Sheila Mac Neil

Keratinocyte-driven contraction of reconstructed human skin.

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose‐6‐phosphate to be ineffective and ascorbic acid‐2‐phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.

Differential protein oxidation in Duchenne and Becker muscular dystrophy

WE describe the use of an immunoblotting technique to investigate the potential role of reaction oxygen species in the pathogenesis of Duchenne muscular dystrophy. Quadriceps femoris muscle biopsy samples were obtained from six patients with Duchenne and six with Becker muscular dystrophy, and from six control subjects. These were analysed for the presence of protein carbonyl moieties (indicative of oxidation to protein) by SDS–polyacrylamide gel electrophoresis and Western blotting, using a commercially available antibody. In all Duchenne and Becker patient samples analysed, a heavily oxidized protein species was identified migrating at 125 kDa. This oxidized species was not present (or was present at very low levels) in normal control samples. Use of the present technique also identified that the various muscle proteins in Duchenne and Becker muscular dystrophy muscle are oxidized to varying degrees, supporting the hypothesis of a differential susceptibility of proteins to oxidation in these disorders. Work from the present study further supports the hypothesis that reactive oxygen species play a role in dystrophic muscle cell pathogenesis.

Use of an in vitro model of tissue‐engineered human skin to study keratinocyte attachment and migration in the process of reepithelialization

To produce a stable epidermis, keratinocytes need to be firmly attached to the basement membrane. However, following wounding, keratinocytes are required to develop a migratory phenotype in order to reepithelialize the wound. To investigate some of the issues underlying reepithelialization, we have developed a three‐dimensional in vitro model of tissue‐engineered skin, comprising sterilized human dermis seeded with human keratinocytes and dermal fibroblasts. Using this model, we have shown that the inclusion of fibroblasts within the model increases the stability of keratinocyte attachment. We have also demonstrated that keratinocyte migration occurs most effectively in the absence of a basement membrane and following the inclusion of fibroblasts in the model. In addition, subjecting the keratinocyte layer to mechanical trauma induces a migratory phenotype. We conclude that this three‐dimensional in vitro wound model can be used to increase our understanding of the factors that enhance keratinocyte migration and hence wound healing in vivo.

CALCIUM, CYCLIC AMP AND HORMONE ACTION

It is now well established that the communication of hormonal signals to the interior of the cell is achieved by the generation of second messengers at the cell membrane level. The concept that intracellular regulators or ‘second messengers’ mediate the effects of hormones that themselves do not enter the cell, was proposed originally by Sutherland et al. (1965). Sutherland and his co-workers found that cyclic AMP played a key role in the response of the liver to adrenalin and to glucagon and discovered the enzyme responsible for the formation of cyclic AMP from ATP, adenylate cyclase. Since then a wide variety of hormones have been shown to stimulate adenylate cyclase in cell membranes. The specificity of the response resides in the specificity of the receptor for a particular hormone on the cell surface. The role of calcium as an intracellular regulator has also been known for many years (Rubin, 1982). However, attention has tended to focus on the importance of calcium in excitable cells such as those of nerve and muscle. As the role of cyclic AMP as an intracellular regulator became increasingly apparent, it was also recognized that this nucleotide does not subserve a primary role in the action of a number of hormones. It soon became clear that a distinction between the action of calcium ions in neuro-secretion and excitation/contraction and cyclic AMP in hormone action in non-excitable tissues was invalid. Indeed, in many excitable and in non-excitable systems calcium ions and cyclic AMP can serve as dual interrelated messengers in determining the cellular responsc to a specific stimulus (Rasmussen, 1980). The purpose of this brief review is to outline the importance of cyclic AMP and of calcium as intracellular regulators, to try to describe how they are generated and terminated as signals and to discuss their interrelated roles in cellular regulation.

A simple in vitro model for investigating epithelial/mesenchymal interactions: keratinocyte inhibition of fibroblast proliferation and fibronectin synthesis

Hypertrophic scarring and graft contracture are major causes of morbidity after burn injuries. It is well established that application of a split‐thickness skin graft reduces scarring and contraction, and cultured epithelial autografts have a similar effect. To investigate the influence of keratinocytes on fibroblast proliferation and fibronectin synthesis, we used an in vitro separated co‐culture model in which epithelial sheets were cultured above fibroblast monolayers without physical contact. We also investigated the response of fibroblasts to keratinocyte‐conditioned medium (KCM) obtained from confluent and subconfluent keratinocyte monolayers. Both cultured epithelial sheets, composed of adherent fully confluent keratinocytes, and their conditioned medium, reduced fibroblast proliferation. However, KCM from subconfluent keratinocytes stimulated fibroblast proliferation at low concentrations while inhibiting it at higher concentrations, indicating that keratinocytes can produce both mitogenic and growth‐inhibiting factors for fibroblasts. KCM, but not epithelial sheet co‐culture, also inhibited fibroblast fibronectin synthesis. This indicates regulation of fibroblast phenotype by soluble factors released by the keratinocyte and also suggests that there is a dialogue between keratinocytes and fibroblasts with respect to fibronectin production. We conclude that this separated co‐culture model is a simple way to study epithelial/mesenchymal communication particularly with respect to the role of the fibroblast in wound healing.

