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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.

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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.

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The existence of a psoriasis susceptibility locus, PSORS1 (HUGO/GDB-approved symbol), in or near the HLA region of chromosome 6 is strongly supported by a lod score analysis of HLA-B and psoriasis in 97 families from 16 published datasets. Families included in the dataset represent all the psoriasis families with usable HLA data that we could find in the published literature through May 1997. The recombination fraction between PSORS1 and HLA-B is estimated to be at or near 0.00, with a maximum two-point lod score of 23.7, assuming a dominant mode of inheritance with low (20%) penetrance at the PSORS1 locus. Although these families are geographically and ethnically diverse, there is no evidence for linkage heterogeneity at the HLA-linked locus in this analysis. We also conclude that the HLA-B17 allele, which is strongly associated with psoriasis, is unlikely itself to contribute directly to psoriasis susceptibility; rather, the HLA-B locus is probably tightly linked to the PSORS1 locus. Finally, we raise the possibility of a two-locus/heterogeneity model as one way to reconcile several findings in the literature.

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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.

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A431 cells grown as three-dimensional spheroids show growth stimulation in response to nanomolar concentrations of EGF in contrast to monolayer cultures that show inhibition. In investigating the alterations in EGF signal transduction that underlie this modification of the proliferative response, we have compared the expression of EGF receptors on A431 cells under these conditions and related our findings to tyrosine phosphorylation and the growth response. EGF receptors were measured by 125I-EGF binding to trypsin-dispersed cells. Unexpectedly, dispersion of the monolayers caused an 80% decrease in surface EGF receptor, although, after dispersion, EGF receptor was digested by trypsin with a half-life of 69 +/- 32 min. No evidence for a comparable loss of cellular EGF receptor was seen on trypsin dispersion of spheroids. After allowing for this effect, we found that the receptor density on nondispersed monolayers (5 x 10(6) per cell) was twentyfold greater than that on spheroids (0.25 x 10(6) per cell). EGF-induced tyrosine phosphorylation was confined to the outermost cells of the spheroid, although the presence of surface-expressed EGF binding sites could be demonstrated throughout the structure and the number of EGF receptors/cell on dispersed spheroid cells showed a single distribution peak by flow cytometry, with no evidence for more than one population. Using RCM-lysozyme as a substrate, tyrosine phosphatase activity in spheroids lay within the range observed in monolayer cultures. Autophosphorylation of the EGF receptor following EGF stimulation in monolayer cultures of A431 cells rose rapidly in the first 10 seconds and then slowly increased for at least 3 h. In spheroids, it reached a maximum within 10 seconds and then declined over 3 h. Since the microenvironment within a tumor resembles that in a spheroid, a similar reduction in surface EGF receptor expression may be expected in tumors relative to monolayer cultures, together with corresponding growth stimulation in response to EGF.

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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.

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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.

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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.

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Following activation with the inflammatory mediator phorbol myristate acetate (PMA), human microvascular endothelial cells (DMEC) is olated from the human dermis (DMEC) rapidly and dramatically convert from a classical epithelioid morphology to a spindle-shaped configuration. This is accompanied by changes in the organization of gap junctions and the vimentin and actin cytoskeletons. This report describes the sequential changes in the expression of four proto-oncogenes, c-fos, c-myc, c-sis and H-ras in DMEC following PMA exposure. The synthesis of c-fos mRNA was transiently induced by PMA from a basal concentration below the limit of detection to a maximum at 60 min., declining to the unstimulated level within 2 hrs. Synthesis of c-myc mRNA declined continuously and reached 37% of control levels over 16 hrs. Expression of c-sis which encodes for the B chain of platelet-derived growth factor, also declined to 34% of the control value over 16 hrs. There was no change in the synthesis of H-ras mRNA nor of beta-actin mRNA which was used as a control. The expression of c-myc in normal DMEC was compared to a human dermal microvascular cell line transformed by SV-40 (TREND). The TREND cell line maintains a permanent spindle-shaped configuration under all growth conditions and multiplies faster than DMEC. In contrast to the non-transformed cell cultures, expression of c-myc in TREND cells was induced by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)

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We have compared the EGF responses of A431 cells when grown as monolayers at a variety of cell densities or as multicellular spheroids in order to investigate the effects of cell contact and 3-dimensional structure on signal transduction. Proliferation of the A431 squamous carcinoma cell line grown in our laboratory was unaffected by EGF when grown in monolayer culture. As 3-dimensional, multicellular spheroids, however, growth was stimulated by EGF. The maximum volume attainable in the presence of EGF was more than 30 times that in its absence. EGF-dependent tyrosine phosphorylation was compared under these conditions by immunohistochemistry and Western blotting. In initial experiments using published procedures, tyrosine phosphorylation was density-dependent in monolayers and undetectable in spheroids. However, the density-dependence was abolished by the addition of high concentrations of protein tyrosine phosphatase inhibitors (1 mM Zn++ and VO4(3)-). The density dependence of EGF-stimulated tyrosine phosphorylation in monolayers was, therefore, largely the result of changes in phosphatase activity rather than kinase. Using high concentrations of phosphatase inhibitors, phosphotyrosine was clearly visible by immunohistochemistry in the outermost cells of spheroids, but it was still not visible in the spheroid center. The lack of response within the spheroid was not related to the presence of EGF receptor nor diffusion of EGF. In companion experiments, we showed that staining for EGF receptor was present homogeneously throughout the spheroid and that EGF penetrated to its center under the conditions of the experiment. Thus, although an increase in tyrosine phosphatase activity was a major factor affecting tyrosine phosphorylation in the outer cells, other factors were important in the inner cells. We concluded that an increase of tyrosine phosphatase activity was the most important component of the adaptation of the EGF signal transduction system to high cell density in monolayer cultures. In spheroids, tyrosine phosphatases are also enhanced, but other factors, such as autocrine synthesis of TGF-alpha and possibly the cellular distribution of EGF receptors and cell shape, play a role.

