Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

<p>Kontaktna senzibilizacija, kao stanje specifične reaktivnosti kože, može biti egzogeni pokretač psorijaze. Rezultat je interakcije endogenih i egzogenih činioca. Jedan od najznačajnijih endogenih faktora kome se pripisuje uloga faktora rizika jeste pol. Cilj istraživanja bio je da se kod obolelih od psorijaze utvrdi učestalost i distribucija kontaktne senzibilizacije u odnosu na pol, razlike u distribuciji kontaktne senzibilizacije po polu&nbsp; između osoba obolelih od psorijaze i osoba pod sumnjom na postojanje kontaktnog alergijskog dermatitisa, povezanost kontaktne senzibilizacije i težine kliničkog nalaza i povezanost kontaktne senzibilizacije i dužine trajanja oboljenja. Hipoteza istraživanja polazila je od pretpostavke da je kontaktni alergijski dermatitis redak kod obolelih od psorijaze, jer hronična inflamacija u koži smanjuje njenu sposobnost specifične senzibilizacije a da je veća učestalost kontaktne senzibilizacije kod osoba ženskog pola rezultat egzogenog faktora tj.ekspozicije, kao i da je učestalost kontaktne senzibilizacije u pozitivnoj korelaciji sa težinom i dužinom trajanja oboljenja. Istaživanje je sprovedeno kod 176 ispitanika koji su bili podeljeni u dve grupe. Eksperimentalnu grupu su činili oboleli od psorijaze, a kontrolnu grupu ispitanici upućeni na alergolo&scaron;ko testiranje pod sumnjom na postojanje kontaktnog alergijskog dermatitisa. Obolelima od psorijaze je ocenjivana težine oboljenja PASI skorom, a svi ispitanici bili su alergolo&scaron;ki testirani epikutanim -pač testom. Pozitivni rezultati alergolo&scaron;kog testiranja su analizirani, u cilju utvrđivanja kliničke relevantnosti istih. Ispitanicima u grupi obolelih od psorijaze je testom skarifikacije određivano prisustvo ili odsustvo Koebnerovog izomorfnog podražajnog fenomena. Istraživanjem je utvrđeno, da se kontaktno reagovanje kod obolelih od psorijaze na najmanje jedan standardni alergen nije statistički značajno razlikovalo od reagovanja osoba kod kojih je postavljena sumnja na postojanje kontaktnog alergijskog dermatitisa, ali je senzitivnost izražena kroz prosečan broj pozitivnih testova po jednom ispitaniku bila statistički značajno niža kod obolelih od psorijaze. Poređenjem kontaktnog reagovanja mu&scaron;karaca i žena nisu utvrđene značajna razlike u reagovanju u odnosu na pol. Težina oboljenja nije uticala na učestalost kontaktne senzibilizacije kod obolelih od psorijaze, ali je učestalost kontaktne senzibilizacije bila u pozitivnoj korelaciji sa dužinom trajanja bolesti. Niža stopa kontaktnog reagovanja utvrđena je kod osoba obolelih od psorijaze koji su imali pozitivan Koebnerov fenomen u trenutku ispitivanja.</p> / <p>Contact sensitization as a state of specific skin reactivity may provoke psoriasis resulting from an interaction between extrinsic and intrinsic factors. One of the most significant factors characterized, as a risk factor, is the sex. The aim of this study was to determinate the frequency and distribution of contact sensitization in patients with&nbsp; psoriasis with respect to their sex, as well as the differences in the distribution of contact sensitization in both sexes, namely&nbsp; with patients with psoriasis and patients&nbsp; suspected to allergic contact dermatitis. Consequently, appropriate attention was paid to the correlation between contact sensitization and disease severity, and between contact sensitization and disease duration. Hypothesis were based on the assumption that allergic contact dermatitis is rare in&nbsp; patients with psoriasis, as a chronic inflammation of the skin reduces its ability specific sensitization, as well as that the higher frequency of contact sensitization in females represent a result of exogenous factors, i.e. exposition, and finally that there is a positive correlation between the incidence of contact sensitization and the disease severity, and