PubMed Health⌕ Search

Biomedical subjects

B A Gilchrest

Publications and source records attributed to B A Gilchrest.

At least 19 recordsLinked to original sources
PubMed1992

Cytoskeletal events underlying dendrite formation by cultured pigment cells.

In contrast to neurite outgrowth, pigment cell dendrite formation is relatively unstudied. Keratinocyte-conditioned medium (KCM) induces a striking dendricity in human melanocytes and B16 melanoma cells that is detectable within 30 min, maximal in 24-48 hr, and quantifiable by computerized image analysis. Cytochalasin B (CB), known to disrupt actin microfilaments, completely blocks dendrite formation if added to cultures before or with KCM. This effect is rapidly reversible, and dendrites appear within 1 hr after refeeding with KCM alone. In contrast, CB treatment fails to disrupt existing dendrites previously induced by KCM. Agents known to cause microtubule disassembly (colchicine, nocodazole, or vinblastine) do not inhibit dendrite formation if added before or with KCM. In contrast, these agents disrupt established dendrites. Inhibition of protein synthesis with cycloheximide or actinomycin D completely blocks dendrite formation, but if cultures are provided fresh KCM lacking protein synthesis inhibitors, dendrites reappear within 24 hr. Actin microfilaments visualized with a monoclonal antibody or rhodamine-phalloidin are poorly organized in untreated cells, but form numerous fibers localized along dendrites in KCM-treated cells. Microtubules visualized with a monoclonal anti-tubulin antibody are localized in the center of dendrites. These cytoskeletal changes occur without altering beta actin or beta tubulin mRNA levels. Taken together, these data implicate actin microfilaments in dendrite outgrowth, but not in maintenance, and conversely microtubules in dendrite maintenance but not in formation. These keratinocyte-induced changes involving beta actin and beta tubulin polymerization appear to require both new protein synthesis and post-translational regulation. The observed similarities between melanocytes and other neural crest-derived cells suggest that cutaneous pigment cells might serve as an alternative model for studies of neurite outgrowth.

Actins↗
PubMed1992

Modulation of mRNA levels during human keratinocyte differentiation.

Cultures of human keratinocytes provide an excellent model system in which to study differentiation. Using the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and calcium, two agents known to induce keratinocyte differentiation in vitro, we examined the expression of the genes encoding c-fos, c-myc, and c-jun; involucrin, a protein precursor of the keratinocyte cornified envelope; and L-7, a ribosomal protein. Overall, at the doses studied, TPA induced a more rapid and profound differentiation than did calcium, as evaluated by culture morphology and northern blot analysis. Our studies showed a constant low level of c-fos and c-jun expression in unstimulated cells with no significant change after addition of either TPA or calcium except when transcript breakdown was inhibited by cycloheximide. The c-myc proto-oncogene, known to have a high constitutive expression in actively proliferating cells, was strongly downregulated by TPA, but calcium had no effect over a 32 hour period, consistent with the greater growth inhibition of TPA in this system. Involucrin was induced about ninefold by both TPA and calcium as early as 8 hours after stimulation, suggesting transcriptional regulation of this gene during differentiation. L-7, recently demonstrated to be downregulated in late passage human fibroblasts in an in vitro model of senescence, was also strongly downregulated by either TPA or calcium, consistent with an interrelationship between the basic cellular processes of aging and differentiation. These finding expand our knowledge of the differentiation process in human keratinocytes.

Calcium↗
PubMed1992

The molecular cloning and expression of two CRABP cDNAs from human skin.

Retinoic acid (RA) is known to have a profound effect on the growth and differentiation of human epidermal cells in vivo and in vitro. One of the proteins thought to be involved in mediating the action of RA is the cellular retinoic acid-binding protein (CRABP). We have used PCR technology to generate cDNAs for two distinct CRABPs from human skin and skin-derived cells. One is highly homologous to the CRABP I cDNAs previously cloned from bovine and murine sources. The second shares extensive deduced amino acid homology with CRABP II, a protein recently described in newborn rat and embryonic chick. Although both mRNAs can be detected in neonatal foreskin, CRABP II mRNA is the predominant one in this tissue, as well as in cultured newborn fibroblasts and keratinocytes. Northern blot analysis showed CRABP II mRNA level was only slightly reduced by addition of 10(-6) or 10(-5) M RA to cultures of neonatal foreskin-derived fibroblasts, as was the CRABP I mRNA level in cultured human gut epithelial cells. In contrast, expression of CRABP II mRNA by cultured neonatal keratinocytes was strongly downregulated by RA. We conclude that CRABP II is the predominant CRABP in human skin, at least in the newborn period, and that it is differentially regulated in fibroblasts versus keratinocytes. Our data are consistent with a role for CRABP in regulating the amount of RA delivered to the nucleus.

