The human eccrine sweat gland is present on most body sites and is crucial for thermoregulation. Yet, little is known on the mechanisms that govern its function and its morphogenesis. The main reason for the lack in research with regards to the human eccrine gland is the difficulty in isolation and maintenance of the glands and cells in vitro. Only one other cell line derived from the human eccrine gland has ever been reported, the NCLSG3 cell line. NCL-SG3 cells do not however, function like native eccrine secretory coil cells, and thus a better cell model was required. In this project, a human eccrine secretory coil cell line, the EC23 cell line, was developed, along with 8 clones derived from said cell line. EC23 cells and their clones express a panel of markers characteristic of the human eccrine sweat gland secretory coil cells. Furthermore, calcium fluxes can be elicited by cholinergic stimulation of the cells suggesting retention of the native secretory cell phenotype unlike NCL-SG3 cells. The EC23 cell line is also responsive to adrenergic stimuli to a higher degree than NCL-SG3 cells, especially clone 2, however all the cell lines responded significantly less than primary eccrine secretory coil cells upon isoproterenol stimulation. It was also found that the mesenchyme has a crucial effect in determining the formation of eccrine like down-growths in Matrigel organotypic models seeded with EC23 cells, where organotypics made with adult fibroblasts failed to form down-growths in comparison to neonatal fibroblasts. Furthermore, the co-culture of EC23 cell with keratinocytes enhanced the amount of downgrowth. EC23 cells have the capacity to form branching structures that resemble native eccrine glands in GFR Matrigel supplemented with EGF and EDA, and to a lesser extent BMP4. In conclusion it was demonstrated that the EC23 cells can be used as a model to study the human eccrine gland, in particular the secretory coil.

The final publication is available at http://dx.doi.org/10.1111/pcmr.12297. Natsuko Okamoto et al. A melanocyte–melanoma precursor niche in sweat glands of volar skin. Pigment Cell & Melanoma Research. Volume 27, Issue 6, pages 1039–1050, November 2014 / 京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12890号 / 論医博第2090号 / 新制||医||1007(附属図書館) / 31644 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 羽賀 博典, 教授 鈴木 茂彦 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

<p>The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease.</p><p>Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells.</p><p>S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. </p><p>Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity.</p><p>Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.</p>

Secretoglobins is a newly described polypeptide family that has gained a lot of interest in human cancer and inflammation research. Although the first secretoglobin polypeptide was discovered more than 30 years ago, their physiological function is still not known. The aim of this thesis was to study the expression of secretoglobins in normal and neoplastic human cells and tissues, and to clarify their possible involvement in human cancer. We established sensitive and specific quantitative real-time RT-PCR assays for uteroglobin, lipophilins A, B, C, mammaglobin, HIN-1, and UGRP1, and developed specific antibodies for lipophilin B and mammaglobin. By using quantitative real-time RT-PCR, immunohistochemistry, Western blotting, and in situ hybridization, we studied secretoglobin expression in normal and neoplastic cells and tissues. In normal tissues, real-time RT-PCR analysis showed high expression of mammaglobin in skin. The mammaglobin expression in skin tissue was further confirmed by in situ hybridization and immunohistochemistry, and the expression was shown to be localized to the coiled gland cells of the eccrine sweat glands and the apocrine sweat glands. In addition, we showed by using Western blotting, that mammaglobin was secreted into perspiration from the eccrine sweat glands. In pituitary gland, immunohistochemical analysis showed that lipophilin B was expressed by approximately half of the cells in the anterior pituitary. By using quantitative real-time RT-PCR it was shown that both lipophilins B and C mRNA were expressed in the pituitary gland, therefore we suggested that lipophilins B and C form heterodimers in human pituitary. In neoplastic tissues, real-time RT-PCR analysis showed dysregulated secretoglobin expression in lung tumors, with down-regulation of uteroglobin and frequent up-regulation of lipophilins A, B, C, and mammaglobin. Immunohistochemical analyses showed down-regulation of mammaglobin in cylindromas versus non-neoplastic eccrine sweat glands and of lipophilin B in pituitary adenomas versus non-neoplastic anterior pituitary. The majority of investigated cell lines showed low, or most often, lack of secretoglobin expression. Nevertheless, it has been shown that mammaglobin is over-expressed in human breast carcinomas. However, ectopic over-expression of mammaglobin and/or lipophilin B had no appreciable effect on cell proliferation rates of Hs578T breast carcinoma cells in vitro. This does not exclude the possibility that secretoglobins could confer some advantage to tumor cells in vivo, but, it indicates that the reported over-expression of mammaglobin is an epiphenomenon not causally involved in breast carcinogenesis. In summary, our major findings were that mammaglobin was expressed and secreted by the sweat glands of the skin and lipophilin B was expressed by the anterior pituitary gland; and, that expression of mammaglobin and lipophilin B were down-regulated in tumors derived from the same tissues, i.e, in cylindromas and pituitary adenomas, respectively. Furthermore, ectopic over-expression of mammaglobin and lipophilin B in breast carcinoma cells had no appreciable effect on cell proliferation rates in vitro.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease. Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells. S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity. Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.

