Macrophages restrain contraction of an in vitro wound healing model.

Significant numbers of macrophages are present during all stages of dermal wound repair, but the functional significance of these macrophages, especially during the later contraction and remodelling stages of repair, remains unclear. We investigated the effect of macrophages on wound contraction using a novel in vitro model based upon the contracting dermal equivalent (DE). Macrophages were found to reversibly restrain DE contraction, a rapid and sustained effect that was enhanced by lipolysaccharide (LPS) treatment of macrophages and partially inhibited by hydrocortisone. Prolonged inhibition of contraction was strongly correlated with an inhibition of fibroblast proliferation. The rapid contraction-inhibiting effect of the macrophages was mediated through activation of protein kinase C (PKC). These results suggest that inflammatory macrophages restrain the later stages of wound repair, namely matrix contraction and remodeling. The novel in vitro model established here provides a useful system for examining fibroblast-macrophage interactions in the healing wound.

Pathological scarring: strategic interventions.

Aberrant cutaneous scar formation is a substantial cause of postoperative morbidity. There is at present no clear consensus on the best way to prevent or treat such scarring, although recently there has been considerable progress in developing an understanding of the mechanisms of tissue repair and scarring. We carried out a literature review using Medline to establish the current understanding of the key events occurring during tissue repair and to identify potential causes of scarring. We now review the key events during tissue repair and the pathogenesis of fibroproliferative disease. Tissue repair is achieved through a multistranded, elegantly coordinated process within which the balance between synthesis and breakdown of matrix is upset during fibrotic disease. Scars form because the signals directing tissue repair are not correctly terminated, and while the initiation and propagation of repair is well understood the signals that direct its cessation have yet to be elucidated.

4-(N-hydroxyphenyl)retinamide can selectively induce apoptosis in human epidermoid carcinoma cells but not in normal dermal fibroblasts.

The retinoid 4-(N-hydroxyphenyl)retinamide (4HPR, fenretinide) has both growth inhibitory and apoptosis-inducing effects on a number of cancer cell lines in vitro and in vivo and has been entered into a number of oncological trials. However, little is known about its mechanism(s) of action or its effects on normal cells such as fibroblasts. In this study, the effects of fenretinide on both epidermoid carcinoma cells of vulva (cell line A431) and normal human dermal fibroblasts, both as monolayers and also grown in 3D cell culture systems, have been investigated. The 3D cell culture system contained normal human fibroblasts embedded in a type I collagen gel with the carcinoma cells seeded on top of the collagen gel, which mimics the epidermoid carcinoma. Fenretinide significantly inhibited the rate of DNA synthesis of carcinoma cells, while there was little effect on fibroblasts on monolayers, at 10(-6)-10(-5) M, which are clinically attainable doses. Fenretinide at 5 x 10(-6) M induced apoptosis characterised by cell shrinkage, membrane blebbing, nuclear condensation and/or fragmentation, and cell detachment in carcinoma cells, but not fibroblasts from monolayers. Fenretinide also reduced the viability of carcinoma cells in the 3D cell culture system without affecting fibroblasts. These data show that fenretinide may preferentially induce apoptosis in epidermoid carcinoma cells.

Exposure of human keratinocytes and fibroblasts in vitro to nickel sulphate ions induces synthesis of stress proteins Hsp72 and Hsp90.

Exposure of the epidermis to nickel-containing compounds is one of the most common causes of contact allergy and may indicate that exposure of skin cells to nickel ions causes stress. In this study, cytotoxicity assays upon human keratinocytes and fibroblasts in monolayer culture indicated a 50% decrease in viability of both cell types at nickel sulphate concentrations in excess of 10(-3) M. To investigate the possible induction of a stress response within these cell types, monolayer cultures were exposed to concentrations of nickel sulphate for 1 h, followed by 35S-methionine labelling. Whole cell lysates were analysed by SDS-PAGE and immunoblotting with specific monoclonal antibodies, or by fluorography. For keratinocytes, increased synthesis of Hsp90 at concentrations of 10(-5) M and above and induction of the stress-inducible Hsp72 at concentrations of 10(-4) M and above were observed. For fibroblasts, increased induction of Hsp90 at all concentrations under test and a dose-responsive increase in Hsp72 synthesis were detected. These results indicate that both keratinocytes and fibroblasts react to the toxic effects of nickel ions by mounting a stress response involving the up-regulation of synthesis of key stress proteins.

Fenretinide and its relation to cancer.

