Human cell-conditioned media produced under embryonic-like conditions result in improved healing time after laser resurfacing.

BACKGROUND: Laser resurfacing procedures are continuing to grow in popularity as patients select less invasive procedures for rejuvenation of photo-damaged and aging skin. However, although physicians have begun exploring options to aid in postlaser healing, currently available treatments have little clinical evidence to support their use for wounded skin. METHODS: When grown under conditions of very low oxygen and suspension, a simulation of the embryonic environment, neonatal cells have been found to produce proteins and growth factors in types and quantities similar to those of fetal cells. The human cell-conditioned media (hCCM) produced by the cells was extracted and formulated into a gel to evaluate its efficacy in the healing of postlaser wounds. RESULTS: A split-face clinical evaluation of the material was performed, with 42 subjects undergoing combination ablative and nonablative laser procedures. Three concentrations of the hCCM were tested (× 0.1, × 1.0, × 10.0), and a dose-response trend was seen in the blinded physician evaluation, particularly in the assessment of crusting. In addition, transepidermal water loss readings showed a significant difference (p ≤ 0.05), indicating a more rapid return to normal skin barrier function with the active treatment. Histopathologic evaluation of subject biopsies showed reduced inflammation and a more normal epidermal appearance in the active treatment sites. CONCLUSIONS: The results of this clinical evaluation support the use of the soluble hCCM produced under embryonic-like conditions to accelerate wound healing after laser resurfacing procedures. The utility of the × 10 concentration appears to promote more rapid, scarless wound healing after resurfacing procedures and more normal skin recovery.

Use of cloned genetically modified human fibroblasts to assess long-term survival in vivo.

Because human fibroblasts are easily brought to tissue culture conditions and can be stably transduced with retroviral vectors encoding transgenes ex vivo, genetically modified fibroblasts are frequently considered in strategies to correct disease with gene therapy. This enthusiasm has been dampened by studies showing that transgene expression by genetically modified fibroblasts diminishes with time in vivo, but not in vitro, for reasons that are unclear. We elected to study this problem using cloned human fibroblasts that had been cloned by limiting dilution and stably transduced with a retroviral vector encoding lacZ ex vivo. These were seeded onto a nonbiodegradable nylon matrix that was transplanted to nude mice. Transgene expression was followed prospectively by histologic exam. Data show that human fibroblasts can withstand the pressure of cloning by limiting dilution. In addition, they can be passaged from 10 to > 20 times, and > 1 x 10(20) of genetically modified fibroblasts can be generated as progeny of one cell. Loss of transgene expression by the cloned genetically modified fibroblasts in vivo occurs in an orderly and progressive fashion, but is not complete by 4 months. Neither the loss nor the persistence of expression appear to be random. These observations are most compatible with the thesis that a major cause of the loss of transgene expression in vivo is secondary to apoptosis of the genetically modified fibroblast. Loss of expression of transgenes in senescent genetically modified fibroblasts occurs more rapidly than in their presenescent counterparts in the age-neutral, in vivo setting of the nude mouse.

Role of transforming growth factor beta in the maturation of human epidermal keratinocytes.

Changes in protein synthesis and phosphorylation in cultured human keratinocytes in response to TGF-beta have been examined by one and two dimensional electrophoresis. Transforming growth factor beta has been shown to cause little change in the rate of methionine incorporation in the concentration range in which growth is reversibly arrested. It does, however, prevent the labeling of certain specific bands detected on gels of triton-soluble proteins after 3 days of treatment. Phosphorylation of triton-soluble proteins is inhibited at concentrations of TGF-beta rather higher than the Kd of its receptor and may represent a nonphysiological effect. Nonetheless, the phosphorylation of certain prominent species is reduced. In keratinocytes cultured in delipidated serum, which show some expression of keratin 1 (67 kd) characteristic of normal maturation, TGF-beta reduces the incorporation of methionine into this keratin 1 and increases labeling of keratins 6 and 16. Transforming growth factor beta thus promotes regenerative maturation, which is normally expressed during wound healing. The ability of TGF-beta to arrest keratinocyte growth in a reversible manner and to stimulate regenerative maturation, supports its physiological role in controlling the balance between cell division, migration and maturation during epidermal wound healing.

The binding of Helix pomatia and Ulex europeus agglutinins to normal and psoriatic skin.

