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Human scalp skins were treated with 20 mM ethylenediaminetetraacetic acid and terminal hair follicles were extracted with the epidermis. Some terminal hair follicles were morphologically preserved well and provided opportunity to examine three-dimensional distribution of CAM5.2 (K8, 52.5 kD) reactive Merkel cells. In anagen terminal hair of the scalp numerous immunoreactive Merkel cells were distributed in the presumptive bulge area. Distinct swelling as in the bulge of human vellus hair was usually absent; however, in rare instances anagen terminal hair demonstrated unilateral prominent swelling with dense aggregation of Merkel cells. In telogen hair the bulge becomes indistinguishable from the regressed end of the club hair follicle but Merkel cells continued to be abundant. We found morphologic variation of the bulge such as formation of knoblike swellings and villous projections. Interestingly, Merkel cells were also located in these structures. Palisading stockade-like nerve endings were observed surrounding the follicular epithelium at the sebaceous gland level. Merkel cells were sparse in this follicular segment. Variable number of Merkel cells were also scattered in the infundibulum of terminal hair in no association with peripheral nerves.

The origin of Merkel cells is uncertain, although current evidence by immunohistochemical keratin marker studies favors an epidermal derivation. We studied the expression of keratin species in Merkel cells of human fetus and adult using 19 anti-keratin antibodies. Epidermal and dermal Merkel cells contained not only simple epithelium-type but also some stratified epithelium-type keratins. Interestingly, expression of some keratins was different between epidermal and dermal Merkel cells, for example, AN3 (50, 58 kD) and CKB1 (50 kD) recognized epidermal Merkel cells, but not dermal Merkel cells. These results suggest that surrounding keratinocytes influence the expression of cytokeratins in Merkel cells or that dermal Merkel cells undergo a modification from keratin-producing epidermal Merkel cells to a more neural cell type by the association with nerve endings in the upper dermis. On the other hand, certain cytokeratin polypeptides recognizable with Ks19.1 (40 kD), CK5 (45 kD), and CAM5.2 (52.5 kD) were expressed in both epidermal and dermal Merkel cells. The expression of simple epithelium-type keratins in Merkel cells remained even after the epidermal basal cells gradually lost their expression.

Tetradecane (TD), testosterone (TS), and dimethyl sulfoxide (DMSO) were separately inuncted on rabbit pinnas once a day; the pinnas were biopsied on days 1, 3, 7, and 28. Untreated pinnas and squalane-treated pinnas were used as controls. Three-dimensional images of sebaceous glands were reconstructed from their serial histologic sections using a computer-image analysis system. By stereometry, the sebaceous gland volumes gradually increased during the course of treatments with TS and DMSO, whereas the volumes of TD-treated sebaceous glands increased, reached the maximum on day 7, and then decreased. The acinus number was increased by all the substances, the DMSO-treated sebaceous glands on day 28 showing the largest number of acini. The volumes of individual acini increased until day 28 with TS treatment, whereas with TD and DMSO treatments they increased, reached the maximum on day 7, and then decreased. Stereographically, each untreated control sebaceous gland was composed of 20-30 pear-shaped acini. On day 28, acini of TS-treated sebaceous glands were swollen and ovoid in shape, DMSO-treated sebaceous glands resembled a bunch of grapes, and acini of TD-treated sebaceous glands were widely scattered and showed globular swelling arising from an elongated tube. Hyperplastic responses of sebaceous glands are quite different according to substances applied topically.

The plucked hairs and biopsied hair follicles of the scalp were obtained from a female patient with monilethrix. By scanning electron microscopy, the plucked hairs showed a typical moniliform feature composed of alternated nodes and inter- nodes. By computer stereography, reconstructed three-dimensional models of in vivo hair structures showed that the diameter of hair shaft was partially reduced in the keratogenous zone and that the reduction was severe in the hair cortex and cuticle layers but mild in the inner root sheath. By transmission electron microscopy, a significant degeneration of hair matrix cells was found, and a zig-zag disarray of cortical tonofibrils and invaginations of the hair cuticle cells into the cortex were noticed in the suprabulbar portion. In some hairs, the hair bulbs showed no degeneration, but a degeneration of cortical cells and invaginations of hair cuticle were seen in some portions in the developing zone of the cortex. This suggests that not every hair matrix is damaged in a synchronized fashion, but individual hairs are affected independently. Furthermore, in all the hair and hair follicles examined, cytoplasmic vacuolations were always seen in the various layers, and abnormal formation of tonofibrils was often observed in the cortex. In monilethrix, a cell abnormality may inherently be present in the hair tissue and, when such abnormality occurs severely, the cortical cells are particularly affected in the hair matrix. This seems to result in. a decrease in number of cortical cells and thinning of the hair shaft.

