£²£²²óÆüËܸ¦µæÈéÉæ²Ê³Ø²ñÁí²ñ¡¦¾¶Ï¿
-199- Poster Discussion 9/5 Room-W
THE EXPRESSIONS OF TRANSGLUTAMINASE 1 IS INDUCED IN HUMAN FIBROBLAST BY RETINOID TREATMENT.
Soo-Youl Kim1, Akiyama Masashi2, Kozo Yoneda3.
1Laboratory of Skin Biology, Pacific R&D Center, Kyonggi-do, Korea. 2Division of Dermatology, Kitasato Institute Hospital, Tokyo, 3Department of Dermatology, Faculty of Medicine, Kyoto University, Kyoto, Japan.
Retinoids play an importnat role in the coodinate program of gene expression in the skin as well functional roles in embryonic development. They are known to be major regulator of growth and differentiation in the skin. It was reported that retinoic acid treatment in vivo, in contrast to in vitro, increases transglutaminase 1(TGase 1) mRNA as well as enzyme activity. To elucidate this discrepancy between in vivo and in vitro results, we investigated the effects of retinoids on TGase 1 and enzyme levels in cultures of normal human fibroblasts and normal human epidermal keratinocytes. Each culture system was treated with retinoids including retinoic acid, retinol, retinaldehyde and retinyl palmitate at 10-6M for 48 hr respectively. Surprisingly we detected the TGase 1 expression in normal human fibroblasts under the normal culture conditions by RT-PCR and immunoprecipitation with TGase 1 antibody. Furthermore, the membrane-bound TGase activity and mRNA expression of TGase 1 were increased about three-fold by retinoid treatment in cultured fibroblast. On the other hand, in keratinocyte system, both of membrane bound TGase activity and TGase 1 mRNA are down-regulated by retinoid as reported. This is the first report of expression of TGase 1 in mesodermal origin tissue although TGase 1 was discovered and thought to be only in keratinocyte. Further study is needed to understand the physiological role(s) of TGase 1 in fibroblast.
-200- Poster Discussion 9/5 Room-W
trichohyalin expression in fetal skin development and various skin disorders.
S.C. Lee, J.B. Lee, J.J. Seo, H.W. Lee, I.K. Chun, Y.H. Won, Y.P. Kim.
Department of Dermatology, Chonnam University Medical School, Kwangju, Korea.
-201- Poster Discussion 9/5 Room-W
EPIDERMAL PROLIFERATION OF THE SKIN IN METALLO- THIONEIN-NULL MICE.
Katsumi Hanada1, Daisuke Sawamura1, Katsuto Tamai1, Isao Hashimoto1, Kazuyuki Kida2, Akira Naganuma3.
1Departments of Dermatology, 2Public Health, Hirosaki University School of Medicine, Hirosaki, 3Molecular and Biochemical Toxicology, Faculty of Pharmaceutical Science, Tohoku University, Sendai, Japan.
For understanding the significant role of metallothionein (MT) in the epidermal hyperplasia, mice with null mutation in their MT-1 and MT-2 genes were used. We compared the epidermal hyperplasia between in MT-null mice and in C57BL/6J control mice after treatments with cholera toxin, TPA and ultraviolet B irradiation for epidermal proliferation. MT-null mice exposed to each stimulator developed significantly low level of epidermal hyperplasia when compared with that in control mice. Metal contents of the skin determined by proton-induced x-ray emission (PIXE) method showed decreased tendency of low content of zinc in MT-null mice after proliferation. These findings indicate that cellular MT is involved in proliferative process of the epidermis through its regulatory capacity of zinc metabolism required for cell growth.
-202- Poster Discussion 9/5 Room-W
EFFECTS OF PMA ON THE INTRACELLULAR DISTRIBUTION OF PROTEIN KINASE C AND M (THE KINASE DOMAIN) ISOZYMES AND KERATIN-PHOSPHORYLATION IN CULTURED KERATINOCYTES.
Chikako Esaki, Mariko Seishima, Kazuko Osada, Yasuo Kitajima.
Department of Dermatology, Gifu University School of Medicine, Gifu, Japan.
We have previously reported that PKMs were detected with antibodies against PKCs ( and (, but not ( and (, in DJM-1 cells (a squamous cell carcinoma line) by immunoblot analysis. In addition, PKM of ( isozyme was distributed in the soluble (cytosol), Triton X-soluble (membrane) and Triton X-insoluble (cytoskeleton-associated) fractions, whereas that of ( was only in the cytosol fraction. Currently, we investigated effects of PMA on the intracellular distribution of PKC/M isozymes and phosphorylation of cytokeratins. Phosphorylation activity of PKM preparations, which were purified from cytosole by DEAE-Sephacel column, was confirmed and this activity was partially inhibited with anti-PKC-( antibody. PKM-( was distributed in all fractions including cytokeratins. PMA caused an increase of PKM-( in cytoskeleton-associated fractions, although cytokeratin-phosphorylation was unexpectedly reduced. These results suggest that various isozymes mediate protein-phosphorylation independently at diverse intracellular compartments and PKC-( and PKM-( downregulate phosphorylation of cytokeratins.
-203- Poster Discussion 9/5 Room-W
EFFECT OF CIS-UROCANIC ACID ON HISTAMINE MEDIATED ACTIVATION OF ADENYLATE CYCLASE AND INCREASE OF INTRACELLULAR FREE CALCIUM.