contact sensitization and the disease duration. The study included 176 patients. They were divided into two groups: the study group included patients with psoriasis, while the control group included patients referred for allergy testing, since they were suspected to allergic contact dermatitis. The severity of psoriasis was evaluated by PASI score. Thereafter,&nbsp; each patient underwent patch testing. The positive results of patch tests were evaluated with the aim to define their clinical relevancy. Subjects from the group of psoriatic patients passed scarification test carried out to indicate the presence or absence of K&ouml;ebner isomorphic phenomenon. This research led us to the conclusion that the positive reaction of psoriatic patients to at least one standard allergen did not indicate a statistically significant different reaction when compared to the reaction of patients suspected to allergic contact dermatitis. From the other side, the sensitivity expressed through the average number of positive&nbsp; tests per one&nbsp; tested&nbsp; patients was&nbsp; significantly lower in&nbsp; patients&nbsp; with psoriasis. Comparison of the contact response of men and women showed no significant differences in response with respect to&nbsp; their sex. The&nbsp; disease severity did not influence the frequency of contact sensitization in patients&nbsp; with&nbsp; psoriasis. At&nbsp; the same time, the frequency of contact sensitization stood in a positive correlation with the duration of disease. The lower rate of contact sensitization was found in patients with psoriasis who have had a&nbsp; positive K&ouml;ebner phenomenon at&nbsp; the time of testing.</p>

The adaptor protein Shb has been implicated in the signaling of several tyrosine kinase receptors and previous studies have suggested a role for Shb in the signal transduction of T cells. Shb associates with the T cell receptor (TCR) and partakes in the signal propagation of activated T lymphocytes. In order to explore Shb’s influence on TCR signaling in vivo, T cell development and function was studied in a Shb knockout mouse. The loss of Shb led to aberrant TCR signaling in both thymocytes and peripheral CD4+ TH cells, with elevated basal phosphorylation of key components in the signal cascade. Shb was found to be dispensable for thymocyte development, but its absence resulted in a TH2 bias in in vitro stimulated peripheral CD4+ TH cells. As imbalances in TH2 responses are linked to allergic diseases, we further explored Shb’s role in immune regulation in a mouse model of atopic dermatitis. Shb knockout mice exhibit more aggravated signs of atopic dermatitis, including increased immune cell recruitment to the affected areas and elevated mRNA levels of typical TH2 cytokines. The effect of Shb on hematopoiesis in general was determined by examining populations of long-term hematopoietic stem cells (LT-HSCs) and hematopoietic progenitor cells in bone marrow of Shb knockout and wild type mice. Shb deficient bone marrow was found to contain significantly fewer relative numbers of LT-HSCs due to a proliferative defect. The reduced cell cycle activity of Shb LT-HSCs could further be linked to an abnormal regulation of the focal adhesion kinase/Rac1/p21-activated kinase pathway. Since alterations in LT-HSC proliferative abilities may have implications for leukemia development, BCR-Abl induced myeloid neoplasia was investigated in the absence of Shb. Shb deficiency confers a more aggressive progression of BCR-Abl induced myeloid neoplasia characterized by an increased peripheral blood neutrophilia and a deregulated cytokine profile. In addition, focal adhesion kinase and STAT3 signaling is hyperactivated in Shb knockout leukemic cells. In conclusion, Shb appears to be a multifunctional signaling mediator that controls several responses in hematopoietic cells, under homeostatic as well as disease conditions.