The mechanism of formation of trichorrhexis invaginata, which is one of the characteristic hair anomalies seen in Nether ton's syndrome, was studied using plucked hairs obtained from 3 patients with the typical clinical features. The biopsy specimens used to examine the hair root were obtained from the scalp of one of the patients and the eyebrow area of the other. The scanning electron microscopic observation of plucked hairs showed trichorrhexis invaginata, tortion nodule, pili torti, and trichorrhexis nodosa. The light microscopic sections of nodules of plucked hairs revealed invaginated hair cuticle into the cortex. Transmission electron microscopy revealed cleavages and electron-dense depositions in the cortex of plucked hair nodules; such changes were not seen in newly formed nodules in follicles in biopsy specimens. In the keratogenous zone, a zigzag pattern of cortical fibers and invaginations of hair cuticle cells into the cortex were observed. Histochemical staining for -SH groups and S-S linkages using N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM) was performed on frozen sections of plucked hairs from patients and normal controls. Keratinized cortical cells of patients possessed more -SH groups than the controls and a strong fluorescence for -SH was sometimes seen in the cortex of nodules. It is concluded that the invagination of affected hairs is caused by softness of the cortex in the keratogenous zone, which may result from an incomplete conversion from -SH to S-S in proteins in cortical fibers.

The hair follicles and the eyes of pallid mice (C57/6J-Pa/Pa) and those of black mice (C57/6J−+/Pa) were examined ultrastructurally, histochemically, and biochemically to determine the cause of pigment dilution. The pigment cells in the hair follicles and the eyes of pallid mice have less mature melanosomes than those of black mice. In the hair follicles the pallid melanosomes were transferred into keratinocytes and became aggregated. In the eyes they were already aggregated within the pigment cells and were digested in acid phosphatase-positive lysosomes. The activity of acid phosphatase, a marker of lysosomal enzymes, was significantly higher in pallid hair follicles and eyes than in black hair follicles and eyes. Dopa reactions at light and electron microscopical levels indicated that the pigment cells in each tissue produced a large amount of Dopa oxidase when compared with those in each black ocunterpart. However, the rate of hydroxylation of L-tyrosine–3,5– 3 H was significantly lower in the pallid eyes than in black eyes, while this rate was significantly higher in pallid hair follicles than in black hair follicles. Immediate digestion of melanosomes within the pigment cells, i.e. autophagocytosis, seemed to explain the low activity in the pallid eyes. The diluted coat and eye colors of pallid mice are, therefore, not related to low Dopa oxidase activity but to immaturity of melanosomes and high activities of lysosomal enzymes; these enzymes seem to digest many of these immature melanosomes and contribute to the diluted coat and eye colors of pallid mice.

A new fluorogenic maleimide DACM, i.e., N(7-dimethyl amino-4-methyl coumarinyl) maleimide, does not fluoresce by itself. It is specifically combined with --SH groups and becomes fluorescent (lambda ex: 400 nm, lambda em: 485 nm). At this range of excitation the frozen skin section has no native fluorescence because the emission maximum of DACM does not overlap with any of aromatic residues of proteins such as tryptophane. S--S groups can be demonstrated with DACM by inhibiting native --SH and then reducing SS to --SH. --Sh was generally abundant at the bulb region. --SH of hair cortex was more concentrated at keratogenous zone. Further up in the follicle, --SH of hair cortex was gradually decreased, although at the region of isthmus, --SH reaction of the hair cortex was still moderately strong. Outer root sheath had --SH from the upper bulb to the surface of the epidermis. One the other hand, no S--S was demonstrable at the bulb region. At the level of keratogenous zone, however, S--S linkages of hair cortex and inner root sheath began to appear and further up in the follicle S--S linkages were increased gradually. Outer root sheath had no S--S linkages by DACM staining up until it is keratinized along the hair canal in the upper follicle. The concentration of --SH and S--S thus seemed to be reciprocal; --SH is found in non-keratinized tissues, whereas S--S is abundant in keratinized areas. These findings are at some variance with conventional data, which suggest, for example, that --SH groups disappear suddenly above the keratogenous zone.

Immunoglobulin heavy and light chains and C3 were detected on amyloid deposits in cutaneous nodular amyloidosis (NA) by a direct immunofluorescence method. These immunoglobulin chains and C3 could be removed except lambda (lambda) light chain after treating sections with 0.05% Tween 20 solution, 0.1 M glycine buffer pH 7.4 or 0.5% Triton X solution. Lambda light chain was detected consistently after these treatments. Amyloid filaments were not altered with these treatments. Immunoelectron microscopy demonstrated persistent antigenicity of lambda light chain on amyloid filaments. It was suggested that lambda light chain is an inherent component of amyloid filaments in NA, whereas other chains and complement represent nonessential absorption on amyloid material.

