Search

Barbara Gilchrest, in her inaugural Editorial for this series on translational research, related that her clinical experiences kindled a desire to pursue laboratory-based training to bridge “basic discoveries at the cellular and molecular level with improvements in patient care” (Gilchrest, 2015). Her approach was bedside to the bench (...and back). My career had just the opposite genesis. I wanted principally to do fundamental biochemistry and cell biology and, indeed, embarked on a postdoctoral career studying tadpole metamorphosis under the tutelage of Arthur Eisen, then head of the Division of Dermatology at Washington University School of Medicine in St. Louis. That I found a career in dermatology, let alone one steeped richly in translational research, is due largely to consummately practical advice from two people.

Over the past 25 years both the quality and quantity of pharmaceutical and biopharmaceutical research has changed. Formerly rigidly separated research efforts in academic institutions and the biopharmaceutical industry have become increasingly transparent to one another. Industry has in some cases scaled down its internal research efforts, while enhancing its outreach to basic research in academic institutions. In parallel, research at academic institutions has-in some cases-added a focus on application of discoveries to patient needs. This porosity between industry and academia has created opportunities for more rapid translation of basic discoveries to patient needs. Additionally, both physicians and fundamental scientists have broadened their career opportunities, and movement between industry and academia-almost unheard of two decades ago-now occurs regularly. At the same time, numerous examples exist of how these translational efforts have benefited not only patients but also investigators and academic institutions as well. Despite many potential advantages of closer interactions between industry and academia, other issues, such as conflicts of interest (both real and perceived), continue to pose challenges.

Dr Ruth Freinkel died 17 May 2014 at the age of 87 at her home in Eugene, Oregon. Born in Hamburg, Germany, in 1926, Ruth immigrated to the United States when she was seven years old with her family (including her father, Paul Kimmelstiel, who described Kimmelstiel–Wilson syndrome/diabetic nephropathy).

Psoralens are a class of pharmaceutical agents commonly used to treat several cutaneous disorders. When irradiated with a mode-locked titanium: sapphire (Ti:sapphire) laser tuned to 730 nm, an aqueous solution of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) emits blue light. The emission spectrum is centered at 452 nm and is identical to that obtained by one-photon excitation with UVA excitation, and its magnitude depends quadratically on the intensity of laser excitation. These results suggest that two-photon excitation occurs to a potentially photochemically active state. To estimate the two-photon absorption cross section, it was first necessary to measure the emission quantum yield of HMT using 365 nm excitation at room temperature that resulted in a value of 0.045 +/- 0.007. The two-photon absorption cross section of HMT at 730 nm is therefore estimated to be 20 x 10(-50) cm4 s (20 Göppert-Mayer). The excited-state photophysics and photochemistry of psoralens suggest potential applications to cutaneous phototherapy in diseases such as psoriasis and dystrophic epidermolysis bullosa.

The development of topical drug delivery systems has recently gained significant interest due to the ease of administration and lesser risks of systemic toxicity. The development of these new technologies utilizes the properties of the structure and function of the skin. The stratum corneum plays the largest role in affecting drug permeation, as the corneocytes and lipid matrix in this layer effectively prevent the diffusion of large molecules. In this review, we introduce the structure and function of the skin as it relates to topical drug delivery.

Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. The most severe, dystrophic (scarring) forms of EB demonstrate blister formation below the cutaneous basement membrane at the level of the anchoring fibrils. Ultrastructural observations of altered anchoring fibrils and genetic linkage to the gene encoding type VII collagen (COL7A1), the major component of anchoring fibrils, have implicated COL7A1 as the candidate gene in the dystrophic forms of EB. We have recently cloned the entire cDNA and gene for human COL7A1, which has been mapped to 3p21. In this study, we describe mutations in four COL7A1 alleles in three patients with severe, mutilating recessive dystrophic EB (Hallopeau-Siemens type, HS-RDEB). Each of these mutations resulted in a premature termination codon (PTC) in the amino-terminal portion of COL7A1. One of the patients was a compound heterozygote for two different mutations. The heterozygous carriers showed an approximately 50% reduction in anchoring fibrils, yet were clinically unaffected. Premature termination codons in both alleles of COL7A1 may thus be a major underlying cause of the severe, recessive dystrophic forms of EB.

We have isolated the basement membrane component nicein and performed rotary-shadow analyses using electron microscopy that showed the presence of two forms (I and II) of the protein. Molecular cloning of the cDNA that codes for the 100-kDa chain of the protein revealed that the sequence matches those independently identified for the 105-155-kDa subunit of kalinin, a recently identified basement membrane component. These data demonstrate that nicein and kalinin contain an identical chain. The length of the open reading frame in the cDNA (approximately 5200 nucleotides) and amino acid sequence obtained from the N-terminus of the 105-kDa kalinin chain showed the occurrence of a precursor polypeptide. This immature polypeptide is probably related to form I, observed by rotary shadowing, while the mature form is related to form II. It is noteworthy that nicein/kalinin subunits share discrete sequence similarities with the B2 chain of human laminin, but with a cleavage occurring within domain III that eliminates domains IV and V from the final product. The sequence of this subunit is nearly identical to that of B2t, a recently described polypeptide supposed to be related to a new laminin variant. Since nicein/kalinin expression is specifically impaired in the severe genodermatosis Herlitz junctional epidermolysis bullosa, the role and structure of this tissue-restricted laminin variant is crucial for the understanding of epidermal-dermal adhesion.