Hiroko Koizumi, Tadamichi Shimizu, Akira Ohkawara.
Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan.
UVB irradiation causes suppression of delayed hypersensitivity. One of the photoreceptors and mediators is cis-urocanic acid formed in the epidermis. Cis-urocanic acid is regarded to act via histamine-like receptor. Histamine induces an activation of adenylate cyclase and an increase of intracellular free calcium on keratinocytes. Thus we investigated the effects of cis-urocanic acid on these transmembrane signaling systems. Cyclic AMP in cultured normal human keratinocytes was measured with radio-immunoassay. Intracellular free calcium was detected with digital imaging system. Cis-urocanic acid did not activate adenylate cyclase, however preincubation with cis-urocanic acid inhibited cyclic AMP accumulation by histamine. Cis-urocanic acid did not increase intracellular free calcium. Preincubation with cis-urocanic acid attenuated the responsibility of the cells against histamine not against epinephrine. The effects of cis-urocanic acid on keratinocytes is revealed through modulation of the effect of histamine.
-204- Poster Discussion 9/5 Room-W
EFFECTS OF Ca2+ CONCENTRATION OF CULTURE MEDIUM ON THE SOD ACTIVITY AND UVB-INDUCED CYTOTOXICITY IN CULTURED HUMAN KERATINOCYTES.
Hiroko Sasaki, Hirohiko Akamatsu, Takeshi Horio.
Department of Dermatology, Kansai Medical University, Osaka, Japan.
We examined the effects of Ca2+ concentration of culture medium on the SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes in order to investigate the relationship between cellular differenciation and antioxidant capacity. Human keratinocytes were incubated in high Ca2+ concentration ( >1 mM ) or low Ca2+ concentration ( < 0.1 mM ) DMEM for 24h at 37¡î in 5% CO2 . Then, we measured SOD activity and also both Cu,Zn- and Mn-SOD activities in keratinocytes separately. Furthermore, after incubation in high or low Ca2+ DMEM, human keratinocytes were irradiated with 10, 30, 50 mJ/cm2 UVB. The quantity of LDH leaked in the supernatant from damaged keratinocytes was measured 24 hr after UVB irradiation. Total SOD activity and Cu,Zn- SOD activity in human keratinocytes cultured in low Ca2+ DMEM were significantly lower than in keratinocytes cultured in high Ca2+ DMEM. In contrast, Mn-SOD activity was not affected. LDH leakage in the supernatant from keratinocytes cultured in low Ca2+ DMEM was significantly higher than that from keratinocytes cultured in high Ca2+ DMEM. These results suggest that cellular differenciation due to the change of Ca2+ concentration of culture medium affects the Cu,Zn-SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes.
-205- Poster Discussion 9/5 Room-W
THE PHOTOPROTECTIVE EFFECT OF 1,25-DIHYDROXYVITAMIN D3 ON ULTRAVIOLET LIGHT B-INDUCED DAMAGE IN KERATINOCYTE AND ITS MECHANISM OF ACTION.
Joo H. Lee1, J.I. Youn2.
1Department of Dermatology, Inha University Medical School, Incheon, 2Department of Dermatology, College of Medicine, Seoul National University, Seoul, Korea.
We investigated the photoprotective effect of 1,25(OH)2D3 on UVB induced skin damage and its probable mechanism. The in vitro and in vivo photoprotective effect of 1,25(OH)2D3 against UVB was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasoliumbromide assay and by the counting of sunburn cells respectively. The mechanism of photoprotection was sought through observing in situ inducibility by 1,25(OH)2D3 for metallothionein( MT) and verifying inherent photoprotective potential of 1,25(OH)2D3. The results of the present study were as follows; 1)In the presence of 1,25(OH)2D3(12nM in culture, 240uM in topical application), the decrease of survival of cultured human keratinocytes by UVB(¡å40mJ/cm2) as well as the formation of sunburn cells were inhibited. 2)The inhibition by 1,25(OH)2D3 of UVB-induced formation of sunburn cells was also observed in glutathione-depleted skin with its photoprotective effect being comparable to glutathione alone. 3)1,25(OH)2D3 induced the expression of MT in basal layer of ICR mice skin. 5)1,25(OH)2D3 neither inhibited peroxidation of plasma lipids nor interacted with superoxide or hydrogen peroxide. These findings suggest that 1,25(OH)2D3 has photoprotective effect and this effect appears not to be related with glutathione or inherent antioxidant effect of the vitamin but to be attributed to the induction of MT in skin.
-206- Poster Discussion 9/5 Room-W
effect of ultraviolet b radiation on lipid peroxidation of the rat skin.
S.C. Lee, H.W. Lee, J.S. Kim, J.W. Jung, I.K. Chun, Y.H. Won, Y.P. Kim.
Department of Dermatology, Chonnam University Medical School, Kwangju, Korea.
-207- Poster Discussion 9/5 Room-W
THREE DIMENSIONAL CULTURE OF HUMAN KERATINOCYTE -AN ALTERED BARRIER FUNCTION MODEL-.
Noriko Yamamoto1, Takeshi Tamura1, Hiroshi Tonogaito1, Kazuhiko Hamada2, Junko Miyasaka1.
