Immunohistochemical study of melanocytic nevus and malignant melanoma with monoclonal antibodies against S-100 subunits.

Immunohistochemical localization of S-100 protein alpha and beta subunits in the cells of melanocytic nevi and malignant melanomas was studied by using monoclonal antibodies directed against each subunit. Although polyclonal anti-S-100 reactivities have been demonstrated uniformly in all nevus cells and melanoma cells, monoclonal anti-S-100 alpha and anti-S-100 beta reactivities were either absent or rarely found in ordinary junctional nevi or junctional nests of ordinary compound nevi. However, in the junctional nests of dysplastic junctional nevi and junctional components of dysplastic compound nevi, monoclonal anti-S-100 alpha reactivity become more frequent, whereas monoclonal anti-S-100 beta reactivity remains negative. In the superficial variety of melanomas such as superficial spreading melanoma and lentigo maligna melanoma, monoclonal anti-S-100 beta is nonreactive until vertical growth or invasiveness begins. Most nodular melanomas are positively stained with both monoclonal anti-S-100 alpha and anti-S-100 beta. It is suggested that monoclonal anti-S-100 alpha can be an indicator of active junctional nevus of melanocytic nevi and the reactivity with monoclonal anti-S-100 beta may be related to vertical progression of superficial spreading melanomas and lentigo maligna melanomas.

Vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) in human eccrine sweat glands: demonstration of innervation, specific binding sites and presence in secretions.

Vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) immunoreactivities have been detected in alcohol extracts of human axillary skin using sensitive and specific radioimmunoassays. VIP immunoreactivity (7.63 + 2.33 pmol/g, x + SE, n = 9) was more abundant than PHM immunoreactivity (3.86 + 0.56 pmol/g, x + SE, n = 9). Immunocytochemistry of sections of skin revealed a network of VIP/PHM immunoreactive nerve fibres around the perimeter of eccrine but not apocrine sweat glands. In vitro autoradiography of skin sections using 125I-labelled VIP and PHM, demonstrated binding sites on the membranes of eccrine gland secretory cells. The binding of each radiolabelled ligand was eliminated by the presence of a large molar excess of appropriate cold peptide but was unaffected when incubated with related peptide, indicating the presence of specific binding sites for both VIP and PHM. Radioimmunoassay of Sep-pak concentrated human sweat identified the presence of both VIP immunoreactivity (30.6 pmol/l) and PHM immunoreactivity (43.4 pmol/l). Reverse-phase HPLC analysis of axillary skin extracts and sweat, followed by radioimmunoassay of fractions, identified single resolved peaks of VIP and PHM immunoreactivity with identical retention times to synthetic standards. Eccrine sweat glands in human axillary skin have VIP and PHM peptidergic innervation and possess specific binding sites for each peptide which are both secreted to the surface of the skin.

Clinical and histologic features of primary oxalosis.

Primary oxalosis should be considered in patients with multisystem disease of the kidneys, heart, peripheral vasculature, and skin. Crystalline deposits can lead to nephrolithiasis with kidney failure, complete heart block, peripheral vasospasm, and livedo reticularis, as in our patient. Crystals were first observed in the myocardial biopsy specimen and then identified as calcium oxalate in skin from an area of livedo reticularis.

Ultrastructural localization of keratin and alpha-L-fucose in human eccrine sweat glands.