A concentração de eletrólitos no suor e a taxa de sudorese estão bem descritas em atletas que treinam na terra e em não atletas. Embora exista alguma pesquisa acerca das respostas termorregulatórias de nadadores, não foi encontrada alguma que tenha verificado a concentração eletrolítica no suor. O objetivo deste estudo foi comparar a taxa de sudorese e a composição eletrolítica no suor de nadadores, corredores e não atletas. Dez nadadores (23 ± 3 anos, 179 ± 6 cm, 75 ± 7 kg), dez corredores (26 ± 3 anos, 178 ± 4 cm, 74 ± 7 kg) e dez não atletas (26 ± 3 anos, 178 ± 6 cm, 79 ± 8 kg) pedalaram em um ciclo ergômetro (CYBEX, The bike,USA) por 30 min a 32oC e 60% de umidade relativa. O esforço estabelecido foi de 10% abaixo do segundo limiar anaeróbico. O suor foi coletado por meio de adesivos absorventes (Tegaderm 3582, 3M, Neuss, Germany) colocados na escápula direita após a limpeza apropriada da pele. Após o término do exercício, os adesivos foram colocados em uma seringa e o suor colocado em um tubo. As concentrações de sódio (Na+), cloro (Cl-) e potássio (K+) no suor foram analisadas através de um seletor de ions (AVL 9180). Média e desvio padrão foram calculados através da estatística descritiva. As diferenças foram estabelecidas usando ANOVA fatorial e teste post hoc de Tukey. A taxa de sudorese foi maior no grupo dos corredores. As concentrações de Na+ e Cl- foram menores no grupo dos corredores do que nos outros grupos. A concentração de K+ não mostrou diferença entre os grupos. As concentrações eletrolíticas do suor dos corredores estão de acordo com os valores previamente publicados para atletas. No entanto, as concentrações de Na+ e Cl- no suor dos nadadores foram similares as dos não atletas, provavelmente porque a regulação da sudorese dentro da água é diferente daquela que ocorre fora dela. / Sweat electrolyte concentration and sweat rate have been reported in athletes trained on land and in nonathletes. Although there is some research on thermoregulatory responses in swimmers, none of them have addressed the sweat electrolyte concentration. The purpose of this study was to compare sweat electrolyte concentration and sweat rate among swimmers runners and non-athletes. Ten swimmers (age 23 ± 3 years, height 179 ± 6 cm, body mass 75 ± 7 kg), 10 runners (age 26 ± 3 years, height 178 ± 4 cm, body mass 74 ± 7 kg) and 10 nonathletes (age 26 ± 3 years, height 178 ± 6 cm, body mass 79 ± 8 kg) cycled on a eletromagnetic-braked ergometer (CYBEX, The bike,USA) for 30 min at 32oC and 60% relative humidity. The work rate was set at 10% below second anaerobic threshold. Sweat was collected by absorbent patches (Tegaderm 3582, 3M, Neuss, Germany) from the scapula. All patches were placed on the right hand side of the body after appropriate cleaning of the skin. After exercise was completed, patches were set into a syringe and sweat was squeezed in a tube. Sweat was analysed for sodium (Na+), cloride (Cl-) and potassium (K+) concentration by ion selector (AVL, 9180). Mean and SD are given as descriptive statistics. Differences were established using ANOVA factorial and Tukey post hoc test. Sweat rate of runners were higher than that of swimmers and nonathletes. Na+ and Cl- concentrations of sweat in the runners group were different from that of the swimmers and nonathletes. K+ concentration did not show difference among the 3 groups. Sweat electrolyte concentrations of runners were within the normal range for athletes. However, sweat Na+ and Cl- concentrations of swimmers were more similar to that of nonatlhetles, and this is probably because regulation of sweating during exercise in water is different from that during exercise on land.