C-Jun is a cellular transcription factor that can control gene expression in response to treatment of cells with phorbol esters, growth factors, and expression of some oncogenes. The ability of c-Jun to catalyze the transcription of certain genes is controlled, in part, by changes in the phosphorylation state of specific amino acids in c-Jun. One of the major sites that is phosphorylated during signal response is Ser73. Here we show that substitution of a negatively charged aspartic acid residue at 73 constitutively increased transcriptional activity of c-Jun. The Asp73 substitution also enhanced its availability to bind to DNA in a whole cell extract without altering its intrinsic DNA binding activity since the intrinsic activity was unaltered for the c-Jun mutant proteins expressed in a bacterial system. The negatively charged Asp substitution may mimic the negative charge of a phosphorylated serine at 73. The substitution of an uncharged alanine at 73 resulted in lowered activities. The N-terminal end of c-Jun containing these substitutions was fused to the DNA-binding region of the bovine papilloma virus E2 protein, and was able to confer the same activation properties to the fusion protein at the heterologous E2 DNA-binding site. Ser73 lies in a region of c-Jun previously proposed to bind an uncharacterized inhibitor, perhaps related to a protein of approximately 17.5 kD that coprecipitates along with our c-Jun or the JunE2 fusion products.

A variety of methods were used to fracture the dermal-epidermal junction (DEJ) of human skin. These included warm and hot phosphate buffered saline, trypsin, cold 1 M salt, potassium bromide and proteolytic digestion with dispase. The localization and sensitivity of basement membrane components (bullous pemphigoid antigen, BM 600/nicein, epiligrin, kalinin, laminin, collagens IV and VII (EBA antigen) and linkin) were determined after the DEJ was fractured by each method. We found that the basement membrane zone proteins, BM 600/nicein, epiligrin and kalinin remained with the dermal side of the DEJ fractured through the lamina lucida by cold salt, phosphate buffered saline and potassium bromide. BM 600/nicein, epiligrin and kalinin were not detected after treatment with trypsin. In contrast, laminin, another glycoprotein in the lamina lucida, was insensitive to all of the procedures, but co-localized to the dermal side of DEJ-fractured skin. We also found that separation of the DEJ with brief exposure of skin to 56 degrees C provided a useful substrate for testing the autoantibodies in the sera of patients with epidermolysis bullosa acquisita (EBA). Heat-separated skin can be prepared in a significantly shorter period of time than salt-separated skin.

Melanoma growth-stimulatory activity (MGSA)/GRO is well characterized as a potent neutrophil chemoattractant. In the present study, we have demonstrated that MGSA induced a dose-dependent decrease in the expression of interstitial collagens by rheumatoid synovial fibroblasts. The decrease was observed over a dose range of 0.6-6.0 nM MGSA. This effect was specific, as MGSA had no demonstrable effect on the expression of collagen-degrading metalloproteinases, nor did it affect the collagenase inhibitor, tissue inhibitor of metalloproteinases. It also had no effect on the proliferation rate of these fibroblasts, unlike its mitogenic effect on melanoma cells. The ability to inhibit collagen expression was also demonstrated by another member of the C-X-C branch of the platelet factor 4 superfamily, interleukin-8 (IL-8), but not by RANTES, MIP-1 alpha, or MIP-1 beta, which belong to the C-C branch. Steady-state levels of expression of MGSA and IL-8 transcripts in normal adult tissues were dissimilar, suggesting that expression may be an important level at which the activity of these cytokines is regulated. Direct binding experiments with 125I-MGSA on synovial fibroblasts have allowed us to identify an MGSA receptor with a KD of 10.1 nM and approximately 75,000 binding sites/fibroblast. 125I-MGSA binding was specific and could not be displaced by unlabeled IL-8. These results suggest that MGSA, as well as IL-8, may play a role other than that of neutrophil chemo-attractant and more specifically, may be important in the regulation of collagen turnover.