ABSTRACT Introduction and Rationale: Atopic Dermatitis (AD) is a common skin condition, characterized by markedly pruritic eczematous lesions, that most often presents in childhood. The majority of children diagnosed with AD will have mild disease and will first present with symptoms to a primary care provider (PCP), however approximately 85% of pediatricians only provide limited initial care followed by a referral to dermatology (Eichenfield et al., 2015). While there are specialty care based treatment guidelines for childhood AD, there are no guidelines available that specifically address primary care management of childhood AD. Purpose and Objective: The primary purpose of this DNP project is to develop an evidence-based clinical practice guideline (CPG) for pediatric PCPs. The secondary purpose is to develop a corresponding atopic dermatitis action plan (ADAP) to be used by children and parents. The objective is to equip PCPs to better manage children with AD in the primary care setting and to guide patients and parents in the importance of daily control measures and in the individualized treatment plan prescribed by the PCP. Methods: The Appraisal of Guidelines for Research & Evaluation II (AGREE II) framework and Social Cognitive Theory (SCT) serve as the theoretical frameworks for CPG and ADAP development. The American Academy of Pediatrics (AAP) process for evidence based policy setting is used as a model for key action statement development. Results: Evaluation of the CPG was completed using the AGREE II tool, a reliable and validated tool for evaluating CPGs. Five of the six domains evaluated, yielded combined scores of at least 90%, with one domain a combined score of 63%. The overall standard deviation was 0.58, indicating an overall low level of user discrepancy Additions and revisions were made based on the results of the AGREE II evaluation scores with specific emphasis on the lowest scoring domain. Conclusion: This DNP Project identified the need for a CPG specific to pediatric primary care. A CPG with accompanying ADAP was developed and evaluated using the AGREE II tool. The CPG was found to meet the recommended standards and recommended for use in pediatric primary care.

The parasites are one of the most important factors that shape relationships in the nature. This thesis deals mainly with developmental stages of trematodes (Platyhelmintes: Trematoda), especially the cercariae of so-called Schistosomes and sporocysts of Leucochloridium paradoxum, as well as with and their intermediate host - freshwater snails. Introductory chapters are focused on general introduction into the topic, such as basic terminology or occurrence issue of trematodes (mainly schistosomes) in the world and in the Czech Republic. In order to determine larval stages of flukes and their morphological types, the research was conducted in four Prague localities with known presence of aquatic snails. Only in one site, Kunratická tůň Pond, the constant presence of echinostomous cercariae (and in lesser extent also furcocercariae) in Radix labiata was recorded during summer season of 2015. In small pond in the Botanical Garden of the Natural Sciences Faculty of Charles University and in Modřanské tůně Ponds, xiphidocercarie in Lymnaea stagnalis were found. No cercarie were detected in the pond in the Genetic garden of Charles University. The presence of Leucochloridium paradoxum in the European Amber Snail Succinea putris was confirmed in the vicinity of Modřanské tůně Ponds. The verified...

A qualidade de vida tem sido objeto de muito interesse nos últimos tempos. A Qualidade de Vida Relacionada à Saúde é considerada como um indicador de saúde, que aborda aspectos físicos, mentais e sociais relativos à saúde. Para sua mensuração necessita-se de instrumentos válidos e confiáveis desenvolvidos ou adaptados para a cultura alvo. Condições crônicas dermatológicas têm sido associadas ao declínio da qualidade de vida dos indivíduos acometidos por estas. O objetivo deste estudo foi adaptar culturalmente para o Brasil e obter as características psicométricas iniciais do instrumento DISABKIDS®-MDA para avaliação da qualidade de vida relacionada à saúde de crianças/adolescentes com Dermatite Atópica e seus pais/cuidadores, instrumento que possui 12 itens e duas dimensões, impacto e estigma. A pesquisa consistiu de uma investigação metodológica quantitativa, que incluiu uma amostra de 70 crianças/adolescentes brasileiros com Dermatite Atópica, na faixa etária de 8 a 18 anos, e seus responsáveis, recrutados no Serviço de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, na cidade de São Paulo, entre os meses de abril e setembro de 2009. O processo abrangeu as fases de tradução-retrotradução, validação semântica e teste piloto, por meio de análise estatística apropriada. A validação semântica mostrou boa aceitação do instrumento pelos participantes, o qual foi considerado como muito bom e composto por itens de fácil compreensão. A confiabilidade de consistência interna foi satisfatória, com