We have compared the staining patterns of Ulex europeus agglutinin (UEA) I-FITC and Helix pomatia agglutinin (HPA)-FITC on normal and psoriatic epidermis in order to follow the production of the binding sites as a function of maturation. We have further characterized them with respect to solvent extraction and enzyme digestion. The bandlike pattern of membrane staining by UEA I in the upper spinous and granular layer cells of normal epidermis is lost in psoriasis. Instead there is a fainter cytoplasmic staining which is largely sensitive to chloroform/methanol extraction, and thus presumably is glycolipid in nature. Epidermal binding of HPA to normal skin sections is mainly seen in cell membranes. It spares the basal layer and then increases from the suprabasal region to the granular layer. HPA staining in the spinous layer is destroyed by extraction with Triton X-100. In contrast, binding to the membranes of cells in psoriatic epidermis is triton-resistant. The result indicates the production of a component early in the maturation pathway which has no counterpart in normal skin. HPA binding to the spinous cells of symptomless skin from psoriatic patients shows decreased sensitivity to Triton X-100 by comparison with normal. The results are discussed in terms of changes in the pathway of keratinocyte maturation in psoriasis.

Reduced expression of surface receptors for synthetic N-formylated chemotactic oligopeptides by stimulated peripheral blood neutrophils in psoriasis.

Receptors for synthetic N-formylated chemotactic peptides on peripheral blood neutrophils were studied by the binding of fluorescein-labeled hexapeptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys) to the cells in vitro at the range of concentrations 0.01-100 nM. Mean fluorescence of neutrophils was quantitated by a flow cytometry using FACS III. Comparison was made between 27 patients with psoriasis vulgaris and 14 normal controls. Various receptor states related to cell activities were induced by different temperatures, by incubation of cells with cytochalasin B and by preincubation with nonlabeled N-formyl-Met-Leu-Phe. This allowed us to distinguish between the specific binding of fluoresceinated hexapeptide to plasma membrane receptor already present (0 degree C), modulation of receptors by peptide and cytochalasin B stimulated degranulation (25 degrees C), and net binding, including internalization of peptide and receptor recycling system (37 degrees C). At peptide concentrations of 1-10 nM, the labeling of neutrophils at 25 degrees C and 37 degrees C, but not at 0 degree C, was found to be about 10-35% lower in psoriatic than in healthy subjects (p less than 0.002). The amount of fluorescein-labeled peptide bound to the cells at 25 degrees C was markedly increased by cytochalasin B, but to a much lower extent in psoriatic patients than in normal controls. Although the number of plasma membrane receptor for chemotactic peptides in the nonstimulated neutrophils was not altered in psoriasis, the receptor up-regulation induced by preincubation of the cells with 1-10 nM of nonlabeled N-formyl-Met-Leu-Phe at 37 degrees C was reduced when measured by subsequent fluoresceinated hexapeptide uptake at 0 degree C. Receptor recycling, as measured by an increase with time (0-30 min) in the binding of chemotactic peptide by neutrophils in which receptors had been down-regulated, was found to be within normal range in patients with psoriasis. These data indicate that nonstimulated, circulating neutrophils have a normal number of chemotactic peptide receptors on the cell surface, but are less able to recruit intracellular receptors to the cell surface. This finding may be related to smaller internal pools or less efficient translocation of these receptors.

Induction of the synthesis of triton-soluble proteins in human keratinocytes by gamma interferon.

Recombinant human gamma interferon (r-IFN-gamma) induces the synthesis and expression of HLA-DR antigen on cultured, normal, human keratinocytes depleted of Langerhans cells. After removal of r-IFN-gamma from the culture medium of keratinocytes that are expressing HLA-DR antigen, the cells continue to express this antigen for at least 2 days. r-IFN-gamma induces, in a dose dependent fashion, the synthesis of several triton-soluble proteins with the most prominent having an apparent molecular weight of 53,000. Whereas normal keratinocytes do not express HLA-DR antigen in vivo, they do express HLA-DR in a variety of skin diseases such as lichen planus, graft-versus-host disease, and mycosis fungoides. We propose that an understanding of lymphocyte-keratinocyte interactions in vivo may be achieved by further studies of the mechanism of action of r-IFN-gamma on cultured keratinocytes and that the results may provide insight into the patho-physiology leading to a number of common inflammatory and neoplastic skin diseases.

Induction of new proteins by gamma interferon in cultured human keratinocytes.

