Enhanced collagen synthesis and transcription by peak E, a contaminant of L-tryptophan preparations associated with the eosinophilia myalgia syndrome epidemic.

The pathogenesis of the eosinophilia myalgia syndrome (EMS) remains unclear. Several abnormal constituents have been found in the L-tryptophan lots responsible for the illness, particularly, 1,1-ethylidenebis[L-tryptophan], also called peak E or EBT, and 3-phenylamino-alanine or peak 5. However, the role of these contaminants in the pathogenesis of EMS and in the development of fibrosis is unknown. We now report that peak E, a dimer of L-tryptophan, is a potent stimulus for human dermal fibroblast DNA and collagen synthesis. Peak E (0.1-1.0 microM) increased DNA synthesis up to four-fold (P = 0.0001) in a dose-dependent manner (r = 0.987). When added to monolayer cultures for 2 to 24 h, peak E (0.5 to 100 microM) caused a progressive, more than threefold increase in alpha 1(I) procollagen mRNA levels and collagenous protein. No increase in procollagen mRNA levels was found after the addition of another major L-tryptophan contaminant, peak 5, or with L-tryptophan itself. Transient transfection with a 2.5-kb alpha 1(I) procollagen promoter-luciferase construct showed that peak E causes a twofold upregulation of promoter activity (P = 0.022). Contraction of collagen gels, consisting of human dermal fibroblasts incorporated into a type I collagen lattice, was enhanced two-fold by exposure to peak E (P = 0.001). We conclude that a major constituent of contaminated batches of L-tryptophan, peak E, is a potent stimulus for fibroblast activation and collagen synthesis. This stimulatory action of peak E may provide a direct mechanism for the development of fibrosis in EMS.

Stanozolol as a novel therapeutic agent in dermatology.

Anabolic steroids are synthetic derivatives of testosterone that were developed in the 1950s in an attempt to dissociate the anabolic and androgenic effects of testosterone. The anabolic steroid stanozolol has been particularly helpful because it has one of the largest anabolic/androgenic ratios. In addition, stanozolol has substantial fibrinolytic properties. We discuss the safety profile and the use of stanozolol for a variety of clinical applications. Stanozolol is approved for use in the treatment of hereditary angioedema, but numerous reports have detailed the effectiveness of this agent in the treatment of urticaria, Raynaud's phenomenon, and, more recently, cryofibrinogenemia and lipodermatosclerosis. Side effects are mostly dose related and are preventable with appropriate follow-up.

Human dermal fibroblast clones derived from single cells are heterogeneous in the production of mRNAs for alpha 1(I) procollagen and transforming growth factor-beta 1.

Fibroblast clonal heterogeneity has been reported for growth and protein synthesis, but quantitative studies of synthetic phenotype at the pretranslational level have been limited because of difficulty in reliably growing large numbers of clonal cells. We have recently shown a unique stimulatory activity of low oxygen tension in the early phases of clonal growth, which can be used to establish clonal fibroblast cultures suitable for Northern analysis. Using this methodology, we have measured mRNA levels of alpha 1(I) procollagen and transforming growth factor-beta 1 (TGF-beta) both at baseline and after TGF-beta stimulation in a total of 43 clones derived from single cells and from seven different cell strains. We report a remarkable baseline heterogeneity, commonly four- to six-fold, in procollagen mRNA levels among clones and between clones and their parent cultures. Conversely, differences in baseline TGF-beta mRNA levels among clones were either not present or less than onefold. The clonal phenotypic expression of alpha 1(I) procollagen mRNA remained stable after eight additional cell passages. TGF-beta stimulation of itself (autoinduction) was highly variable among clones (range of increases 30% to 150%), and up-regulation of procollagen mRNA levels after TGF-beta stimulation was detected in only 15 (54%) of 28 clonal cultures (range of increases 30% to 353%). A notable lack of correlation was found between baseline mRNA levels of TGF-beta and alpha 1(I) procollagen in clonal cultures. In conclusion, fibroblast clonal populations are remarkably heterogeneous in their baseline procollagen mRNA levels and in their response to TGF-beta.