coeficiente Alfa de Cronbach aceitáveis para as dimensões constantes no instrumento (0,7024/0,8124 e 0,7239/0,8604). A análise MAP para validade convergente mostrou valores maiores que 0,30 para todos os itens. Quanto à validade discriminante, a análise revelou resultados satisfatórios. A concordância entre as versões self e proxy foi acessada pelo coeficiente de Correlação Intra-Classe, com valores de 0,8173 para impacto e 0,7629 para estigma. Diante dos resultados encontrados neste estudo considera-se que o instrumento DISABKIDS®-MDA possa vir a ser utilizado por pesquisadores brasileiros depois de finalizado seu processo de validação no país, por seus resultados iniciais apontarem ser um instrumento válido e confiável. / Quality of Life has been object of many interesting nowadays. Health Related Quality of Life is considered as a health indicator, which approaches physical, mental and social aspects. Its measurement involves instruments with validity and reliability developed or adapted to a specific culture. Dermatologic chronic conditions has been associated with patients quality of life decrease. The aim of this study was to do the cultural adaptation to Brazil and to obtain DISABKIDS®-MDAs preliminary psychometric properties to measurement of health related quality of life of children/adolescents with Atopic Dermatitis and their parents/caregivers. The instrument has 12 items and two dimensions, impact and stigma. The research consisted of a quantitative methodological investigation, that included a sample of 70 Brazilian children/adolescents with Atopic Dermatitis, aged 8 to 18, and their parents/caregivers, recruited at Dermatology Service from Clinic Hospital from College of Medicine from University of Sao Paulo, at Sao Paulo, between April and September of 2009. The procedure comprised translation, back-translation, linguistic validation and pilot test, with appropriate statistic analysis. On the linguistic validation the instrument was well accepted by the participants, and it was considered very good and has comprehensible items. Reliability of internal consistency was adequate, with Cronbachs Alpha acceptable to instruments dimensions (0,7024/0,8124 and 0,7239/0,8604). The MAP analysis to convergent validation displayed values higher than 0,30 for all items. In relation to discriminant validation, the analysis presented acceptable outcomes. The agreement between self and proxy versions was accessed by ICC, with values of 0,8173 to impact and 0,7629 to stigma. By the outcomes from this research, DISABKIDS®-MDA, after its complete validation procedure at the country, may be used by Brazilian researchers, once its initial outcomes appoint to be a valid and reliable instrument.

INTRODUÇÃO: A dermatite atópica é uma doença inflamatória crônica da pele que possui alta prevalência e etiopatogenia multifatorial. Os ácaros são alguns dos desencadeadores das crises desta enfermidade. OBJETIVO: avaliar a freqüência de positividade ao teste de contato, utilizando extratos do ácaro Aleuroglyphus ovatus em doentes com dermatite atópica, comparando-a à dos doentes portadores de alergia respiratória e à dos indivíduos sem atopia. MÉTODOS: Cento e vinte e quatro indivíduos (48 doentes com dermatite atópica, 47 com alergia respiratória e 29 sem atopia) foram avaliados através do teste de contato contendo extrato de Aleuroglyphus ovatus nas concentrações de 0,1%, 0,5%, 1,0%, 1,5%, 2,0%. O teste de puntura com leitura imediata (“prick test") e a dosagem da IgE sérica também foram avaliados. RESULTADOS: seis doentes com dermatite atópica, 4 com alergia respiratória e 1 indivíduo sem atopia responderam positivamente ao teste de contato alérgico com Aleuroglyphus ovatus. Não houve diferença estatística significativa entre os grupos estudados. CONCLUSÃO: O Aleuroglyphus ovatus possui papel relativo na elicitação das crises de dermatite atópica, podendo o teste de contato alérgico com Aleuroglyphus ovatus ser usado em caso de dúvida no diagnóstico etiológico. / INTRODUCTION: Atopic dermatitis is a chronic, inflammatory disease of the skin with high prevalence and complex etiopathogenesis. Mites are known to cause flares of this disease. OBJECTIVE: Evaluation of the epicutaneous test response with Aleuroglyphus ovatus antigen in patients with atopic dermatitis. METHODS: One hundred and twenty four subjects were patch tested with Aleuroglyphus ovatus antigen in concentrations of 0.1%, 0.5%, 1.0%, 1.5%, 2.0%. Forty eight patients with atopic dermatitis, 47 with respiratory allergy and 29 healthy subjects were studied. Prick test and total serum IgE were also evaluated. RESULTS: six patients with atopic dermatitis, 4 with respiratory allergy and 1 healthy subject had positive responses to Aleuroglyphus ovatus atopy patch test. No statistical differences among the studied groups were found. CONCLUSION: This mite must have a relative role in the atopic dermatitis flares and Aleuroglyphus ovatus atopy patch test should be reserved for etiopathogenic uncertainties cases.

