Immunohistochemical demonstration of keratin 19 expression in isolated human hair follicles.

We examined keratins 19 and 8 in extracted human hair follicles using monoclonal antibodies Ks19.1 and CAM5.2, respectively. Ks19.1 reactivity was found in the bulge and infundibulum. Ks19.1(+) 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 Ks19.1(+) cells was confirmed in transverse sections. Infundibular Ks19.1(+) 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 Ks19.1(+) cells but were less dense. These patterns of distribution and staining density were not influenced by different phases of hair cycle. Sequential staining of Ks19.1 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.

Merkel cells of the terminal hair follicle of the adult human scalp.

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.

Use of chromogenic substrate S-2251 for determination of plasminogen activator in rat ovaries.

A simple specific and reproducible method for determination of plasminogen activator activity in rat ovaries has been developed by using the chromogenic substrate S-2251. The two steps of enzymatic reactions, i.e. activation of plasminogen and subsequent hydrolysis of the substrate was performed in one step incubation. A linear relationship was observed between the amount of chromogen produced and activator activity in the range of the optical density form 0.05 to 1.20 for 30 min's incubation. Endogenous activity of non-specific proteases, plasmin or plasmin inhibitors which might be contained in rat ovaries turned out not to interfere with the specificity of a standardized assay procedure. Reproducibility was firmly established with coefficient of variation not exceeding 10%. Using this method, a marked increase followed by a drastic decrease in the activator activity was shown with rat ovaries around the time of ovulation after the injection of human chorionic gonadotropin.

Co-operation of progesterone and prostaglandins in ovulation induced by human chorionic gonadotrophin in immature rats primed with pregnant mare serum gonadotrophin.

Serial injections of a mixture of prostaglandin (PG) E2 and F2 alpha 0, 2, 4, and 6 h after simultaneous injection of human chorionic gonadotrophin (hCG) and indomethacin incompletely restored the ovulation that would have been blocked by indomethacin in immature rats treated with pregnant mare serum gonadotrophin followed by hCG. Serial injections of another mixture of PGE2 and PGF2 alpha 6, 8, 10 and 12 h after simultaneous injection of hCG and indomethacin similarly reversed, in part, the inhibitory effects of indomethacin on hCG-induced ovulation. In contrast, serial injections of the mixtures of PGE2 and PGF2 alpha 0, 2, 4, 6, 8, 10 and 12 h after simultaneous injection of hCG and indomethacin completely restored the indomethacin-blocked ovulation, suggesting that the prostaglandins mediate the action of hCG on ovulation both in the earlier and later stages of the preovulatory process. Six hours after simultaneous injection of hCG and indomethacin serial injections of a mixture of PGE2 and PGF2 alpha reproduced the acute and temporary increase in concentrations of progesterone and testosterone in plasma which would have been abolished by indomethacin. Progesterone given concurrently with hCG and indomethacin partially antagonized the inhibitory action of indomethacin on ovulation. Serial injections of a mixture of PGE2 and PGF2 alpha 6, 8, 10 and 12 h after concurrent administration of progesterone with hCG and indomethacin completely restored the indomethacin-blocked ovulation, suggesting that progesterone can substitute the action of prostaglandins injected serially in the first half of the preovulatory process. It was concluded that the co-operation of progesterone in the earlier stage and of prostaglandins in the later stage of the preovulatory interval is required to mediate the action of hCG on ovulation.

A progesterone-dependent step in ovulation induced by human chorionic gonadotrophin in immature rats primed with pregnant mare serum gonadotrophin.

In immature rats primed with pregnant mare serum gonadotrophin, antiserum to progesterone could prevent or reduce ovulation in response to injected human chorionic gonadotrophin (HCG). To be effective, antiserum treatment had to be within 6 h of gonadotrophin treatment; antiserum given 9 h after HCG was ineffective. Progesterone restored the antiserum blocked ovulation completely or incompletely when administered intravenously within 6 h of treatment with HCG. The first 6 h was shown to be a progesterone-dependent step in the ovulatory process in this experimental system.

Inhibition by indomethacin of ovulation induced by human chorionic gonadotrophin in immature rats primed with pregnant mare serum gonadotrophin.

