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Biomedical subjects

B A Gilchrest

Publications and source records attributed to B A Gilchrest.

At least 145 records · Page 8Linked to original sources
PubMed1989

Cultured allogenic keratinocyte grafts in the management of wound healing: prognostic factors.

Cultured allografts derived from neonatal foreskin provide a potent stimulus to wound healing in a wide variety of wounds. Their application is a simple outpatient procedure involving no discomfort for the patient. In contrast to autografting, no biopsy is necessary, and growth of newborn keratinocytes in cultures is more rapid than that of adult cells. Use of cultured allogeneic cells offers immediate graft availability and the possibility of stockpiling and preserving the graft for future use. Cultured epidermal allografts may be valuable in accelerating healing by second intention in surgical wounds, as well as being a helpful addition to chronic ulcer management. In venous disease, the outcome is at least comparable to other forms of skin grafting. Ulcers due to connective tissue disorders fared less well and deep chronic ulcers (down to fascia or tendon) were not significantly improved by cultured allograft application. Surprisingly, patient age did not influence outcome.

Adult↗
PubMed1989

Calcium, lanthanum, pyrophosphate, and hydroxyapatite: a comparative study in fibroblast mitogenicity.

Calcium-containing crystals and elevated levels of calcium chloride (CaCl2) and lanthanum chloride (LaCl3) have been previously reported to enhance the proliferative activity of cultured fibroblasts. We have investigated the relative mitogenicity of these agents, whether they function via precipitation on the cell surface and whether they interact with one another. Confluent cultures of newborn foreskin fibroblasts provided with fresh medium containing 10% fetal bovine serum (FBS) in the presence of hydroxyapatite (HA), pyrophosphate (PPi), LaCl3 (La), or additional CaCl2 (Ca) were all stimulated more than control cultures provided with fresh medium and 10% FBS alone as assessed by cell counts 5 days later. Increases in cell yield above the original confluent cell density were 316% for La, 271% for Ca, 189% for HA, 131% for PPi, and 45% for controls. Addition of fresh medium containing 10% FBS and epidermal growth factor or fresh medium containing 20% FBS as additional points of reference yielded increases of 204 and 107%, respectively, over original confluent density. Stimulation induced by La or Ca was significantly greater (P less than 0.001) than the stimulation induced by each of the other treatments. The same treatments added to confluent cultures without a change of medium also renewed mitotic activity, with La and Ca again the most mitogenic and approximately doubling the pretreatment cell yields. Cultures incubated in an inverted position to avoid cell contact with precipitates in the medium were also stimulated by La and Ca, but not by HA and PPi. When added to confluent cultures simultaneously supplemented with optimal additional Ca, La decreased Day 5 cell yields in a dose-dependent manner at low concentrations (0.03-0.2 mM) but increased cell yields over those obtained with 0.2 mM LaCl3 again in a dose-dependent manner at higher concentrations. Thus, while HA and PPi act via precipitation on the cell surface, the more mitogenic agents La and Ca function in solution and appear to stimulate cell division by different nonadditive mechanisms. These findings suggest multiple mechanisms of membrane participation in mitogen responsiveness and in density-dependent inhibition of growth.

Calcium Chloride↗
PubMed1988

Inositol is a required nutrient for keratinocyte growth.

Bovine hypothalamus is known to contain a growth-promoting activity for human epidermal keratinocytes. By sequential purification, the substance was isolated and found to be myo-inositol. The identity of the substance as myo-inositol was confirmed by ion modified partition, gas liquid, thin layer chromatography, by mass spectrometry, and quantitative bioassay. The inositol content of the crude hypothalamic extract and of an active acetone precipitate (the first step in the purification) was determined to be sufficient to account for their observed bioactivity. At an optimal concentration of 55 microM (10 micrograms/ml), myo-inositol approximately tripled keratinocyte yield compared to paired cultures in basal medium containing 0.3 microM, although this yield was only half that produced by a crude saline extract of hypothalamus, suggesting that there are additional growth-promoting activities in the tissue extract. No other skin-derived cell type tested was stimulated by supplemental inositol. These results establish that the inositol requirement for cultured human keratinocytes is markedly higher than for any other normal or malignant cell type investigated to date, and expand the list of brain-derived phospholipid precursors known to stimulate epithelial proliferation in vitro. These data suggest that inositol may subserve quantitatively or qualitatively different functions in the keratinocyte than in other cell types.

