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Human dermal fibroblasts were cultured in a hydrated type I collagen lattice. When collagen fibers were arranged in one direction, fibroblasts were arranged in the same direction. Cell proliferation was markedly suppressed in the collagen lattice as compared with that on plastic, with growth being arrested after day 5. No differences in proliferation were observed between aligned cells and randomly oriented cells. Flow cytometry with DNA staining was performed to analyze each phase of the cell cycle of fibroblasts. Among the 10,000 cell population, S phase cells on day 2 of culture accounted for 43% on plastic but were markedly inhibited to 25% in the lattice. On day 4, S phase cells accounted for 33% on plastic but only for 10% in the lattice. These findings suggest that cell advancement to the S phase is markedly inhibited in the collagen lattice, resulting in accumulation of most of cells in the G0G1 phase. The present study clearly showed that culture in the collagen lattice allowed alignment of fibroblasts with a definite orientation as observed in vivo and produced a status resembling that in vivo in terms of proliferation and cell cycle phase composition.

The influence of topical PUVA on the activity of ornithine decarboxylase (ODC) and its gene expression was investigated in the skin of hairless mouse. After 1 h of application of 0.3% 8-methoxypsoralen, irradiation of 3 J/cm2 of ultraviolet A (UV-A) was administered. The ODC activity markedly increased and peaked at 24 h following UV-A irradiation. The ODC raRNA level analyzed in dot-blot analysis elevated to about 5 times that of control at 24 h after treatment. These results show that the PUVA-induced ODC activity is due in part to an increase in ODC gene expression.

腫瘍間質と腫瘍細胞との相互作用をin vitroで研究する目的の第一歩として, B16メラノーマ細胞をI型コラーゲン·ゲル上およびゲル内で培養し, その形態, 増殖, チロジナーゼ活性, メラニン産生量について検討し, 通常のプラスチック上で培養した場合と比較した。ゲル内での細胞の形態は, プラスチック上に比べて, より細長く樹枝状となつた。増殖は, ゲル上およびゲル内ともに著明に抑制され, 培養7日後の細胞数は, プラスチック上と比較して, それぞれ約15%と10%となつた。チロジナーゼ活性は, ゲル上およびゲル内では促進され, プラスチック上での場合に比べて約3倍に増加した。それにともなつて, メラニン合成量もゲル上およびゲル内では, 約2倍にまで増加した。したがつて, 今回の結果よりコラーゲンとメラノーマ細胞との相互作用の存在が示唆された。

I型コラーゲン·ゲル上およびゲル内でヒト真皮線維芽細胞を培養し(再構成真皮モデル), その増殖を検討するとともに, propidium iodideによるDNA染色と, S期細胞に取り込ませたbromodeoxyuridineに対するFITC標識モノクローナル抗体を使つたtwo color flow cytometryを用いて, 線維芽細胞の細胞周期上の位相分布分析を行つた。通常のプラスチック上での培養に比べて, ゲル上では線維芽細胞の増殖は抑制され, 10日目に約50%の飽和密度で増殖を停止した。ゲル内では, さらに抑制され, 4∼7日以内にほぼ増殖を停止した。培養後5日目では, プラスチック上での培養に比べて, ゲル上においてはS期細胞は49.3%に減少し, G0G1期細胞は79.8%の増加を見た。一方, ゲル内では, S期細胞は非常に少数で(総細胞数の4.3%), 大多数はG0G1期に蓄積した。したがつて, コラーゲンとの相互作用により大多数の線維芽細胞は, G0G1期で停止することが示された。以上より, コラーゲン·ゲルを用いた再構成真皮モデルは, 増殖度と細胞周期位相分布の面からも生体真皮に近いことが示された。

ポリアミン合成の律速酵素であるオルニチン脱炭酸酵素(ODC)は, マウス実験的皮膚発癌におけるプロモーション過程のマーカー酵素として知られている。アルビノ·ヘアレスマウスに中波長紫外線を照射すると, この酵素活性は著明に上昇した。このメカニズムを分子生物学的に検索する目的で, マウス皮膚よりmRNAを抽出し, ODC cDNAをプローブとしてノーザン·ブロット分析を行つた。その結果, 90 mJ/cm2の中波長紫外線照射により, コントロールに比べて, 4時間後で2.0倍, 24時間後では2.6倍のODC mRNAレベルの増加を認めた。酵素活性の上昇とmRNAレベルの増加度とは並行せず, 転写後の調節の可能性も示唆されたが, 中波長紫外線照射によるODC活性の促進には, ODC mRNAレベルの増加が関与することが示された。