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The expression of transforming growth factor (TGF beta 1) protein in human and porcine skin has been analyzed by immunohistochemistry with two polyclonal antibodies (anti-CC and anti-LC) following cutaneous injury. The anti-LC antibody binds intracellular TGF beta 1 constitutively expressed in the nonproliferating, differentiated suprabasal keratinocytes in the epidermis of normal human skin, while the anti-CC antibody does not react with the form of TGF beta 1 present in normal skin as previously shown. TGF beta 1 may play a role in wound healing as suggested by its effect on multiple cell types in vitro and its acceleration of wound repair in animals. We have evaluated the natural expression and localization of TGF beta 1 protein in situ during initiation, progression, and resolution of the wound healing response in two models of cutaneous injury: the human suction blister and the dermatome excision of partial thickness procine skin. Anti-CC reactive TGF beta 1 in the epidermis is rapidly induced within 5 minutes following injury and progresses outward from the site of injury. The induction reflects a structural or conformational change in TGF beta 1 protein and can be blocked by the protease inhibitor leupeptin or by EDTA, suggesting a change in TGF beta 1 activity. One day post-injury anti-CC reactive TGF beta 1 is present in all epidermal keratinocytes adjacent to the wound including the basal cells. This corresponds temporally to the transient block of the basal keratinocyte mitotic burst following epithelial injury. Three to 4 days post-injury anti-CC reactive TGF beta 1 is localized around the suprabasal keratinocytes, in blood vessels, and in the papillary dermis in cellular infiltrates. The exclusion of TGF beta 1 from the rapidly proliferating basal cells and its extracellular association with suprabasal keratinocytes may represent physiological compartmentation of TGF beta 1 activity. Anti-CC staining is strong in the leading edge of the migrating epithelial sheet. The constitutive anti-LC reactivity with suprabasal keratinocytes seen in normal epidermis is neither relocalized nor abolished adjacent to the injury, but anti-LC staining is absent in the keratinocytes migrating within the wound. As the wound healing response resolves and the skin returns to normal, anti-CC reactive TGF beta 1 disappears while constitutive anti-LC reactive TGF beta 1 persists. Thus, changes in the structure or conformation of TGF beta 1, its localization, and perhaps its activity vary in a spatial and temporal manner following cutaneous injury and correlate with physiological changes during wound healing.

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The time course of appearance and the dynamic changes of immunocompetent cells were assessed in human skin following sterile suction blister would healing. During epidermal regeneration, a local increase in Langerhans cells (LC) and the appearance of a mononuclear cell infiltrate were observed. Initially, T cells were exclusively of the T helper/inducer subtype, whereas during the later stages of the healing process an increasing number of cytotoxic/suppressor T cells (up to 30%) was observed. Keratinocytes of neither the adjacent nor the newly formed epidermis expressed HLA-DR over the course of the wound-healing process. Our results suggest that immune-competent cells such as T cells and LC may play an important role in the regulation of the wound-healing process.

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Transforming growth factor-beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGF beta 1-specific polyclonal antibodies (anti-LC and anti-CC) to determine the presence of TGF beta 1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti-TGF beta 1 staining was observed primarily in the epidermis. Anti-LC TGF beta 1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti-CC TGF beta 1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti-LC and anti-CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGF beta 1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGF beta 1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGF beta 1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGF beta 1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.

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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.

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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.

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A murine monoclonal antibody, VM-1, which binds to basal cells of normal human epidermis, reduces the ability of human squamous cell carcinoma cells (SCL-1) derived from the skin to attach and spread on collagen by about 50% and causes cell rounding. Similar effects have been previously shown using normal human keratinocytes. The attachment of cell lines derived from human lung squamous cell carcinomas (SW1271 and SW900), melanoma A375, glioblastoma 126, and fibrosarcoma HT1080 is also inhibited by this antibody. VM-1 antibody does not bind to normal human fibroblasts, benign nevus cells, or the human B-cell-derived line 8866. VM-1 antibody inhibits the growth of SCL-1 cells in vitro as measured by cell numbers and [3H]thymidine ([3H]TdR) incorporation. It is not cytolytic in the presence of complement as measured by 51Cr release. Repeated treatment of SCL-1 cells with VM-1 antibody significantly reduces the proportion of SCL-1 cells that attach to collagen. In addition, after treatment of SCL-1 cells with VM-1 antibody, several proteins can no longer be demonstrated by gel electrophoresis of the cell-free supernatant. The VM-1 antibody effect on attachment and spreading is partially reversed by pretreatment of the collagen surface with laminin and fibronectin, but not with the carbohydrates chondroitin-6-sulfate or hyaluronic acid or with the protein lysozyme. By fluorescence staining, the antigen recognized by VM-1 antibody is membrane-bound and Triton X-100 extractable. The VM-1 antigen is excluded from Bio-Sil TSK-400 and sediments at about 10.5 S. It has a covalent molecular weight on the order of 10(6). Proteinase K digestion produces VM-1 antibody reactive fragments, assumed to be polysaccharides, with a polydisperse molecular weight distribution in the range 5000 to 30,000. The VM-1 antigen is partially lost from solution on boiling and is no longer detectable in the aqueous or organic phase after chloroform-methanol extraction. The properties of the VM-1 antigen are consistent with those of a proteoglycan involved in attachment and spreading of keratinocytes and certain tumor cells on collagen.

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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.

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