Cells, Cultured↗
PubMed1988

Autocrine growth stimulation of human keratinocytes by epidermal cell-derived thymocyte-activating factor: implications for skin aging.

The monocyte-derived cytokine interleukin-1 (IL-1) has growth-promoting activity for a variety of cell types, including lymphocytes and fibroblasts. We have previously shown that the epidermal cell-derived thymocyte-activating factor (ETAF) strongly resembles IL-1 in terms of biological, biochemical, and molecular biological properties. Because some lymphokines are known ot alter epidermal cell growth and differentiation and because cultured epidermal keratinocytes are capable of autocrine growth stimulation in vitro through "conditioning" of their culture medium, we sought to evaluate the effect of ETAF on keratinocyte growth. While there was marked donor variability in the responsiveness of keratinocytes to ETAF, partially purified preparations of ETAF showed substantial ability to stimulate the growth of keratinocytes, particularly those of newborn donors. In addition, in conditioned media there appeared to be activities distinct from ETAF that also promoted keratinocyte growth. Keratinocytes in serum-free medium secreted large amounts of ETAF, as reported previously, and keratinocyte cultures derived from newborn donors secreted significantly more than did those derived from adult donors. These results are consistent with an autocrine growth regulatory role of ETAF in human epidermis and with an age-associated loss of this phenomenon.

Adult↗
PubMed1988

Induction of nerve growth factor receptors on cultured human melanocytes.

Normal differentiation and malignant transformation of human melanocytes involve a complex series of interactions during which both genetic and environmental factors play roles. At present, the regulation of these processes is poorly understood. We have induced the expression of nerve growth factor (NGF) receptors on cultured human melanocytes with phorbol 12-tetradecanoate 13-acetate and have correlated this event with the appearance of a more differentiated, dendritic morphology. Criteria for NGF receptor expression included protein accumulation and cell-surface immunofluorescent staining with a monoclonal antibody directed against the human receptor and induction of the messenger RNA species as determined by blot-hybridization studies. The presence of the receptor could also be induced by UV irradiation or growth factor deprivation. The NGF receptor is inducible in cultured human melanocytes, and we suggest that NGF may modulate the behavior of this neural crest-derived cell in the skin.

Cell Line↗
PubMed1988

Demonstration of a choline requirement for optimal keratinocyte growth in a defined culture medium.

Nutrient requirements for proliferation and differentiated function of individual cell types can be determined using cell culture methodologies. Human epidermal keratinocytes are stimulated to grow by choline supplementation in the presence of myo-inositol when grown in a commercial nutrient medium containing six other defined supplements. The optimal range of choline concentrations varied among donor cell lines, but consistently fell between 36 microM and 180 microM. Addition of 72 microM choline increased cell yield to 250 +/- 38% of that produced by myo-inositol supplementation alone and 92 +/- 8% of that produced by addition of a highly mitogenic hypothalamic extract, which was previously required for good growth in this culture system. Supplementation of the basal medium with both the extract and choline resulted in 165 +/- 13% of the cell yield observed with the extract addition alone. Supplementation with other phospholipid precursors did not further increase keratinocyte growth. Neither dermal fibroblasts nor epidermal melanocytes were stimulated by supplementation with choline, suggesting the high keratinocyte requirement is unusual. This completely defined culture medium for keratinocyte growth should prove useful in analyzing the role of phospholipids and other nutrients in human epidermis.

Cell Count↗
PubMed1988

Vitamin D, its precursors, and metabolites do not affect melanization of cultured human melanocytes.