A 7-year-old girl with alopecia universalis had dystrophy of all 20 nails. A nail biopsy specimen disclosed features of lichen planus. The patient also had ichthyosis vulgaris and hypogammaglobulinemia. We are not aware of any previous reports of these associations, which we believe to be noncoincidental.

A case of subcorneal pustular dermatosis (SPD) is reported in a 48-year-old man with a 2-year history of hyperthyroidism (Graves' disease). SPD has been reported in association with IgA monoclonal gammopathy, but to our knowledge it has not been associated with hyperthyroidism. The coexistence of hyperthyroidism and SPD suggests that immunologic factors may play a role in the pathogenesis of SPD.

A 14-year-old boy with linear localized scleroderma had a dramatic improvement in contractures after treatment with N-(3',4'-dimethoxycinnamoyl) anthranilic acid (tranilast, Rizaben). The observation that this anti-allergic drug was effective in localized scleroderma lends further support to the concept that mast cells play a role in increased collagen synthesis in this disease.

Summary Four cases of solitary glomus tumour are reported, three of the angiomatous type, and one of the epitheliomatous type. In all cases, mucinous degeneration was seen, the extent of which correlated with the number of glomus cells.

Background. Rhinophyma is an end stage of acne rosacea. It results in a large nose due to a proliferation of sebaceous glands and fibrous tissue. Many cases of rhinophyma have been reported in the Western world; however, in Japan, rhinophyma has been an uncommon disease. Methods. We present two patients associated with rhinophyma who were treated by cross-shaped full-thickness excision followed by direct closure and compare the rhinophyma in Japan with that in the West. Results. To date, only 20 rhinophyma cases including our 2 cases have been reported in Japan. Epidemiologic factors of rhinophyma between the West and Japan do not differ except for location, malignancy and surgical treatment. Conclusions. In Japan, almost all cases are located on the lower half of the nose, treated by full-thickness excision followed by application of either skin grafts or direct closure. None have been malignant.

Summary Secondary cutaneous T-cell lymphoma may present with various types of skin lesions, hut rarely produces ulceration in contrast to primary cutaneous T-cell lymphoma. We report a case of malignant lymphoma of the tonsil that recurred as a giant cutaneous ulcer on the back of a 45-year-old man, Biopsy of the ulcer revealed a malignant lymphoma of diffuse, mixed-cell type. Surface marker analysis of the tumour cells showed suppressor/cytotoxic T-cell characteristics. The skin lesions were treated by electron beam therapy.

A 45-year-old woman presented with persistent headache which was of acute onset and occurred daily. Laboratory findings included thrombocytosis, elevated levels of antinuclear antibody, rheumatoid factor, and antibodies to Epstein-Barr virus (EBV). Magnetic resonance image of the head, left selective external carotid angiography, temporal arterial biopsy, and cerebrospinal fluid revealed no abnormal findings that could explain her headache. Her headache was compatible with NDPH, which has been reported by Vanast, and was thought to be related to EBV reactivation.

Summary Articular tissue was obtained at surgery for a femoral neck fracture in a patient with psoriasis without arthritis. The proteoglycan of the cartilage of the sample was analysed biochemically. Normal cartilage is known to produce two types of proteoglycan monomers (fast- and slow-sedimenting groups), which are distinguishable by density-gradient ultracentrifugation. In the psoriatic cartilage analysed in the present study, it was shown that the former group was absent and only the latter group remained.

Summary A case of a solitary pigmented neurofibrosarcoma involving the lateral side of the face in a 19-year-old female is described. Clinically it resembled a haemangioma, and angiography showed hypervascularity of the tumour. Light-microscopic study revealed that the major cellular element of the tumour was composed of bizarre neoplastic cells with large pleomorphic, hyperchromatic nuclei, conspicuous nucleoli and moderate to abundant amounts of conspicuous cytoplasm. Mitotic figures and multi-nucleate cells were present in most fields. Neurofibrosarcoma is one of the most highly malignant tumours; however, in the present case, there was no evidence of recurrence during a follow-up period of 5 years.