Cross-banded filamentous aggregations (CBFA) with a periodic banding pattern (850–1250 A) have been found frequently in the stroma of squamous cell carcinomas and malignant melanomas of the skin. In these tumors, the aggregations were commonly associated with the basal lamina and abnormal fibrils. No CBFA was found where collagen fibrils did not show degradative changes. Less frequently, CBFAs were found in the skin lesions of primary and secondary amyloidoses, in inflammatory foci of a halo nevus, and in other skin lesions. CBFAs were similar to the Luse's bodies which have previously been found in normal and abnormal tissues of the central nervous system. Although some investigators have equated the aggregates with the fibrous-long-spacing (FLS) type of collagen, FLS collagen prepared in our laboratory showed a longer periodicity (2,200 A) than that of CBFA. Staining with ruthenium red significantly increased the electron density of CBFA, suggesting that CBFA contains acid mucopolysaccharides.

Histochemically identifiable amyloid associated with actinic keratosis was studied with the electron microscope. Typical amyloid filaments forming islands identical to collagen strands were found in the upper dermis. Beyond the lesion of actinic keratosis, no amyloid was found. In the healing wound of a previously biopsied lesion, amyloid was regenerated only when the epidermis covered the wound. In such epidermis-covered granulation tissue, electron-dense amorphous substance was initially formed in the upper dermis and amyloid filaments subsequently emerged within it. In both original and regenerated lesions, perivascular spaces were free from amyloid, and actinic elastosis was absent. It was concluded that secondary amyloidosis associated with actinic keratosis is produced by local fibroblastic cells under an abnormal influence of the epidermis.

We studied the recovery phase of immune response-associated (Ia)-positive or ATPase-positive epidermal Langerhans cells (ELCs) after ultraviolet B (UVB)-induced depletion by using mouse ear epidermal sheets. An area 3 mm in diameter was irradiated with 300 nm UVB light (40 mJ/cm2). A time sequence study was carried out to 56 days. During this period the Ia-positive ELC population increased stepwise, i.e., first a rapid increase between day 7 and day 14, which we called the early recovery phase, and next a gradual increase between day 42 and day 56, which we called the late recovery phase. During the early recovery phase, we found polymorphous ELCs in the irradiated area which were giant or normal in size, dendritic or round in shape, and single or paired in distribution. Electron microscopy revealed some of round and some of paired ATPase-positive ELCs to be in metaphase or telophase of mitotic division. Within the entire observation period of our study, there was no evidence suggesting migration of ELCs from hair follicles or from the nonirradiated epidermis. These findings indicated that mitosis of ELCs contribute to their repopulation during the early recovery phase.

The mouse monoclonal antibody AFH1 was produced using formalin-fixed, sham paraffin-embedded human melanoma cell culture line A375 as immunogen. Reactivity of this antibody was assessed by immunohistochemical techniques against formalin- or acid alcohol-fixed paraffin-embedded tissue as well as formalin- or acid alcohol-fixed unembedded lesions. Ninety-seven nevomelanocytic lesions, neurofibromas, epithelial lesions, and a plasmacellular infiltrate were evaluated. AFH 1 was immunoreactive on 54 of 55 nevocytic lesions (98.2%), 15 of 16 primary melanomas (93.7%), a lentigo maligna, and nests in 21 of 21 dysplastic nevi (100%). Of 100 consecutive basal melanocytes of intraepidermal melanoma cells counted in each lesion, mean AFH1 immunoreactivity for nonnested basal melanocytes in nevocellular nevi was 3.8%; for dysplastic nevi, 13.8%; and for intraepidermal melanoma cells, 78.0%. When nonnested basal melanocytes were subdivided into cytologically normal and abnormal cell groups, AFH1 immunoreactivity was 9.4% and 72.6%, respectively. AFH1 recognition of the lentiginous portion of dysplastic nevi corresponds statistically to the appearance of abnormal melanocyte cytology, nest formation, or both. Using 50% immunoreactive nonnested melanocytes as the criterion, AFH1 seems to distinguish primary melanoma from dysplastic nevi with a sensitivity of 93.8% and a specificity of 95.8%.

A new monoclonal antibody NKH-1 was developed using human subepidermal basement membrane zone substances as immunogen. NKH-1, IgG1 kappa light chain, labeled proteins in the subbasement membrane zone in a linear fashion. It also labeled oxytalan fibers and elaunin fibers in the papillary dermis. Mature elastic fibers were labeled only in their peripheral microfibrils (elastofibrils) and the center core of elastin was nonreactive. Basal lamina itself was not decorated with NKH-1 even at the immunoelectron microscopic level. Skin appendages such as eccrine and apocrine glands, arrector pili muscle, hair follicle, and sebaceous gland were surrounded with NKH-1 -positive substances. This substance was in linear fashion closely associated with the basal lamina but deposited linearly outside of it. Species specificity tests were performed only in nonprimates: mouse and guinea pig skins were nonreactive with NKH-1. NKH-1 seems to recognize a new substance in the sub- basal lamina region (subbasal lamina proteins) which cross-react with elastic fiber microfibrils.

The apocrine duct extends from the apocrine secretory segment to the pilosebaceous follicle. Because apocrine glands are limited to a few areas, and even in these areas are few in number, the structure of their ducts has been largely ignored in descriptions of the ultra-structure of the epidermis and its appendages. Charles (1) and Zelickson (2) have briefly described the apocrine duct in their studies which were concerned primarily with the apocrine secretory segment.