Amino Acid Sequence↗
PubMed1992

Induction of c-fos but not c-myc in S-91 cells by melanization signals.

Synthesis of the pigment melanin is a complex differentiated function performed by pigment cells in response to a variety of stimuli. The possible roles of the proto-oncogenes c-fos and c-myc in the control of pigmentation were studied using subconfluent, actively proliferating Cloudman S-91 murine melanoma cells stimulated to synthesize melanin by melanocyte stimulating hormone (MSH) or forskolin. Stimulation caused a significant increase in melanin synthesis when compared to control cells, but had no effect on cell growth. Northern analysis of total cellular RNA demonstrated rapid, transient induction of c-fos mRNA as early as 30 min after stimulation with MSH or forskolin. In contrast, there was no effect on the high constitutive expression of c-myc in these actively proliferating cells. These data strongly suggest that the induction of c-fos mRNA is an early genetic event in stimulation of melanin synthesis and thus this proto-oncogene may play a major role in the regulation of this differentiated function, as reported for other forms of cellular differentiation. In contrast, c-myc expression is unaffected and instead correlated with cellular proliferative capacity. These results are consistent with the hypothesis that the down-regulation of c-myc frequently observed during cell differentiation is not a necessary event, but rather reflects an associated decrease in cell growth rate. The S-91 melanoma system appears to provide a convenient model for study of the regulation for a single, well defined differentiated function that is independent of growth rate.

Animals↗
PubMed1992

Histopathologic differences in the photoaging process in facial versus arm skin.

We used clinical criteria to study skin biopsy specimens with mild to moderate photoaging taken from the face and dorsal forearms of 74 Caucasian volunteers between the ages of 30 and 50. Facial skin had a greater number of granular cell layers, a higher degree of keratinocytic atypia, and more often showed a compact stratum corneum than arm skin. Furthermore, the dermis of facial skin had a more extensive perivascular and perifollicular lymphocytic infiltrate, more perifollicular fibrosis, a greater number of mast cells and melanophages, and thinner vascular walls than forearm skin. This study demonstrated that the photoaging process is different for face and arm skin. Appreciation of these differences should permit more refined studies of photoaging and the development of more efficient therapies.

Adult↗
PubMed1992

Effect of aging and habitual sun exposure on the genetic response of cultured human keratinocytes to solar-simulated irradiation.

Aging and chronic sun exposure are known to be associated with decreased cutaneous immune function, changes in the balance between epidermal proliferation and differentiation, and a greatly enhanced risk of photocarcinogenesis. However, their specific effects on the response of human keratinocytes to ultraviolet (UV) irradiation are unknown. We therefore asked whether aging and photoaging modulate the response at the mRNA level to UV-inducible genes implicated in immunomodulation and/or growth control. Cultured human keratinocytes derived from newborn, young adult, and old adult donors were exposed to a single physiologic dose of solar-simulated UV or sham irradiation and harvested at 1, 4, 8, 24, and 48 h post-irradiation for northern blot analysis. Specific mRNA was detected using cDNA probes encoding the proto-oncogenes c-fos and c-myc and the growth-arrest and DNA damage (GADD153) gene, all recently shown by our laboratory to be modulated by UV in newborn keratinocytes; interleukin (IL)-1a, IL-1b, and the IL-1 receptor antagonist (IL-1ra), two keratinocyte cytokines and their competitive inhibitor, implicated in the immunomodulatory effect of UV; and SPR2, a recently cloned gene known to be induced during normal keratinocyte differentiation and by lethal UV-C irradiation. Our data suggest that aging alone strikingly increases the baseline expression of SPR2 and IL-1ra but has relatively little effect on the response to UV for the other genes examined. In contrast, the combination of aging and habitual sun exposure, so-called photoaging, markedly increases c-fos inducibility and decreases baseline expression of SPR2 and IL-1ra relative to that in cells from sun-protected skin of the same donors. The implied alterations in signal transduction and differentiation state observed in cells derived from habitually sun-exposed sites of old adults may explain in part the predisposition to photocarcinogenesis in photoaged skin.