A Qualidade de Vida Relacionada à Saúde é definida como um indicador de saúde, que compreende fatores físicos, mentais e sociais e os impactos que uma determinada condição de saúde pode trazer à vida do indivíduo. Instrumentos válidos e fidedignos são ferramentas de grande valia para a mensuração deste construto. A Dermatite Atópica é uma condição crônica dermatológica associada a inúmeros prejuízos nos diversos aspectos da vida de uma pessoa, especialmente em crianças e adolescentes. O objetivo deste estudo foi validar para o Brasil o instrumento DISABKIDS®- Módulo Dermatite Atópica, para mensuração da Qualidade de Vida Relacionada à Saúde de crianças e adolescentes, com Dermatite Atópica, em idade escolar. O Instrumento compreende 12 itens e duas dimensões, Impacto e Estigma, e duas versões, self e proxy. A amostra foi composta por 200 sujeitos, 100 crianças e adolescentes brasileiros com Dermatite Atópica, entre 8 a 18 anos de idade, e seus pais ou cuidadores, recrutados no Serviço de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, na cidade de São Paulo, em dois momentos, entre os meses de abril e setembro de 2009 e julho de 2012 a junho de 2013. A fidedignidade do instrumento foi satisfatória tanto em relação à sua consistência interna, mensurada segundo o Coeficiente Alfa de Cronbach (self: ?Cronbach = 0,855; 0,853 e proxy: ?Cronbach = 0,905; 0,858, dimensões Impacto e Estigma, respectivamente), quanto à sua reprodutibilidade, mensurada segundo Coeficiente de Correlação Intra-Classe (self: ICC = 0,952; 0,971 e proxy; ICC = 0,960; 0,972, dimensões Impacto e Estigma, respectivamente). A análise Multitraço-multimétodo para teste de validade convergente mostrou valores de coeficientes de correlação lineares de Pearson entre cada item e sua dimensão maiores que 0,40. Quanto à validade discriminante, a análise revelou resultados igualmente satisfatórios, com índices de ajuste iguais a 87,5% e 100% para a versão self e a 75% e 100% para a versão proxy, dimensões Impacto e Estigma, respectivamente. Houve concordância entre as versões self e proxy, com valores para Coeficiente de Correlação Intra-Classe iguais à 0,610 para a dimensão Impacto e 0,595 para a dimensão Estigma. A Análise Fatorial Confirmatória mostrou que o instrumento adaptado para o Brasil manteve a estrutura assumida para o construto no instrumento original (self: RMSEA=0,083; CIF=0,932 e proxy: RMSEA=0,098; CIF=0,936). Considera-se que o instrumento DISABKIDS®- Módulo Dermatite Atópica encontra-se validado para crianças e adolescentes brasileiros em idade escolar com diagnóstico de Dermatite Atópica apresentando características de boa fidedignidade de consistência interna e reprodutibilidade, boa validade de construto convergente e discriminante, boa correlação e entre as versões self e proxy, e com a estrutura fatorial do construto inalterada em relação à inicialmente assumida no instrumento europeu. / The Health-related Quality of Life is defined as an indicator of health, including physical, mental and social factors and the impacts that a certain health condition can bring to an individual\'s life. Valid