Vascular endothelial growth factor (VEGF) is a 45kDa secreted peptide that has potent mitogenic activity specific for endothelial cells in vitro and the ability to induce a strong angiogenic response in vivo. In the present study, 24 h treatment with VEGF resulted in a stimulation of expression of the metalloproteinase, interstitial collagenase, at the protein and mRNA levels 2.5-3.0-fold in human umbilical vein endothelial cells but not in human dermal fibroblasts. The dose response curve for collagenase induction was biphasic with the peak stimulatory response obtained by treatment of cells with 10-100 ng/ml (0.2-2 nM) VEGF. The dose response curve for collagenase induction overlapped with, but was not identical to, the response curve for proliferation, which showed VEGF mitogenic activity between < or = 0.1-50 ng/ml (< or = 0.002-1 nM). There was no induction seen in expression of other members of the matrix metalloproteinase family, including the 72kDa type IV collagenase, the 92kDa type V collagenase, or stromelysin. Expression of transcripts for the major metalloproteinase inhibitor, tissue inhibitor of metalloproteinases, was also unaltered by treatment with VEGF (1-200 ng/ml). These studies demonstrate that in addition to stimulating proliferation of endothelial cells, VEGF can also induce the expression of the only metalloproteinase that can initiate degradation of interstitial collagen types I-III under normal physiological conditions. Both responses are likely to contribute to the angiogenic potential of this peptide.

In order to identify structures in human skin that bind collagenase, sections from frozen or paraffin-embedded skin were incubated with either procollagenase or activated collagenase. After washing, bound procollagenase or collagenase was detected by immunofluorescence microscopy. In normal skin, procollagenase bound only to isolated granular dermal cells that were identified as mast cells on the basis of staining with fluoresceinated avidin and pinacyanol erythrosinate. When mast cells were degranulated by exposure to the ionophore A23187, extracellular granules bound procollagenase. Of various pathologic conditions examined, the highest binding of procollagenase occurred in specimens of urticaria pigmentosa. Procollagenase bound to granular cells and to abundant granules scattered throughout the dermis. Binding could be abolished by pre-treatment of tissue sections with heparinase or by pre-incubation of procollagenase with soluble heparin, suggesting that heparin is the binding agent in the granules. Activated collagenase also bound to dermal mast cells but in addition bound strongly to the dermal collagen. Enzymatic activity of activated collagenase was not inhibited by heparin in concentrations up to 10 mg/ml. There is evidence that mast cell tryptase can contribute to procollagenase activation. This study further supports a role for mast cells in collagenolysis by demonstrating that heparin from mast cells binds procollagenase and possibly serves as a reservoir for procollagenase, which may then subsequently be activated.

Epidermolysis bullosa (EB) is a group of mechanobullous diseases of the skin and mucous membranes characterized by the development of blisters and erosions following minor trauma. Hereditary EB exists in four major patterns: (1) intraepidermal blistering in autosomal dominant EB simplex; (2) junctional blistering in autosomal recessive junctional EB; (3) dermal blistering in autosomal dominant dystrophic EB; and (4) dermal blistering in autosomal recessive dystrophic EB. Electron microscopic examination of the skin is the current standard for diagnosis, although a number of immunologic reagents have been developed that are diagnostic tools and probes to pathogenesis. Particularly important are the antibody probes that have been developed for use in junctional and dystrophic forms of EB, since several of the monoclonal antibody preparations also confer at least some information about the likely genetic pattern and prognosis. Antibody preparations have also been used to screen complementary DNA (cDNA) expression libraries for clones representing candidate genes in EB. In EB simplex, mutations in the keratin 14 gene have been discovered. Since keratin 14 combines with keratin 5 to form a heterodimer, the existence of these mutations results in poor keratin filament formation in vitro. In recessive junctional EB, antibody staining patterns suggest involvement of the proteins of the anchoring filaments, such as BM-800/nicein. A partial cDNA clone representing one of the subunits of BM-800/nicein has been developed, and this protein represents an isoform of laminin, suggesting the existence of a family of laminin-like molecules that are important for adhesion of epidermis in the basement membrane zone of the skin. In dominant dystrophic and recessive dystrophic EB, often there are decreased numbers and abnormal appearance of the anchoring fibrils suggesting involvement of type VII collagen. Genetic linkage has been established to the type VII collagen gene in dominant dystrophic EB families. In recessive dystrophic EB, abnormalities in anchoring fibrils may be due to mutation(s) in the type VII collagen gene and/or due to excessive degradation by increased amounts of collagenase. Spontaneous, transgenic, and xenograft models of EB exist. Among the spontaneous models is recessive dystrophic EB in white alpine sheep, which show no staining for type VII collagen in the skin. A new xenograft model of recessive dystrophic EB has also been developed using EB skin grafted onto severe combined immunodeficient mice. A transgenic model has been developed for the herpetiform type of EB simplex resulting from the insertion of a truncated form of the keratin 14 gene.