1Biochemistry Lab., R&D Division, Pias Corporation, Kobe, 2ACSEINE Corporation, Osaka, Japan.
To develop an in vitro model for the skin that shows unusually enhanced proliferation and the altered barrier function, we applied three-dimensional culture system supplement with interleukin-6(IL-6). IL-6 is one of autocrine growth factor for keratinocytes and recognized to be located in atopic dermatitis(AD). In this model, total amount of protein was unchanged whereas that of DNA was increased significantly, indicating the increase of cell-number and the reduction of cell-size. Moreover, its barrier function was defective and it had large interstitium in this histological structure. In addition, the marked reduction of cholesterol level was observed regarding composition of the polar lipids from this model which was agreed with that of atopic dermatitis. These results suggests that this model with IL-6 is useful for an altered barrier function model.
-208- Poster Discussion 9/5 Room-W
MOUSE LANGERHANS CELLS DO NOT EXPRESS HIGH AFFINITY IgE RECEPTOR.
Satoru Hayashi1,2, Hironori Matsuda1, Kou Okumura1, Chisei Ra1.
1Department of Immunology, Juntendo University School of Medicine, Tokyo, 2Department of Dermatology, Yamanashi Medical University, Yamanashi, Japan.
The epidermal Langerhans cells play an important role in the allergen specific T cell activation in atopic dermatitis. The FceRI on human Langerhans cells may influences the functional property of T cell activation. Since an IgE bearing Langerhans cell mouse model would be useful in studying the pathophysiology of atopic dermatitis, we determined whether FceRI was also present on murine Langerhans cells. Monoclonal antibody to murine FceRIa chain reveals absence of FceRI a chain protein on Langerhans cells. And also the mRNA of FceRI a, b chain also absent in epidermal Langerhans cells. These date indicate that the murine epidermal Langerhans cells do not express the high affinity receptor for IgE.
-209- Poster Discussion 9/5 Room-W
FUNCTIONAL EXPRESSION OF FAS ANTIGEN(CD95) ON MURINE LANGERHANS CELLS.
Tatsuyoshi Kawamura1,2, Miyuki Azuma3, Hideo Yagita2, Ko Okumura2, Shinji Shimada1.
1Department of Dermatology, Yamanashi Medical University, Yamanashi, 2Department of Immunology Juntendo University, Tokyo, 3Department of Immunology, National Children's Medical Research Center, Tokyo, Japan.
In this study, we demonstrated that murine epidermal Langerhans cells(LC) constitutively express Fas antigen, and their expression was upregulated by IFNg. We examined that epidermal LC underwent apoptosis by the addition of both recombinant soluble FasL and IFNg, but not by FasL alone. Further we have shown that antigen(Ag)-bearing LC in the draining lymph nodes after skin painting with FITC also express Fas and the ratio of Ag-bearing LC in Fas negative lpr/lpr mice was significantly increased as compared to normal mice. Our data suggested that the migrated Ag-bearing LC may undergo apoptosis through Fas-mediated pathway in the draining lymph node.
-210- Poster Discussion 9/5 Room-W
REGULATION OF THE EXPRESSION OF ADHESION MOLECULES ON HUMAN MICROVASCULAR ENDOTHELIAL CELLS.
Toshimitsu Kawashima, Akihiko Shibaki, Hiroyuki Matsue, Hideki Nakamura, Hitoshi Kobayashi, Akira Ohkawara.
Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan.
Adhesion molecules expressed on endothelial cells are thought to play important roles in the pathogenesis of various skin disorders such as psoriasis. We first examined the effect of cytokines on the expression of adhesion molecules on human microvascular vein endothelial cells (HMVEC). HMVEC were cultured in the presence of various cytokines, and the expression of ICAM-1 and ELAM-1 were analyzed by cellular ELISA and FACS. ICAM-1 expression on HMVEC was enhanced by IFN-g, TNF-a and IL-1b. On the other hand, ELAM-1 expression was induced by TNF-a and IL-1b. We then examined the effect of various anti-psoriatic drugs on TNF-a-induced ICAM-1 expression on HMVEC. Retinoic acid enhanced TNF-a-induced expression on HMVEC, but vitamin D3 and cyclosporin did not. Thus it is shown that retinoic acid can modulate cytokine-induced adhesion molecule expression on HMVEC.
-211- Poster Discussion 9/5 Room-W
VARIOUS CHEMICAL AND PHYSICAL STIMULI INDUCE THE UNIQUE PATTERNS OF CYTOKINE mRNA EXPRESSION IN KERATINOCYTES (KC), VASCULAR ENDOTHELIAL CELLS (MVEC) AND DERMAL FIBROBLASTS (FB).
Atsushi Terunuma, Setsuya Aiba, Hachiro Tagami.
Department of Dermatology, Tohoku University School of Medicine, Sendai, Japan.