Human eccrine sweat glands were embedded in Lowicryl K4M. Cytokeratin proteins and blood group H antigen were localized by applying a postembedding immunogold method using a monoclonal antikeratin antibody and the lectin Ulex europaeus I. The antikeratin antibody labeled intermediate filaments in the secretory coil and dermal duct. Within dark secretory cells bundles of filaments criss-crossing the cell were labeled. Within the luminal cells of the dermal duct filaments arranged parallel to the cell surface and lying in the apex of the cell were labeled, too. The association of keratin filaments with desmosomes was visualized demonstrating their subcellular connection with other cell organelles. The desmosomes themselves remained unlabeled. The lectin Ulex europaeus I is a blood group H specific lectin and binds to alpha-L-fucosyl-containing glycoproteins. Dark cells of the secretory coil reacted with the lectin. Here the secretory granules, the lateral cell membranes, and the microvilli membranes were labeled. The endoplasmatic reticulum, the Golgi complex, and transport vesicles were not labeled, although the glycoprotein synthesis is considered to be located in the Golgi complex. Thus, either the number of alpha-L-fucose molecules in the Golgi is too low to be detected by the technique employed or the determinant of blood group H antigen is released after the secretory granules and transport vesicles leave the Golgi complex.

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.

Mixed tumors of the skin. A histological and immunohistochemical study.

Sixty-four specimens of mixed tumors of the skin were studied by conventional microscopy. Sections from all 64 specimens were stained by hematoxylin and eosin, and sections from 18 of those specimens were stained by immunoperoxidase techniques for the presence of S-100 protein, carcino-embryonic antigen (CEA), keratin, actin, vimentin, epithelial membrane antigen (EMA), and gross cystic disease fluid protein-15 (GCDFP-15). Two distinctive histopathological patterns of mixed tumors of the skin became apparent, namely, apocrine and eccrine. Mixed tumors with apocrine features are by far the most common. Immunoperoxidase techniques, in our experience, do not enable differentiation between apocrine and eccrine types of mixed tumors.

[An immunohistochemical study of normal sweat glands using an antibody to the breast cyst fluid protein (GCDFP-15)].

In this study, we applied the antibody against the molecular weight 15,000 protein purified from breast cyst fluid (GCDFP-15) to cutaneous tissue, especially to sweat glands. Breast cyst fluid was obtained by needle aspiration from patients with gross cystic disease. DEAE-cellulose column chromatography was performed and followed by SDS-polyacrylamide gel electrophoresis. Antisera against the purified protein were prepared in rabbits. We stained specimens of normal skin by the PAP method. The normal apocrine gland cells were strong positive and eccrine gland dark cells were positive. Eccrine gland clear cells were weak positive or negative. Duct cells of both glands were negative. From these observations, we suggest that this antibody is useful for detecting seromucous cells of sweat glands, including both the apocrine gland cells and eccrine dark cells in the skin.

An NADH-linked luciferase assay for glycogen: the preparation of glycogen-free, viable human eccrine sweat glands.

A glycogen assay based on bacterial NADH luciferase is described. It is free of tissue interference. The detection limit is 0.12 nmol glycogen, and the coefficient of variation is 5.5%. A method of depleting human eccrine sweat glands while retaining their viability is described. This depends on their incubation in 10(-5) M acetylcholine and 1 mM pyruvate. This method may be applicable to other tissues. The evidence for the viability of glycogen-depleted human eccrine sweat glands is reported and includes tissue contents of ATP and the rates of oxidation of glucose, pyruvate, beta-hydroxybutyrate, and palmitate.

Diagnosis of Lafora disease by skin biopsy.

The Lafora type of progressive myoclonus epilepsy is a rare and fatal familial disease characterized by seizures, myoclonus, and dementia. This diagnosis was confirmed in 2 patients by demonstrating the presence of intracytoplasmic polyglucosan bodies, or Lafora bodies, in the peripheral portion of the eccrine sweat gland duct. Exclusive use of the periodic acid-Schiff stain is recommended for demonstrating these diagnostic inclusions. Electron microscopy reveals fine pale-staining filaments, fine dark-staining granules, and dark-rimmed vacuoles within these non-membrane-bound inclusions. Skin biopsy is the preferred method of confirming the diagnosis of Lafora disease.

Immunohistochemistry of gross cystic disease fluid protein (GCDFP-15) in 65 benign sweat gland tumors of the skin.