Retinoids, natural or synthetic substances which have vitamin A activity, have a well-known reputation for their antitumour and differention-inducing activity in vitro and in vivo. More than 1500 retinoids have been tested so far but very few of them have been entered into clinical trials because of their side-effects. All-trans-N-(4-hydroxyphenyl)retinamide (4HPR or fenretinide) is a synthetic retinoid that is reported to have fewer side-effects compared to naturally occurring retinoids such as all-trans retinoic acid (ATRA) and 9-cis retinoic acid. In addition, fenretinide has been shown to induce cell death (apoptosis) even in ATRA-resistant cell lines. Although the mechanism by which fenretinide acts is not entirely known it is considered to be a promising drug and seems to induce apoptosis via different pathway(s) from classical retinoids. In this review, we discuss possible mechanisms of fenretinide action and summarize results of clinical trials.

Induction of glutathione S-transferase activity in Triatoma infestans

Several synthetic pesticides and allelochemicals used to treat Triatoma infestans adults by topic application showed some degree to cytosolic glutathione S-transferase (GST) induction. General inducers of detoxication systems such as phenobarbital and 3-methylcholantrene topically applied on T. infestans resulted in no GST induction. Meanwhile, general insecticide synergist such as piperonyl butoxide (160 µg/insect) increased the GST-activity in the range of 120-140 per cent. Insects injected with reduced glutathione (300 mg/insect) presented at the forth day elevated GST activity.

Glutatión S-transferasas en no-vertebrados y en mamíferos: su importancia en la detoxificación de insecticidas / The Glutathione S-transferases (GST) of non-vertebrate organisms and mammals

Las Glutatión S-transferasas (GST) de organismos no-vertebrados no han sido estudiadas con la misma intensidad que las de mamíferos. El interés en las GST en no-vertebrados radica en su importancia como protección bioquímica de los organismos expuestos a compuestos químicos ambientales. En efecto, se ha observado que niveles elevados de GST podrían estar asociados con la tolerancia a pesticidas. La intención de esta actualización es revisar el nivel de conocimiento actual sobre estas enzimas en no-vertebrados, comparándolas con las de mamíferos. Evaluar la contribución de estos estudios al conocimiento del rol de las glutinatión transferasas en general, e intentar discernir la dirección de las futuras investigaciones en este campo

Development of a multilayered in vitro model for studying events associated with wound healing.

Simplified in vitro models such as cellular monolayer cultures have only limited usefulness in the study of cutaneous wound repair processes. This has stimulated the investigation of three-dimensional tissue equivalent systems such as the dermal and skin equivalent models. With the use of a wound system constructed of rat tail type I collagen and human dermal fibroblasts, experimental wounding was accompanied by problems with mechanical scoring of the plastic substratum which prevented cell migration. These problems were overcome with the use of a multilayered model in which a punch biopsy-wounded dermal equivalent (bilayered model) or skin equivalent (tri-layered model) was placed onto an acellular collagen lattice and fixed in place with polymerizing collagen. This model permitted observation of the process of cellular repopulation of the "wound space," into which both fibroblast and keratinocyte migration commenced within 1 day. The number of fibroblasts in this space increased dramatically over a period of 9 days, the cells appearing to migrate both over and through the acellular lower collagen layer. Keratinocyte reepithelialization of the "wound space" was completed after 5 days. With the model it was shown that platelet-derived growth factor--AB and epidermal growth factor had positive effects in increasing fibroblast number within the wound space. In conclusion, the model described here should facilitate the study of fibroblast and keratinocyte responses to a wound stimulus in vitro and be a plausible in vitro system for evaluating agents which may have a potential stimulatory or inhibitory effect on numerous cellular responses associated with wound healing.

Are stress proteins induced during PUVA therapy?

Heat shock or stress proteins are produced in practically all cell types when they are exposed to temperatures a few degrees above normal. Measurement of the skin temperature of patients undergoing psoralen and ultraviolet A (PUVA) cabinet treatment for psoriasis revealed that the outer layers of the skin experience a mean temperature rise of 5.3 degrees C. However, this did not produce a detectable stress response in epidermal samples taken after PUVA treatment. In vitro exposure of epidermis from biopsies or of cultured keratinocytes to a 5-7 degrees C temperature rise produced a heat shock response, as measured by an increase in the production of proteins of the HSP90 and HSP70 families. These results were confirmed by the use of specific monoclonal antibodies. The corresponding mRNAs were also analysed using labelled probes. In an in vitro system, following simulated PUVA treatment of cultured keratinocytes, increases in the synthesis of HSP90 and HSP70 were detected but these increases did not correlate with changes in mRNA levels.

The effect of calcipotriol on lesional fibroblasts from patients with active morphoea.