Cutaneous inflammation induced by electrophilic compounds involves irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD). Reduced glutathione (GSH) and related thiols have been postulated to play important roles in detoxification of electrophilic xenobiotics, protection of tissues against reactive oxygen species, and modulation of immunologic functions in normal and diseased subjects. The dynamic aspects of GSH metabolism, however, and its significance in patients with ICD and ACD remain to be clarified. The current study was carried out to elucidate the pathogenesis and possible involvement of GSH in both types of inflammation. Normal mice and mice sensitized with dinitrochlorobenzene (DNCB) were challenged by cutaneous administration of DNCB, and changes in GSH metabolism in skin and liver were determined. Kinetic analysis revealed that 24 h after challenge with DNCB, levels of hepatic glutathione and its secretion increased more markedly in the sensitized mice than in the unsensitized animals. Administration of buthionine-L-sulfoximine (BSO), a specific inhibitor of GSH synthesis, inhibited the increase in glutathione levels in the liver and the skin of both groups. Histologic examination revealed that cutaneous inflammation was enhanced by BSO more significantly in mice with ACD than with ICD. These results suggest that GSH might play an important role in the suppression of the immune reaction in mice with ACD.

A 21-year-old male patient had localized papules on the chest and arms. Biopsy of a lesion showed it to be a malignant lymphoid neoplasm, and immunohistochemical studies confirmed a lymphoblastoma origin. Aberrant lymphoid cells were noted in the cutaneous blood vessels, but simultaneous examination of the peripheral blood showed no evidence of leukemia. One month elapsed before observable leukemic cells were found in the blood and sternal marrow, confirming the diagnosis of acute lymphocytic leukemia. Examination of a cutaneous biopsy specimen led to early diagnosis of the disease.

Tumor necrosis factor-alpha (TNF-alpha) has been demonstrated to selectively decrease the production of type I and type III collagens in human dermal fibroblasts. The effects of the commonly used food antioxidants, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol, propyl gallate, superoxide dismutase (SOD), and catalase on TNF-alpha-induced growth enhancement and collagen metabolism were evaluated in the present study. BHA at concentrations of both 5 x 10(-5) and 10(-4) M inhibited cell proliferation and DNA synthesis induced by 10 ng/ml TNF-alpha in human dermal fibroblasts, while other antioxidants had minimal effects. Further, BHA (5 x 10(-5) M and 10(-4) M) significantly blocked TNF-alpha-induced decreases in collagen synthesis. These results suggest that antioxidants such as BHA may be involved in the modulation of collagen synthesis by TNF-alpha in human dermal fibroblasts.

Lysyl oxidase (LOX) initiates the crosslinking of the lysine-derived aldehyde and plays an essential role in maturation of collagen, for example in wound healing. Although the activity of this enzyme has been examined in various disorders, and a further intriguing aspect of the relationship between LOX and tumorigenesis has recently emerged, its gene expression pattern in tissues is still unknown. We examined LOX gene expression during wound healing in rat skin. In addition, type III collagen gene expression was studied to determine the formation of fibrils. The LOX mRNA level reached a peak by day 3 after injury, which was earlier than that of type III collagen, and continued at a high level until day 22. The type III collagen mRNA level began to rise from day 3 and had increased intensely by day 22. In situ hybridization revealed grains corresponding to LOX mRNA in the fibroblasts of the granulomatous tissue. These results suggest that LOX is produced before collagen synthesis in preparation for crosslinking in the early phase of wound healing.

S-carboxymethylated (SCM) fibrous proteins (FPs) from the scalp hairs of the three different hereditary hair abnormalities (trichorrhexis invaginata in Netherton's syndrome, pili trianguli et canaliculi in uncombable hair syndrome, and fine hair in anhidrotic ectodermal dysplasia) were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the SCM FP compositions of the three hair abnormalities with that of the normal revealed that the SCM FP compositions of these three abnormal hairs were all electrophoretically different from each other and that of the normal.