Recessive dystrophic epidermolysis bullosa (RDEB) is a subgroup of hereditary blistering diseases characterized by repetitive wounding and healing with subsequent extensive scarring. The purpose of this study was to establish a xenograft model that retains the RDEB phenotype and thus might be used as an experimental in vivo model to explore the molecular and biochemical mechanisms of the chronically wounded phenotype of RDEB. Full-thickness, tumor-free RDEB skin tissues were grafted onto the dorsum of severe combined immunodeficiency (SCID) mice. At 4, 8, 12, and 24 weeks after grafting, the xenografts were removed for examination. Immunofluorescence studies were performed using species-specific antibodies to human class I antigen, mouse class I antigen, human type IV and VII collagens and with cross-reacting antibody against bullous pemphigoid antigen (BPA). Staining with the antibody to human class I antigen, W6/32, and with the antibody to mouse class I antigen, 20.8.4s, confirmed the species-specific results obtained with the type IV and type VII collagen and laminin antibodies. The RDEB grafts showed essentially no staining with the type VII collagen antibody. Antibodies against laminin and BPA showed normal staining patterns in RDEB grafts. There was an overall paucity of anchoring fibrils in the grafts when examined with electron microscopy. Blisters could be induced in these grafts with minor trauma and showed a sublamina densa separation by immunomapping and electron microscopy. As late as 24 weeks post-transplantation, the RDEB grafts remain human, are not significantly replaced by mouse cells, and retain the RDEB disease phenotype.

The expression of collagenolytic activity by cells represents the rate-limiting step in the turnover of collagen during remodeling. The collagenase gene is transcriptionally activated in normal dermal or rheumatoid synovial fibroblasts by interleukin-1 beta (IL-1 beta), resulting in secretion of trypsin-activatable procollagenase measuring in the range of 2.0-5.0 units/10(6) cells/48 h in the 14C-fibril assay. The addition of interferon-gamma (IFN-gamma; 50-100 units/ml) inhibits the expression of collagenase activity by 45-80% in these cells. The IL-1 beta induction of procollagenase protein was not altered by IFN-gamma, as judged by Western blot analysis using a monoclonal antibody to collagenase and by gelatin zymography, and procollagenase mRNA was also unaltered, as assessed by Northern blot analysis. Because collagenolytic activity is also controlled by the quantity of tissue inhibitor of metalloproteinases present, its expression was examined by Western blot analysis using a polyclonal antibody to tissue inhibitor of metalloproteinases and by reverse gelatin zymography. Tissue inhibitor of metalloproteinase protein was found to be unaltered or slightly less abundant in conditioned media from cultures treated with IL-1 beta and IFN-gamma when compared with that from cultures treated with IL-1 beta alone. However, the expression of the metalloproteinase activator of procollagenase, stromelysin, was found to be significantly inhibited by the addition of IFN-gamma. Addition of purified activated stromelysin to these conditioned media completely reconstituted collagenolytic activity. These observations demonstrate in an intact system that stromelysin is a specific activator necessary for the development of collagenolytic activity. Despite stromelysin's lack of catalytic activity against collagen, its expression can serve as a control point in the regulation of collagenolysis.

Inherited epidermolysis bullosa encompasses a number of diseases, with the common finding of blister formation, after minor mechanical trauma to the skin. In some forms significant, if not eventually fatal, extracutaneous disease activity may occur. In recent years application of newer technologies has contributed substantially to an overall understanding of this collection of inherited diseases. Concurrently, many new phenotypes have been recognized, in part the result of ongoing prospective patient registries in the United States and abroad. Unfortunately, this has resulted in a massive literature that may appear to be confounded by seemingly excessive or arbitrary subdivision of epidermolysis bullosa variants. With these concerns in mind a subcommittee was established by the National Epidermolysis Bullosa Registry to summarize the current literature and to make recommendations as to the best clinical and laboratory criteria for the practical diagnosis and subclassification of patients with inherited epidermolysis bullosa.

Activation of procollagenase constitutes a crucial event in collagenolytic activity regulation. In this study we have purified by DEAE-cellulose, Ultrogel AcA-44, and zinc chelate sepharose chromatographies, a procollagenase-activator from the culture medium of the guinea pig carrageenin granuloma model. On SDS-PAGE, the activator migrates as a principal band of Mr approximately 44,000. The molecule activates procollagenase from human lung fibroblasts in a concentration dependent manner and an enhancement of collagenase activity of trypsin-treated crude culture medium was observed. A loss of about 50% of its activity occurs after heating. In addition, this activator degrades gelatin and casein. All these data suggest that this procollagenase-activator might be stromelysin. The activator was found in both phases of the granuloma, at 7 days when collagen is actively deposited and an important proportion of the collagenolytic activity remains in latent form; and at 14 days, when this enzymatic activity is fully expressed.

BACKGROUND: Since publication in 2000 of the Second International Consensus Report on Diagnosis and Classification of Epidermolysis Bullosa, many advances have been made to our understanding of this group of diseases, both clinically and molecularly. At the same time, new epidermolysis bullosa (EB) subtypes have been described and similarities with some other diseases have been identified. OBJECTIVE: We sought to arrive at a new consensus of the classification of EB subtypes. RESULTS: We now present a revised classification system that takes into account the new advances, as well as encompassing other inherited diseases that should also be included within the EB spectrum, based on the presence of blistering and mechanical fragility. Current recommendations are made on the use of specific diagnostic tests, with updates on the findings known to occur within each of the major EB subtypes. Electronic links are also provided to informational and laboratory resources of particular benefit to clinicians and their patients. LIMITATIONS: As more becomes known about this disease, future modifications may be needed. The classification system has been designed with sufficient flexibility for these modifications. CONCLUSION: This revised classification system should assist clinicians in accurately diagnosing and subclassifying patients with EB.