This study was undertaken in order to describe the fundamental differences between normal skin and the oral mucosa mainly from the standpoint of keratinization.A brief survey of the literature dealing with the human oral mucosa reveals that Sognnaes and Albright (1,2), Fasske and Themann (3), and Zelickson and Hartmann (4) have done electron microscopic work on the human oral epithelium, Listgarten (5) and Kurahashi and Takuma (6) on the human gingival epithelium, Melcher on the oral connective tissue (7), and Frithiof and Wersall on some organelles (8). Until now, however, no electron microscopic work has been done on the posterior buccal mucosa of the human oral cavity, although the anterior buccal mucosa has been studied by Zelickson and Hartmann (4); and there has been no comparative study of the human mucous membrane with either adult or embryonic human skin.In this study it was found that the normal human posterior buccal mucosa is very similar to the immature embryonic epidermis in all aspects; and that despite a complete lack of keratinization, the plasma membranes of the posterior buccal epithelial cells in upper layers become considerably thicker than those of the lower layers, thus suggesting that this thickening process is a phenomenon independent of keratinization.MATERIALS AND METHODSBiopsy specimens were taken from the normal human buccal mucosa, posterior or lateral to thesecond molar tooth, of ten healthy white men and women between the ages of 17 and 50. Five specimens were obtained from the oral lesions of pemphigus vulgaris, and one from the lesion of stomatitis of the posterior buccal mucosa. All specimens were immediately sliced into small pieces, fixed in 1% osmic acid in veronal buffer (pH 7.2) containing 4.5% sucrose. After fixation for one hour, the tissues were dehydrated through a series of graded alcohols and propylene oxide, and were embedded in Araldite. Thin sections were then cut with an LKB Ultrotome at 400-600 A. The sections were stained first with 1% uranyl acetate in 50% etha-nol, and then, before completely dried, re-stained with lead citrate (9). Some specimens were stained for acid phosphatase before osmic acid fixation using the method of Barka and Anderson (10).RESULTSBasement membrane and basal layer. The basement membrane measured approximately 600 A in thickness. Occasionally, phagocytes, lymphocytes, and leukocytes were seen crossing the basement membrane (Fig. 1). In such areas the subepithelial reticular fibrils were thick, and re-duplication of the basement membrane, as reported by Caulfield and Wilgram in erythema multiforme (11), was observed (Fig. 1). The basal cells here contained fewer tonofilaments than the epidermal basal cells (Fig. 1). Intercellular spaces were rather wide, with numerous slender villi of the basal cells projecting into them (Fig. 1). Some basal melanocytes contained well-developed, rough-surfaced endoplasmic reticulum and were actively producing all gradients of maturation of melanin granules in their melanosomes (Fig. 2). The dendritic processes of these melanocytes, found between the basal cells and squamous cells, contained a greater number of more mature (larger and denser) melanin granules than did the body of the melanocytes. This phenomenon can be explained by assuming that the melanocytes inject their melanin granules into the surrounding cells as soon as the granules reach maturity. The basal cells and the squamous cells contained varying numbers of injected melanin granules (Fig. 2).As the basal cells moved up into the squamous512ULTRASTRUCTURES OF THE BUCCAL MUCOSA513*-i

With the advent of the electron microscope, investigators of elastic fiber have used two major approaches: first, the method of teasing out tissue fragments with or without additional shadow casting technics (replication method) (1-7), and second, that of using tissue sections (8-14). Several workers in each group have shown interest in the elastic tissue of the skin, either normal (2-4, 6, 10), or abnormal (9, 11-13).

Calcifying epithelioma is a rare, solitary tumor which often occurs in young individuals (1), most frequently on the face and the arms (1, 2). Since Malherbe and Chenantais (3) described “calcified epithelioma of sebaceous glands” in 1880, the histogenesis of this tumor has been attributed to various skin appendages such as sebaceous glands (3, 4) and hair (2, 5, 6, 7). Lever and Griesemer (7) regarded the basophilic cells in calcifying epithelioma as primary epithelial germ cells with a tendency to differentiate into keratotic hair cells. Forbis and Helwig (1), in their recent study on 240 specimens, agreed with the opinion of Lever (2) and Lever and Griesemer (7) that calcifying epithelioma arises from primitive cells of the hair matrix. They pointed out that the broad dense transitional zone from basophilic to shadow cells of this tumor, where sulfhydryl groups are abundant, is analogous to the kera-togenous zone of the hair. They also demonstrated that hyalinized shadow cells in some instances show a brilliant birefringence, as does the keratogenous zone of the hair.

As an extension of our histochemical studies of the skin (1, 2), the distribution and activity of cholinesterases were studied in the skin of the rat. The development of cholmesterase activity was followed during the histogenesis of the skin, with special reference to the nerve elements.