We report a 22-year-old man with acquired dermal melanocytosis on the hand which developed without any history of previous inflammation. He had no history of contact with or oral administration of any drugs or metals which might have caused pigmentation. Histopathologically, spindle-shaped dermal melanocytes were observed running parallel to the collagen bundles and scattered in all portions of the dermis. The dopa reaction of these cells was positive. Ultrastructurally, dermal melanocytes containing numerous melanosomes (stages II to IV) which were surrounded by the extracellular sheath were observed. Differential diagnosis of other dermal melanocytosis was discussed.

Seven patients who had surgery for malignant skin tumors of the heel were followed up for several years. The reconstructive procedure performed was repair of the large skin defect after excision of a tumor, by using a flexor digitorum brevis muscle flap and an overlying free graft. Although this procedure is a good method without any serious complications, no long-term follow-up data supporting this opinion have been reported. In this study, plantar flexion of the toes and flattening of the plantar arch appeared 3 months after surgery and developed for 1 year, and the patients complained of easy fatigability in walking and difficulty in going up stairs. These sequelae were probably due to sacrifice of the flexor digitorum brevis muscle. We feel that this procedure should be replaced by another procedure that does not sacrifice any muscle, for example, that using a medial plantar flap.

The patient, a 34-year-old Japanese woman who noticed worsening of her rash after using topical corticosteroid preparations on her neck, was patch tested for both commercial preparations and corticosteroids themselves. The patch lest results revealed that she had a contact allergy to gold, oxytetracycline, and 2 types of corticosteroid (acetonides and esters) in 7 compounds (betamethasone valerate and dipropionate, hydrocortisone butyrate and hydrocortisone butyrate propionate, ameinonide, budesonide, and fluocinonide).

Ultraviolet-B and PUVA share several biological events with phorbol ester tumor promoters. The effects of ultraviolet-B irradiation and topical PUVA treatment on ornithine decarboxylase activity, DNA synthesis, and protein kinase C activity, which are known to be induced or activated by phorbol ester tumor promoter, were investigated in hairless mouse skin. Ornithine decarboxylase activity was remarkably enhanced by ultraviolet-B and PUVA. Although PUVA did not affect DNA synthesis significantly, ultraviolet-B stimulated epidermal DNA synthesis approximately 5-fold over control values at 48 h. However, unexpectedly, neither cytosolic nor membrane-bound protein kinase C activity showed any change during the 2 h after either treatment. These results suggest that the protein kinase C system is not involved in the initial signal transduction system of ultraviolet-B or PUVA, unlike the case with phorbol ester tumor promoter.

Myasthenogenic regions in the acetylcholine receptor (AChR) alpha-subunit were studied in view of the conformation-dependent B-cell epitope expected at beta-turn and the MHC class II-restricted T-cell epitope expected at alpha-helix. Torpedo AChR alpha 67-76 and alpha 107-116 were synthesized as the main immunogenic region and the site specific for T-cell epitope in Lewis rat, respectively. Model peptides, synthesized by combining these natural sequence segments or by intervening the segment aligned as Asn-Pro-Gly-Gly (NPGG) in natural sequence segments, were tested in terms of antigenic conformation. The model peptide, alpha 107-116.alpha 67-76.alpha 107-116, was immunogenic in the induction of the animal model of myasthenia, accompanied by the anti-peptide antibody cross-reactive with the native AChR. High antigenicity in antibody assays for various peptide- and native AChR-immunized rats was found when the model peptides, alpha 107-116.alpha 67-76 and/or alpha 107-116.NPGG.alpha 67-76 were used for measurement as antigens. Antigenic conformation for the induction of the disease may thus be different from that for the reactivity to antibody.

Summary Herpes simplex viral DNA was detected in biopsy specimens obtained from four patients with Kaposi's varicelliform eruption using an in situ hybridization technique with btotinylated complementary DNA probes. Herpes simplex viral DNA was consistently found almost exclusively in the nuclei of giant cells and balloon cells in vesicles during day 2, 5 and 7 of the disease. In the nuclei, the viral DNA staining pattern was granular or agglomerate. No positive staining was observed in the cytoplasm.