Low-dose cyclosporine therapy for severe plaque psoriasis is effective. Most side effects can be controlled by patient monitoring, with appropriate dose adjustment or pharmacologic intervention, or both, if indicated. Prevention or reversibility of laboratory and chemical abnormalities may be achieved by discontinuation of therapy after the induction of clearing. However, relapse occurs rapidly on discontinuation. Maintenance therapy with cyclosporine after induction has not been fully evaluated.Our purpose was to compare a regimen of 3.0 mg/kg per day of oral cyclosporine with placebo in maintaining remission or improvement in patients with psoriasis.After a 16-week unblinded induction phase in which 181 patients received cyclosporine, 5.0 mg/kg per day (an increase up to 6.0 mg/kg per day and a decrease to 3.0 mg/kg per day were allowed, if required, to achieve efficacy or tolerability, respectively), those patients showing a 70% decrease or more in involved body surface area (BSA) entered the 24-week maintenance phase and were randomly assigned to either placebo, cyclosporine, 1.5 mg/kg per day, or cyclosporine, 3.0 mg/kg per day. Patients were considered to have had a relapse when BSA returned to 50% or more of the prestudy baseline value. Clinical efficacy, adverse effects, and laboratory values were monitored regularly throughout both study phases.During induction, cyclosporine at approximately 5.0 mg/kg per day produced a reduction in BSA of 70% or more in 86% of the patients. During maintenance, the median time to relapse was 6 weeks in both the placebo and cyclosporine 1.5 mg/kg per day groups, but was longer than the 24-week maintenance period in the 3.0 mg/kg per day group (p0.001 vs placebo). By the end of the maintenance period, 42% of the patients in the 3.0 mg/kg per day cyclosporine group had a relapse compared with 84% in the placebo group. Changes in laboratory values associated with the higher induction dosage generally exhibited partial or complete return toward mean prestudy baseline values during the maintenance phase, with the greatest degree of normalization in the placebo group.Cyclosporine, 3.0 mg/kg per day, adequately and safely maintained 58% of patients with psoriasis for a 6-month period after clearing of their psoriasis with doses of approximately 5.0 mg/kg per day.

Herlitz junctional epidermolysis bullosa is an autosomal recessive disorder characterized by generalized blistering at the lamina lucida of the cutaneous basement membrane. The monoclonal antibody GB3 has been used as a diagnostic probe because of its lack of reactivity in patient skin. The antigen recognized by GB3 has been identified as laminin-5, a glycoprotein consisting of three subunits (alpha 3, beta 3 and gamma 2). To identify the laminin-5 protein chain that contains the epitope recognized by GB3 and to determine if chain assembly is required for antibody recognition, we expressed a gamma 2 protein constructed from a full-length gamma 2 cDNA. Radioimmunoprecipitation of the culture medium from 293 cells revealed that both GB3 and anti-gamma 2 polyclonal antibodies were capable of directly precipitating recombinant gamma 2 without coprecipitation of other proteins. In immunodepletion experiments, each antibody removed most of the protein that was reactive with the other antibody. The epitope recognized by GB3 is present only when the complex is in the native conformation because GB3 reacted only with the non-reduced laminin-5, but not the reduced laminin-5 in immunoblots. Moreover, because GB3 reacted with laminin-5 of SCC25 cells (gamma 2 in the heterotrimer) but not recombinant gamma 2 in 293 cells (gamma 2 alone) during indirect immunofluorescence staining, this epitope may be dependent upon a less stable conformation of gamma 2. We conclude that GB3 recognizes the gamma 2 chain of laminin-5 and that the epitope is entirely contained in the native form of the gamma 2 chain.

Laminin-5 is a heterotrimer composed of alpha 3, beta 3, and gamma 2 chains, produced by keratinocytes and the human squamous cell carcinoma line (SCC-25), and is one of the candidate proteins for the genetic lesion in junctional epidermolysis bullosa. Two-dimensional SDS-polyacrylamide gel electrophoresis (first dimension, nonreducing conditions; second dimension, reducing conditions) revealed that the immunoprecipitated laminin-5 from a SCC-25 cell fraction consisted of alpha 3, beta 3, and gamma 2 monomers, a beta 3 gamma 2 heterodimer, and an alpha 3 beta 3 gamma 2 heterotrimer. The presence of the beta 3 gamma 2 heterodimer, but not heterodimers containing an alpha 3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a beta 3 gamma 2 heterodimer to an alpha 3 beta 3 gamma 2 heterotrimer. We showed, by cotransfection experiments using full-length recombinant beta 3 and gamma 2 chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of alpha 3 chain expression. In the SCC-25 cell fraction, the alpha 3 monomer pool was the smallest of the monomers. Pulse-chase experiments using the cell fraction also indicated that the heterotrimer was assembled after a 10-min pulse and was nearly absent after a 24-h chase. These results are consistent with the synthesis of alpha 3 being limiting for heterotrimer assembly, with rapid association of the alpha 3 chain with beta 3 gamma 2 heterodimers to form complete heterotrimers. Treatment with tunicamycin reduced the size of each of the laminin-5 subunits, indicating that all chains are glycosylated, but that N-linked glycosylation is not necessary for chain assembly and secretion.