Although there have been several electron microscopic studies of human axillary apocrine glands (1, 2, 3, 4) the exact mechanism of apocrine secretion remains an enigma. Hibbs (1) denied true apocrine secretion in normal human axillary apocrine glands because he was unable to find the secretory cells in the process of extruding its contents. In addition, he could not visualize any secretion product within the lumen. He assumed, therefore, that the principal mode of secretion is by droplet formation at the apex of the cell and that secretion by disruption of the cell probably occurs only under extreme conditions, if at all. Montagna (2) suggested that the secretory granules are dissolved in the apical cytoplasm and excreted either by a fragmentation of the luminal microvilli or by oozing out through the plasma membrane. Charles (3) also failed to see cellular debris in the lumen, but did report disruption of the apical membrane through which he thought the contents of the secretory cell had been extruded. Observations similar to Mon-tagna's, e.g., that the secretory granules are dissolved in the apical cytoplasm, were made by Kurosumi et al. (4) ; they further described the formation of expanded apical secretory projections and were inclined to defend the classical decapitation secretion theory. They denied that secretion occurred through fragmentation of the luminal microvilli. The following report will be concerned primarily with the maturation of the apocrine secretory granules and their mechanism of excretion from the cell. A multiplicity of secretory mechanisms of the apocrine glands was found in this investigation, including decapitation secretion.

A glomus tumor of the multiple type was examined by electron microscopy. Except for a single layer of endothelial cells lining the vascular lumen, the remainder of the tumor cells are smooth muscle cells. What have been called “glomus” or “epithelioid” cells in the past are transversely sectioned smooth muscle cells. There is no ultrastructural similarity to the hemangiopericytoma.

Electron microscopic studies were performed on the hair cone and intraepidermal hair canal formation in the scalp and eyebrow skin using 11 Negro embryos of the menstrual ages of 9 to 18 weeks. While most of the previous observations of light microscopists were confirmed, information on several fine structural features was added. (1) The hair cone formation represented the very initial keratinization of the cells of future Henle and Huxley layers. These cells were connected by desmosomes, tight junctions, and intercellular cement. (2) The intraepidermal portion of the hair canal was formed by keratinization and vacuolization of the organized hair canal matrix cells. Lysosome-like dense bodies were present and thought to play a role in self-destruction of the matrix cells. (3) The cellular envelopes of the hair cone cells and of the wall cells of the hair canal became twice as thick as the plasma membranes of their matrix cells. An 80–100 A thick cytoplasmic band, i.e., the newly synthesized marginal band, was found to be responsible for this thickening.

Merkel cells identical to those described in the skin were found in the gingival mucosa and in the vermilion border of the lip. They were often clustered in rete ridges but also found individually in the basal layer. Unmyelinated axons (neurites) of presumably a sensory afferent nerve surrounded the basal half of these cells within the epithelium. Occasional desmosomes connected the Merkel cell to the neighboring keratinocytes, but neither desmosomes nor other junctional devices were found between the Merkel cell and the neurite. The Merkel cell is characterized by membrane-surrounded dense granules, measuring between 800 and 2000 A, which seemed to derive from Golgi saccules. Some Merkel cells contained lysosome-like dense bodies and transferred melanosomes.

Animal Material. Twenty-five rats of the Wistar strain were used. Twelve of these were embryos (3 each on the 12th, 14th, 16th and 18th days of gestation), 2 were new-born (the period of gestation varied from 19 to 21 days), 3 were ten-day-old, 3 were fifteen-day-old and 5 were adults. The adult rats weighed approximately 100 g. New-born and older rats were killed by a blow on the neck without anesthesia. Hair was shaved off the back, and skin specimens were taken from there and cut into small square pieces. These pieces were immediately immersed for about five minutes into ice-cold 0.1 M Sorensen's phosphate buffer and then used for frozen sections. (The buffer had a pH of 7.6 for the study of the succinic dehydrogenase system and a pH of 7.4 for the study of the malic and lactic dehydrogenase systems). In embryos, skin was removed from the back while they were still alive. Frozen sections, approximately 20 in thickness, were made from the fresh skin specimens. Histochem feat Demonstration of the Succinic Dehydrogenase System (SDS). Ogawa and Zimmerman's method (7) was employed to demonstrate the succinic dehydrogenase system. As a rule, nitroneotetrazolium chloride (nitro-NT) was used as an electron acceptor. Occasionally, however, nitro-blue tetrazolium (nitro-BT) was used, instead.

Perifollicular nerve endings of the human lanugo hairs have been studied with the electron microscope. The endorgans are arranged in palisade along the periphery of the follicle and are composed of an axon and an enveloping Schwann sheath. Spaces between the endorgan are largely free from collagen and cellular elements. This arrangement of endorgans perpendicular to the follicle may be most efficient to detect the movement of the follicle, and the empty spaces between the organs permit a maximum deformation of the terminal axon. The axon produces a subcytomembrane dense layer at the follicular end but is, with rare exceptions, separated from the external root sheath cells by the basal lamina. The endorgan can attach to any portion of the follicle including the cellular junctions, indicating that there is no specialization on the follicular side at the site of attachment. In fact, half-desmosomes and subbasal dense plaques are regularly seen on the basal surface of the follicular basal cells.