and reliable instruments are valuable tools for the measurement of this construct. Atopic Dermatitis is a chronic dermatological condition associated with numerous losses in various aspects of a person\'s life, especially in children and adolescents. The objective of this study was to validate for Brazil the DISABKIDS® - Atopic Dermatitis Module, for measurement of Health- related Quality of Life of children and adolescents, with atopic dermatitis, school aged. The instrument comprises 12 items and two dimensions, Impact and Stigma and two versions, self and proxy. The sample was composed of 200 people, 100 Brazilian children and adolescents with atopic dermatitis, between 8 to 18 years of age, and their parents or caregivers, recruited at the Dermatology Service of the Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo, in the city of Sao Paulo, in two moments, between the months of April and September 2009 and July 2012 to June 2013. The reliability of the instrument was satisfactory in relation to its internal consistency, measured according to Cronbach\'s alpha Coefficient (self: ?Cronbach = 0,855; 0,853 e proxy: ?Cronbach = 0,905; 0,858, dimensions Impact and Stigma, respectively), as to its reproducibility, measured according to Intra-class Correlation Coefficient (self: ICC = 0,952; 0,971 e proxy; ICC = 0,960; 0,972, Impact and Stigma dimensions, respectively). Multitraço- Multimethods Analysis to convergent validity test showed values of Pearson Linear Correlation Coefficients between each item and its size greater than 0.40. As to the validity, discriminant analysis showed also satisfactory results with adjustment indexes equal 87,5 and 100 for self and 75 and 100 for the proxy version, dimensions, Impact and Stigma, respectively. There was agreement among self answers and proxy, with values for Intra-class Correlation Coefficient equal to 0,610 Impact dimension and 0,595 Stigma. The Confirmatory Factor Analysis showed that the instrument adapted to Brazil kept the structure assumed for the original instrument (self: RMSEA=0,083; CIF=0,932 e proxy: RMSEA=0,098; CIF=0,936). It is considered that the DISABKIDS® - Atopic Dermatitis Module is validated for Brazilian children and adolescents of school age with a diagnosis of Atopic Dermatitis presenting features of good internal consistency for reliability and reproducibility, good discriminant and convergent construct validity, good correlation and between self and proxy versions, and with the factorial structure of the construct unchanged in relation to the initially assumed in the European instrument

目的:銀屑病是一種慢性炎症性皮膚疾病，特點是角質形成細胞過度增生。角質形成細胞過度增生在銀屑病家族中被證實與位於染色體6p2 1. 3 上的編碼人類白細胞抗原的基因組區有關。通過全基因組掃描，至少確定了10 個銀屑病易感基因位點(PSORS 卜的ORS10) ，而6p21 區的PSORS1 被認為是銀屑病最主要的易感位點，而HLA-C， CCHCRl (coiled coil alpha-helixrod homolog) 和CDSN(corneodesmosin) 是該位點上的重要的侯選基因。然而，這三個基因之間的連鎖不平衡很難將他們的個體效應分開。我們的目標是研究CCHCCRl 基因以及它的相關蛋白在皮膚上皮細胞增生過程中的作用。 / 方法:本研究應用免疫螢光技術'免疫印跡分析和即時反轉錄聚合臨鏈反應 (Real Time RT-PCR) 三種方法比較CCHCRl 基因在銀屑病人的皮損區與正常人的皮膚細胞的表達。其次比較了CCHCRl 蛋白在喜樹鹼(拓撲異構酪I 抑制劑/凋亡誘導劑)的作用下在三種細胞(永生化的人皮膚角質形成細胞-HaCaT '人直結腸癌細胞-HT29 和人子宮頸癌細胞- HeLa) 內的表達。