A concentração de eletrólitos no suor e a taxa de sudorese estão bem descritas em atletas que treinam na terra e em não atletas. Embora exista alguma pesquisa acerca das respostas termorregulatórias de nadadores, não foi encontrada alguma que tenha verificado a concentração eletrolítica no suor. O objetivo deste estudo foi comparar a taxa de sudorese e a composição eletrolítica no suor de nadadores, corredores e não atletas. Dez nadadores (23 ± 3 anos, 179 ± 6 cm, 75 ± 7 kg), dez corredores (26 ± 3 anos, 178 ± 4 cm, 74 ± 7 kg) e dez não atletas (26 ± 3 anos, 178 ± 6 cm, 79 ± 8 kg) pedalaram em um ciclo ergômetro (CYBEX, The bike,USA) por 30 min a 32oC e 60% de umidade relativa. O esforço estabelecido foi de 10% abaixo do segundo limiar anaeróbico. O suor foi coletado por meio de adesivos absorventes (Tegaderm 3582, 3M, Neuss, Germany) colocados na escápula direita após a limpeza apropriada da pele. Após o término do exercício, os adesivos foram colocados em uma seringa e o suor colocado em um tubo. As concentrações de sódio (Na+), cloro (Cl-) e potássio (K+) no suor foram analisadas através de um seletor de ions (AVL 9180). Média e desvio padrão foram calculados através da estatística descritiva. As diferenças foram estabelecidas usando ANOVA fatorial e teste post hoc de Tukey. A taxa de sudorese foi maior no grupo dos corredores. As concentrações de Na+ e Cl- foram menores no grupo dos corredores do que nos outros grupos. A concentração de K+ não mostrou diferença entre os grupos. As concentrações eletrolíticas do suor dos corredores estão de acordo com os valores previamente publicados para atletas. No entanto, as concentrações de Na+ e Cl- no suor dos nadadores foram similares as dos não atletas, provavelmente porque a regulação da sudorese dentro da água é diferente daquela que ocorre fora dela. / Sweat electrolyte concentration and sweat rate have been reported in athletes trained on land and in nonathletes. Although there is some research on thermoregulatory responses in swimmers, none of them have addressed the sweat electrolyte concentration. The purpose of this study was to compare sweat electrolyte concentration and sweat rate among swimmers runners and non-athletes. Ten swimmers (age 23 ± 3 years, height 179 ± 6 cm, body mass 75 ± 7 kg), 10 runners (age 26 ± 3 years, height 178 ± 4 cm, body mass 74 ± 7 kg) and 10 nonathletes (age 26 ± 3 years, height 178 ± 6 cm, body mass 79 ± 8 kg) cycled on a eletromagnetic-braked ergometer (CYBEX, The bike,USA) for 30 min at 32oC and 60% relative humidity. The work rate was set at 10% below second anaerobic threshold. Sweat was collected by absorbent patches (Tegaderm 3582, 3M, Neuss, Germany) from the scapula. All patches were placed on the right hand side of the body after appropriate cleaning of the skin. After exercise was completed, patches were set into a syringe and sweat was squeezed in a tube. Sweat was analysed for sodium (Na+), cloride (Cl-) and potassium (K+) concentration by ion selector (AVL, 9180). Mean and SD are given as descriptive statistics. Differences were established using ANOVA factorial and Tukey post hoc test. Sweat rate of runners were higher than that of swimmers and nonathletes. Na+ and Cl- concentrations of sweat in the runners group were different from that of the swimmers and nonathletes. K+ concentration did not show difference among the 3 groups. Sweat electrolyte concentrations of runners were within the normal range for athletes. However, sweat Na+ and Cl- concentrations of swimmers were more similar to that of nonatlhetles, and this is probably because regulation of sweating during exercise in water is different from that during exercise on land.

