Premenstrual flares of pyoderma gangrenosum controlled with use of a combined oral contraceptive and antiandrogen (ethinyl estradiol/drospirenone).

The effect of sex hormones on pyoderma gangrenosum (PG) has not been reported. We report the case of a 34-year-old woman with chronic PG leg ulcers who was found to have recurring, premenstrual flares of PG. Her PG flares were controlled with the use of ethinyl estradiol/drospirenone.

New horizons for cutaneous microbiology: the role of biofilms in dermatological disease.

Human skin is colonized by bacteria. The development of new genomic microbiological techniques has revealed that the bacterial ecology of human skin is far more complex than previously imagined and includes many fastidious or noncultivable bacterial species which are found on both normal and diseased skin. In nature, the predominant bacterial phenotype on epithelial surfaces is that of organisms organized within a biofilm. This contrasts with the widely held belief that bacteria are planktonic, i.e. free-floating single cells. Biofilms are sessile bacterial communities encased in an extracellular matrix that have a well-developed communication system and can regulate bacterial growth and metabolism, confer resistance to antimicrobials and to host inflammatory cells, and alter host metabolism. Biofilms have been observed on healthy skin and in a number of dermatological conditions, including some that were previously thought not to have an infectious aetiology. Here we review the concept of biofilms and their role in cutaneous health and disease.

Degradation of the epidermal-dermal junction by proteolytic enzymes from human skin and human polymorphonuclear leukocytes.

The degradation of normal human skin by the human polymorphonuclear leukocyte proteinases cathepsin G and elastase, and by a human skin chymotrypsin-like proteinase that appears to be a mast cell constituent, was examined. Enzymes were incubated with fresh, split-thickness skin for up to 8 h; the tissue was examined ultrastructurally and immunohistochemically using antibodies to known basement membrane constituents. In all cases, the primary damage observed was at the epidermal-dermal junction. Elastase degraded the lamina densa leaving scattered and disorganized anchoring fibrils, dermal microfibril bundles, and normal-appearing collagen fibers. Immunohistochemically, type IV collagen, laminin, KF1 antigen, and EBA antigen were absent. The bullous pemphigoid antigen was present and localized on the basal cells. Epidermal-dermal separation produced by the chymotrypsin-like proteinases, cathepsin G, and the human skin proteinase, was confined to the lamina lucida. The lamina densa and sub-lamina densa fibrillar network remained intact. The human skin chymotrypsin-like proteinase produced extensive epidermal-dermal separation, while cathepsin G, at comparable concentrations, produced only focal separations. Immunohistochemically, all antigens were present after incubation with enzyme. The bullous pemphigoid antigen, however, was found on the epidermal side of the split, while laminin was found on the dermal side. These results show that the epidermal-dermal junction is highly susceptible to neutral serine proteinases located in mast cells and polymorphonuclear leukocytes. Although all the proteinases produce epidermal-dermal separation, the patterns and extent of degradation are different. The distinctive patterns of degradation may provide a clue to the involvement of these proteinases in skin diseases.

Anti-cell surface pemphigus autoantibody stimulates plasminogen activator activity of human epidermal cells. A mechanism for the loss of epidermal cohesion and blister formation.

Binding of anti-cell surface pemphigus autoantibodies to cultured human epidermal cells stimulates synthesis and secretion of plasminogen activator (PA). Increases in PA activity were detected within 6 h of the addition of IgG and stimulation was dependent upon IgG concentration. Stimulation of PA activity was inhibited by cycloheximide, which indicates that synthesis of protein was necessary. Pharmacological doses of dexamethasone also prevented IgG-induced stimulation of PA. Electrophoretic profiles of PA secreted by cultured human epidermal cells in the presence or absence of pemphigus IgG were similar. The majority of the PA activity comigrated with the higher-molecular-weight species of human urokinase (approximately 55,000). Explants of normal human skin incubated with pemphigus vulgaris IgG displayed loss of epidermal cohesion similar to that observed in patient biopsies. The histologic changes were potentiated by the inclusion of human plasminogen. Loss of epidermal cohesion in normal skin explants incubated with pemphigus foliaceous IgG was dependent upon the addition of plasminogen and was inhibited by aprotinin or lima bean trypsin inhibitor, which indicated that plasmin is the active enzyme in producing acantholysis. These data support the hypothesis that stimulation of PA by the anti-cell surface autoantibodies of pemphigus results in a localized increase in plasmin, which through proteolysis produces the loss of epidermal cohesion characteristic of pemphigus.

Immunoinhibition of intracellular protein digestion in macrophages.

