Skin zinc concentrations in patients with varicose ulcers.

The concentration of zinc in the skin has been determined noninvasively in patients with varicose vein ulcers. The examinations were performed with the use of diagnostic x-ray spectrometry, a method based on x-ray fluorescence for in vivo noninvasive evaluation of trace elements. Four skin foci were examined: at the periphery of the ulcer and control areas in a nonulcerated area in the diseased leg, in the noninvolved leg, and in the proximal inner surface of the arm. Zinc levels around the ulcer (mean +/- SD, 9.8 +/- 4.0 micrograms of zinc in 1 g of wet tissue) were higher than those in the nonulcerated skin in the diseased leg (6.9 +/- 3.0 micrograms/g, p greater than 0.05) and those in the noninvolved leg (5.4 +/- 2.0 micrograms/g, p less than 0.01). The concentration of zinc in the inner proximal surface of the arm (9.8 +/- 2.8 micrograms/g) was significantly higher than those of a control group (5.3 +/- 1.9 micrograms/g, p less than 0.01). These results suggest a defect of zinc distribution in patients with varicose vein ulcers.

[Comparison of the distribution sites and diagnostic significance of three antibodies as the melanoma markers].

S-100 and its subunits have subsequently been shown to be present in a wide variety of human neoplastic tissues. Three antibodies, monoclonal S-100, polyclonal S-100 and HMB-45, were used for comparison of the positive rates of different sites of melanomas. The results showed that HMB-45 was positive for those melanoma cells in epidermis and negative for those in dermis. Monoclonal S-100 was negative for melanoma cells in epidermis and positive for those in dermis. They are useful for the diagnosis of melanoma in surgical pathology.

Intraepidermal neutrophilic IgA dermatosis.

We report a patient with a vesiculopustular eruption with features distinct from typical subcorneal pustular dermatosis. Clinically, well-formed pustular lesions, which were flowerlike in appearance, were present. Histopathologically, early vesicular lesions showed intraepidermal bullae containing numerous neutrophils, a few eosinophils, and acantholytic cells. Direct immunofluorescence study revealed IgA deposits in the intercellular space of the epidermis. The patient's serum, however, did not contain circulating antibodies reactive with the epidermis. We consider this eruption an immunologically mediated, intraepidermal blistering disease, similar to intraepidermal neutrophilic IgA dermatosis.

Distribution of fodrin in the keratinocyte in vivo and in vitro.

Distribution of fodrin in the keratinocyte, both in vivo and in vitro, was examined by immunofluorescence microscopy. In the rat epidermis in vivo, fodrin was localized in the cell periphery of the spinous layer of all the skins studied. In only the basal layer of the thick skin, however, fodrin was seen intensely in the cytoplasm. As in vitro keratinocytes, a mouse cell line (Pam 212) cultured in low (0.06 mM) as well as standard (1.87 mM) Ca2+ was examined. In low Ca2+, fodrin was observed throughout the cytoplasm without marked accumulation irrespective of the cell density. The cytoplasmic labeling in low Ca2+ looked filamentous and became aggregated when cells were treated with cytochalasin B; at least some of the aggregates coexisted with those of F-actin. In contrast, fodrin distribution was not affected with colchicine. On the other hand, in standard Ca2+, the protein became concentrated along the cell periphery and less conspicuous in the cytoplasm as the cells reached confluency. When cells were transferred from low to standard Ca2+, the distribution of fodrin changed accordingly within 180 min. The present results indicate that fodrin in the keratinocyte is likely to be associated with actin filaments and that it takes two different ways of distribution both in vivo and in vitro. The peripheral and the cytoplasmic labeling of in vivo and in vitro cells are likely to correspond. It may be that fodrin changes its localization according to the cell's proliferative activity.

Keratin expression in equine normal epidermis and cutaneous papillomas using monoclonal antibodies.