Human epidermal keratinocytes incubated with recombinant gamma interferon (r-IFN-gamma) show, on two-dimensional gel electrophoresis, both the appearance of new proteins and the loss of others. Among [35S]methionine-labeled proteins, which are induced in an actinomycin- or alpha-amanitin-sensitive manner, is a prominent group with an apparent relative molecular mass of 53,000 and pI of 5.3-5.8. The synthesis of these proteins continues for at least 4 days in the presence of gamma interferon (IFN-gamma). Over the concentration range tested, up to 670 pM, there is no inhibition of protein synthesis, so the appearance of these proteins cannot be explained by overall inhibition of protein synthesis. Furthermore, at 4 pM we found only minor inhibition of DNA (21%) and RNA (29%) synthesis. Half-maximal induction of the prominent 53 kD proteins occurs at an interferon concentration of 0.8-3.5 pM which may be compared with a range of 1.5-30 pM for HLA-DR induction. The same prominent proteins are also induced by type I interferons. The 53 kD protein complex appears to consist of at least 4 different proteins, one of which is phosphorylated and another one of which is not induced in fibroblasts treated with IFN-gamma. We could obtain no evidence that the proteins were related by glycosylation. The presence of these proteins provides a sensitive means of identifying keratinocytes responding to interferons. Lack of these proteins in normal epidermis indicates that interferon does not play a major role in the control of keratinocyte behavior in sound skin.

Histologic distribution of staining by a monoclonal antibody (psi-3) in psoriasis and occurrence of psi-3 antigen in other cutaneous diseases.

psi-3 is a monoclonal antibody that recognizes a 135,000 molecular weight structural component of maturing keratinocytes in psoriasis (the psi-3 antigen) but fails to bind to any constituent of keratinocytes in normal epidermis. This paper describes the occurrence of the psi-3 antigen in a variety of dermatopathologic conditions using immunoperoxidase (biotin-avidin-peroxidase) and immunofluorescence methods which show excellent concordance. In 35 of 36 specimens of psoriasis vulgaris, psi-3 antibody consistently immunolabels the cytoplasm of keratinocytes above the basal layer. At the edges of psoriatic plaques, psi-3 antibody staining extends for a variable distance into lesion-free epidermis. A similar pattern has been found in a certain number of other conditions described in the paper, including squamous cell carcinoma and condyloma acuminatum, but not Darier's disease, basal cell carcinoma, nor lamellar ichthyosis. In all but one condition, the outermost or basal layer of cells is never stained. The only disease in which the lowermost cell layer is stained is a lichen planus-like lesion. The occurrence of psi-3 antigen cannot be correlated with any histologic feature of psoriasis such as acanthosis, loss of the granular layer, or hyperproliferation. The antigen appears to be a unique keratinocyte constituent which is expressed in certain pathologic conditions and which is not detected by any other histologic or immunophenotyping method. It is a potentially valuable addition to the panel of antibodies available for characterizing epithelial cells.

Evidence for an alternative pathway of keratinocyte maturation in psoriasis from an antigen found in psoriatic but not normal epidermis.

We have isolated a murine monoclonal antibody, called psi-3, which immunolabels maturing keratinocytes in psoriatic skin but not in normal epidermis. The staining is cytoplasmic and is not extractable with 1% Triton X-100, which suggests that the psi-3 antigen is a structural component of the keratinocyte. Neither basal cells nor invading inflammatory cells are stained in psoriatic skin and the antigen appears to be associated specifically with maturing and not proliferating keratinocytes. Keratinocytes cultured in vitro from skin from nonpsoriatic individuals display the antigen in a granular pattern in differentiated cells. The antigen is also expressed after tape-stripping of normal skin and, therefore, represents an inducible product of normal keratinocytes. The antigen is destroyed by proteinase K and appears to be a protein. On discontinuous sodium dodecyl sulfate-gel electrophoresis, the antigen has been found to have a molecular weight of 135,000. The psi-3 antigen is interpreted as a new keratinocyte product expressed in psoriasis, culture, wound healing, and certain other pathologic skin conditions. The synthesis of such a new antigen would not be expected if keratinocyte maturation in psoriasis is a truncated version of the normal system and supports the hypothesis that psoriatic keratinocytes are following an alternative pathway. Results using experimental injury suggest that the psoriatic pathway is normally expressed during wound healing.

Effects of recombinant interleukin 1 and interleukin 2 on human keratinocytes.