The formation of the intradermal eccrine sweat duct lumen began in the 15-week-old embryo with a dissolution of the desmosomal attachment plaques, followed by the separation of apposing inner cells and subsequent formation of microvilli at the luminal surfaces. The luminal cells possessed numerous microvilli and crypts and a periluminal band of tonofilaments. In the secretory portion of older embryos (22 weeks old), columnar secretory cells extended from the basement membrane to the luminal border. The presence in these secretory cells of microvilli, Golgi elements, mitochondria, small secretory vesicles, dense secretory granules and abundant endoplasmic reticulum, implies that preparation for secretory functioning begins in embryonic life. The pyramidal myoepithelial cells differentiated from the basal cells seen in younger embryo, and rested upon a basement membrane. In the oldest embryo examined (22 weeks) there was as yet neither intercellular canaliculi nor sufficient difference in the amount of glycogen to enable one to distinguish between dark and light secretory cells. Isolated cilia of 9 + 0 type were occasionally found in the luminal cells of the duct and the secretory segment.

In 1952 Lutz (1) described a peculiar serpiginous keratotic eruption that was present on the sides of the neck of a 21-year-old man for 8 years. He named the lesion keratosis follicularis serpiginosa. Histologic studies of the keratotic wall showed that it was composed of a cystic structure containing horny lamellae situated just beneath the epidermis and that it appeared to have arisen from the hair follicles. Under the same title, Beening and Ruiter (2) in 1954 described the eruption in a 13-year-old girl who had developed ring-formed and semi-circular lesions on the side and nape of her neck. When first seen 3 years before, these lesions consisted of a number of closely-set hailstone-sized, hard, reddish papules. The summit of each papule revealed a keratotic plug which when removed revealed a deep depression. The center of the rings appeared depigmented but not atrophic. Three years later most of the lesions had undergone involution. Histologic study of specimens removed by biopsy showed a markedly acanthotic epidermis which contained horny lamellae. Serial sections demonstrated that the horny masses penetrated into the cutis in some places. Where they remained in contact with connective tissue, inflammatory infiltrate containing small round cells, histiocytes and occasional giant cells were found. The hyperkeratosis appeared to have a direct relationship to the hair follicle and the cyst formations were apparently connected with the sweat glands. Neither of the aforementioned authors elucidated the true nature of the horny lamellae found in the cysts.

A study was undertaken to clarify the origin of sweat gland myoepithelial cells using monoclonal antibodies EKH1, EKH4, and AN3. EKH1 recognizes all classes of intermediate filaments. EKH4 and AN3 recognize keratin type intermediate filaments. Since within the skin, only epithelial cells of ectodermal origin contain keratin, EKH4 and AN3 could be used as ectodermal markers within the skin. Sweat gland myoepithelial cells were labeled by all three antibodies. In contrast, arrector pili muscle and vascular smooth muscle were recognized only by EKH1, but not by EKH4 and AN3. This study demonstrated that myoepithelial cells of sweat glands contain keratin type intermediate filaments and suggested their ectodermal origin. On the other hand, arrector pili muscle and vascular smooth muscle did not contain keratin type intermediate filaments, despite their ultrastructural similarity to myoepithelial cells. Electron microscopic studies using human fetal and adult skin revealed that myoepithelial cells are developed from basal cells of the coiled tip of fetal gland and not from mesenchymal cells. In order to determine the time of appearance of myoepithelial cells during fetal development, embryonic and newborn mouse skin was also examined. It was found that sweat gland myoepithelial cells first appear around 20 weeks of gestation in humans and after birth in mice.

Phagocytic cells are present at all levels of the outer root sheath of mouse vibrissa (sinus hair) follicles. Occasionally these cells are seen in the process of engulfing other cells; more often phagocytic cells are seen which contain one or more vacuoles whose contents are in various stages of digestion. Most of the phagocytosed cells contain many tonofilaments and a reduced amount of cytoplasm. A few of the vacuoles contain no filaments. As one vibrissa after another grows from the follicle without an intervening rest period, the outer root sheath changes in size and shape to accommodate both the club hair and actively growing vibrissa. Phagocytosis appears to play a role in the continuous reshaping of the outer root sheath.

The biochemical characteristics of cyclic 3',5'-nucleotide phosphodiesterase were studied in homogenates of male albino rat skin using preparations which were predominantly epidermal. Enzymatic activity was detected in both the particulate and soluble fractions of these skin homogenates. Two kinetically distinct phosphodiesterase (PDE) activities were detected in the soluble fraction (100,000 times g supernatant). This 100,000 times g supernatant contains at least two distinct protein bands that hydrolyze cyclic AMP as demonstrated by gel electrophoresis. Divalent cations (Mg-++ or Mn-++) and 2-mercaptoethanol were required for maximal enzymatic activity. Epinephrine, dibutyryl cyclic AMP, and methylxanthines inhibited while imidazole and histamine phosphate stimulated the cyclic AMP phosphodiesterase activity at high and low cyclic AMP concentrations. Cyclic GMP competitively inhibited hydrolysis of low, but not high, concentrations of cyclic AMP. Hydrocortisone phosphate in pharmacologic concentrations blocked PDE denaturation by heat. These studies indicate that there are complex interrelationships between cyclic nucleotides and PDE in rat skin.