Keratin expressions in normal equine epidermis and experimentally induced equine papillomas were studied by immunohistochemical methods with three different human cytokeratin monoclonal antibodies, 34 beta B4 (directed against component 1), 34 beta E12 (directed against components 1, 5, 10, 11) and 35 beta H11 (directed against component 8). Staining patterns with 34 beta B4 and 34 beta E12 in the normal equine epidermis did not differ from those in the normal human epidermis. In the early developing papilloma, keratinocytes showed an abnormal suprabasal staining pattern and expressed an additional 56 kD keratin protein detected by 34 beta E12. In the advanced papilloma, cytolytic cells in the outer spinous and the granular layers did not stain positively with any of the three antibodies used. In both early and advanced papillomas, the expression of high molecular weight keratin proteins, as detected by 34 beta B4 and 34 beta E12, did not correlate with the degree of keratinization. By electron microscopy, keratinocytes in the advanced papilloma showed a marked decrease of tonofibrils and desmosome-tonofilament complex. These alterations may result from an abnormality in both proliferation and functional terminal differentiation of keratinocytes in the papilloma. There were obvious differences in staining patterns with 35 beta H11 between the normal human and equine epidermis; 54 kD keratin protein was expressed in suprabasal layers of the equine normal and papillomatous epidermis. Thus, this keratin protein may be regarded as a "permanent" marker for the equine epidermis.

Identification of specific human epithelial cell integrin receptors as VLA proteins.

Cell adhesion to extracellular matrix is mediated by a set of heterodimeric cell surface receptors called integrins. We have examined the expression of the very late antigens or alpha beta 1 group of integrins in human epithelial cells. The six known members of this group share a common beta 1 subunit but have distinct alpha subunits that confer selective affinity toward collagen, fibronectin, and laminin essentially. Using a panel of specific antibodies we showed that freshly harvested human epidermal basal cells express VLA-2 and VLA-3 receptors, a low amount of VLA-5, but fail to express VLA-4. The findings reveal that these receptors are characterized by the alpha subunits which associate with a beta subunit different in weight (Mr 110,000 reduced) from that normally seen (Mr 130,000). Moreover, immunoprecipitates of VLA-2 contained additional proteins of Mr 80,000 and Mr 40,000 and immunoprecipitates of VLA-3 contained an additional protein of Mr 90,000. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to extracellular matrix revealed that cell attachment to type IV collagen was completely inhibited by antibodies to VLA-2 alpha chain, that antibody to VLA-3 alpha chain significantly blocked attachment to fibronectin while antibodies to both VLA-2 and VLA-3 partially inhibited attachment to type I collagen. Cell attachment to types I and IV collagen and to fibronectin was not affected by antibodies to VLA-4 and VLA-6. These results show that multiple VLA receptors function in combination to mediate epidermal basal cell adhesion to extracellular matrix. This cooperation function of multiple VLA receptors and their differential expression could be considered to be one of the controlling points in the localization of epithelial basal cells in the epidermis.

"Activated" keratinocyte phenotype is unifying feature in conditions which predispose to squamous cell carcinoma of the skin.

While some cutaneous squamous cell carcinomas (SCC) arise from predisposing conditions such as burn scars, draining sinuses, and chronic, nonhealing wounds, the vast majority of these tumors arise from actinically damaged epidermis. It has been shown previously that keratinocytes within healing wounds show an "activated" immunophenotype when stained with antibodies to psi-3, involucrin, filaggrin, and cytokeratins. A similar pattern has been seen in keratinocytes from patients with recessive dystrophic epidermolysis bullosa (RDEB), in whom the incidence of cutaneous SCC is markedly increased. We tested the hypothesis that actinic keratoses (AK), recognized as precursors in the development of the majority of SCC, would show a similar activated immunophenotype when stained with the antibody panel described above. We examined 10 AK, biopsied from the facies and extremities of ten patients, ages 60 to 80, with antibodies to psi-3, involucrin, filaggrin, and AE1. All lesions examined had an immunostaining pattern indistinguishable from that seen in keratinocytes from patients with RDEB or within healing wounds. There was suprabasilar staining of keratinocytes with antibodies to psi-3 and AE1. Involucrin and filaggrin was expressed by all keratinocytes above the midstratum spinosum. Within the acrosyringia and acrotrichia, the staining pattern was that of the normal epidermis, i.e., AE1 staining of basal keratinocytes, granular layer staining of involucrin and filaggrin, and absence of psi-3 expression. These data suggest that an activated keratinocyte phenotype is a unifying feature in conditions which predispose to development of cutaneous SCC.

Detection of pemphigus vulgaris and pemphigus foliaceus antigens by immunoblot analysis using different antigen sources.

