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The effects of Brazilian green propolis ethanol extract on Cry j1-induced cys-leukotrienes and histamine release from peripheral leukocytes of patients with allergic rhinitis were investigated. One of the key mechanisms for the anti-allergic properties of the extract was revealed to be the suppression of cys-LTs release. Furthermore, a series of propolis components and their phenethyl esters were synthesized and evaluated as inhibitors of cys-LTs release. Artepillin C, baccharin, and kaempferide were the major active components of the ethanol extract. The inhibitory activity of artepillin C phenethyl ester was comparable to that of existing LT synthesis inhibitors.

Lignins are major cell wall components formed by the dehydrogenative polymerization of three monolignols, p-coumaryl, coniferyl and sinapyl alcohols. We prepared lignin–carbohydrate complexes (Fr. VI and Fr. VII) from pine cones by acid and ethanol precipitation, and investigated which part of these molecules is essential for expression of biological activities. They showed potent antiviral activity upon direct interaction with the virus. The antiviral activity of Frs. VI and VII required the higher-order structure of polyphenols without polysaccharides. Pretreatment of mice with Fr. VI or VII induced higher antiparasite activity than those of natural and chemically modified antitumor polysaccharides. Fr. VI or VII at higher concentrations enhanced the radical intensity and cytotoxic activity of vitamin C, whereas tannins counteracted the effect of vitamin C. Fr. VI at lower concentrations enhanced the O 2 - -scavenging activity of vitamin C. Frs. VI and VII stimulated mouse macrophage-like cells Raw 264.7 to produce nitric oxide (NO), citrulline (CIT) and asparagine (ASN), via the enhanced expression of iNOS and ASN synthetase, whereas phenylpropenoid monomers and polymers inhibited NO/CIT/ASN production. These data suggest that the polymerized structure of phenylpropenoids in lignin–carbohydrate complexes is required for the induction of antiviral activity, and that the higher-order structure of phenylpropenoid polymers and polysaccharides is required for immunopotentiation, including macrophage activation.

Terminal hair follicles of the human scalp of all ages showed apoptotic pocket-like structures in the outer root sheath of the bulge area at anagen, but not telogen phase. The occurence of these hole structures was roughly estimated in 15% of anagen terminal hair follicles of the human scalp. The size of these apoptotic pockets was variable, ranging from pin hole-like spaces to larger structures filled with homogeneous black materials. These unusual variations were often co-localized with apoptotic degenerations and exclusively present in the presumptive bulge of anagen terminal hair follicles where arrector pili muscles were seen in the vicinity. In fact, these vacuolated structures tended to be present on the side where the major part of the arrector pili muscles anchored.

Etiology of connective tissue disease is unknown. The association of infectious agents has been suspected serologically, ultrastructurally and recently by means of molecular biological techniques. We extracted RNA from lesions of 25 discoid lupus erythematosus, 9 systemic LE, and 3 systemic sclerosis biopsies, and reverse transcription-polymerase chain reaction was performed for all paramyxoviruses known to infect human beings. None of the samples yielded positive signal for any of the viruses. The existence of paramyxovirus in the skin of connective tissue disease is very unlikely.

Extracted human terminal and vellus hair follicles were prepared by EDTA and examined by light and electron microscopes. These samples were morphologically well preserved and enabled us to observe 3-dimensional (3-D) views of the outer surfaces of hair follicles. Interestingly, the bulge areas of terminal and vellus hair follicles showed several morphological variations, such as knob-like swellings and villous projections, independent of hair cycle. Moreover, skirt-like structures were furnished in the small vellus hair follicles independent of hair cycle, but not terminal and large vellus hair follicles. These morphological variations of human hair follicles were confirmed to genuinely represent 3-D views by means of the conventional transverse and vertical sections of the human skin.

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.

Mouse monoclonal antibodies (MoAbs), IKH-1 and IKH-2, were produced against cloned human melanoma cells, KHm-6, which were cultured with 12-O-tetradecanoylphorbol-13-acetate (TPA) and processed by formalin fixation and alcohol dehydration (FFAD). According to the biochemical analysis, antigenic substances which reacted with IKH-1 were 34.0-60.0 kDa glycoproteins, and those which reacted with IKH-2 were 33.5, 34.5 and 36.0 kDa glycoproteins. Immunoelectron microscopy revealed that reaction products of IKH-1 were seen in some membranous vesicles, premelanosomes and cell membrane of TPA-treated KHm-6 cells, while IKH-2 recognized only premelanosomal structures. Immunohistochemical tests revealed that IKH-1 and IKH-2 have a high sensitivity (94.0% and 85.0%, respectively) to formalin-fixed, paraffin-embedded (FFPE) tissue sections of malignant melanomas. IKH-1 had a high specificity and IKH-2 and 100% specificity to FFPE tissue sections of melanocytic lesions.

Using Merkel cell specific antikeratin antibodies and neurofilament antibody the nerve-Merkel cell relationship was studied with a double staining method on frozen sections. Merkel cells were stained with monoclonal anti-cytokeratin CK-5 and CAM 5.2 which react against human cytokeratin polypeptide 45 kDa and 52.5 kDa, respectively. Peripheral nerves were stained with monoclonal murine antibody Neurofilament 70 and 200 kDa. Epidermal Merkel cells were recognized in 12-week fetuses in the plantar skin. In 15-week fetuses dermal Merkel cells were found. Most of the dermal Merkel cells initially lacked a close association with immunoreactive nerve fibers. In 16-week fetuses immunoreactive small nerves reached the epidermis and a few dermal Merkel cells became heavily entangled with the meshwork of nerve endings. Nerve-Merkel cell complex in the dermis was confirmed by electron microscopy. The appearance of epidermal Merkel cells preceded the attachment of immunoreactive nerve endings to the epidermis. It an analysis of 448 dermal Merkel cells the nerve-Merkel cell complex became more frequent as the age of fetus advanced. It was concluded that Merkel cells do not arrive at the epidermis with peripheral nerves. Rather, the peripheral nerves are attracted to the dermal Merkel cells which originated in the epidermis.