Specific anti-(rabbit cathepsin D) serum, previously shown to inhibit cathepsin D, arrested the intracellular digestion of sheep IgG and radiochemically labeled hemoglobin and proteoglycan in rabbit alveolar macrophages. In the presence of antiserum, cells remained viable, but became very vacuolated. Both sheep IgG and hemoglobin were demonstrated immunocytochemically in vacuoles most of which could also be shown to contain cathepsin D. When the antiserum was removed, cells regained their normal morphology, and digestion of endocytosed proteins returned to normal. These results indicate that cathepsin D can be inhibited within lysosomes of viable cells, in which it plays a major role in the intracellular digestion of certain proteins.

Uninvolved skin from psoriatic patients develops signs of involved psoriatic skin after being grafted onto nude mice.

Clinically involved psoriatic epidermis maintains its histological appearance, increased labeling index, and increased level of plasminogen activator after being grafted onto athymic nude mice. Uninvolved psoriatic epidermis develops increases in plasminogen activator activity after being grafted onto athymic nude mice; this is accompanied by an increased labeling index. Thus, psoriatic skin can develop markers of psoriasis independent of the host.

Polymorphonuclear leukocytes: possible mechanism of accumulation in psoriasis.

Extracts of involved and uninvolved skin from nine patients with untreated psoriasis were studied for chemotactic activity. Psoriatic plaque contains increased amounts of a complement-dependent chemotactic factor that is inhibited by diisopropyl fluorophosphate. This factor may be human skin serine proteinase.

Human granulocyte collagenase.

A collagenase, operative at neutral and alkaline pH, has been extracted from the granule fraction of human granulocytic leukocytes. It digests reconstituted collagen fibrils and reduces the viscosity of collagen solutions. Cleavage of collagen in solution with purified enzyme produces the discrete products characteristic of other animal collagenases.

Development of pemphigus vulgaris-like lesions in severe combined immunodeficiency disease mice reconstituted with lymphocytes from patients.

Pemphigus vulgaris is an autoimmune blistering disease that is induced by binding of antibodies to a 130/85-kD protein complex on epidermal keratinocytes. An in vivo experimental model of this disease was developed by reconstituting severe combined immunodeficiency (SCID) mice with 1-10 x 10(7) PBL from patients with naturally occurring pemphigus vulgaris. Of 49 reconstituted mice, 34 (69%) produced human IgG levels of > 0.1 mg/ml. Circulating anti-pemphigus antibodies were found in 20 of the 34 successfully reconstituted mice; 44% of these animals had deposits of human IgG in their own skin after it was traumatized by either heat or cold. Spontaneous pemphigus vulgaris-like blisters associated with human IgG deposits were rarely found in mouse skin. By contrast, allogeneic human skin grafted to 10 to 12 mice before reconstitution with patients' PBL developed pemphigus vulgaris-like lesions containing human IgG deposits. These results demonstrate that SCID mice can serve as a model of an antibody-mediated human autoimmune skin disease.

Degradation of collagen by a human granulocyte collagenolytic system.

This report suggests a mechanism for collagen degradation mediated by human granulocytic leukocytes. A specific collagenase, which is extractable from human granulocytes, has been partially purified by DEAE chromatography. This collagenolytic enzyme is operative at physiological pH and is inhibited by EDTA, cysteine, and reduced glutathione but not by human serum. The enzyme cleaves the collagen molecule into two specific products, without loss of helical conformation. Electron micrographs of segment long spacing aggregates indicate that the cleavage occurs one-quarter of the length from the carboxy terminal end of the molecule. Experiments with crude extracts from granulocytes suggest that the specific products of granulocyte collagenase activity are then degraded by other proteases present in the human granulocyte.

Proteinases of human epidermis; a possible mechanism for polymorphonuclear leukocyte chemotaxis.

Three neutral proteinases (EC 3.4.--.--) and cathepsin D have been identified in human epidermis utilizing a highly sensitive radioactive method. The proteinases were extracted in 1.0 M KC1 and 0.1% Triton X-100 and separated by Sephadex G-75 chromatography. The neutral proteinase peaks were all inhibited by diisopropyl fluorophosphate and thus were serine proteinases. Incubation of the enzyme fractions with [3H] diisopropyl fluorophosphate followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the two larger molecular weight proteinases were enzyme mixtures. The small molecular weight [3H] diisopropyl fluorophosphate proteinase migrated as a single band. Injection of the small molecular weight neutral proteinase into rabbit skin produced a polymorphonuclear leukocyte infiltration and edema. The reaction was not observed with the diisopropyl fluorophosphate-inhibited enzyme fraction. The release of neutral proteinases may be one of the signal events in the epidermal inflammatory response.