We examined whether immunoglobulin (Ig) and complement (C) components of amyloid and colloid bodies were inherent parts of these substances or were present due to nonspecific absorption only. In direct immunofluorescence (IF) studies without pretreatment, skin-limited and systemic amyloid and colloid bodies in all cases showed positive staining for Ig or C. When these sections were pretreated with 0.1 M glycine buffer (pH 7.2) or 0.05% Tween-20 solution, Ig and C in skin-limited amyloid deposits were negative or weakly positive. In contrast, positive fluorescence of colloid bodies and systemic amyloid masses was not influenced by pretreatment. Existence of amyloid masses before and after pretreatment were confirmed by Thioflavin-T and Dylon stains. In addition, pretreatment did not alter disulfide bonds by DACM staining or the reactivities of amyloid with monoclonal antikeratin antibody EKH4. These results suggest that skin-limited amyloid can be differentiated from systemic amyloid or colloid bodies by these methods. We can infer from the present studies that most of the Ig and C in skin-limited amyloid masses are a result of nonspecific absorption due to penetration of serum, which is different from Ig in systemic amyloid and colloid bodies in as much as in these conditions immunobinding is specific.

Previous studies of loss of heterozygosity (LOH) have revealed distinct patterns of allelic loss in some skin tumors. In basal cell carcinomas (BCCs) loss of heterozygosity is virtually restricted to chromosome 9, whereas in squamous cell carcinomas (SCCs) and actinic keratoses loss is more widespread involving chromosomes 3, 9, 13, and 17. Because there are histological similarities between BCCs and some appendageal tumors, and because some lines of evidence suggest that BCCs are appendageal in origin, we carried out a limited allelotype in 41 appendageal tumors. The overall frequency of allelic loss was low (4 out of 247 informative loci; 1.6%). LOH was seen in a proliferating trichilemmal cyst (l7p), a sebaceous epithelioma (l7q), an eccrine porocarcinoma (17q), a trichoepithelioma (9q), and in two basal cell carcinomas showing eccrine or granular cell differentiation that were originally misdiagnosed (9q). The pattern of loss in this mixed group of appendageal tumors shows differences from both BCCs and SCCs, and further emphasizes the unique genetic profile and behavior of BCCs. The finding of 9q loss in BCCs with eccrine or granular cell differentiation shows that 9q loss occurs in different histological subtypes of BCCs.

We examined keratins 19 and 8 in extracted human hair follicles using monoclonal antibodies Ks l9.1 and CAM5.2, respectively. Ksl9.1 reactivity was found in the bulge and infundibulum. Ksl9.1(+) cells were dense in the bulge of vellus and intermediate hair follicles. The intact bulge of terminal hair could not be extracted, but the presence of Ksl9.1 (+) cells was confirmed in transverse sections. Infundibular Ksl9.1(+) cells exhibited a dense network pattern of staining in terminal hair follicle, but only a few cells were labeled in vellus and intermediate hair follicles. CAM5.2(+) cells, i.e., Merkel cells, were found in the same locations as Ks 19.1 (+) cells but were less dense. These patterns of distribution and staining density were not influenced by different phases of hair cycle. Sequential staining of Ksl9.1 and CAM5.2 in the same hair follicle demonstrated that the same cells could be reactive for both. However, considering the large number of Ks19.1(+) cells and rather small number of CAM5.2 in the same locations, it was assumed that only a subset of Ks19.1(+) cells are Merkel cells. It was postulated that the bulge area of human adult hair follicles houses embryonic pluripotential cells characterized by stem cells and post-stem cells and that the Merkel cells in the bulge area arise from these immature cells and may play a role in the maintenance and stimulation of this group of immature cells.

Normal human skin was cultured with blister fluid (BF) from 2 patients with epidermolysis bullosa letalis (EB letalis). After 12–24 h incubation, initial separation was observed at the dermal-epidermal junction. Complete separation was seen after 48–72 h incubation. Epidermal and dermal structures themselves were well preserved up to 72 h. Electron microscopy revealed that the separation took place through the lamina lucida. These findings corresponded to in vivo histologic pictures of the skin lesions. The activity in BF was lost by lowering (pH 4) or elevating (pH 9) pH of the media or by prolonged autoclaving (5 h). Boiling and dialization did not affect the activity of BF. Certain protease inhibitors, such as α 2 -macroglobulin, aprotinin (trasylol), soybean trypsin inhibitor, phenylmethylsulfonylfluoride, or 50% fetal bovine serum, inhibited or depressed the separation, while triamcinolone acetonide, n-ethylmaleimide, or EDTA did not. Patient's serum (case 2) and normal human sera partially inhibited cleft formation. It was proposed that in EB letalis some factors in BF, probably protease(s), play an important role in the induction and/or spread of blister.