Adult↗
PubMed1992

Retinoids and photodamage.

Extensive well-controlled clinical studies performed over the past 5 years have demonstrated a consistent, dose-dependent, statistically significant improvement in the appearance of photodamaged skin after 3-6 months of daily treatment with topical 0.001-0.1% tretinoin cream. Clinical changes included decreases in surface roughness, irregular pigmentation, fine and coarse wrinkling, and sallowness. Actinic keratoses have also been reported to decrease in size and number. Blinded analysis of biopsies from more than 500 subjects showed that there was compaction of the stratum corneum, an increase in the number of granular layers, thickening of the epidermis and a decrease in epidermal melanin. There were no detectable histological changes in any dermal parameters. The specific cellular mechanisms by which retinoic acid (RA) exerts its beneficial effect on photodamaged skin are currently the subject of intensive investigation. It is well established that RA enters the nucleus where it binds to an RA receptor (RAR), and that the RA-RAR complex then binds to specific RA response elements in the DNA, modulating the expression of target genes. It is thus likely that RA improves at least some aspects of photoageing by modifying cellular differentiation programmes, as retinoids have been shown to do during embryogenesis, in malignantly transformed cells and in skin affected by certain dermatoses.

Dose-Response Relationship, Drug↗
PubMed1992

Ageing and photoageing of the skin: observations at the cellular and molecular level.

It is now well established that ageing occurs at the level of individual cells in the skin and other organ systems. Changes in cell behaviour, protein production and gene expression in response to standardized stimuli are readily observed in cultured cells derived from young vs old donors and from photoaged vs sun-protected body sites. Whether these changes are best viewed as a cause or a consequence of ageing cannot be determined at present. Nevertheless, available data now provide cellular and molecular correlates for the well-known differences in clinical responsiveness between newborn, adult and photoaged skin. From this basis, it will hopefully be possible to develop a more comprehensive understanding of cutaneous ageing processes.

Adult↗
PubMed1992

Evidence that filopodia outgrowth is a common final pathway for fibroblast growth inhibition in vitro.

To identify events associated with fibroblast growth inhibition, the effect of two known inhibitors, interferon-alpha and all-trans retinoic acid, on the growth and surface morphology of cultured fibroblasts was examined. Interferon-alpha administered at seeding reduced both growth rate and saturation density; all-trans retinoic acid reduced only saturation density. However, both negative growth modulators were associated with an increase in filopodia outgrowth and an increase in intracellular filamentous actin in a time course corresponding to onset of growth inhibition by these agents. In combination with earlier findings, these data suggested that, regardless of etiology, cultured fibroblast growth restriction is mediated in part by an actin-dependent outgrowth of filopodia that augment intercellular contact.

Cell Division↗
PubMed1992

Clinical applications of cultured epithelium.

Techniques that allowed the successful serial subcultivation of human keratinocytes into sheets of epithelium suitable for grafting have made possible a variety of clinical applications for cultured epithelium. Following the first description of this technique in 1981 to treat third-degree burns, cultured keratinocytes derived from a small biopsy of the patient's normal skin (autografts) have been used in centres throughout the world to provide permanent wound coverage for extensive burns. Over the years, applications have expanded to include the treatment of leg ulcers and blistering skin disorders. A further development in this field has been the use of cultured epithelium derived not from the patients own skin, but from an allogeneic donor (cultured allograft). Cultured allografts have also been widely used in the treatment of burns, leg ulcers, the donor sites for split-thickness grafts, and other dermatological disorders. These allografts seem to act as a potent stimulus to wound healing, but do not survive permanently on the wound bed. Their postulated mechanism of action is through release of multiple cytokines that stimulate epithelialization from the wound periphery as well as from adnexal elements within the wound bed. Allograft application is a simple outpatient procedure which involves no discomfort for the patient. No skin biopsy is necessary and cryo-preservation of grafts for future use is possible.

Burns↗
PubMed1991

Evidence for nerve growth factor-mediated paracrine effects in human epidermis.

Nerve growth factor (NGF) is critical to the development and maintenance of the peripheral nervous system, but its possible roles in other organ systems are less well characterized. We have recently shown that human epidermal melanocytes, pigment cells derived from the neural crest, express the NGF receptor (p75 NGF-R) in vitro (Peacocke, M., M. Yaar, C. P. Mansur, M. V. Chao, and B. A. Gilchrest. 1988. Proc. Natl. Acad. Sci. USA. 85:5282-5286). Using cultured human skin-derived cells we now demonstrate that the melanocyte p75 NGF-R is functional, in that NGF stimulation modulates melanocyte gene expression; that exposure to an NGF gradient is chemotactic for melanocytes and enhances their dendricity; and that keratinocytes, the dominant epidermal cell type, express NGF messenger RNA and hence are a possible local source of NGF for epidermal melanocytes in the skin. These combined data suggest a paracrine role for NGF in human epidermis.