Treatment of immature rats with pregnant mare serum gonadotrophin followed by human chorionic gonadotrophin (HCG) caused an acute and temporary increase in concentrations of progesterone, testosterone and oestradiol in plasma with maximum levels 3 h after the administration of HCG. Concurrent injection of indomethacin and HCG reduced, in a dose-dependent manner, the mean number of ova shed and this was accompanied by a dose-dependent decrease in concentrations of plasma progesterone and testosterone but not of oestradiol when they were measured 3 h after the injection of HCG. The minimum effective dose that blocked ovulation completely at 0 h abolished the acute increase of progesterone and testosterone, suggesting that prostaglandins act on ovulation by stimulating steroidogenesis at an early stage in the preovulatory process. The anti-ovulatory action of the minimum effective dose at 0 h became progressively less potent as the time between injection of HCG and administration of indomethacin was increased, although plasma concentrations of progesterone and testosterone measured at autopsy 18 h after treatment with HCG had not changed appreciably. When indomethacin was administered 10 h after HCG, the relationship between the dose of indomethacin and the mean number of ova differed from that observed when simultaneous injections of indomethacin and HCG were given, and the minimum effective dose that prevented ovulation was much higher than that at 0 h, suggesting that prostaglandins act differently on ovulation in the later stage of the preovulatory process. It was concluded that prostaglandins may mediate the action of HCG on ovulation through two mechanisms which operate at different stages of the preovulatory process.

Perifollicular clear space under skirt-like epithelial structure of human small vellus hair follicle.

Human hair follicles, especially small vellus hair follicles, showed characteristic structures as follows: (I) small vellus hair follicles were surrounded with skirt-like epithelial structures or perifollicular connective tissue capsule; (II) perifollicular clear space was present between the outer root sheath and skirt-like structure, or the outer root sheath and perifollicular connective tissue capsule; (III) perifollicular clear space was filled with a loose, alcian blue positive mucinous substance and elongated fibroblasts and mast cells; (IV) perifollicular nerve endings were attached to the follicular wall in a palisading arrangement within the clear space under the skirt; (V) perifollicular nerve end organs and connective tissue capsule were positively immunostained for CD34 and protein gene product 9.5 (PGP9.5). These specialized structures of human small vellus hair follicles seem to correspond to the blood sinus of the sinus hair follicles, where similar structural elements were found.

Two- and three-dimensional observations of human terminal and vellus hair follicles.

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.

Apoptotic pocket-like structures of the bulge of the terminal hair follicles of the human scalp.

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 occurrence 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.

A new method for preparation of pure zonae pellucidae in large quantities from porcine ovaries.

A new method for preparation of large quantities of zona substance in pure form from porcine ovaries has been described. In addition to the previously reported techniques of glass wool treatment and sieving by saran meshes, the following three significant improvements have been introduced: disruption of ovaries by an electric machine equipped with two multi-needed disks, which resulted in a considerably accelerated recovery of follicular oocytes with a high yield; low-speed centrifugation at 170 X g for 15 s was found to be an obligatory step to eliminate light particulate material from the crude oocyte suspension; use of 50% sucrose solution in discontinuous density gradient centrifugation permitted complete separation of homogeneous samples at two stages of the final preparation of zona-encased oocytes or of oocyte-free zonae. Microscopic examination revealed no contaminating components in the zona preparation. With this method 93 mg of lyophilized zona preparation were obtained from 24 553 porcine ovaries. Analysis of a solubilized zona substance by Sephacryl S-200 column chromatography showed the presence of two major glycoproteins which could not be separated completely from each other. By analysis of the two components with SDS-PAGE, only a single, but broad, band of glycoprotein was found, indicating the successful isolation of a major component(s) from porcine zonae.

Characterization of glycoproteins isolated from porcine zonae pellucidae.

Two glycoproteins, tentatively designated as PZ-alpha and PZ-beta, have been isolated and purified to homogeneity from porcine zonae pellucidae by a simple purification procedure producing a high yield. The procedure included the dissolution of the zona material in 0.1 M sodium borate buffer pH 10.0, Sephadex G-100 column chromatography and preparative SDS-polyacrylamide gel electrophoresis. The purified glycoproteins gave a single band on polyacrylamide gel and had molecular weights of 60 000 (PZ-alpha) and 96 000 (PZ-beta). Glutamic acid was detected as the NH2-terminal residue in both glycoproteins, using the dansyl chloride method. Though their amino acid compositions were similar, their carbohydrate contents were slightly different (PZ-alpha: 24.9%; PZ-beta; 19.6%), but these components contained the same types of monosaccharides: fucose, mannose, galactose, NAcGlc and sialic acid. The antigenic properties of the two glycoproteins were indistinguishable by immunodiffusion tests. The PZ-beta could be converted in part to smaller molecular weight components, though not to PZ-alpha, by treatment with beta-mercaptoethanol. Thus clear differences between PZ-alpha and PZ-beta could not be detected by chemical or immunological analyses except for the difference in the behaviour on SDS-polyacrylamide gel electrophoresis.

A method for specific detection of autoantibodies to the zona pellucida in infertile women.