Various chemical and physical stimuli can induce unique inflammatory responses in the skin. To understand the physiological roles of three major cutaneous components, i.e., KC, MVEC and FB, in those inflammatory responses, we stimulated these cells with various chemicals, e.g., NiCl2, dinitrochlorobenzene (DNCB) and sodium lauryl sulfate (SLS), and oxidative stress by H2O2 or hyperosmolar stress by 0.5 M NaCl solution. After these treatment, we extracted total cellular RNA and examined mRNA expression levels of the following cytokines using reverse transcriptase-polymerase chain reaction (RT-PCR): IL-1a, IL-1b, IL-6, IL-8, IL-10, TNF-a, GM-CSF, TGF-a, TGF-b and ICAM-1. Treatment with 0.5 M NaCl solution decreased the expression of IL-1b mRNA by KC whereas it increased that by FB. H2O2 decreased IL-6 mRNA in KC while it increased that in MVEC and FB. The expression of IL-6 mRNA was also increased in MVEC and FB by NiCl2, which did not affect that in KC. DNCB decreased IL-6 mRNA in KC and increased that in MVEC. SLS increased IL-1b mRNA in KC and FB whereas it decreased that in MVEC. These results indicate that these three major components of the skin respond to different stimuli by unique combination of cytokine mRNA expression, and that even the same stimulus induces different patterns of cytokine mRNA expression in these cells. The orchestration of a variety of cytokines produced by cutaneous cells after different chemical or physical stress may play a crucial role in cutaneous inflammation.
-212- Poster Discussion 9/5 Room-W
IMMUNOHISTOCHEMICAL STUDY OF EXPRESSION OF VASOACTIVE INTESTINAL POLYPEPTIDE ON THE EPIDERMIS IN PATIENTS WITH ATOPIC DERMATITIS.
Maki Kakurai, Toshio Demitsu, Satoru Murata, Hideo Yaoita.
Department of Dermatology, Jichi Medical school, Tochigi-ken, Japan.
Immunoreactivity of vasoactive intestinal polypeptide ( VIP ) in the patients with atopic dermatitis ( AD ) was studied using indirect immunofluorescence. ¡¡Polyclonal VIP-immunoreactivity ( VIP-IR ) was expressed in the skin from the patients with AD. VIP-IR was strongly observed in the intercellular spaces of the epidermis, whereas VIP-IR was not observed in the normal skin epidermis. Positive immunoreaction against monoclonal VIP was observed in the intracellular spaces of the whole epidermis in the skin from the patients with AD. In the normal skin controls, monoclonal VIP-IR was strongly found in the intracellular space of the only upper epidermis . ¡¡The localization of VIP in the keratinocyte suggests a possible physiological role for VIP. The presence of VIP-IR in the keratinocyte is consistent with the possibility that this peptide may relate to the exacerbation of AD.
-213- Poster Discussion 9/5 Room-W
THE MECHANISMS OF IMPAIRED TH1-IMMUNITY IN ATOPIC DERMATITIS PATIENTS.
Takashi Matsuyama1, Kazushi Urano1, Muneo Ohkido1, Akio Ohta2, Takashi Nishimura2.
1Department of Dermatology, 2Department of Immunology, Tokai University School of Medicine, Isehara, Japan.
We have already reported that CD4+CD45RO+ T cells from atopic dermatitis(AD) patients showed lower levels of IFN-¦Ã production and cell proliferation in response to anti CD3 mAb plus IL-2 stimulation compared with healthy donor's PBMC. In the present study, we investigated the cytoplasmic cytokine expression pattern using freshly isolated PBMC. It was demonstrated that CD4+ T cells from AD patients expressed lower levels of intracellular IFN-¦Ã than that from healthy donors. No significant differences cytoplasmic IL-4 expression was demonstrated between CD4+ T cells from AD patients and healthy donor's. We also demonstrated that the decreased response to PPD in AD patients was derived from the disfunction of PPD-reactive CD4+CD45RO+ memory T cells. These results suggested that abnormally suppressed Th1 type cytokine was considered as one of the causative factors in impared cellular immunity in AD.
-214- Poster Discussion 9/5 Room-W
IMMUNOMODULATION INDUCED BY AVENE SPRING WATER IN PATIENTS WITH ATOPIC DERMATITIS.
Pierre Portales1, Patrick Dupuy3, Jocelyne Pinton2, Catherine Hernandez-Pion2, Yvon Gall3, Jacques Clot1.
1Department of Immunology, Montpellier, 2Av»Ïe Medical Spa Center, Av»Ïe, 3Pierre Fabre Research Institute, Toulouse, France.
Patients suffering from atopic dermatitis are well known to have abnormalities of some immune response parameters such as low lymphoproliferative responses to mitogens, imbalance production of interleukin-2 (IL-2), -4 (IL-4) and interferon-¦Ã(IFN-¦Ã). Clinical status of these patients can be improved by medical cure, using the poorly mineralized spring water. Furthermore, various in vitro tests for IL-2, IL-4, IFN-¦Ãand IL-10 production were shown to be modulated by incubating peripheral blood mononuclear cells (PBMC) in a culture medium containing different amounts of Av»Ïe water. In the present work, we investigated 10 patients with atopic dermatitis before and 20 days after the Av»Ïe spring water treatment. Patients treated with non systemically absorbed corticosteroids or antihistamines within 2 weeks prior to entry to the study were excluded from the study. In all patients, the lymphoproliferative response to various mitogens (phytohemagglutinin, phorbol myristate acetate, staphylococcus superantigen and anti-CD3 monoclonal antibody), and the IL-2, IFN-¦Ã, IL-4 and IL-10 production were measured in parallel experiments using a culture medium containing or not 50 % of Av»Ïe water. At the end of the water cure, the SCORAD clinical severity index was decreased by 33 %, compared to baseline (mean ¡Þ SEM : 18¡Þ 3.5 vs 26.8¡Þ4.8, p < 0.03). Av»Ïe spring water was able to enhance mitogen responsiveness in vitro, as well as increase the lymphoproliferative response in vivo which was low before treatment. The increased IL-2 production in patients before treatment decreased to normal values at the end, but no in vivo effect was noted on IL-4 and IFN-¦Ã synthesis. However, Av»Ïe spring water was able to reduce IL-4 production induced by superantigen stimulation in vitro. Similarly, Av»Ïe water decreased IL-10 production before the treatment in vitro, but increased this cytokine after 20 days of treatment in vivo. Such data are in favour of an in vitro as well as an in vivo immunomodulating effect of Av»Ïe spring water on atopic dermatitis.