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.

Crystalloid inclusion bodies (CIB) of the endothelial cells (EC) were investigated in blood vessels of human fetal skin and the umbilical cord by electron- and immunoelectron microscopy. They were found in up to 15% of the investigated EC in various types of vessels. Their sizes ranged from 0.2 μm to 0.6 μm in the largest diameter. Most frequently we observed a laminated pattern of the crystalloid structure with a regular periodicity of dark and light bands. Additionally a honeycomblike pattern was also seen. Measurement of the CIB laminated structure revealed similar dimensions to Weibel-Palade bodies (WPB). In EC of all vessel types we found numerous WPB differing in electron density, shape and size from those of WPB found in adult blood vessels. WPB were found much less frequently in EC with CIB, suggesting that CIB is a precursor of WPB. After incubation with monoclonal antibody against von Willebrand factor (vWf) both WPB and large organelles were labeled. Because of their shape and size the labeled large organelles seemed to represent inadequately preserved CIB. After incubation with anti-lysozyme only the large organelles were labeled. A possible relationship of CIB to WPB is thus suggested. The presence of lysosomal enzymes such as lysozyme suggest that CIB are lysosomal organelle and participate in the uptake of vWf. The crystalloid pattern of CIB may represent an accumulation of a highly condensed form of vWf.

Dermal reactions to primary intradermal or appendageal sensitization are compared to predominantly dermal reactions to standard patch tests and to intradermal antigen tests. In contrast to epicutaneous spongiotic contact dermatitis, HLA-DR was only seen on skin appendages and nearby basal keratinocytes in indurated tissue reactions with the exception of the reactions with focal basal cell layer disruption and an indurated patch test performed one week post angry back syndrome. Other intradermal skin tests showed only minimal epidermal HLA-DR expression despite spongiotic epidermal changes. Predominantly dermal hypersensitivity reactions can be induced by intradermal or epicutaneous routes. They can evoke hypersensitivity responses which do not cause most epidermal keratinocytes to express HLA-DR.

Desmoplakin I and II are important components of the attachment plaque of the desmosome which mediates cell to cell adhesion, in epithelial cells. In this study we used well-characterized antibody against desmoplakin I and II immunohistochemically and immunoelectron microscopically on two cases of pemphigus vulgaris and one case of pemphigus erythematosus and two cases each of Hailey-Hailey's disease and Darier's disease. In the normal human epidermis the desmosomes were demonstrated in a dotted pattern along cell periphery. In pemphigus vulgaris and pemphigus erythematosus acantholytic cells and the perilesional cells exhibited normal dotted pattern along the cell periphery. In Hailey-Hailey's disease and Darier's disease, the dotted pattern is lost in acantholysed and perilesional areas and anti-desmoplakin I + II positive proteins were observed diffusely in the cytoplasm. Immunoelectron microscopical findings correspond to these light microscopical observations. It is concluded that in autoimmune acantholytic disease such as pemphigus vulgaris and pemphigus erythematosus, desmoplakins are intact even in acantholytic cells, whereas in genodermatoses such as vulgaris and pemphigus erythematosus, desmoplakins are intact even in acantholytic cells, whereas in genodermatoses such as Hailey-Hailey's disease and Darier's disease primary or secondary abnormalities abnormalities of desmosomes may be involved in their pathogenesis.

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 present study was performed to investigate the ultrastructural localization of the binding sites of the lectin Ulex europaeus agglutinin, Type I (UEA) and the antibody against factor VIII related antigen (F VIII RAG), both widely used as vascular markers in human skin capillaries at light microscopy level. In addition the ultrastructural localization of vimentin, the major protein of the endothelial intermediate filaments, was demonstrated in human vascular endothelial cells (EC) in situ. The low temperature postembedding technique with Lowicryl K4M with subsequent application of gold labelled antibodies provided both well preserved antigenicity and morphology. The antigenic site of UEA, alpha- l -fucose containing glycohydrate residues, was primarily found in or near the luminal plasma membrane of EC. Gold particles representing the sites of F VIII RAG were located in the Weibel-Palade bodies (WPB) of EC. The ultrastructural staining pattern of the anti-vimentin antibody showed an exclusive labelling of the intermediate filaments of EC.

A case of papillary eccrine adenoma was analysed with immunohistochemical and ultrastructural methods regarding their direction of differentiation. It was found that the majority of the structures show either eccrine ductal or glandular differentiation. There were some segments, particularly in those exhibiting papillary growth, where cells similar to eccrine secretory (clear) cells or cells with characteristics of both ductal basal cells and glandular myoepithelial cells were present.

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 thermodynamic parameters of the CO-equilibria of isolated chains of hemoglobin A and of two α-chains in hemoglobins M Milwaukee-I and Saskatoon at 25°, pH 7.0 were determined. The parameters for the binding of the first CO molecule to the hemoglobins M were ΔH′=−17 and −18 kcal/mole heme and ΔS′=−30 and −29 e.u. for hemoglobins M Milwaukee-I and Saskatoon, respectively. In contrast to this the characteristics of the second step of the binding were ΔH′=+5.9 · and +4.3 kcal/mole and ΔS′=+51 and +49 e.u. These values for the second step were also significantly different from those of the isolated α-chain (ΔH′=−15 kcal/mole and ΔS′=−11 e.u.).

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.