Type I collagenase plays an important role in both tumor metastasis and the remodeling of connective tissue in normal human skin, during wound healing, for example, and may participate in the pathophysiology of some dermatological diseases such as skin cancer and a chronic blistering disease, recessive dystrophic epidermolysis bullosa. In an effort specifically to inhibit collagenase expression, we have designed phosphorothioate antisense oligonucleotides, linked at the 5' ends with photoreactive 4'-(hydroxyethoxymethyl)-4,5',8-trimethyl-psoralen (HMT), and directed them against the 5' end of the collagenase mRNA. Two antisense-HMT molecules targeting a region overlapping the initiation codon were compared. Only one contained the HMT moiety targeting a 5'TpA on its complementary sense strand, and we observed greater than 50-fold improvement on the cross-linking of this antisense oligonucleotide to its target sequence after ultraviolet A (UVA) irradiation. Likewise, sequence complementary to the 5'TpA target was also required to demonstrate specific inhibition of in vitro translation of collagenase mRNA. Tissue culture experiments, conducted by incubation of collagenase-specific antisense-HMT oligonucleotides with fibroblasts in monolayer or in 3-dimensional dermal equivalents, showed lowered collagenase levels 24 h after UVA irradiation as compared to controls. Initial screening of antisense oligomers for specific hybridization and photo-cross-linking is a useful step in the design of antisense oligonucleotides, and allowed us to design an HMT-linked antisense phosphorothioate oligonucleotide that specifically inhibits the expression of fibroblastic collagenase.

Recessive dystrophic epidermolysis bullosa (RDEB) is a mutilating disease of the skin characterized by recurrent blistering and erosions that result from compromised integrity of the basement membrane zone. In this study, fibroblasts derived from the skin of RDEB patients were characterized for expression of the major metalloproteinases, particularly interstitial collagenase. Consistent with previous reports on increased collagenase protein levels in fibroblasts from some RDEB patients, we found that steady-state levels of collagenase mRNA were significantly increased in fibroblast strains derived from three of five RDEB patients compared to fibroblasts obtained from normal donors. Stromelysin mRNA was elevated in the same three fibroblast strains, whereas expression of neither the 72- nor the 92-kDa type IV collagenases was different from that of controls. Tissue inhibitor of metalloproteinases was expressed in RDEB fibroblasts at levels similar to those observed in normal fibroblasts. To investigate the mechanism behind the steady-state elevation in collagenase and stromelysin expression, AP-1 expression and activation were studied. Although levels of Jun expression were not different from those seen in normal fibroblasts, AP-1 activity, as assessed by ability to bind to a TPA response element-containing oligonucleotide, was endogenously elevated in RDEB fibroblasts compared to normal fibroblasts. Transfection studies using a plasmid construct containing the collagenase promoter linked to a CAT reporter gene demonstrated that RDEB fibroblasts were able to support active transcription of the promoter compared to normal fibroblasts. These studies support the hypothesis that RDEB fibroblasts contain chronically activated AP-1, and perhaps other transactivating factors, that contribute to the cellular phenotype of collagenase and stromelysin overexpression.

Fibroblasts derived from the involved skin of scleroderma patients frequently display a phenotype of supernormal collagen expression when cultured. Fibroblasts displaying this phenotype derived from seven patients were treated with relaxin (1-100 ng/ml) and interferon-gamma (1-100 U/ml), individually and in combination, to assess the relative abilities of these cytokines to down-modulate collagen synthesis and secretion. Scleroderma fibroblasts displayed varying sensitivities to both relaxin and interferon-gamma. Relaxin (100 ng/ml) decreased expression of collagen by six of seven lines tested from 8 to 59% compared to untreated cultures. Interferon-gamma (100 U/ml) depressed collagen secretion by all seven lines in a range from 7 to 89%. When relaxin and interferon-gamma were used in combination, relaxin augmented IFN-gamma-induced decreases in collagen secretion in four of seven lines. In three of these lines, the use of relaxin in conjunction with suboptimal doses of interferon-gamma resulted in decreases equivalent to or greater than that seen with a tenfold higher concentration of interferon-gamma. This study demonstrates the ability of relaxin to directly alter the excessive collagen-producing phenotype of scleroderma fibroblasts. In addition, in some cases, combining relaxin and interferon-gamma resulted in a cooperative effect in decreasing collagen expression by scleroderma cells in vitro.