Characterization of a chemotactic and cytotoxic proteinase from human skin.

A proteinase (EC 3.4.-.-) active at physiological pH has been isolated from human skin utilizing gel filtration and affinity chromatography techniques. The proteinase has a molecular weight of approx. 28 000 and it is inhibited by alpha 2-macroglobulin, alpha 1-antitrypsin, C-1 inactivatory, soybean trypsin inhibitor and diisopropyl fluorophosphate. 2njection of 1 ng of purified proteinase into rabbit skin induces polymorphonuclear leukocyte infiltration of the cutis. Inhibition of enzyme activity with diisopropyl fluorophosphate inhibits the chemotactic effect. Addition of 0.2 microgram/ml of purified proteinase to fibroblast cultures kills the cells within minutes. This proteinase may play a significant role in modulating the inflammatory response after cellular injury.

Involvement of urokinase-type plasminogen activator in acantholysis induced by pemphigus IgG.

Pemphigus IgG induces acantholysis in skin organ culture without the involvement of complement. Urokinase-type plasminogen activator, a proteolytic enzyme, has been implicated in the development of acantholysis. To test this hypothesis, we prepared a rabbit anti-urokinase antibody, which inhibited the plasminogen activator activity in normal human epidermis and in cultured keratinocytes. When added to skin organ cultures along with pemphigus IgG, anti-urokinase IgG completely prevented the development of acantholysis. Normal or preimmune rabbit IgG had no effect on pemphigus IgG-induced acantholysis. Plasminogen activator converts the zymogen plasminogen to its active form plasmin, a broad specificity serine proteinase. When high concentrations of plasminogen alone were added to skin organ culture, acantholysis of the pemphigus foliaceous type was induced. Anti-urokinase antibody also inhibited plasminogen-induced acantholysis. These results strongly support a pivotal role for plasminogen activator in the development of acantholysis.

Autodegradation of 125I-labeled human epidermal cell surface proteins.

Triton X-100 extracts of cultured human epidermal cells exhibited proteolytic activity as measured by the hydrolysis of [3H]-casein at neutral pH. The majority of endogenous proteolytic activity was inhibited by parahydroxy mercuribenzoate and by mersalyl acid, indicating the enzyme(s) was a thiol class proteinase(s). Crude Triton X-100 extracts were prepared from epidermal cells following labeling of proteins with 125I. Autodegradation of labeled proteins at 37 degrees C was detected as early as 1 hr and reached a plateau level by 4 hr. Degradation was inhibited by thiol class proteinase inhibitors. Among the detergent-solubilized radiolabeled proteins a polypeptide chain of Mr 155,000 was particularly sensitive to degradation by endogenous thiol proteinase(s).

Labeling of peripheral blood polymorphonuclear leukocytes with indium-111: a new method for the quantitation of in-vivo accumulation of PMNLs in rabbit skin.

A precise method for quantitation of polymorphonuclear leukocyte (PMNL) accumulation in skin in vivo, has been developed so that the proinflammatory effects of various agents can be compared. This method can also be used to evaluate the effect of therapeutic agents on PMNL accumulation in vivo. Rabbit PMNLs were purified from heparinized blood by dextran sedimentation, hypotonic lysis, and separation on Ficoll-Hypaque. The PMNLs were labeled with 3-5 microCi per 10(6) cells of 111In oxine and reinfused coincidentally with different concentrations of different chemotactic and proinflammatory materials injected intradermally into the back. In some experiments, varying concentrations of acetic acid were applied topically. Four to 18 hours later, the rabbits were sacrificed. Eight-millimeter punch biopsies were obtained from the injection sites and counted in a gamma counter. The number of PMNLs infiltrating the dermis was also quantitated in histologic sections. A significant correlation was found between the percent increase in radioactivity and the percent increase in PMNL accumulation morphologically. Dose-response curves were generated using such proinflammatory materials as formyl-methionyl-leucyl-phenylalanine, lipopolysaccharide, activated serum, trypsin, glycogen, and acetic acid. These curves were highly reproducible from animal to animal. Using this assay, we found that as little as 1 microgram of trypsin induced detectable PMNL accumulation. This is 2-3 logs more sensitive than injecting mice intraperitoneally with trypsin. Diisopropyl fluorophosphate-inactivation of trypsin inhibited PMNL accumulation. This sensitive and quantitative bioassay of PMNL accumulation permits evaluation of multiple agents in the same animal, which decreases animal to animal variation.