A concentração de eletrólitos no suor e a taxa de sudorese estão bem descritas em atletas que treinam na terra e em não atletas. Embora exista alguma pesquisa acerca das respostas termorregulatórias de nadadores, não foi encontrada alguma que tenha verificado a concentração eletrolítica no suor. O objetivo deste estudo foi comparar a taxa de sudorese e a composição eletrolítica no suor de nadadores, corredores e não atletas. Dez nadadores (23 ± 3 anos, 179 ± 6 cm, 75 ± 7 kg), dez corredores (26 ± 3 anos, 178 ± 4 cm, 74 ± 7 kg) e dez não atletas (26 ± 3 anos, 178 ± 6 cm, 79 ± 8 kg) pedalaram em um ciclo ergômetro (CYBEX, The bike,USA) por 30 min a 32oC e 60% de umidade relativa. O esforço estabelecido foi de 10% abaixo do segundo limiar anaeróbico. O suor foi coletado por meio de adesivos absorventes (Tegaderm 3582, 3M, Neuss, Germany) colocados na escápula direita após a limpeza apropriada da pele. Após o término do exercício, os adesivos foram colocados em uma seringa e o suor colocado em um tubo. As concentrações de sódio (Na+), cloro (Cl-) e potássio (K+) no suor foram analisadas através de um seletor de ions (AVL 9180). Média e desvio padrão foram calculados através da estatística descritiva. As diferenças foram estabelecidas usando ANOVA fatorial e teste post hoc de Tukey. A taxa de sudorese foi maior no grupo dos corredores. As concentrações de Na+ e Cl- foram menores no grupo dos corredores do que nos outros grupos. A concentração de K+ não mostrou diferença entre os grupos. As concentrações eletrolíticas do suor dos corredores estão de acordo com os valores previamente publicados para atletas. No entanto, as concentrações de Na+ e Cl- no suor dos nadadores foram similares as dos não atletas, provavelmente porque a regulação da sudorese dentro da água é diferente daquela que ocorre fora dela. / Sweat electrolyte concentration and sweat rate have been reported in athletes trained on land and in nonathletes. Although there is some research on thermoregulatory responses in swimmers, none of them have addressed the sweat electrolyte concentration. The purpose of this study was to compare sweat electrolyte concentration and sweat rate among swimmers runners and non-athletes. Ten swimmers (age 23 ± 3 years, height 179 ± 6 cm, body mass 75 ± 7 kg), 10 runners (age 26 ± 3 years, height 178 ± 4 cm, body mass 74 ± 7 kg) and 10 nonathletes (age 26 ± 3 years, height 178 ± 6 cm, body mass 79 ± 8 kg) cycled on a eletromagnetic-braked ergometer (CYBEX, The bike,USA) for 30 min at 32oC and 60% relative humidity. The work rate was set at 10% below second anaerobic threshold. Sweat was collected by absorbent patches (Tegaderm 3582, 3M, Neuss, Germany) from the scapula. All patches were placed on the right hand side of the body after appropriate cleaning of the skin. After exercise was completed, patches were set into a syringe and sweat was squeezed in a tube. Sweat was analysed for sodium (Na+), cloride (Cl-) and potassium (K+) concentration by ion selector (AVL, 9180). Mean and SD are given as descriptive statistics. Differences were established using ANOVA factorial and Tukey post hoc test. Sweat rate of runners were higher than that of swimmers and nonathletes. Na+ and Cl- concentrations of sweat in the runners group were different from that of the swimmers and nonathletes. K+ concentration did not show difference among the 3 groups. Sweat electrolyte concentrations of runners were within the normal range for athletes. However, sweat Na+ and Cl- concentrations of swimmers were more similar to that of nonatlhetles, and this is probably because regulation of sweating during exercise in water is different from that during exercise on land.

Palmoplantar pustulosis (PPP) is a chronic disease of unknown pathogenesis. Most of the patients were smokers. High prevalence of a number of autoimmune diseases was observed among the patients (thyroid disease 14%, gluten intolerance 8%, diabetes type 1 3%). Eosinophils and neutrophils were found in large numbers in the pustules. Massive infiltrates of lymphocytes and mast cells in the dermis below the pustule and an abnormal acrosyringial pattern indicate that the acrosyringium is the target for the inflammation. Immunofluorescence (IF) revealed decreased innervation of the sweat gland, outward migration of substance P-positive granulocytes in the acrosyringium and an increased number of contacts between mast cells and nerve fibres in the dermis. Distributions of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied, since they regulate the level of acetylcholine, the main inducer of sweating. The most intense AChE-like immunoreactivity (LI) was observed in the acrosyringium in the lowest part of the stratum corneum, corresponding to the site of the pustule in PPP. ChAT-LI in granulocytes and AChE-LI in mast cells were demonstrated, which may have implications for inflammatory processes in general. Nicotinic acetylcholine receptors (nAChR) are activated by acetylcholine but also by nicotine. Immunohistochemstry of α-3 and α-7 subtypes of the nAChRs showed that the nAChR expression in healthy skin was influenced by smoking. A highly abnormal α-7 nAChR distribution in PPP skin was observed. The levels of nAChR antibodies were elevated in 42% of the PPP sera, and 68% of these sera gave specific endothelial IF in the papillary dermis in skin from non-smokers. Positive IF in the acrosyringium was also noted in skin from smokers. Conclusions: Smoking seems to induce up-regulation of an antigen in palmar skin. The results indicate that PPP is an autoimmune disease and that nicotine might have a role in the onset of the inflammation.