The basement membrane zone (BMZ) of the skin is defined as the ultrastructurally identifiable region that includes the basal keratinocytes, the lamina lucida, the lamina densa, and the uppermost papillary dermis. The lower dermal “boundary” of the BMZ is unclear but must extend at least to the depth of the anchoring fibrils where they insert into anchoring plaques.1

Epidermolysis bullosa dystrophica (EBD) is a heterogeneous group of inherited mechanobullous diseases that produce separation in the deep portion of the basement membrane zone beneath the lamina densa (dermolytic separation) [1, 2] (Figure 16.1). Dystrophic scarring results from repeated blistering and serves as a clinical marker of these diseases. Traditionally, EBD is subdivided into autosomal recessive and autosomal dominant types. Cases of recessively inherited epidermolysis bullosa dystrophica vary significantly in the severity and extent of the disease that they manifest.

† Background.— Blue-gray pigmentation of the skin can be elicited by several medications. We report the first case (to our knowledge) of desipramine-induced photosensitive blue-gray pigmentation. Observations.— Diffuse blue-gray pigmentation on sun-exposed surfaces was noted in a healthy 48-year-old woman who had been taking desipramine hydrochloride for 8 years. Ultrastructural studies demonstrated the presence of melanin and homogeneous electron-dense material in the dermis. Conclusions.— We conclude that tricyclic antidepressant agents represent another class of medications responsible for blue-gray cutaneous pigmentation. ( Arch Dermatol. 1993;129:474-476)

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.

We have determined the complete sequence of the cDNA clone representing the full size human skin collagenase mRNA. Collagenase is synthesized in preproenzyme form, Mr 54,092, with a 19 amino acid long signal peptide. The primary secretion products of the enzyme consist of a minor glycosylated form, Mr 57,000, and a major unmodified polypeptide of predicted Mr 51,929. Proteolytic activation of human skin procollagenase results in removal of 81 amino acid residues from the amino-terminal portion of the proenzyme. Both potential N-glycosylation sites are contained within the proteolytically activated form of the enzyme. The primary structure of the coding region of the presented clone is homologous to an oncogene-induced rat protein whose function is still unknown, although preliminary observations suggest that it is not rat skin collagenase.

Verruciform xanthoma is a rare, granular, plaque-like lesion which occurs on mucosal surfaces and is characterized by acanthosis with xanthoma cells in the dermis between rete pegs. A case of verruciform xanthoma is reported which occurred on the sacral region in a patient with recessive dystrophic epidermolysis bullosa (RDEB). Electron microscopic studies suggested that many of the xanthoma cells were fibroblasts whose vacuolated appearance was expressed in cell culture and persisted for several passages. The occurrence of a verruciform xanthoma in a patient with severe recessive dystrophic epidermolysis bullosa suggests that repeated epidermal and/or dermal damage gave rise to a reactive process involving the formation of lipid-laden cells.

The dermis, like other structures composed of connective tissue, is comprised largely of collagen. Indeed, collagen constitutes more than 70% of the dry weight of the skin. 1 Although its major biologic function in the skin is to provide the structural framework underlying the epidermis and overlying the subcutaneous tissue and musculature, the critical tensile properties necessary for structural integrity are achieved by specific interactions with other collagenous proteins, as well as with proteoglycans, glycoproteins, and elastin. The cellular processes involved in the synthesis and degradation of these macromolecules are now sufficiently well-understood to allow characterization of certain cutaneous disorders in terms of specific biochemical abnormalities. In this review, we focus on two cutaneous disorders: one inherited, the other representing an apparent sporadic congenital anomaly. Each of these disorders is characterized clinically by excessive accumulation of one component of the connective tissue with resultant compromise of function of the skin in the affected areas. By utilizing skin fibroblast cultures derived from these patients, we have gained biochemical insights into the aberrations that characterize these diseases of the skin.

Various dermal fibrotic conditions, such as progressive systemic sclerosis, localized morphea and familial cutaneous collagenoma, are characterized by excessive deposition of collagen in the skin. In the present study, we examined the possibility that a circulating serum factor(s) is responsible for increased collagen production in these diseases. The effects of human serum on the synthesis of procollagen were examined by incubating normal human dermal fibroblasts with [ 3 H]proline and varying concentrations of dialyzed heat-inactivated serum. The synthesis of procollagen was measured as formation of nondialyzable [ 3 H]hydroxyproline and collagenase-digestible 3 H-polypeptides. In the absence of serum little procollagen was formed but the synthesis was markedly stimulated by the addition of normal serum in a concentration-dependent manner. The ratio of genetically distinct 3 H-procollagens of type I and type III, assayed by DEAE-cellulose chromatography and SDS-polyacrylamide gel electrophoresis after limited pepsin proteolysis, was unaffected by the addition of serum. Thus, normal human serum contains a nondialyzable factor(s) which stimulates the synthesis of procollagens type I and type III equally. Sera from 5 patients with progressive systemic sclerosis, 3 with localized scleroderma, and 2 with familial cutaneous collagenoma were also tested. Sera from these patients failed to stimulate 3 H-procollagen production more than sera from healthy age-matched controls. Therefore, no increased quantities or qualitatively aberrant factors were shown to be present in the sera of these patients.