In an immunoblot analysis with human epidermal extract as a source of antigens, all (28/28) pemphigus vulgaris (Pv) sera showed a specific reactivity with a 130-kD protein. Several, but not all, Pv sera reacted with similar antigens in both a bovine muzzle desmosome preparation and extract of cultured human squamous carcinoma cells. On the other hand, some pemphigus foliaceus (Pf) sera exhibited reactivity with a 150-kD protein, which is most likely desmoglein I, in both the human epidermal extract and the bovine desmosome preparation, but no Pf serum reacted with this antigen in the squamous carcinoma cell extract. Furthermore, 4/16 Pv sera also reacted with a 150-kD protein in the desmosome preparation, which seemed to be the same as Pf antigen. These results show a relationship between antigens of both Pf and Pv and desmosomes, as well as heterogeneities of both Pv and Pf antigens in terms of antigenic molecules or epitopes. Furthermore, this study presents the possibility that immunoblot analysis can be routinely used for differentiation of Pv and Pf antibodies.

Characterization of cellular elements in healed cultured keratinocyte autografts used to cover burn wounds.

Biopsy specimens from unburned skin were obtained from three severely burned patients and placed into tissue culture. After 2 to 3 weeks, the cultured keratinocytes were released from the Petri dishes and transplanted onto the patient's burn wound, which had been completely excised down to muscle fascia, thereby removing all cutaneous elements. Healing cultured autografts were found to become repopulated with Langerhans cells within 3 to 6 weeks. A neodermis rich in fibronectin rapidly formed between the autografts and muscle fascia. However, using monoclonal antibodies to cytokeratins as markers of differentiation, we found that the autograft keratinocytes expressed an abnormal pattern of differentiation that was similar to the differentiation seen in hyperproliferative states such as psoriasis. In contrast, healed split-thickness graft donor sites and reepithelialized interstices of mesh grafts maintained the basal keratinocyte staining pattern of normal skin with the AE-1 monoclonal antibody.

Cornified envelopes in congenital disorders of keratinization.

A morphological and biochemical analysis was made of cornified envelopes isolated from patients with different congenital disorders. Nomarski contrast microscopy of the envelopes showed that their morphology was not greatly altered in several types of keratoderma and parapsoriasis, but it was grossly modified in ichthyotic disorders. The various types of ichthyoses, keratoderma palmoplantare, KID syndrome and parapsoriasis showed, after cyanogen-bromide cleavage, peptide patterns similar to those obtained from healthy subjects. In contrast, envelopes from patients with Darier's disease, congenital pachyonychia and erythrokeratoderma variabilis showed markedly different peptide patterns.

Keratinocyte and epidermal leukocyte expression of CD36 (OKM5) in benign and malignant skin diseases.

CD36 recognizes a 88 Kd glycoprotein, found on different cell populations involved in immunoregulation and are induced on keratinocytes by in vitro treatment with gamma-interferon. Therefore, we obtained skin biopsies from 48 patients with various dermatological diseases and from 5 healthy volunteers and stained these with monoclonal antibodies OKM5 (CD36), anti-HLA-DR and OKT6 (CD1a) using a three stage immunoperoxidase method. In normal skin, CD36 expression was not observed. In contrast, keratinocytes in diseased skin were CD36+. In most cases, the staining was restricted to the stratum granulosum and the stratum spinosum, but in psoriasis, squamous cell carcinoma and lymphomatoid papulosis, more extensive staining of keratinocytes was seen. In addition, CD36+ epidermal leukocytes were found in allergic patch-test infiltrates and in mycosis fungoides. The findings of CD36 expression by epidermal cells within a broad spectrum of dermatological diseases indicate a role for these cells in the regulation of immune reactions in the skin.

Protooncogene expression in normal and psoriatic skin.

The expression of the c-myc, c-fos, c-jun, c-erbB, and c-Ha-ras protooncogenes was compared by Northern blot analysis of total RNA extracted from keratome biopsies of normal skin and psoriatic plaques. Isolation of intact RNA from frozen tissue required careful attention to technique during the early stages of extraction. Densitometric analysis revealed 1.5- to 2.5-fold elevations of c-myc transcript levels in lesional psoriatic relative to normal epidermis. Similar increases in cyclophilin and lipocortin II transcripts were also observed and may reflect characteristic differences in RNA preparations from normal and psoriatic epidermis. C-myc, c-jun, c-erbB, c-fos, and c-Ha-ras transcript levels were not significantly increased in lesional psoriatic epidermis when protooncogene mRNA levels were normalized to those of the cyclophilin or lipocortin genes. In contrast, transforming growth factor-alpha (TGF-alpha) transcripts were significantly increased (10- to 20-fold) with or without prior normalization. C-myc, c-fos, and c-jun transcripts were significantly induced over in vivo levels 2-4 h after organ culture of normal or psoriatic keratome biopsies, demonstrating that these genes can be highly expressed in the context of tissue injury. Our results suggest that overexpression of these protooncogenes per se is not central to the pathogenesis of psoriatic epidermal hyperplasia.