-215- Poster Discussion 9/5 Room-W
RELATIONSHIP LYMPNOCYTE CYCLOSPORINE SENSITIVITY AND CLINICAL PROGRESS OF PSORIASIS.
Yoshinori Umezawa, Tsunao Oh-I, Michiyuki Koga.
Department of Dermatology, Tokyo Medical College, Tokyo, Japan.
The cyclosporine (CYA) dose in psoriasis is determined based on the blood trough level, but its efficacy differs according to the individual patient. We tried to elucidate whether the difference in efficacy might be related to the characteristics of lymphocytes in different patients. Venous blood was taken from 36 patients with psoriasis vulgaris before CYA administration. We evaluated individual CYA sensitivity by determining the CYA concentration that would give 50% suppression of lymphocyte blastogenesis in vitro (IC50). It was classified into three group by IC50 value. The psoriasis area and severity index (PASI) scores of the patients and CYA dose were observed in each group throughout the treatment. All patients were given CYA at an initial dose of 3-5 mg/kg/day, and the dose was regulated over 16 weeks. We investigated the relationship between the group IC50 value and the recovery rate of the PASI score, and also between the group IC50 value and CYA dose throughout the treatment. There was a negative correlation between IC50 and the PASI recovery rate. The CYA dose of low sensitivity group (10 ng/ml¡åIC50) increased in comparison with the high sensitivity group (0.1 ng/ml¡åIC50¡ã1.0 ng/ml). These results suggest that the efficacy of CYA may be due to the characteristics of the lymphocytes of each patients.
-216- Poster Discussion 9/5 Room-W
SKIN pH AS AN INDEX OF SKIN CLEANNESS DEGREE IN ATOPIC DERMATITIS.
Kaoru Endo, Toshiyuki Aoki.
Department of Dermatology, Habikino Hospital of Osaka Prefecture, Osaka, Japan.
We measured skin pH in patients with atopic dermatitis (AD) and healthy controls (HC) to show the low resistance of infection, especially by Staphylococcus aureus which increased with the severity of AD. The concept of defense against microorganisms due to skin surface acidity has long been a subject of controversy. 'Rheinheitgrad' in gynecology has been published by Schroder in 1928. We applied this to AD and introduced the concept of skin cleanness degree for the first time. (Subjects) We selected randomly 125 AD patients (55 males and 70 females) with a mean age of 24.4 years and 76 HC (33 males and 43 females) with a mean age of 24.4. (Methods) The point of flat electrode of handy pH Meter PE-2CN by Sensornics Japan, co. was soaked in distilled water by which we washed the subjects' surface for ten seconds, and we recorded stabilized pH. We measured skin pH on four areas, namely the cubital fossa, the forearm, the forehead and the cheek. The clinical severity of each area was classified to mild, moderate and severe. We classified the perspiration level of the subjects to much, moderate and little. Furthermore we asked HC about the complication of allergic diseases.
(Results) Skin pH in AD was 4.47 on the cubital fossa, 4.67 on the forearm, 4.77 on the forehead and 4.99 on the cheek, while skin pH in HC was 4.06, 4.33, 4.36 and 4.62 respectively, which showed skin pH was significantly higher in AD and in order of the above tested areas. Skin pH was significantly higher in females than in males and increased in proportion to the clinical severity. Skin pH was significantly higher in those subjects with little sweat. And healthy controls with the past history of allergic diseases had higher skin pH.
(Conclusion) The concept of skin cleanness degree which consists of the number of S. aureus and the skin pH can be used for the evaluation of AD.
-217- Poster Discussion 9/5 Room-W
COMPARATIVE STUDY OF BACTERIAL FLORA ON THE FACIAL SKIN OF ATOPIC DERMATITIS AND HEALTHY SUBJECTS.
Susumu Morimatsu1, Shuichi Higaki1, Masaaki Morohashi1, Takayoshi Yamagishi2.
1Department of Dermatology, Toyama Medical and Pharmaceutical University, Toyama, 2Department of Laboratory Sciences, School of Health Sciences, Faculty of Medicine, Kanazawa University, Kanazawa, Japan.
To study the differences of characteristics of Staphylococci on the skin of atopic dermatitis and healthy subjects, the bacterial flora of these two groups were sampled by a contact-plate technique. The Staphylococci were selectively isolated on mannitol salt agar medium, and identified according to 20 biochemical reactions of the API staph system. The results indicated that face of atopic dermatitis was dominantly colonized by S.aureus. The number of Staphylococci on atopic dermatitis was higher than that on the healthy subjects. The value of MIC50 of ten kinds of antibiotics to Staphylococci on atopic dermatitis were generally higher than those on healthy subjects. The suscep-tibility among these ten antibiotics tested, gentamycin was the least susceptible. It was considered that increase in number of multiple resistant-Staphylococci might be seen because of much use of them. Though the correlation between the degree of rash and skin flora was observed, it was also necessary to study the correlation between skin surface lipids and skin flora.