The effects of recombinant interleukin 1 alpha and beta, as well as recombinant interleukin 2, on human keratinocyte proliferation were studied in serum-containing as well as defined media. Both interleukin 1 preparations did not stimulate keratinocyte growth; interleukin 2 also did not stimulate keratinocyte growth. To determine whether interleukin 1 beta binds to keratinocytes, a cell membrane assay was developed for these cells. Iodinated interleukin 1 beta binds to keratinocytes with a kD of 6.2 nm and 2500 receptors per cell. To determine the effects of interleukin 1 beta on protein synthesis, the molecular patterns of radiolabeled cell extracts of interleukin 1 beta-treated and nontreated keratinocytes were compared using two-dimensional polyacrylamide gel electrophoresis. No significant changes in the molecular pattern of newly synthesized proteins were detected. Finally, none of these lymphokines induced HLA-DR expression by keratinocytes.

Penetration of lucifer yellow into human skin: a lateral diffusion channel in the stratum corneum.

We investigated the penetration of Lucifer Yellow into human and murine epidermis in 4-mm punch biopsies by incubation in dye solution. Lucifer Yellow was taken up freely by the dermis but penetrated only slightly into keratinocytes of the basal and suprabasal layers. However, progressive lateral diffusion was observed in the lowest layers of the stratum corneum, extending a distance of 1 mm in 6 hr. Under high magnification, Lucifer Yellow appeared to lie within rather than between corneocytes of this layer. Control samples stained with Lucifer Yellow after sectioning showed no preferential binding of the dye in this region. We concluded that the localization of staining was the result of diffusion from the cut edge of the stratum corneum. Lucifer Yellow penetration was insensitive to PMSF, 1:10 phenanthroline, or N-ethyl maleimide and was also observed in an in vivo injury, indicating that it was not an artifact of proteolytic or degenerative changes. In contrast, horseradish peroxidase failed to penetrate, suggesting molecular size limitation to channel entry. Diffusion of Lucifer Yellow beneath the stratum corneum marks a pathway for the lateral movement of small molecules of potential importance in the normal physiology of the skin, drug delivery, and pathology.

Correlation of the ability of retinoids to inhibit promoter-induced anchorage-independent growth of JB6 mouse epidermal cells with their activation of retinoic acid receptor gamma.

Retinoids inhibit the biological effects induced in mouse epidermal cells by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Specific nuclear retinoic acid receptors (RARs) have been identified in the epidermis, but the specific receptor that mediates the inhibitory response by retinoids is not established. Retinoic acid and six conformationally restricted retinoids were evaluated in an in vitro bioassay using the JB6 mouse epidermal cell line. These activities were then compared with the ability of these retinoids to activate the RARs in transient transfection assays for transcriptional activation to identify the retinoid receptor involved in inhibiting TPA-induced anchorage-independent growth. The retinoids inhibited TPA-induced colony formation of JB6 cells in semisolid medium at concentrations that were not toxic based on colony formation of attached cells. These concentrations ranged from less than 10(-9)-10(-6) M. 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethylanthracen-2-yl)benzoic acid (TTAB) was the most potent retinoid, with an EC50 of 0.8 nM. Both RAR alpha and RAR gamma were expressed in JB6 cells. Expression of RAR beta was not detected in these cells using a polymerase chain reaction assay, consistent with its extremely low level in mouse skin. Inhibition of the TPA response by these retinoids in JB6 cells correlated only with their transcriptional activation of RAR alpha, but not with that of RAR alpha. These results suggest that RAR gamma is most probably the receptor that mediates the chemopreventive effects of retinoids in mouse epidermis.

Evidence for an NMDA receptor subunit in human keratinocytes and rat cardiocytes.

Receptor binding studies have demonstrated the presence of an [3H]MK-801 ([3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate) binding site in human keratinocytes. The affinity found in keratinocytes was lower than that found in brain membranes. Northern blots identified mRNA in human keratinocytes and rat cardiocytes, as well as rat brain, that hybridized with high stringency to a probe for NMDAR1, an NMDA receptor subunit. In each tissue, mRNA that hybridized to another glutamate binding protein that might be part of an NMDA receptor complex, was also present. The presence of NMDA or NMDA-like receptors in keratinocytes and rat cardiocytes together with the low affinity [3H]MK-801 binding suggests that this protein may be a general channel forming protein that is present in many tissues, and forms specific receptors by interacting with additional subunits.

Human-based tissue-engineered implants for plastic and reconstructive surgery.

The emerging field of tissue engineering focuses on the creation of living tissues and organs for use in tissue repair and transplantation. Human cells are seeded onto biocompatible scaffolds and grown under physiologic conditions to produce all-human biointeractive implants. Tissue-engineered skin implants have shown efficacy in a variety of wound applications. Near term products, including injectable human matrix for contour defects and tissue-engineered cartilage, are proving to be important tools for plastic and reconstructive surgery.