Effects of hydroxychloroquine on 'band test' in discoid lupus erythematosus.

Ten cases of localized and generalized discoid lupus erythematosus are reported in which previously untreated patients were given hydroxychloroquine sulphate 600 mg daily for 10 days followed by 400 mg for 20 days. The purpose of the study was to evaluate the effect of this drug on the 'lupus band' before and after treatment, in diseased, unaffected sun-exposed, and unaffected non sun-exposed skin. A good response from both the clinical and immunopathologic (i.e. reduction or disappearance of the immune reactants) standpoint was evident in 6/10 patients; in another 3 patients a good clinical but not immunopathologic response was recorded, while in 1 case a clinical worsening corresponded to an immunofluorescence improvement. In 5/10 cases (4 females, 1 male) one or more immunoglobulin classes which were present in the 'lupus band' before therapy remained at the dermoepidermal junction after treatment.

Immunohistochemical investigations in epidermal Merkel cells--a common phenotype with eccrine sweat gland epithelium.

Mono- and polyclonal antibodies were used for immunofluorescence investigations in cytoskeletal and associated antigens on frozen sections of adult human skin. Epidermal Merkel cells were identified in the stratum basale. Their phenotype was compared with other epithelial cells of human skin. The inner duct epithelium of eccrine sweat glands showed an immunoreactivity similar to that of epidermal Merkel cells. A common origin of both cell types is suggested.

Recessive dystrophic epidermolysis bullosa skin displays a chronic growth-activated immunophenotype. Implications for carcinogenesis.

Epidermolysis bullosa represents a grouping of inherited skin diseases characterized by epidermal fragility and frequently wounded skin. The recessive dystrophic subtype of epidermolysis bullosa (RDEB) is characterized by extensive dermal scarring after healing of repeated epidermal injuries and by an unusually high incidence of squamous cell carcinoma occurring in chronically wounded skin. In contrast, the simplex form of epidermolysis bullosa usually heals without scarring and does not predispose to malignant neoplasms of the skin. The differences in scarring and the neoplastic potential of these two forms of epidermolysis bullosa prompted us to investigate growth activation and differentiation characteristics in epidermal keratinocytes in individuals with these disorders. The expression of filaggrin, involucrin, cytokeratins, and the growth activation marker psi-3 was examined by immunohistochemistry in skin biopsy specimens from four individuals with epidermolysis bullosa simplex and six individuals with RDEB. Previous experiments using this technique have demonstrated that these antibodies are good markers for identifying growth-activated keratinocytes in wounded and hyperplastic epidermis. All biopsy specimens of healed wounds in skin from patients with RDEB showed epidermis that reacted with antibodies to filaggrin, involucrin, specific cytokeratins, and psi-3 in a growth-activated pattern. This growth-activated phenotype was maintained in keratinocytes from previously wounded skin that had been healed for more than 2 years. The RDEB growth-activated phenotype detected by immunohistochemistry was not associated with microscopically detectable epidermal hyperplasia. In contrast, all cases of epidermolysis bullosa simplex examined showed an epidermal phenotype similar to that of keratinocytes in normal skin. Thus, healing with dermal scar formation in RDEB is associated with a persistent growth-activated immunophenotype of epidermal keratinocytes. This chronic growth activation state or failure of cells to differentiate in a normal fashion may be directly linked to the high incidence of squamous cell cancers in individuals with RDEB.

Immunoblot analyses of Brazilian pemphigus foliaceus antigen using different antigen sources.

We investigated the Brazilian pemphigus foliaceus (BPf) antigen applying the immunoblotting method to two different antigen sources using 27 patients' sera. Twelve BPf sera reacted specifically with a 150 kD protein in extract of dispase separated human epidermis, while 18 sera yielded a similar protein band in bovine muzzle desmosomal preparation. The diversity of staining intensities between the two samples suggested the heterogeneity of BPf antigens in terms of epitopes. Japanese sporadic pemphigus foliaceus (Pf) sera showed similar results but Japanese pemphigus vulgaris (Pv) sera recognized different antigens of 130 kD or 135 kD, suggesting that BPf is similar to Japanese Pf but is distinct from Pv in respect to the antigenic substance. Furthermore, the present study showed that immunoblot analysis using different antigen sources should be a valuable tool to determine clinical types of pemphigus.