Biomedical subjects
R Dover
Publications and source records attributed to R Dover.
Cultured composite skin grafts: biological skin equivalents permitting massive expansion.
After assessment in mice, a biological skin equivalent consisting of cultured fibroblasts in a collagen gel overlain with cultured keratinocytes was applied to three patients who had had tattoos excised. In all patients the grafts took well with good cosmetic results and little or no contracture. A biopsy at 4 weeks showed that the central graft area had a fully differentiated epidermis and a mature dermis without adnexae.
A comparative study of protein synthesis by keratinocytes and fibroblasts in vitro using two-dimensional gel electrophoresis and dual isotope autoradiography.
A procedure is described for the detection of keratinocyte specific proteins. Fibroblasts and keratinocytes were isolated from human skin and radiolabelled in vitro. Samples were separated by two-dimensional polyacrylamide gel electrophoresis to compare the proteins synthesised by the different types of cultured skin cells. Dual label autoradiography of samples radiolabelled with [35S]methionine and [75Se]selenomethionine was used to identify keratinocyte specific proteins. We report 45 keratinocyte-specific components and identify some of these proteins. The differential expression of these proteins and their relevance to epidermal differentiation are discussed.
Heterogeneity and cell cycle analyses from time-lapse studies of human keratinocytes in vitro.
We have analysed the behaviour of cultured epidermal keratinocytes using time-lapse video recordings. We have found evidence for heterogeneity in the behaviour of the cells. Some lines underwent extensive self-renewal, thus expanding the population, while others produced daughter cells that migrated suprabasally and are presumed to have undergone terminal differentiation. We also present kinetic data on the cell cycle times, mitotic durations and post-mitotic residence times. The latter is the time between a cell's birth and eventual suprabasal migration. The data suggest that the 'decision' to migrate is in some way 'programmed' but that the actual migration is a stochastic process. Time-lapse analysis is a very powerful technique for lineage and cell kinetic analysis.
Factor X-activating activity in patients with colorectal carcinoma.
The ability of malignant tissue from 50 patients with colorectal carcinoma to activate blood coagulation factor X directly was compared with samples of adjacent, macroscopically normal colonic mucosa from the same patients, and tissue from four patients with non-malignant bowel disease. The resected tissue was homogenized and incubated with purified factor X and calcium ions. The subsequent generation of activated factor X was measured spectrophotometrically with a chromogenic substrate. Results were expressed as absorbance units, and as the ratio of tumour and normal activities. Factor X-activating activity (FXAA) was present in all normal and malignant tissues tested. FXAA was significantly greater (P less than 0.001) in the tumour homogenates than in the uninvolved tissue. The tumour:normal ratio was significantly (greater than 1.2) elevated in 38 patients (76 per cent). FXAA was not correlated with the degree of differentiation of the tumour, the Dukes' classification of the disease or the exact site of the tumour. There was no difference between the FXAA content of non-involved tissue from the colorectal cancer group and colonic mucosa from patients with non-malignant bowel disease. It is concluded that colorectal carcinomas contain significantly more FXAA than adjacent, non-malignant colonic mucosa from the same subject, but there is no direct evidence for a relationship between procoagulant levels and the extent of malignancy in these patients.
How does skin expansion work?
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Measurement of the rate of epidermal terminal differentiation: expression of involucrin by S-phase keratinocytes in culture and in psoriatic plaques.
At present little is known about the control mechanisms involved in coordinating cell production and maturation in epidermis. To investigate this, we have measured the rate of transit from the proliferative to the terminally differentiating compartment in confluent low-calcium cultures of normal epidermal keratinocytes, using involucrin as a marker of terminal differentiation. We estimate a rate of transit of 3.58 cells/5000 cells/h and a differentiation probability of 0.017, indicating a bias toward self-renewal. Surprisingly, some cells in culture synthesized DNA and expressed involucrin simultaneously. In psoriatic plaques, involucrin expression begins closer to the basal layer than in normal epidermis, and here too we found S-phase involucrin-positive cells. We also observed occasional mitotic involucrin-positive cells in psoriatic epidermis, although we were unable to detect them in culture. Our experiments show that temporal separation of proliferation and terminal differentiation is not obligatory, and thus, the kinetic organization of epidermis may be less rigid than some models imply.
Congenital non-bullous ichthyosiform erythroderma--cell kinetics before and after treatment with etretinate.
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Radiosensitivity of normal human epidermal cells in culture.
Using an in vitro culture system we have derived radiation survival curves for the clonogenic cells of normal human epidermis. The culture system used allows the epidermal cells to stratify and form a multi-layered sheet of keratinizing cells. The cultures appear to be a very good model for epidermis in vivo. The survival curves show a population which is apparently more sensitive than murine epidermis in vivo. It remains unclear whether this is an intrinsic difference between the species or is a consequence of the in vitro cultivation of the human cells.
Cell cycle kinetics of cultured human epidermal keratinocytes.
When stratified epithelia maintained in culture are used for autoradiographic studies of labeling index, the emulsion is usually placed over the uppermost strata of the culture. In many cases the distance from the basal cell nucleus to the emulsion exceeds the average pathlength of beta-particle emissions from 14C or 3H. We describe a technique for inverting the cultures so that the emulsion can be brought into close association with the basal cells. Attempts to label cultured human epidermal keratinocytes using a pulse of [3H]- or [14C]-thymidine produced labeling only at the periphery of the colonies. This was noted when emulsion was laid on top of the colonies but also when the emulsion was in close contact with the "basal cells" adhering to the plastic culture vessel. Continuous labeling of the cultures produced nearly 100% labeling of all the basal layer, i.e., central and peripheral, indicating that the central cells were also in rapid cell cycle. The results are interpreted as indicating the presence of an efficient barrier to free diffusion over the center of the colonies, presumably due to the presence of several layers of corneocytes. Percent labeled mitoses (PLM) studies produced an unusual PLM curve with a well-defined third peak which showed a higher PLM than the second peak. These results may indicate that the cultures contain discrete cell populations with different cell kinetic phase durations.
Cell kinetics of the human hair follicle.
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