Endothelin-1, a potent vasoconstrictor peptide that has recently been isolated from the supernatant of cultured porcine aortic endothelial cells (1), is involved in some vascular disorders (2). Mal...

The effects of Ryo-kan-kyomi-sin-ge-nin-to (RKSG) extract, a medicinal agent traditionally used in China and Japan for treatment of asthma, on the degranulation of and histamine release from rat mast cells were studied. At a concentration of 5 mg/ml RKSG, degranulation of mast cells stimulated either by antigen (DNP-Ascaris) or compound 48/80 was markedly suppressed. At a concentration of 1-5 mg/ml RKSG, histamine release from mast cells due to application of either antigen or compound 48/80 was inhibited in a dose-dependent fashion. These results suggest that RKSG may be useful for the treatment of type I allergy-related diseases.

Melanoma cells were cultured on type I collagen gel, and the infiltration of of those cells into the gel was observed. B16 murine melanoma cells initially adopted a spherical form on the gel, but they assumed a dendritic form after infiltration into the interior. The degree of infiltration increased very rapidly and was time-dependent. No correlations between the growth rate or melanogenic activity and infiltrative potential were observed. When Syrian hamster and human melanoma cell lines were cultured, the degrees of infiltration varied. This culture system using collagen gel is considered to be a useful in vitro model of tumor cell invasion.

The effects of human recombinant tumor necrosis factor-alpha (TNF-alpha) on human keratinocytes cultured in a serum-free medium were investigated. TNF-alpha markedly suppressed cell growth. The growth-inhibitory effect was reversible and cytostatic at a concentration of 1-5 U/ml, but appeared to be irreversible and cytocidal at 10 U/ml. The growth suppressive effect was more marked when TNF-alpha was added in the late growth phase or preconfluent phase than when it was added in early or mid-growth phases. No effects of TNF-alpha on cell adhesion to the substrate were observed. These results indicate that TNF-alpha is a very potent anti-proliferative agent for human keratinocytes.

As the first step in developing an in vitro model of melanoma cells infiltrating the dermis, B16 murine melanoma cells were cultured on and in type I collagen gels. Under these conditions, the melanoma cell adopted an elongated or dendritic form. Cell proliferation was suppressed in the culture system using the collagen gel as compared with the conventional monolayer culture on plastic. Microcinematographically, this suppression was found to be due to an extension of the cell cycle time of each individual cell. On the other hand, there were no appreciable differences in proliferation pattern between the cells cultured on type I and IV collagen film and those cultured on plastic. These results suggest that there are interactions between type I collagen in the gel form and melanoma cells, especially with respect to cell growth.

We report a case of microcystic adnexal carcinoma (MAC) occurring on the upper lip of an 82-year-old woman. Microscopically the tumor showed both pilar and sweat gland differentiation, involved the entire dermis and subcutaneous tissue, and invaded perineural spaces. Immunoperoxidase studies revealed carcinoembryonic antigen to be present in the ductal lining cells and in the amorphous content in the lumen, confirming sweat gland differentiation. The S-100 protein was positive in dendritic cells within the solid cell nests, but negative in cells lining cystic spaces. Ultrastructural study confirmed that the neoplasm was composed of two components, with pilar and eccrine differentiation. The former showed concentric layers of squamous epithelial cells with well-developed desmosomes and cytofilaments. The latter had ductal and alveolar structures; the ultrastructural features included: i) numerous villous folds of plasma membrane to interdigitate each other by focal desmosomes, ii) aggregates of cytofilaments, and iii) basally located myoepithelial cells which were separated from the surrounding stroma by rather thick basement membrane. In addition, distinct amyloid deposition was also observed on ultrastructural examination. To our knowledge, amyloid deposition has not been previously reported in MAC.

A case of generalized granuloma annulare associated with temporal arteritis is described. The patient, a 79-year-old man, noticed numerous asymptomatic lesions on his trunk and extremities for 3 months. Four months later, he suffered from headache and loss of vision. Both were successfully treated by oral administration of prednisolone.

Ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine synthesis, is a well-established indicator of tumor promotion [7]. The mechanism of ODC induction has been intensively studied using Northern blot analysis, especially in 12-O-tetradecanoylphorbol-13-acetatetreated (TPA-treated) skin [4, 10]. Following ultraviolet (UV) irradiation, ODC activity is markedly induced in the skin [5]. We have previously demonstrated a UVinduced increase in ODC gene expression using Northern blotting of mRNAs extracted from the whole skin of the mouse [6]. In the present study, we examined the spatial distribution o fODC mRNA in UV-irradiated mouse skin using an in situ hybridization technique. Female Charles River CD-1 mice aged 7 to 10 weeks were used in all experiments. The dorsal skin of the mouse was shaved 3 to 4 days before the experiments. The dorsa of the mice were irradiated with 40 mJ/cm 2 UV using two fluorescent lamps [FL20SE-30, Toshiba, Japan: UV emission between 280 and 320 nm (peak, 305 nrn)]. The dorsal skin was excised 24 h after irradiation, fixed in 4% paraformaldehyde (PFA), embedded in OCT compound (Lab-Tek, Naperville, Ill., USA), frozen in liquid nitrogen and sectioned at 4 ~tm. The sections were processed for in situ hybridization using a modification of methods previously described [3, 8]. In brief, sections were treated with proteinase K, post-fixed in 4% PFA, acetylated in acetic anhydride and dehydrated in an ethanol series. The samples were pre-hybridized for 30 rain at 42~ in a solution containing 50% formamide, 0.6 M NaC1, 20% dextran sulfate, 200 ~tg/ml bovine serum albumin, 0.02% polyvinylpyrrolidone, 250 I, tg/ml yeast tRNA, 100 ~tg/ml salmon sperm DNA, 0.02% Ficoll

Acanthosis nigricans (AN) is generally classified into the malignant, benign and pseudo-types. The malignant type is the most frequently reported and 65% of the benign type are associated with diabetes mellitus (218 of 337 reports, 1969-1995, our investigation). Moreover, familial reports are not frequent, and previous reports have suggested that AN is endocrinological or hereditary in origin, but the cause remains unclear. Familial polyposis coli (FPC), on the other hand, is a rare autosomal dominant intestinal disease that progresses to cancer in many cases. Both AN and FPC are autosomal dominant, but no common human leukocyte antigen (HLA) typing between these diseases has been reported previously.

We report a 19-year-old woman with the associated disorders of generalized pruritus, hypertrichosis and anorexia nervosa, all of which had been present for approximately 4 months. Psychotherapy induced both weight gain and resolution of the pruritus. We propose that anorexia-associated pruritus be considered one of the important cutaneous signs in anorexia nervosa.

The effects of antitumor agents are usually assayed on soft agar culture or monolayer culture on plastic. However, such an environmental condition cannot be found in vivo and could be too severe for cells to survive. Recently, studies have indicated that culture of cells in or on a collagen gel provides a matrix-like environment and enables the cells to exhibit fully their differentiating functions not observed on plastic [1, 4, 9, 11, 12]. To evaluate the effect of antitumor drugs under the conditions resembling those occurring in the process of infiltrating the dermis in vivo as much as possible, we cultured B16 melanoma cells in type I collagen gel and compared the effects of dacarbazine (DTIC) [3, 8] and all-trans-retinoic acid (RA) [7] with those on the conventional plastic dishes. Three-dimensional culture in type I collagen gel was performed according to the method of Elsdale and Bard [4] after modification as described previously [6]. Briefly, 0.3% pepsinized type I collagen solution (derived from porcine tendon, Nitta Gelatine, Osaka, Japan), HEPES/ NaOH neutralizing solution, concentrated Eagle's medium, cell suspension, and fetal calf serum (FCS) were mixed at below 4 ~ C. One millilitre of the mixture was poured into a 35-ram bacteriological dish (Falcon) and incubated at 37 ~ C. The final collagen concentration was 2.0 mg/ml and the initial cell density was 2 x 10 s cells/ml. After gelling, 2 ml of Eagle's medium supplemented with 10% FCS was added. Addition of DTIC or RA was started on day 4. DTIC was activated with exposure to white light [8]. Culture medium supplemented with FCS and these reagents was changed every other day. At indicated times gels were digested by collagenase at 37 ~ C for 40 rain and released viable cells were counted with a hemocytometer using the dye exclusion test.