Base Sequence↗
PubMed1991

A novel approach to analysis of transcriptional regulation in human cells: initial application to melanocytes and melanoma cells.

An assay system for transcriptional profile analysis of cultured eukaryotic cells has been developed to simultaneously handle multiple samples in a rapid, sensitive, and internally controlled manner. The methodology incorporates a microtiter plate assay system, a rapid cell-harvest enzyme-assay technique, and the bacterial reporter genes beta-glucuronidase and beta-galactosidase. We demonstrate, using beta-actin and SV40 (late) transcription promoting sequences, that this technically refined microtiter-triton-lysate (MTL) assay methodology can readily differentiate between the transcriptional states of human melanocytes before and after pharmacologic stimulation and malignantly transformed versus normal cell environments. Differences in the transcriptional environments are revealed by the relative expression of transcription element probes. The transcriptional activity ratio of the beta-actin compared to the SV40 late transcription promoting sequences was approximately 1:2 in primary cultured melanocytes, 2:1 in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-treated melanocytes and 1:4 in the Tang melanoma cell line. Because this MTL assay methodology can accommodate a panel of transcription element probes, we anticipate that the resultant transcriptional profiles will prove useful in deciphering the diverse transcriptional changes that occur within normally regulated and malignantly transformed cells.

Actins↗
PubMed1991

Pigment content of cultured human melanocytes does not correlate with tyrosinase message level.

Tyrosinase is considered to be the rate-limiting enzyme for the biosynthesis of melanin in epidermal melanocytes, and thus tyrosinase activity is thought to be a major regulatory step in melanogenesis. To determine whether the rate of pigment production was controlled at the level of tyrosinase gene expression, we developed a culture system capable of generating large populations of pure human melanocytes and then measured both melanin content as determined spectrophotometrically by absorption at 475 nm and mRNA levels as detected by hybridization with cloned cDNA Pmel 34, encoding human tyrosinase. We examined the relationship between pigment content and tyrosinase mRNA levels among human melanoma and melanocyte lines with very different levels of basal pigmentation; between two clones of a single human melanoma line, one pigmented and one amelanotic; and sequentially in melanocytes before and after simulation with isobutylmethylxanthine to increase melanin content per cell. Using Northern blot analysis and in-situ hybridization we found no correlation between tyrosinase message levels and melanin content, suggesting that posttranscriptional regulation of tyrosinase and/or other events determine the rate of pigment synthesis in human melanocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Immediate and delayed molecular response of human keratinocytes to solar-simulated irradiation.

The clinical and histologic events in human skin after a single ultraviolet exposure are well documented; and both in vitro and in vivo studies have established immunologic and mutagenic effects of ultraviolet rays on cells. However, little is known about the ultraviolet damage/repair response at the level of gene induction. This study examines the genetic response in cultured human keratinocytes exposed to a single solar-simulated ultraviolet dose and harvested at 1, 4, 8, 24, and 48 hours postirradiation for northern blot analysis using c-DNA probes to well studied stress related genes, heat shock protein 70 and ubiquitin; to a recently cloned gene associated with growth arrest and DNA damage (gadd 153); to c-myc and c-fos, two proto-oncogenes known to be associated with cell growth and differentiation; and to glyceraldehyde phosphate dehydrogenase, known to be involved in cellular intermediary metabolism, gadd 153 and c-fos mRNA levels are increased early (1-4 hours) in keratinocytes, at a time when the cells appear indistinguishable from nonirradiated controls. At the same early time points, the c-myc mRNA level is decreased. Heat shock protein 70 and ubiquitin transcripts are detectable under basal conditions but are not increased by ultraviolet exposure; although at later time points (24-48 hours), when there is morphologic evidence of cell damage and downregulation of the otherwise constitutively expressed gene glyceraldehyde phosphate dehydrogenase, their unchanged steady state levels of mRNA constitute relative over expression. Irradiation of cells with the same solar-simulated ultraviolet dose filtered to remove the shortest and most damaging wavelengths results in a similar pattern of early gene induction, even though cell growth and survival are unaffected. These data suggest that two classes of genes participate in the response to ultraviolet rays: early genes whose mRNA levels are increased (gadd 153, c-fos) or decreased (c-myc) before or independent of any morphologic evidence of cell damage and late genes that are relatively increased (heat shock protein 70, ubiquitin) or decreased (glyceraldehyde phosphate dehydrogenase) in expression when morphologic cell damage is present. The early events may be a direct response to ultraviolet irradiation, whereas the late events may represent a secondary response to stress.