Exposure of the skin to sunlight results in both tanning and vitamin D3 production. It has therefore been suggested that vitamin D3 or its active metabolite 1,25-dihydroxyvitamin D3 may be the mediator of UV-induced melanogenesis. To test this hypothesis, newborn foreskin-derived melanocytes were cultured in paired dishes in hormone-supplemented medium with 2% serum containing no detectable vitamin D3 or in the same medium containing 10(-8) or 10(-10) M of either provitamin D3, lumisterol, previtamin D3, vitamin D3, 25-hydroxyvitamin D3, or 1,25-dihydroxyvitamin D3. After 10 days, cell number in cultures containing vitamin D compounds was 93%-140% of unsupplemented controls and melanin content was 60%-120% of control, with no significant difference in either parameter for any compound tested. In separate experiments, human melanocytes and Cloudman S91 melanoma cells were repeatedly irradiated with physiologic doses of simulated sunlight and incubated between irradiations with provitamin D3, previtamin D3, vitamin D3, or 1,25-dihydroxyvitamin D3. Irradiated cultures had a 90%-95% inhibition of cell growth associated with a 200%-800% increase in melanin content per cell relative to controls, but there was no effect of any vitamin D compound on either cell type. Neither cultured human melanocytes nor S91 cells showed evidence of the cytosolic 1,25-dihydroxy-vitamin D3 receptor binding by sucrose density gradient analysis using radiolabeled 1,25-dihydroxyvitamin D3. The combined data strongly suggest that neither vitamin D3 nor its precursors or metabolites directly mediate melanogenesis in these cells.

Cells, Cultured↗
PubMed1988

An extract of bovine thymus stimulates human keratinocyte growth in vitro.

An extract prepared from newborn calf thymus stimulated proliferation of human keratinocytes cultured from newborn foreskins and from skin biopsies of 26 adult volunteers aged 19 to 70 years. Growth over the 7-day assay period in the basal medium was age-dependent, with newborn cultures achieving a 10-fold increase in cell number over seeding density, old adult cultures barely maintaining their seeding density and young adult cultures intermediate in proliferative capacity. Maximally stimulatory extract concentration was 5-fold higher for newborn than for adult keratinocytes, with adult cultures experiencing toxicity at doses still growth-promoting for newborn cultures. At optimal extract concentration the maximal average increase in cell yield (66.3% for newborn, 53.6% for young adult, and 18.1% for old) indicated decreased mitogen responsiveness or increased inhibitor sensitivity with increasing donor age. Stimulation of cholera toxin-treated cultures was equally high, ranging from 39.4% to 145.9%, suggesting that the extract acts through a cyclic AMP-independent pathway. Thymic extract did not increase colony forming efficiency. Our findings provide further support for the concept of functional interactions between the skin and the immune system, in addition to the recognized morphologic similarities between thymic cells and keratinocytes. Furthermore, these data confirm earlier findings of an inverse relationship between mitogen responsiveness and donor age for cultured cells.

Adult↗
PubMed1988

Normal human keratinocytes contain an interferon-like protein that may modulate their growth and differentiation.

Epidermal growth and differentiation is a complex process which depends upon a balance between positive and negative growth signals, and in normal skin the majority of the cells in the germinative basal layer do not proliferate unless stimulated. Using the indirect immunofluorescent method, it can be demonstrated that purified polyclonal epidermis in cross sections of normal skin and to the basal layer of cultured keratinocyte colonies. Furthermore, extracts of keratinocyte cultures contain interferon bioactivity. With Western blot analysis, antibodies to interferon recognize a band of approximately 40 kD both in keratinocyte lanes and in recombinant interferon lanes that give in addition a band of approximately 20 kD. Addition of interferon to rapidly growing keratinocytes inhibits their growth by as much as 90% and promotes their terminal differentiation. The growth inhibitory effect of interferon is completely reversible. These data demonstrate that interferon or a closely related protein is present in human epidermis and suggest that this protein may act as a physiologic modulator of keratinocyte growth and differentiation.

Antibodies↗
PubMed1988

Human epidermal keratinocytes in culture convert thyroxine to 3,5,3'-triiodothyronine by type II iodothyronine deiodination: a novel endocrine function of the skin.