A specific and sensitive indirect inhibition enzyme-linked immunosorbent assay (ELISA) was develop for human skin collagenase. Using an antiserum dilution of 1 : 8000, the ELISA could detect 0.2 ng of enzyme, which was approximately 10 times more sensitive than the previously described radioimmunoassay for human skin collagenase. The assay was also highly reproducible. In comparative studies, bacterial, tadpole and crab collagenases did not react in the ELISA. Rat uterine collagenase and collagenases from bovine gingival explants and fibroblasts displayed approximately 0.1% of the reactivity found with human skin collagenase. Human synovial and gingival collagenases and collagenase from skin fibroblasts from patients with recessive dystrophic epidermolysis bullosa showed almost complete identity with the normal human skin fibroblast enzyme.

We administered phenytoin (diphenylhydantoin) by mouth to 17 unselected patients to assess its ability to reduce blistering in recessive dystrophic epidermolysis bullosa (RDEB). Therapeutic response was correlated with blood levels of the drug. Although there was a decrease in blistering of 53 +/- 6 per cent (mean +/- S.E.) among all patients at levels of more than 8 microgram of phenytoin per milliliter, the response was variable, with 12 of 17 patients having a decrease in blistering of more than 40 per cent. Since increased collagenase in human skin has been implicated in the pathogenesis of blistering in RDEB, we examined the effect of phenytoin on this enzyme. Although the drug did not inhibit collagenase activity directly, its addition to human-skin explant and fibroblast cultures produced a 50 to 60 per cent decrease in collagenase activity and immunoreactive protein concentrations. These in vitro studies suggest that phenytoin inhibits synthesis or secretion of collagenase of both, and that the favorable clinical response can be explained by this inhibition.

Human skin collagenase was quantitated by radioimmunoassay in 40 patients with various forms of epidermolysis bullosa to compare levels of the enzyme in blistered and clinically unaffected skin. Immunoreactive human skin collagenase was significantly elevated in the blistered skin of patients with both recessive and dominant forms of dystrophic epidermolysis bullosa (DEB). In addition, patients with generalized recessive DEB manifested a 4-fold increase in collagenase protein in normal-appearing skin, and patients with localized recessive DEB or epidermolysis bullosa letalis showed a 3-t to 3.5-fold elevation in the enzyme. However, patients with dominantly inherited DEB failed to displays a statistically significant increase in immunoreactive collagenase in nonblistered skin. Although it cannot be definitely stated whether the elevated collagenase content in the blistered skin represents a primary or secondary event, such as part of a wound healing response, the demonstration of markedly increased levels of collagenase in normal-appearing skin could, in part, provide an explanation at the molecular level for the formation of blisters in this disease.

Tissue and cultured fibroblasts derived from one patient with the classical findings of lipoid proteinosis have been used to examine pathologic mechanisms in the disease. Ultrastructural examination of the skin revealed not only extracellular deposits of finely granular, moderately electron dense material, but in addition the dermal fibroblasts characteristically demonstrated marked cytoplasmic vacuolization. Phase contrast microscopy of the cultured skin fibroblasts also showed strikingly abnormal cells with many inclusions, which by electron microscopy were delimited by a single membrane. Membranous lamellar material was also increased in these cells. Biochemical analysis of the fibroblasts revealed a 3- to 4-fold elevation in intracellular hexuronic acid. These morphologic and biochemical findings suggest certain similarities with known storage diseases and support the postulate that lipoid proteinosis may represent a lysosomal storage disease.

H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.

Platelet-derived growth factor (PDGF) is both chemoattractant and mitogenic for stromal cells. Here, we examined the effects of PDGF on collagenase expression by normal human skin fibroblasts. Culturing cells for 24 hr in the presence of PDGF at 0-180 ng/ml resulted in a dose-dependent, saturable increase in collagenase activity in the culture medium that was paralleled by equal increases in immunoreactive collagenase protein, suggesting enhanced synthesis of a catalytically unaltered enzyme. The specificity of this effect was demonstrated by comparing the collagenase-stimulatory effect with that on total protein synthesis and DNA synthesis. Under in vitro conditions that produced a 2.5-fold increase in collagenase synthesis, there was an approximately equal to 20% increase in total protein synthesis and no change in DNA synthesis. In addition, platelet factor 4, another platelet-derived protein, caused a less than 20% increase in collagenase expression. In time-course studies, stimulation of collagenase synthesis was first observed 8-10 hr after exposure to the growth factor. Conversely, when cells were primed with PDGF for approximately equal to 24 hr and the stimulator was then removed, an increased rate of synthesis was seen for an additional approximately equal to 6 hr, after which the rate reverted to control levels. Since the kinetic data suggested a possible pretranslational effect, fibroblasts cultured with PDGF were used to prepare mRNA. In cell-free translation, total protein synthesis was essentially unaltered; however, the growth factor caused a greater than 2-fold increase in translatable collagenase mRNA. The data suggest that PDGF specifically modulates collagenase synthesis, possibly through a series of events that lead to increased transcription or preferential translation of collagenase mRNA.