Simplifying the Delivery of Melanocytes and Keratinocytes for the Treatment of Vitiligo Using a Chemically Defined Carrier Dressing

Obtaining pigmentary function in autologous skin grafts is a current challenge for burn surgeons as is developing reliable robust grafting strategies for patients with vitiligo and piebaldism. In this paper, we present the development of a simple methodology for delivering cultured keratinocytes and melanocytes to the patient that is of low risk for the patient but also user friendly for the surgeon. In this study, we examined the ability of keratinocytes and melanocytes to transfer from potential cell carriers under different media conditions to an in vitro human wound bed model. The number of melanocytes transferred, their location within the neoepidermis, and their ability to pigment were evaluated as preclinical end points. Two inert substrates (polyvinyl chloride and silicone sheets) and three candidate plasma-polymerized coatings with controlled surface chemistry deposited on these substrates were explored. Two media for expansion of cells, Greens, currently used clinically (but which contains fetal calf serum), and a serum-free alternative, M2 (melanocyte medium), were explored. Reproducible transfer of physiologically relevant numbers of melanocytes capable of pigmentation from the coculture of melanocytes and keratinocytes was obtained using either Greens medium or M2 medium, and a silicone carrier pretreated with 20% carboxylic acid deposited by plasma polymerization.

Calmodulin in human serum and the specific release of calmodulin from calmodulin-rich platelets

Calmodulin(CaM)-like activity was detected in human serum and foetal calf serum, with an activity i0 times more than that detectable in plasma. Serum CaM was largely accounted for by release from human platelets as confirmed by both radioimmunoassay and sodium-dodecyl-sulphate/polyacrylamide-gel electrophoresis of CaM partially purified from platelet releasate obtained in response to thrombin. Lactate dehydrogenase release was unaffected by thrombin. Platelet CaM content was very variable (1.3 to 11.3 pg/mg protein; n=15). It is suggested that intact platelets are rich in CaM and that release of CaM during preparation explains the variation in CaM content reported here and in the literature.

Stimulation of the adenylate cyclase of a B16 melanoma cell line by pro-opiocortin-related peptides — A structure-activity study

The ability of alpha-melanotrophin (alpha-MSH or ACTH 1-acetyl-13 amide) and other structurally related peptides derived from the common precursor, pro-opiocortin, to stimulate adenylate cyclase activity in a pigmented B16 mouse melanoma was investigated. The peptides ACTH 1-39, ACTH 1-24, alpha-MSH, ACTH 1-13 amide and beta-MSH all stimulated the enzyme to a similar maximal extent and with similar potency (ED50 = 1.3 . 10(-6) M) except that ACTH 1-39 was slightly less potent (ED50 = 5 . 10(-6) M). ACTH 4-10 (ED50 = 4 . 10(-5) M) and gamma-MSH (ED50 = 5 . 10(-6) M) were partial agonists. ACTH 1-10 was no more effective than ACTH 4-10 in stimulating the enzyme whereas ACTH 1-13 amide was a full agonist. The peptides beta-endorphin and its derivatives, Met-enkephalin and melanotrophin potentiating factor (MPF), failed to stimulate the enzyme. We suggest that the B16 melanoma requires not only the sequence ACTH 4-10 but also some part of the sequence ACTH 11-13, or a similar sequence in the terminal portion of beta-MSH, for full activation of the receptor-linked enzyme.

Evidence that calmodulin may be involved in phytohaemagglutinin-stimulated lymphocyte division

Phytohaemagglutinin-stimulated and non-stimulated incorporation of [3H]thymidine into human peripheral blood lymphocytes is inhibited by the calcium antagonist PY 108–068 and by the calmodulin antagonists trifluo-perazine andN-(6-aminohexyl)-5-chloro-l-naphthalene sulphonamide (W7). It is argued that calmodulin may be involved in both non-stimulated [3H]thymidine uptake in lymphocytes and also in the lymphocyte response to phytohaemagglutinin.

Soluble cyclic AMP-dependent protein kinases from chick kidney effects of dilution and non-protein inhibitors

Cyclic AMP-dependent protein kinases prepared from crude cytosols of chick kidney, rat kidney and rat liver were found on dilution to exhibit complex kinetics. Dilution of the cytosols appears to increase the state of activation of the enzymes. This effect was due to the presence of inhibitory agents in the cytosol which had a greater inhibitory effect on the cyclic AMP-dependent than on the cyclic AMP-independent enzyme. Two types of inhibitory activity were found by column chromatography, one resistant to trichloroacetic acid precipitation and boiling but affected by trypsin digestion and the other resistant to boiling and trypsin digestion but precipitated by trichloroacetic acid. Inhibitory activity corresponding to the former characteristics has been described previously but the presence of additional soluble inhibitory agents in the cytosol has not been documented. The complete characterisation of this previously undescribed inhibitory activity requires further investigation. The relevance of such cytosolic inhibitory activity to the interpretation of states of activation of protein kinase enzymes is discussed.