Human antibodies to porcine erythrocytes were found to bind to porcine but not to human zonae pellucidae, whereas human isohemagglutinins bound to human but not to porcine zonae. On the basis of the binding behavior of agglutinins, it was found that the immunofluorescence of porcine zonae produced by selected sera from infertile women was due to an antibody of different specifity from that which agglutinated pig red blood cells. However, no serum component other than heteroagglutinin was contained in selected sera from control subjects which fluoresced porcine zonae. Since the component was present in the immunoglobulin G fraction, bound to human zonae, and was reactive with zona-specific antigen(s), it was judged an autoantibody to zonae. Thus a method for specific detection of autoantibodies to zonae has been developed by indirect membrane immunofluorescence using porcine ova as targets.

Steatocystoma multiplex. A facial papular variant.

A 32-year-old man had multiple asymptomatic, yellowish to normal skin-colored, moderately firm papules that were disseminated on the forehead, temple, and periauricular region. The histologic, enzyme histochemical, and ultrastructural findings were those of steatocystoma multiplex.

Hyperthermia in the treatment of psoriasis.

The effectiveness of commercially available, chemically generated, topical exothermic pads that elevate skin surface temperature from 42 to 43 degrees C was stressed in 22 patients with psoriasis. Control sites were treated with conventional modalities such as Goeckerman's regimen, as well as with occlusion with nonexothermic pads. Skin lesions in 19 patients disappeared after the use of hyperthermia. The average time required for complete regression in the treated areas was 27 days with hyperthermia, compared with 44 days with Goeckerman's regimen. There were no hyperthermic side effects. Seventeen patients whose skin lesions disappeared with the use of both hyperthermia and Goeckerman's regimen were subsequently reexamined. The hyperthermia produced an equal or longer duration of remission than did Goeckerman's regimen.

Psoralen-UVA-treated psoriatic lesions. Ultrastructural changes.

Psoralen-ultraviolet light (PUVA)-treated psoriatic lesions were studied for ultrastructural changes. In early stages of treatment, sunburn cells in the epidermis and bizarre giant cells in the dermis were more frequently observed. When clinical improvement was apparent, these changes had subsided. Dermal abnormality in long-term therapy consisted of a thick perivascular coat of amorphous substance. No abnormality was found in the epidermal keratinocytes in long-term therapy, except a clustering and giant cell formation of melanocytes, a heavy melanization of keratinocytes, and hyperkeratosis. Low-dose initiation and slow increment of both 8-methoxypsoralen and UVA is probably a reasonable regimen for benign dermatoses such as psoriasis because it will allow enough time for the skin to become more protected, while the therapeutic results are as satisfactory as in a high-dose schedule.

Importance of cytotoxic T lymphocytes in the rejection of transplanted hepatocellular carcinoma.

Fischer rats became resistant to syngeneic hepatocellular carcinoma (FAA-HTC1) cells on repeated sensitization with mitomycin C-treated FAA-HTC1 cells. In contrast, FAA-HTC1 cells injected into the liver killed normal control Fischer rats within 2 months. Histopathological studies revealed massive accumulation of mononuclear cells in the tumor tissues of sensitized rats that rejected syngeneic FAA-HTC1 cells, whereas very few mononuclear cells were found in the tumor tissues of control rats. Cell populations infiltrating the tumor tissues were identified by flow cytometric analysis. Mononuclear cells found within the regressing tumors of the sensitized rats were identified as mostly T cells, and two-thirds of these T cells were CD8-positive. Compared with the activity in control rats, the killer activity of mononuclear cells infiltrating tumors was significantly increased in the sensitized rats 7 days after tumor inoculation. Depletion of CD8(+) T cells significantly reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from sensitized rats. In contrast, depletion of CD16(+) cells reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from both control and sensitized rats. Furthermore, the CD16(+) cell-depleted fraction of mononuclear cells infiltrating tumors showed significant cytotoxicity against FAA-HTC1 cells, but failed to show cytotoxicity against other syngeneic tumor cells or allogeneic hepatoma cells.

Two- and three-dimensional demonstrations of morphological alterations of early anagen hair follicle with special reference to the bulge area.