-218- Poster Discussion 9/5 Room-W
RESULTS OF PATCH TESTING WITH SYNTHETIC PRIMIN.
Mariko Sugiura, Ritsuko Hayakawa.
Department of Dermatology, Daiko Medical Center, Nagoya University Hospital, Nagoya, Japan.
Primin was synthesized from 3,4-dihydro-2H-pyran and guaiacol according to L.W. Bieber's method and has been distributed among members of the Japanese Society for Contact Dermatitis. We studied whether positive reaction to synthetic primin is relevant to primula dermatitis. Using Finn Chamber and Scanpor tape, we carried out 48h closed patch testing with synthetic primin 0.01%pet on 267 cases over 13 month period from February, 1996 to March, 1997. Readings were made at 24h after removal according to ICDRG recommendations. Two cases with primula dermatitis and one case having past history of primula dermatitis showed ¡Ü¡Ü reactions, and 1 case with neither primula dermatitis nor past history of primula dermatitis showed ¡Ü reaction. We considered that ¡Ü reaction might be irritant reaction. Among subjects with - and ¡Ü? reactions there were no cases with primula dermatitis nor with past history of primula dermatitis. We concluded that synthetic primin is a suitable allergen for patch testing and that optimum concentration is 0.01%pet.
-219- Poster Discussion 9/5 Room-W
STUDY FOR SKIN TESTING TO DETERMINE CAUSATIVE AGENTS OF OCCUPATIONAL CONTACT DERMATITIS.
Zhenlin Xie1, Ritsuko Hayakawa2, Mariko Sugiura2.
1Department of Hygiene, Nagoya University School of medicine, 2Department of dermatology, Daiko Medical Center, Nagoya University Hospital, Nagoya, Japan.
Over a 2 year period from January 1995 to December 1996, 66 patients (21 males and 45 females)¡¡were diagnosed as having occupational dermatitis based on their occupational exposure histories, onset situation, clinical features and the results of skin testings such as closed patch testing, open testing and scratch testing. The patients comprised 21 beauticians and a barber, 13 factory workers, 6 dental technicians and a dentists, 7 agriculture-related workers, 4 cooks, 4 nurses, and 9 others. The most frequently involved area was hands and forearms at 69.7% (46/66). Forty six cases were diagnosed as allergic contact dermatitis (ACD) , 11 cases as irritant contact dermatitis (ICD), 6 cases as contact urticaria (CU), and 3 cases as CU accompanied by ACD. There was no significant difference in atopic predisposition ratio between ACD and ICD. The causative agents were determined in 62 (93.9%) out of 66 cases: 20 (90.9%) of the 22 barber and beauticians, 13 (100%) of the factory workers, 5 (71.4%) of the 7 dentist and dental technicians, all 7 (100%) agriculture-related workers, all 4 (100%) cooks, all 4 (100%) nurses, and all 9 (100%) others. We concluded that skin testing is useful for determining the causative agents of work-related contact dermatitis.
-220- Poster Discussion 9/5 Room-W
INDUCTION OF SUBCUTANEOUS TISSUE FIBROSIS IN NEWBORN MICE BY SIMULTANEOUS APPLICATION OF TRANSFORMING GROWTH FACTOR B AND BASIC FIBROBLAST GROWTH FACTOR.
Mikio Shinozaki, Shigeru Kawara, Kazuhiko Takehara.
Department of Dermatology, Faculty of Medicine, Kanazawa University, Kanazawa, Japan.
To establish an appropriate animal model of skin fibrosis by exogenous application of growth factors, we have investigated the in vivo effects of subcutaneous coinjection of TGF-beta and other growth factors such as b-FGF,PDGF,and EGF in new born mice. Striking induction of granulation tissue and fibrosis was observed at the injection site after 3 consecutive days or 7 consecutive days coinjection of TGF-beta and b-FGF compared with that of injected with TGF-beta alone. Further, this change persisted for at least 2 weeks. Thus, we succeeded in establishing an animal model of skin fibrotic disorders.
-221- Poster Discussion 9/5 Room-W
DETECTION OF SPECIFIC ANTIBODIES TO VASCULAR ENDOTHELIAL CELLS IN THE SERA OF PATIENTS WITH BEHCET'S DISEASE.
M.S. Cha1, Dongsik Bang1, E.-S. Lee2, S. Lee2, Kwan H. Lee1.
1Department of Dermatology, Yonsei University College of Medicine, Seoul, 2Department of Dermatology, Ajou University School of Medicine, Suwon, Korea.