We examined the responsiveness of cultured dermal fibroblasts from biopsies of 6 patients with active morphoea and a similar number of matched controls to the cell proliferation inhibition activity of calcipotriol. Cultured fibroblasts from controls showed no significant response to calcipotriol (at concentrations of 1 x 10(-8) to 1 x 10(-4) M). However, calcipotriol did inhibit the proliferation response of morphoea fibroblasts at all concentrations when compared with controls. There was 4- to 20-fold inhibition in 2 of the morphoea patients when compared with control samples. Four other morphoea samples showed inhibition but to a lesser extent compared with controls.

Quantification of cellular proliferation in acne using the monoclonal antibody Ki-67.

The mechanism by which ductal hypercornification occurs in acne is uncertain. We investigated proliferation in normal and acne follicles and in the interfollicular epidermis using the monoclonal antibody Ki-67, which reacts with a nuclear antigen expressed by cells in the G1, S, M, and G2 phases of the cell cycle. Cryostat sections of biopsies from the interscapular region from acne patients and from normal volunteers were stained with Ki-67 antibody and counterstained with 2% methyl green. The number of Ki-67-positive nuclei in the basal layer were counted and expressed as a percentage of the total number of basal nuclei in the ductal or interfollicular epithelia. The data was expressed as mean percent +/- SD. In normal follicles from acne-affected sites 17.40% +/- 1.86% (n = 8) of the nuclei were Ki-67 positive. This was significantly higher (p < 0.01) than follicles from an area of skin unaffected by acne (11.01% +/- 6.16%, n = 8). In the follicular epithelia of non-inflamed lesions, the percentage of Ki-67 positive nuclei was 23.44% +/- 8.36% (n = 15). It was impossible to count the nuclei of follicular epithelium of inflamed lesions because little of this remained intact. In normal interfollicular epidermis, Ki-67-positive nuclei represented 5.33% +/- 3.36% (n = 8) of the total. This value was not significantly different from the value obtained for interfollicular epidermis near non-inflamed lesions (10.46% +/- 4.45%, n = 15). However, the number of Ki-67-positive nuclei in the interfollicular epidermis near inflamed lesions was significantly higher than either of these two values: 25.26% +/- 6.83%, n = 13, p < 0.05. Our results with Ki-67 confirm that ductal hyperproliferation occurs in acne and shows that normal follicles from acne skin may be "acne-prone."

Cold shock induces the synthesis of stress proteins in human keratinocytes.

Heat shock proteins or stress proteins are synthesized when cells are exposed to a wide variety of physiologic stresses. The stress response is evolutionarily highly conserved, suggestive of an essential function(s) for the survival of organisms, protecting them from harmful trauma. Exposure to cold induces a stress response in organisms such as Drosophila melanogaster and Sarcophaga crassipalpis and this led us to determine whether or not cold shock responses occur in human skin after exposure to cold such as might occur during cryopreservation of tissues or cryosurgery. Biopsies taken from fresh human skin at chest surgery were exposed to 4, 15, 20, and 37 degrees C (control) for 60 min and then allowed to incorporate 35S-methionine at 37 degrees C for up to 3 h. Proteins from the epidermis were extracted and analyzed by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis. At 15 degrees C and below there was increased synthesis of 90 and 72 kD proteins 2 h after shocking. The 72-kD protein was identified as a heat shock protein using a monoclonal antibody to HSP72 and it is proposed from electrophoretic evidence that the 90-kD protein is also a heat shock protein. Clearly, cold shock stimulates a stress response in human epidermis altering the spectrum of proteins expressed and inducing the synthesis of heat shock proteins.

Simple assays of retinoid activity as potential screens for compounds that may be useful in treatment of psoriasis.

Human epidermal cell cultures were used to study the effects of retinoids on keratinocyte differentiation. Keratin profiles were studied by quantitative gel electrophoresis of culture extracts, whereas the extent of envelope formation was assessed in an enzyme-linked immunosorbent assay (ELISA) using an antibody that specifically recognizes keratinocyte envelopes. Exposure of cultures to a variety of different retinoids produced both dose-dependent decreases in keratin 16 with consequent increases in the keratin 14: keratin 16 ratio, and a decrease in envelope formation. The order of activity in both assays was similar: arotinoid ethyl ester (Ro 13-6298) greater than or equal to arotinoid acid (Ro 13-7410) much greater than all trans retinoic acid (Ro 1-5488) greater than acitretin (Ro 10-1670) greater than or equal to etretinate (Ro 10-9359), the only difference being that acitretin was slightly more active than etretinate in the keratin assay whereas these retinoids were equi-active in the envelope assay. Analysis of the lesional keratins of psoriasis patients showed that etretinate caused a reduction in keratin 16 and an increase in the keratin 14:keratin 16 ratio, although the magnitude of these changes and their correlation with clinical improvement was variable. As the in vitro assays reported here are simple and quick, they allow rapid screening of compounds for retinoid-like activity.