During late telogen to early anagen secondary hair germ is newly formed by the downgrowth of a clubbed column which is indistinguishable from the bulge. Serial vertical sections demonstrated that the early anagen terminal hair follicle formed the new secondary hair germ associated with a lateral protuberance of basaloid cells which could be considered as the bulge of the new hair follicle. Interestingly, the arrector pili muscle bundle was divided into two branches, one inserted into the original clubbed end and the other into this protuberance of the secondary hair germ. CAM5.2-reactive Merkel cells were present not only in the clubbed ends of the old follicle but also in the protuberance of the new hair germ. The formation of the lateral protuberance of the new hair germ preceded the appearance of CAM5.2-reactive Merkel cells in this location. Ks19.1 immunoreactivity was observed from the clubbed end to the upper half of the new hair germ. These phenomena occurred in early anagen before the club hairs were shed. It is postulated that the early anagen hair follicle formed the area within the new hair germ equivalent to the bulge and Merkel cells either moved from the bulge of the old hair follicle or differentiated de novo from immature epithelial cells. Merkel cells or their products in the bulge may serve as attractants for the readjusting arrector pili muscle to anchor to the bulge of the new hair follicle.

Epithelial skirt and bulge of human facial vellus hair follicles and associated Merkel cell-nerve complex.

Many morphological variations of bulge areas, such as knob-like swellings, were found in extracted human facial vellus hairs. In anagen vellus hair of the face bulge areas including these knobs had a band-like dense aggregation of CAM5.2 (K8, 52.5 kDa) reactive Merkel cells. In telogen hair the bulge became indistinguishable from the clubbed or regressed end of the follicle but Merkel cells continued to be abundant. The epithelial hood at sebaceous gland level showed most commonly a skirt-like structure but variations were also observed; these were bamboo joints, tulip flower, and long apron configurations. Merkel cells were found sparsely in these structures. Palisading stockade-like nerve endings were observed surrounding the follicular epithelium under the skirt and around the bulge areas including the knobs. Merkel cells were sparse in the follicular segment corresponding to the attachment of stockade nerve endings.

Scanning electron microscopic observations of extracted terminal hair follicles of the adult human scalp and eyebrow with special references to the bulge area.

Scanning electron microscopic studies of human terminal hair follicles of the scalp and eyebrow have previously been limited to the hair shaft. In this study we investigated EDTA-treated extracted whole hair follicles in which most of the basal cell surface of the outer root sheath was well preserved. In the bulge area of scalp follicles there were many knob-like or villous projections. These were located in some specimens on one side of the follicle, while in others they were located around the entire circumference of the follicle. These projections were thought to represent the anchoring points of the branched follicular end of the arrector muscles. Ring-like elevations with groove-like depressions above and below were also observed surrounding the entire follicle. These were thought to represent the track of circumfollicular arrector muscles which depressed the outer root sheath when they repeatedly contracted. Most anagen eyebrow follicles showed morphological variations in the bulge area such as lattice-window-like structures and undulation patterns. In telogen follicles, the bulge became indistinguishable from the clubbed end. The lower end of these telogen follicles showed irregularly shaped bulge areas, but did not show lattice-window-like structures or undulation patterns as observed in anagen follicles. Interestingly, a hole was found in some bulge areas of both anagen and telogen follicles. Serial vertical sections of follicles revealed invaginated areas, which seemed to correspond to the openings seen in whole mounts. In vertical sections of eyebrow follicles some keratinocytes of the outer root sheaths of the bulge area were seen to be melanized to various extents. This phenomen was independent of hair cycle phase.

A high concentration of Merkel cells in the bulge prior to the attachment of the arrector pili muscle and the formation of the perifollicular nerve plexus in human fetal skin.

The distribution of Merkel cells in human fetal hair follicles was studied using whole mounts of separated epidermis with attached hair follicles. The technique had the advantage of enabling the elucidation of the spatial relationships of Merkel cells with other cells in the skin. In a 16-week-old fetus the hair anlagen had formed one or two epithelial swellings of variable size. In a 17-week-old fetus sebaceous glands and the bulge of the hair follicle were recognizable and immunoreactive. Merkel cells were present in the bulge and surrounding the acrotrichium (intraepidermal follicular canal). In a 20-week-old fetus the sebaceous gland and bulge were well formed and immunoreactive Merkel cells were concentrated in the bulge and infundibulum. In vertical sections of a 20-week-old fetus immunoreactive Merkel cells were also situated in the vicinity of the bulge. Arrector pili muscles were first observable in a 24-week-old fetus being weakly stained with anti-desmin antibody. In a 24-week-old fetus, nerves were also stained within the arrector pili muscles with S-100 protein antibody. In the presumptive arrector pili muscle immunoreactivity for S-100 protein developed before or at the same time as immunoreactivity for desmin. Merkel cells or their products in the bulge may serve as attractants for the growing arrector pili muscle which contain peripheral nerves. Following our report that dermal Merkel cells influence the formation of the dermal nerve plexus, perifollicular Merkel cells near the bulge may also play an inductive and growth-stimulative role for the perifollicular nerve plexus.