Autoantibodies that bind to endothelial cells have been identified in patients with several forms of vasculitis. We have detected circulating antibodies to cultured human dermal microvascular endothelial ceIIs(HDMEC) in the sera of Behcet's disease(BD) and other vascular disorders including systemic lupus erythematosus(SLE) by ELISA, SDS-PAGE and Western blotting and have observed the difference in activity of antibody from BD compared with that from other vascular disorders. IgM antibody reacted with HDMEC surface antigen in the patients with BD. In SLE, both lgG and lgM antibodies reacted with endothelial cell surface antigens. In Western blotting, IgG-positive BD serum reacted with high molecular weight antigen greater than 100kDa. IgG-positive SLE serum reacted with two protein bands of 30kDa and 66kDa. BD serum containing anti-endothelial cell lgM antibody reacted with 44kDa and SLE serum with 8lkDa. In dot blotting and ELISA using 44kDa HDMEC surface antigen, only lgM-positive BD sera reacted with the antigen. The results indicate that there are anti-endothelial cell lgM antibodies in the sera of BD, reacting with 44kDa endothelial cell surface antigen and suggest that vasculitis in BD may be caused by vascular damage due to specific antibodies against endothelial cells.
-222- Poster Discussion 9/5 Room-W
GENETIC ANALYSES OF SKIN TUMOURS DEVELOPED IN A PATIENT WITH HEREDITARY NONPOLYPOSIS COLORECTAL CANCER (HNPCC).
Minoru Takata1, Naohito Hatta1, Fumiaki Shirasaki1, Kazuhiko Takehara1, Hiromi Nagasaki2, Yoshimitsu Akiyama2, Yasuhito Yuasa2.
1Department of Dermatology, Kanazawa University School of Medicine, kanazawa, 2Department of Hygiene and Oncology, Tokyo Medical and Dental University School of Medicine, Tokyo, Japan.
Defective mismatch repair underlies the genome instability in a patient with HNPCC. We carried out genetic analyses of peculiar multiple keratotic tumours (KT), a keratoacanthoma (KA), and disseminated superficial actinic porokeratosis (DSAP) developed in a patient with HNPCC. PCR amplifi-cation for 7 separate microsatellites revealed prominent microsatellite instability (MI) in 6 of 7 KTs and a KA, suggesting that replication errors (RER) play a role in the development of these tumours. However, PCR-SSCP analysis of 2 MI+ KTs did not reveal any mutation in TGF-¦Â type II receptor gene, the known target for RER normally found in MI+ gastro-intestinal cancers. By contrast, 2 DSAP lesions examined did not show MI, and there seemed to be no pathogenic link between DASP and RER.
-223- Poster Discussion 9/5 Room-W
ANTI CANCER DRUG-INDUCED APOPTOSIS IN CULTUERD MELANOMA CELLS.
Hajime Takagi1, Kazufumi Yoneda2, Hideki Kamiya1, Miyuki Funahashi1, Yasuo Kitajima1.
1Department of Dermatology, Gifu University, School of Medicine, 2Depatment of Dermatology, Gifu Municipal Hospital.
Induction of apoptosis in tumor cells is an important mechanism of chemotherapy-induced cell death. We have evaluated the effects of a variety of anti cancer drugs on apoptosis in melanoma cells. To determine an apoptotic morphology, cells were stained with the Hoechst 33258. DNA fragmentation assay and flow cytometry were performed. Both strains we used showed the generation of apoptosis in dose- and time-dependent manner for CDDP. But sensitivity varied a lot among the strains and drugs. Twelve hours after stimulated with CDDP for 1 hour, we observed nucleic fragmentation. After 24 hours, a DNA ladder was shown. Both strains were found to be less-sensitive to IFN-beta. The mechanisms for anti cancer drugs-induced apoptosis, including the combination of CDDP and IFN-beta were also examined.
-224- Poster Discussion 9/5 Room-W
HISTOGENESIS OF MIXED TUMOR OF SKIN, APOCRINE TYPE: IMMUNOHISTOCHEMICAL STUDY OF KERATIN EXPRESSION.
Takamitsu Ohnishi, Shinichi Watanabe.
Department of Dermatology, Teikyo University School of Medicine, Tokyo, Japan.
To investigate the histogenesis of mixed tumor of the skin. apocrine type, the immunophenotypes of 10 cases were examined using 19 different monoclonal anti-keratin antibodies, and antibodies against carcinoembryonic antigen (CEA) and involucrin. With light microscopy, four epithelial elements in this tumor were characterized: tubular branching structures with lumina lined by cuboidal epithelium, those with lumina lined by columnar epithelium, keratinous cysts, and solid aggregates of epithelial cells. The immunohistochemical patterns of cytokeratin expression suggested that cuboidal and columnar cells differentiated respectively toward the ductal and secretory cells of apocrine glands, while keratinous cysts revealed follicular infundibular differentiation. Furthermore, CEA expression, a marker for sweat gland differentiation, was present not only on tubules' luminal surfaces but also on the inner surfaces of keratinous cysts. The simultaneous co-expression of CEA and cytokeratins specific for follicular infundibulum in the keratinous cysts, while perplexing, suggested that keratinous cysts may contain some cells differentiating toward the intrafollicular portion of apocrine ducts which enter infundibulae, rather than eccrine ducts which have no infundibular association. We conclude that apocrine type of mixed tumors of the skin demonstrate differentiation toward all components of apocrine units.
-225- Poster Discussion 9/5 Room-W
THE EFFECT OF UVB IRRADIATION ON APOPTOSIS IN ARSENIC INDUCED BOWEN'S DISEASE.
Chee-Yin Chai1, Hsin-Su Yu2, Shih-Chung Ko2.
1Department of Pathology,Kaohsiung Medical College,Kaohsiung, 2Department of Dermatology,Kaohsiung Medical College, Kaohsiung, TAIWAN.