Expression of plasminogen activator enzymes in psoriatic epidermis.

The plasminogen activators, tissue type and urokinase type (tPA and uPA, respectively) have been identified in human skin under normal conditions and in various inflammatory dermatoses, including psoriasis. By Northern blot analyses, mRNA for uPA, but not for tPA, has been previously identified in epidermal extracts from normal skin, whereas in psoriasis, mRNA for tPA is readily detected. To further characterize uPA and tPA expression in psoriasis, the localization of uPA and tPA mRNAs was evaluated by in situ hybridization. Studies were conducted using lesional and nonlesional skin of patients with psoriasis as well as normal skin. Additionally, in situ zymography using casein gel overlays was utilized to assess enzymatic activity. In psoriatic lesional skin, both uPA and tPA mRNAs were demonstrated by in situ hybridization. Message for tPA was observed throughout the epidermis with areas of accentuation in the superficial stratum spinosum. Message for uPA was more focal and was localized primarily in the basal layer. Zymography showed tPA activity was coordinately increased in psoriatic lesions. Uninvolved skin of psoriatic patients was similar to that of normal skin with respect to expression of plasminogen activators. In normal epidermis, neither tPA nor uPA mRNA could be detected by in situ hybridization. Activity for uPA, but not tPA, was observed by zymography. These studies suggest that alterations in plasminogen activators expression may contribute to the pathogenesis of psoriasis.

Migrating keratinocytes express urokinase-type plasminogen activator.

When confluent keratinocyte cultures were wounded by cutting with a blade, the cells rapidly retracted from the wounded site, leaving an area denuded of cells. Within 3-4 h of wounding, keratinocytes began to migrate from the edges and gradually reepithelialized the entire denuded area. Mitomycin C did not prevent the reepithelialization but did dramatically inhibit [3H]thymidine incorporation into the leading edge of cells. These results indicate that cell proliferation was not required for reepithelialization. Using a rabbit antibody against urokinase-type plasminogen activator (u-PA) and an avidin-biotin-peroxidase detection method, we localized u-PA in the keratinocytes at the leading edge of the migrating cultures. Cytochalasin B dramatically inhibited the extent of migration and also altered cell morphology; nonetheless, urokinase was detected in the limited number of cells that moved into the wounded area, even in the presence of cytochalasin B. A small but consistent enhancement (36% +/- 9) of plasminogen activator activity was observed in the supernatant of wounded cultures. These data suggest that plasminogen activator may be involved in the migration of keratinocytes that occurs during wound healing.

Transplantation of psoriatic skin onto nude mice.

Involved psoriatic epidermis maintains its histologic appearance, increased labeling index, and increased level of plasminogen activator after grafting onto athymic nude mice. Epidermis from clinically uninvolved psoriatic skin develops an increase in plasminogen activator activity after grafting for 6 weeks on nude mice and demonstrates an increased labeling index. Normal control skin maintains its low level of plasminogen activator and labeling index after grafting. These results indicate that psoriatic skin can maintain and develop markers of psoriatic skin independent of the host.

Transplantation of Psoriatic Skin onto Nude Mice.

Involved psoriatic epidermis maintains its histologic appearance, increased labeling index, and increased level of plasminogen activator after grafting onto athymic nude mice. Epidermis from clinically uninvolved psoriatic skin develops an increase in plasminogen activator activity after grafting for 6 weeks on nude mice and demonstrates an increased labeling index. Normal control skin maintains its low level of plasminogen activator and labeling index after grafting. These results indicate that psoriatic skin can maintain and develop markers of psoriatic skin independent of the host.

Urokinase plasminogen activator is immunocytochemically detectable in squamous cell but not basal cell carcinomas.

The presence of plasminogen activators (PA) in a variety of solid tumors appears to correlate, in a number of instances, with enhanced invasive or metastatic capabilities. In the present study, we have immunocytochemically examined basal cell (BCC) and squamous cell carcinomas (SCC) comprising a spectrum of histologic subtypes for the presence of urokinase-type (uPA) and tissue-type (tPA) PA. Neither uPA nor tPA was noted in any BCC, whether of the nodular, infiltrative, morpheaform, or basosquamous variety. uPA but not tPA was seen in 12 of 16 SCC examined; the tumors lacking uPA were all histologically well differentiated. No relationship between uPA expression and depth of invasion was noted, and uPA was not preferentially expressed at tumor borders. We conclude that uPA presence in SCC may relate to the degree of differentiation.