operated for a unilateral breast tumor, divided into two groups, according to the number of metastases and tumor size. Cathepsin D activity was determined using the method of Anson, while alpha(1)-antitrypsin activity was determined according to the Eriksson method. RESULTS: Both groups of patients with mammary carcinoma were found to have higher activity of cathepsin D before the treatment compared to healthy females. After the surgery the enzyme activity increased significantly, whereas 6 months after the surgery it generally decreased. The activity of alpha(1)-antitrypsin was significantly lower in patients before the treatment than in the controls, while after 6 months an increase in alpha(1)-antitrypsin activity was observed. The correlation between activity of cathepsin D and alpha(1)-antitrypsin was revealed. High enzyme activity and low alpha(1)-antitrypsin activity may result from the stage of neoplastic transformation. CONCLUSION: The determination of cathepsin D activity together with alpha(1)-antitrypsin activity may serve as useful biochemical marker in monitoring of malignant changes in breast tumor.