Base Sequence↗
PubMed1991

Protective effect of alpha-tocopherol in carrier liposomes on ultraviolet-mediated human epidermal cell damage in vitro.

We have examined the postulated protective effect of antioxidants against ultraviolet (UV)-induced epidermal damage in an in vitro model system using either a well-differentiated human squamous cell carcinoma line SCC12F2 or human newborn keratinocytes. Cell were maintained in a serum-free defined medium only or with carrier liposomes (LPs) alone or LPs containing 1 microgram/ml alpha-tocopherol (AT/LP) or 1 microgram/ml alpha-tocopherol in oil (AT) alone. Cultures were irradiated once using a dose of solar-simulated light (25 mJ/m2, metered at 285 nm) determined in preliminary studies to decrease cell yields after 72 h by approximately 50% compared with sham-irradiated controls under basal conditions. Compared with their LP control, UV-irradiated AT/LP-treated SCC cells were significantly protected by an average of 25% against UV-mediated cell death or growth arrest as determined by the ratio of 72-h cell yields for UV-irradiated versus sham-irradiated cells. Ratios for cells treated with AT, LPs alone, or AT/LP did not differ significantly from those for untreated cells. Newborn keratinocytes had higher overall ratios of cell yields for UV-irradiated vs sham-irradiated cells, but results were otherwise similar, with AT/LP again providing significant protection, averaging 29%, compared with LP controls. UV irradiation of cells in basal medium induced the gene for heat shock protein (HSP 70), presumed to be involved in cellular response to stress; and keratinocytes treated with alpha-tocopherol in carrier liposomes showed a further increase in expression of HSP 70.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1991

Cultured epidermal allografts as biological wound dressings.

Recent advances in cell culture technology permit the generation of large stratified epithelial sheets appropriate for wound coverage. Autografts (sheets prepared from the patient's own skin) have proven life-saving in the treatment of large third-degree burns and have been successfully employed in the management of chronic ulcers. Allografts (sheets prepared from the skin of an unrelated donor) have also been used. In our experience, cultured allografts derived from neonatal foreskin provide a potent stimulus to healing in a variety of partial thickness wounds. Their application is a simple outpatient procedure which involves no discomfort for the patient. In contrast to autografting, no biopsy is necessary and use of cultured allogenic cells permits immediate grafts availability and possibility of stockpiling and preserving grafts for future use. Preparation of epithelial sheets suitable for grafting is also faster and easier with newborn than with adult donor cells. Newborn allografts have caused rapid healing of most previously refractory ulcers with long-term results comparable to those obtained with conventional split thickness grafting. We postulate that cultured allografts act by providing a temporary wound covering while releasing multiple cytokines that synergistically promote permanent reepitheliazation by previously quiescent host keratinocytes then stimulated to divide and migrate. Whether allografts are immunologically rejected in a clinically undetectable reaction or simply replaced stochastically by host keratinocytes is presently unknown.

Biological Dressings↗
PubMed1991

Effects of tretinoin on photodamaged skin. A histologic study.

The histologic effects of topical tretinoin therapy on photodamaged facial skin were investigated in two 24-week, double-blind, randomized, vehicle-controlled studies involving 533 subjects at eight US centers. Three concentrations of tretinoin (0.05%, 0.01%, and 0.001%) in a new emollient cream were studied. Pretherapy and posttherapy biopsy specimens from the periorbital (crow's foot) area were examined by conventional light microscopy and computerized image analysis. Four significant dose-dependent differences from vehicle were found in the tretinoin groups: increased epidermal thickness, increased granular layer thickness, decreased melanin content, and stratum corneum compaction. There was no significant difference between 0.001% tretinoin and the vehicle, and no obvious dermal changes were detected in any group. The four epidermal changes in tretinoin-treated skin establish the biologic activity of the new emollient cream formulation and may partially account for the clinical improvements in photodamage observed in the same group of subjects.

Administration, Cutaneous↗