Cultured human keratinocytes converted T4 to T3 by type II iodothyronine deiodination. Homogenates of keratinocytes cultured from neonatal foreskin or adult arm skin had similar mean T4 5'-deiodinating activities. Conversion of T4 to T3 by intact cells was demonstrable in cultures from neonatal and adult donors. Only phenolic ring deiodination occurred in the cultured cells and their homogenates, the apparent Michaelis constant for T4 was 12 nmol/L, and T4 and rT3 each inhibited 5'-deiodination of the other. T4 5'-deiodination was unaffected by addition to the assay mixture of 1 mumol/L T3, but was inhibited less than 10% by 1 mmol/L 6-n-propyl-2-thiouracil, 50% by 270 nmol/L iopanoic acid, 50% by 9.4 mumol/L 3,5-diiodo- 3',5'-dimethyl-L-thyronine, and 33% by 42 mumol/L amiodarone. When keratinocytes were cultured for 3-4 days in medium containing iodothyronine-free fetal calf serum, the T4 5'-deiodination rates in homogenates doubled; this increase was prevented by restoring a physiological free T4 concentration, but not by a supraphysiological T3 concentration. Homogenates of fresh whole skin or fetal cadaveric epidermis did not convert T4 to T3 in measureable amounts, although one epidermal homogenate had low level T3 typrosyl-ring deiodinating activity. These results suggest that human epidermal type II iodothyronine deiodination in man might conceivably contribute to the intracellular T3 content of the skin and even to serum T3 concentrations, especially in hypothyroidism.

Cells, Cultured↗
PubMed1988

Human nevocellular nevus cells are surrounded by basement membrane components. Immunohistologic studies of human nevus cells and melanocytes in vivo and in vitro.

Dermal nevus cells in the skin are surrounded by electron-dense deposits resembling a basement membrane (BM), and epidermal melanocytes rest on the epidermal BM. Using antibodies directed against various BM components, we have determined that the BM-like structure surrounding nevus cells in vivo contains type IV collagen, laminin, and BM-1 proteoglycan, analogous to BM throughout the body, but not bullous pemphigoid antigen or epidermolysis bullosa acquisita antigen that are keratinocyte-associated proteins present in the epidermal BM. Moreover, in vitro, both nevus cells and melanocytes derived from adult donors display intracellular and extracellular fibronectin, BM-1 proteoglycan, type IV collagen, and laminin. In contrast, newborn melanocytes maintained under identical culture conditions display none of these BM components, emphasizing the influence of donor age on cell behavior. The data suggest that dermal nevus cells manufacture a BM in vivo, as do certain other neural crest-derived cells. The apparent shared ability of cultured nevus cells and melanocytes to synthesize BM components, coupled with other previously noted behavioral and morphologic similarities in vitro, suggests that these cell types are very closely related; and that morphologic or histochemical differences present in vivo are the result of environmental influences rather than intrinsic differences.

Adult↗
PubMed1987

Use of strontium to separate calcium-dependent pathways for proliferation and differentiation in human keratinocytes.

The role of extracellular calcium (Caex) in modulating keratinocyte differentiation has been well documented, but its role in proliferation has been harder to define due to the confounding effect of terminal differentiation. Because strontium (Sr) does not induce terminal differentiation in murine keratinocytes but does mimic the stimulatory effect of Caex on DNA synthesis in chick fibroblasts, experiments were undertaken to determine if Sr could be used to separate the presumably opposing effects of Caex on the proliferation and differentiation of cultured human keratinocytes. In response to additions of SrCl2, keratinocytes in a serum-free hormone-supplemented basal medium containing 0.03 mM Ca showed a dose-dependent increase in day 7 cell yields. Cell yield in the optimal concentration of SrCl2 (1.8 mM) was approximately twice that obtained in any concentration of CaCl2. Maximally stimulatory additions of CaCl2 varied from 0.05 to 1.8 mM, but 0.03 and 0.05 mM additional CaCl2 always increased cell yield relative to unsupplemented controls. Keratinocytes grown in low levels of CaCl2 or any level of SrCl2 have minimal contact with each other regardless of cell density in contrast to the colonies of tightly apposed and stratified cells grown in 1.8 mM CaCl2. Transmission electron micrographs of vertically sectioned confluent cultures in low or high levels of SrCl2 or in low levels of CaCl2 revealed abundant ribosomes and keratin filaments but no stratification or desmosomes, while cultures in 1.8 mM CaCl2 were stratified with numerous desmosomes. These results suggest that Caex may separately stimulate keratinocyte proliferation and terminal differentiation and that Srex can substitute for Caex in the former but not the latter process.