Sixty-five cases of benign sweat gland tumors of the skin were studied for the expression and localization of gross cystic disease fluid protein-15 (GCDFP-15) by immunoperoxidase methods. There was positive staining of tumors of probable apocrine differentiation in 10 of 11 cases of apocrine hidrocystoma and five of five cases of hidradenoma papilliferum. There was no immunoreactivity for GCDFP-15 for tumors of probable eccrine differentiation, including five cases of eccrine hidrocystoma, five cases of eccrine poroma, five cases of eccrine spiradenoma, 10 cases of clear cell hidradenoma, and nine cases of syringoma. There was variable positive staining of tumors of more uncertain histogenesis, including eight of eight cases of syringocystadenoma papilliferum, one of four cases of cylindroma, and two of two cases of chondroid syringoma (mixed tumor). The above data support a functional differentiation of the expression of GCDFP-15 by eccrine compared to apocrine glandular epithelium with benign tumor development.

Immunohistochemical identification of epithelial membrane antigen in sweat gland tumors by the use of a monoclonal antibody.

A total of 78 cases of adnexal tumors of the skin were examined with the use of monoclonal antibody against epithelial membrane antigen (EMA). The EMA reaction was confined to luminal surfaces and lateral borders of sweat glands, both eccrine and apocrine, being usually absent in ductal segments. Neoplastic lesions of all the adenomatous tumours and mixed tumours of sweat glands showed specifically positive EMA staining of luminal surfaces and lateral borders of tubular, duct-like, and cystic structures. However, solid foci of those tumours were negative for EMA. Tumours of ductal origin, e.g. syringomas and poromas, exhibited positive EMA staining in their plasma membrane, although normal intact keratinocytes did not stain for EMA. Immunohistochemical distribution of EMA in skin adnexal tumours was classified into two types: one in which the luminal surfaces and lateral outer borders were positive, similar to that of the normal secretory coil, and the other in which the plasma membrane of neoplastic cells of ductal origin was positive.

Lectin-binding sites in Paget's disease.

The presence and distribution of lectin-binding sites on neoplastic cells of Paget's disease was studied using fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA), and FITC-conjugated wheatgerm agglutinin (WGA), and compared with such lectin-binding sites on keratinocytes, and cells of eccrine glands, apocrine glands, and mammary glands. Neoplastic cells of both mammary and extramammary Paget's disease showed cytoplasmic staining with both lectins. There were however fewer stained cells in mammary Paget's disease than in extramammary Paget's disease. The cytoplasmic staining of lectin-binding sites in cells of apocrine glands was in sharp contrast to the cell-surface staining seen on keratinocytes, or cells of eccrine glands or mammary glands. These results indicate that the lectin-binding sites of neoplastic cells of Paget's disease more closely resemble those of cells of apocrine glands than of keratinocytes, cells of eccrine glands or cells of mammary glands.

Immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues.

Epidermal growth factor (EGF) stimulates the growth of many tissues and inhibits stimulated gastric acid secretion. Its primary tissue of origin in man is still unknown. We used polyclonal anti-human EGF sera in the peroxidase-antiperoxidase immunocytochemical staining technique to identify immunoreactive human EGF (ihEGF) in tissue sections from 29 subjects ranging from fetuses to 63 years in age. In addition to acinar cells in the submandibular salivary glands and cells of Brunner's duodenal glands, previously reported to contain ihEGF, we found ihEGF in most anterior pituitary glycopeptide hormone-secreting cells, in gastric and pyloric gland cells of the stomach, and in bone marrow cells that resembled mononuclear phagocytes in subjects of all ages. The eccrine sweat glands in the skin of adults also contained ihEGF. Cells containing ihEGF were found singly or in clusters in the trachea of the fetus only. No fetal pancreatic islet cells stained, but occasional cells in neonates and a majority of islet cells in older subjects contained ihEGF; there was no constant association with insulin, glucagon, or somatostatin. Only the lactating breast contained ihEGF. In adults, outer adrenomedullary cells contained ihEGF. Intense immunostaining was observed in the renal medulla, apparently limited to the extracellular area between the renal tubules, and increased with age; the cortex was devoid of ihEGF. No ihEGF was detected in posterior pituitary gland, thyroid gland, heart, lung, or liver at any age. An adult prostate contained ihEGF only in an area of local injury, and some primordial follicles from the ovary of a newborn appeared to contain ihEGF. Thus, many tissues appear to synthesize hEGF, which may exert exocrine, endocrine, or paracrine functions in different tissues and at different ages.