Low oxygen tension increases mRNA levels of alpha 1 (I) procollagen in human dermal fibroblasts.

Dermal fibroblasts exposed to low oxygen tension show upregulated synthesis of transforming growth factor-beta 1 (TGF-beta 1), an established stimulatory peptide in the formation of extracellular matrix proteins. In this report, procollagen synthesis was measured in cultures of confluent adult human dermal fibroblasts exposed to either standard (20%) or low (2%) oxygen tension. By Northern blot analysis the steady state levels of alpha 1 (I) procollagen mRNA were increased by 75 to 150% of control (standard oxygen) as early as 12 hours and more than 200% 96 hours after exposure of cells to low oxygen. Similar increases in procollagen mRNA levels were obtained in hypoxic fibroblast cultures in a collagen lattice. The stimulatory effect of hypoxia on procollagen mRNA levels in fibroblast monolayers was diminished by antibodies to TGF-beta, and could not be augmented further by the addition of TGF-beta 1, evidence that hypoxic fibroblasts may already be maximally stimulated by TGF-beta 1. We conclude that low oxygen tension enhances steady state mRNA levels of alpha 1 (I) procollagen, and that this effect is mediated at least in part by TGF-beta 1.

Gene expression in low oxygen tension.

Low oxygen tension is a feature of many physiologic and pathologic conditions, including wound healing, fibrosis, and neoplasia. Increasing evidence suggests that low oxygen tension induces the transcription of a number of genes, and that this process depends on the cellular context. The proteins synthesized from these genes enable cells to adapt to the hypoxic environment and/or to fulfill their functional roles. The regulatory regions responsible for the induction of erythropoietin gene transcription and synthesis in response to hypoxia/anemia appear to be cis-acting deoxyribonucleic acid sequences located within the 5' and 3' flanking regions of the erythropoietin gene. Other proteins induced by hypoxia include cytokines (platelet-derived growth factor-beta chain, endothelin-1, transforming growth factor-beta), enzymes (tyrosine hydroxylase, glycolytic enzymes), and stress proteins. The molecular mechanisms of the hypoxia-induced expression of these genes are poorly understood. A heme protein may act as the oxygen tension sensor, or the redox state of certain nuclear transcription factors may function as second messengers.

Studies on the repopulation of Langerhans cells in partial-thickness wounds. Air exposed and occlusively dressed.

BACKGROUND AND DESIGN: The use of occlusive dressings on partial-thickness wounds has been shown to promote early epithelization and connective tissue regeneration. Because Langerhans cells (LC) have been implicated in epidermal homeostasis we studied the rate of repopulation of LC in air-exposed vs occlusively dressed wounds. Partial-thickness wounds on the backs of pigs were treated with occlusive dressings (Tegaderm) for 3 days or left air exposed. On days 3, 5, 7, and 11 after keratome wounding, epidermal sheets from the regenerating wounds were isolated and stained for LC using indirect immunofluorescence. The LC populations were quantified in the interfollicular regions and expressed as average number of cells per square millimeter of epidermis. RESULTS: Normal skin control had 1024 +/- 93 LC/mm2 distributed uniformly. On day 3 after wounding occlusive-dressing-treated wounds had an LC repopulation of 46% of the original value. Langerhans cells in air-exposed skin could not be evaluated until epithelization occurred at day 5. Langerhans cells in both air-exposed and occlusive-dressing-treated wounds were 46% to 51%, 65% to 71%, and 91% of normal value, respectively, on days 5, 7, and 11. CONCLUSIONS: We conclude that at least in regenerating epidermis, the degree of repair of the new epidermis apparently plays a limited role in the migration of LC, as does the earlier growth of blood vessels.