The aim of this study is to evaluate the effect of UVB irradiation on apoptosis in arsenic induced Bowen's disease. Material and Methods: Four Bowen's disease with skin phototype of IV-V, all from endemic area of arsenical skin cancer. Biopsied specimen were obtained before irradiation and two weeks after UVB irradiation with a total dose of 750mJ/cm2 (75mJ/cm2 x 5times/wk). The skin obtained from two patients without skin disease were taken as normal control. Immunohistochemical stain of Bcl-2 (1:100, DAKO, Denmark) was performed and apoptosis was detected by using TUNEL(TdT-mediated dUTP nick end labeling) method. The apoptotic index was counted under high power magnification of light microscopy. Results: The expression of Bcl-2 is decreased after the Bowen's disease is irradiated with the total dose of 750mJ/cm2. In addition, the apoptotic index of the preirradiated specimen (10.78 + 4.65)(p<0.05, paired t-test). Conclusion: In this study, we clearly demonstrate that UVB irradiation inducing apoptotic effect on the arsenic induced Bowen's disease as shown by
decreased Bcl-2 expression and increased the apoptotic index. The apoptotic
effect of UVB irradiation on arsenic induced Bowen's disease provides us with
other areas of study. For example, the possible use of UVB in inhibiting
proliferation of the neoplastic epidermal cells, which may help to prevent
multicentric Bowen's disease progress to overt skin cancer. Undoubtedly,
the possible dangers of UV carcinogenesis should also be considered.
-226- Poster Discussion 9/5 Room-W
SEARCH FOR HUMAN T-CELL LEUKEMIA VIRUS-I ANTIBODY IN THE PEOPLE OF NORTHEAST DISTRICT OF CHINA.
Geng Long1,3, Zai Ning3, Xiao Yi3, Song Fang-Ji3, Akito Tanaka1, Lou Hong2, Shunro Sonoda2, Tamotsu Kanzaki1.
1Department of Dermatology 2Department of Virology, Kagoshima University Faculty of Medicine, Kagoshima, Japan. 3Department of Dermatology, China Medical University, Shenyang, China.
Human T-lymphotropic virus-I (HTLV-I) is thought to be the causative agent for adult T-cell leukemia(ATL). This virus infection is endemic in several small areas in the world. Southwestern Japan is highly prevalent with this virus but very few, if any, in Korean Peninsula and Chinese continent. In order to find the roots of this virus and ancestors of aboriginal Japanese HTLV-1 carriers (Kumaso and Ainu ?), we examined anti-HTLV-I antibody in sera from more than 1600 Chinese inhabitants in the Northeast District of China by the methods of gelatin particle agglutination (PA), immunofluorescence (IF), ELISA and Western bloot analysis. Surprisingly, no one had anti-HTLV-I antibody in 1600 persons. This suggests that Japanese HTLV-1 carriers were not related with Korean, Chinese nor Manchurian.
-227- Poster Discussion 9/5 Room-W
INHIBITION OF POLY-L-LYSINE ON THE GROWTH OF CULTURED CELL LINES.
Kenichiro Chikakane,Hiroko Satoh,Yuka Nakamura,Shinichi Watanabe, Hisashi Takahashi.
Department of Dermatology, Teikyo University School of Medicine, Tokyo, Japan.
The microbicidal activities of poly-L-lysine against Escherichia coli were documented to be dependent on the degree of polymerization of lysine residues. Highly polymerized poly-lysine inhibited the cell growth more potently. We already reported the different growth inhibitions of poly-L-lysines in various molecular weights against yeast cells. In this study,¡¡we investigated the growth inhibition of poly-L-lysines against LL/2(muse lung cancer) and 3T3(muse normal fibroblast ) using Alamar Blue assay. The results were that similar growth inhibitions were recognized against both cells were follows by poly-L-lysines of more than 15 kDa. While more potent inhibition was recognized for LL/2 as compared to 3T3 within 4-15 kDa.
-228- Poster Discussion 9/5 Room-W
SKiN PHYSIOLOGY: MEN VS. WOMEN.
E. Tur, S. Brenner.
Dept of Dermatology, Sourasky Medical Center, Tel Aviv, Israel.
The skin reflects differences between women and men. Genetic and hormonal differences affect skin structure and function, resulting in variations between women and men and causing these gender variations to change with age. In addition, exogenous factors play a role, since they may differ according to differences in life style between the sexes. It is now possible to substantiate impressions, like the impression that women's skin is lighter than men's, and to distinguish between fact and fantasy. During the last few decades, quantitative measurement techniques used in dermatological research have improved substantially, providing means of objective evaluation of skin function and characteristics. Differences between women and men, however, were not yet systematically studied, and most of the available data on differences between genders are a by-product of studies having a different focus. This poster outlines the various aspects of physiological differences in the skin of women and men, with special emphasis on structural and anatomical characteristics, biochemical composition, mechanical properties, functional differences, differences in response to exogenous triggers, cutaneous microvasculature, sensory functions, skin color, hormonal influence, and the pilosebaceous unit. Since the maintenance of skin health is an intricate orchestration of many variables, additional studies of skin physiology and its relation to gender should prove useful in identifying some of these variables and in the understanding of their importance. We hope this work will trigger further investigation of the subject.