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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.

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.

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.

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%.

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).

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.

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.

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.

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.

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.

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.

Electron microscopic examination of a verruca vulgaris has revealed the intranuclear inclusion of tonofibrils. Although many intracellular inclusions may be related directly to the virus, nonviral inclusions may also be present.