Calcium↗
PubMed1987

Ultraviolet radiation directly induces pigment production by cultured human melanocytes.

In humans the major stimulus for cutaneous pigmentation is ultraviolet radiation (UVR). Little is known about the mechanism underlying this response, in part because of the complexity of interactions in whole epidermis. Using a recently developed culture system, human melanocytes were exposed daily to a physiologic range of UVR doses from a solar simulator. Responses were determined 24 hours after the last exposure. There was a dose-related increase in melanin content per cell and uptake of 14C-DOPA, accompanied by growth inhibition. Cells from donors of different racial origin gave proportionately similar increases in melanin, although there were approximately tenfold differences in basal values. Light and electron microscopy revealed UVR-stimulated increases in dendricity as well as melanosome number and degree of melanization, analogous to the well-recognized melanocyte changes following sun exposure of intact skin. Similar responses were seen with Cloudman S91 melanoma cells, although this murine cell line required lower UVR dosages and fewer exposures for maximal stimulation. These data establish that UVR is capable of directly stimulating melanogenesis. Because cyclic AMP elevation has been associated in some settings with increased pigment production by cultured melanocytes, preliminary experiments were conducted to see if the effects of UVR were mediated by cAMP. Both alpha-MSH and isobutylmethylxanthine (IBMX), as positive controls, caused a fourfold increase in cAMP level in human melanocytes and/or S91 cells, but following a dose of UVR sufficient to stimulate pigment production there was no change in cAMP level up to 4 hours after exposure. Thus it appears that the UVR-induced melanogenesis is mediated by cAMP-independent mechanisms.

Cells, Cultured↗
PubMed1987

Syndromes of premature aging.

The classic premature aging syndromes are rare disorders, all of which clinically differ a great deal from the normal aging process. None is well understood at the biochemical level, and no specific treatment exists for any of these syndromes. Genetic counseling, preventive measures in some instances, and symptomatic treatment are available. The interest of gerontologists and geriatricians in these disorders derives in part from the expectation that such presumed single-gene mutations might contribute to our understanding of the molecular basis for normal aging. Unfortunately, to date, this expectation has not been realized.

Adolescent↗
PubMed1987

A survey of skin problems and skin care regimens in the elderly.

In an attempt to provide clinically relevant data regarding both dermatologic disease and skin care needs in the elderly, 68 noninstitutionalized volunteers, aged 50 to 91 years (average age, 74 years), were enrolled in a study consisting of a 33-item questionnaire and a total cutaneous examination. Two thirds of the entire group and 83% of the 23 octogenarians reported medical concerns regarding their skin, with pruritus as the most frequent complaint. On examination, all subjects had at least one cutaneous abnormality, and symptomatic and/or medically significant disorders were present in 64.7%. In decreasing order of prevalence, disorders for which dermatologic therapy was judged desirable included actinic keratoses, tinea pedis, contact dermatitis, seborrheic dermatitis, stasis dermatitis, and skin cancer. Overall, there was rather poor correlation between the subjects' complaints and perceptions and objective physical findings. Further, despite a high prevalence and long average duration of dermatologic concerns, very few subjects had consulted a physician for these problems, and no complaints other than "rashes" and pruritus had ever been discussed with any health care professional. Review of skin care regimens revealed substantial limitations with regard to bathing, shampooing, and nail care, particularly for subjects aged 80 years or older. Despite a small sample size and possibility of selection bias among the subjects, these data strongly suggest that skin problems are common among the elderly and that at present their dermatologic needs are largely unmet.

Aged↗