In a previous communication (1), it was pointed out that the keratotic papular lesion, elastosis perforans serpiginosa, that has masqueraded under a variety of names, appears to be a lesion involving primarily the elastic tissue. Clinically, the eruption is found largely among young male adults, is asymptomatic, and has a self-limited course. It does not appear to have an hereditary basis. Isolated case reports with special staining procedures of biopsy material indicating the nature of the lesion have been supplemented by elastase digestion experiments in several laboratories (1-3).

The dermal-epidermal (DE) junction areas of skin specimens obtained from 16 patients with either lichen amyloidosis or macular amyloidosis were studied. In the dermal papillae where amyloid was deposited, elastic fibers frequently were absent, but periodic acid-Schiff reaction after diastase digestion was homogenously positive. Ultrastructural studies revealed that a basal lamina-like substance with anchoring fibrils was present between and within amyloid deposits. By indirect immunofluorescence technique using an anti-basement membrane zone antiserum obtained from a patient with bullous pemphigoid, specific linear fluorescence occurred at the DE junction, and in a reticular pattern in dermal papillae. It seemed that apoptotic keratinocytes of the epidermis brought down basal lamina and fine fibrous components attached to it when these cells dropped down to the papillary dermis and became the source of amyloid. These findings support the hypothesis that epidermal keratinocyte degeneration plays an important role in the histogenesis of cutaneous amyloidoses.

Light and electron microscopic observations were made on seventeen palmar or plantar pits of the nevoid basal cell epithelioma syndrome. In two specimens, typical basal cell epitheliomas were seen arising from the epithelium at the base of the pits; one of them was a pigmented basal cell epithelioma. The epithelium under the pit was stained only weakly with azure B; this was attributed to a very poor development of tonofibrils. Keratohyaline granules were small and the discharge of the cementsomes was incomplete. Premature desquamation of the horny cells took place along the intercellular spaces and was not due to degeneration of horny cells. It was thought that incomplete discharge of cementsomes, perhaps due to shortened transit time of keratinocytes, failed to provide enough intercellular cement. Surrounding the rete ridges and particularly surrounding the fully grown tumor, collagen stroma showed various degrees of dissolution.

A granular structure was found in the keratogenous zone of the cortex in human hair follicles. This granule was a round, relatively electron-dense, homogeneous structure 0.1-0.3 micron in diameter and not membrane-limited. It appeared in cortical cells in the keratogenous zone, fused to the tonofilaments, and disappeared before the complete keratinization of the cortex. Thus, this granule was considered to be a trichohyaline granule. Similar granules were found in the nuclei of cortical cells in the keratogenous zone. A comprehensive survey of various human hairs confirmed this observation. Trichohyaline granules were also present in the cortical cells of guinea pig hair in the keratogenous zone. However, a similar survey on mouse hair cortex did not reveal trichohyaline granules in this species.

Two cases of lichen amyloidosus and 8 cases of macular amyloidosis were examined by electron microscopy. Epidermal keratinocytes showed variable degrees of focal degeneration in the basal or lower Malpighian layer. The primary change was seen in cells which contain fibrillar (30nm in thickness) cytoplasmic inclusion. The following developments seemed to lead to filamentous degeneration (colloid bodies): (1) aggregation of tonofilaments within the granular or fibrillar cytoplasm, (2) filamentous cells, which are composed of bundles of 7-nm thick filaments surrounded by cell membrane and desmosomes, and (3) filamentous masses composed of bundles or whorls of tightly packed 7-nm thick filaments in the intercellular spaces. At the dermo-epidermal junction, some of the filamentous masses were surrounded by the basal lamina of the epidermis and others were dropping into the dermis. Occasionally, loosened filaments (similar to amyloid filaments) were about to drop into the dermis. Early formation of amyloid islands consisted of electron-dense and electron-light parts. They were located directly beneath the epidermis. In the deeper portion of the papillary dermis and in the upper reticular dermis, the majority were electron-light masses. Electron dense parts were the densely packed 7-nm thick filaments, whereas electron-light parts were the typical straight amyloid filaments. Small tubular filaments were seen in common in the filamentous cells, filamentous masses , and amyloid islands . It is concluded that some of the amyloid substance in primary localized forms of cutaneous amyloidoses derive from the epidermal cells through filamentous degeneration.

As a continuation of our histochemical studies on the skin (1), the changes in the succinic, malic and lactic dehydrogenase systems during the embryonic development of the skin have been investigated. Practically no work has been done as yet on the activity of any of these dehydrogenase systems during the embryonic development of the skin; and only the succinic dehydrogenase activity has been investigated in adult skin by several authors (2-6).