[Morphological study on biopsied skin materials of Lafora disease].

Skin biopsy of a patient with Lafora disease was performed. The specimen showed numerous PAS-positive materials in the eccrine glands as proposed by Carpenter and Karpati. The patient was a 25-year-old man, and his two brothers and sister were diagnosed as Lafora disease by clinical and autopsy findings. The patient has shown typical clinical course of Lafora disease. Light microscopic findings of the skin revealed 5-7 microns oval or round PAS-positive materials in most of the glandular cells in eccrine glands. Several histochemical stainings were applied to the specimens so that the storage materials in the glandular cells were considered as glucose-polymer (polyglucosan). Electronmicroscopic observation showed the materials adjacent to the nucleus without limiting membrane. The materials were composed of numerous glycogen-like particles associated with a small number of fine filaments and vesicles. It have been not clarified why these materials are present in the sweat glands in Lafora disease. Recently skin biopsies have been underwent in patients of storage disease involving the central nervous system. Especially it is much useful for diagnosis of the disease with no reliable enzymatic assays. Pathogenesis of Lafora disease is still unknown. Therefore, skin biopsy might be convenient to differentiate from other types of myoclonus epilepsy.

Immunohistochemical staining characteristics of epidermal appendages (hair follicles and eccrine sweat glands) to anti-epidermal keratin antisera.

The cellular characteristics of the epidermal appendages and their differentiations in relation to those of the epidermis have been chiefly studied from the morphological points in the past. We investigated them from the immunohistochemical characteristics of the constituent keratin protein to the antiserum against total keratin isolated from human plantar stratum corneum (TKA) which stains the whole epidermis, and to the antiserum against 64K keratin separated from total keratin (64KA) which stains the whole epidermis except the basal layer. In the hair follicles, medulla and cortex of the hair shaft and inner root sheath were positively stained with both types of the antisera only at the keratogenous zone. The staining pattern of the outer root sheath with 64KA were variable at different levels of the hair follicle. The secretory portion of the eccrine glands showed a heterogeneous staining pattern as compared with the ductal portions which were stained homogeneously by both antisera. In eccrine poroma, all of the tumor cells were stained positively with TKA, while negatively or positively stained cells were intermingled with 64KA, suggesting that only some tumor cells showed mature keratinization.

Distribution of glycoconjugates in normal human skin using biotinyl lectins and avidin-horseradish peroxidase.

Lectin binding patterns in normal human skin were studied using five different biotinyl lectins and avidin-horseradish peroxidase. The staining pattern was specific for each lectin. In the epidermis, peanut agglutinin (PNA) and soybean agglutinin (SBA) preferentially stained the cell membranes of keratinocytes in the spinous and granular cell layers, indicating changes in the saccharide residues during keratinocyte differentiation. In the secretory segment of an eccrine sweat gland, the superficial cells gave a strong granular staining with Ricinus communis agglutinin (RCA). Dolichos biflorus agglutinin (DBA) and SBA, on the other hand, strongly stained the basal cells. With these lectins, two types of cells in the secretory segment were clearly distinguished. These results show that (1) PNA and SBA binding sites increase during the course of keratinocyte differentiation, and (2) RCA, DBA, and SBA are good markers to distinguish two types of cells in the secretory segment of an eccrine sweat gland.