最後，我們應用了皮膚器官樣培養物( OTC) 研究HaCaT 細胞於皮膚角質化過程中CCHCRl 蛋白的表達。 / 結果:✹HCR1 蛋白在正常皮膚和銀屑病人皮損皮膚表達模式不同，銀屑病人皮損皮膚有五種CCHCR1 蛋白染色模式，而正常人皮膚只有兩種染色模式。免疫印跡分析顯示CCHCR1 蛋白在正常皮膚含量較銀屑病人高。免役螢光顯微技術顯示角蛋白17 (K17) ，一種細胞增生的標誌性蛋白，只表達在正常皮膚基底層的角質細胞中，而在銀屑病人皮膚， K17 從基底層到顆粒層都有顯著表達，並且和CCHCR1 蛋白表達區域相同。這種現象也存在於OTC 發育過程中所有角質細胞中，而CCHCR1 蛋白表達隨OTC 的培養時間從10 天到21 天呈上升趨勢，說明CCHCR1 蛋白與細胞增殖有一定的關係。當細胞生長在培養血表面形成細胞與細胞接合全面的細胞層時， CCHCR1 的信使核糖核酸水平大幅升高，顯示CCHCR1 的信使核糖核酸表達當細胞生長速度呈負相關。CCHCR1 的信使核糖核酸水平在同步化的HaCaT 細胞G2/M 期輕度升高，而蛋白水平在G1 期達到最高。在G2/M 期，高爾基體出現崩解，而CCHCR1 蛋白和其他高爾基蛋白一樣分散到胞漿。這種現象同樣地出現在經2 州喜樹鹼刺激48小時的HeLa 和HaCaT 細胞內，免疫細胞化學染色顯示CCHCR1 和golgin-97 分散到胞漿。CCHCR1 信使核糖核酸水平在HeLa 和HaCaT 細胞都升高，而蛋白在HaCaT 細胞明顯上調。這說明CCHCR1 蛋白量的上調和細胞生長停滯有關。應用siRNA 或shRNA 抑制CCHCR1 蛋白表達後， golgin-97 分散到胞漿，但是HeLa細胞可以繼續增殖。這說明CCHCR1 蛋白可能有助於維持高爾基複合體的完整，喜樹鹼對細胞生長的抑制作用可能與CCHCR1 蛋白的上調相關。 / 結論: CCHCR1 蛋白在正常皮膚表皮細胞和銀屑病人皮損區細胞胞漿內都有表達，但是正常皮膚表達較銀屑病人高，在OTC 增生後期和細胞生長緩慢或停滯期也明顯升高，說明CCHCRl 基因的上調可能抑制細胞的增生。經喜樹鹼刺激後，細胞生長受到抑制，細胞停滯在Gl 期並誘導細胞凋亡， CCHCRl 的信使核糖核酸和蛋白都上調，表明CCHCRl 與細胞的增生抑制有關。同時，它的下調也造成高爾基複合體的崩解，說明CCHCRl 蛋白可以維持高爾基複合體的結構完整性。因此， CCHCRl 不僅可以保持高爾基複合體的完整而且和細胞的增生有關。另一方面，大量證據表明長期性的高血糖會導致胰島　細胞功能紊亂。鑒於此，揭示胰島功能調節的潛在機理并闡明胰島功能与高血糖症之間的關係變得尤為重要。 / Aim: Psoriasis is one of the common chronic inflammatory skin disorders characterized by keratinocyte hyperproliferation, T lymphocyte-mediated inflammation, and abnormal differentiation. Genome-wide scans have revealed that at least ten different susceptibility loci, PSORS1-PSORS10, were linked to psoriasis, among which PSORS 1 on chromosome 6p21.3 was unambiguously associated with families with psoriasis. Three strongly psoriasis-associated susceptibility alleles have been identified in PSORS1, namely HLA-C, CCHCR1 (coiled-coil alpha-helical rod protein 1 ), and CDSN ( comeodesmosin); their strong linkage disequilibrium, however, makes it very difficult to distinguish their individual genetic effects. Among them, we were interested in the CCHCR1 gene, and its possible roles with associated proteins in the process of skin epidermal cell proliferation were studied in this thesis. / Methods: The cellular expressiOn of CCHCR1 protein in the epidermis was compared between human skins from healthy donors and psoriasis patients by immunofluorescence microscopy and immunoblotting analysis. Its intracellular expression was investigated in cultured human immortalized skin keratinocyte cell line (HaCaT), human colorectal cancer cell line (HT29) and human cervical carcinoma HeLa cell line, as well as in the skin keratinocyte-fibroblast organotypic culture (OTC). The responses of these cells to camptothecin (CPT), a topoisomerase I inhibitor, were compared in HaCaT cells, HT29 cells and HeLa cells. In cell experiments, CCHCR1 expression was studied by immunofluorescence microscopy, immunoblotting analysis, and real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). / Results: The immunofluorescence staining patterns for CCHCR1 protein were different between normal and psoriasis skin biopsies. There were at least five patterns (Patterns I, III-VI) in psoriatic skins, but only two (Patterns I and II) in the normal skin, implicating that the expression of CCHCR1 protein was changed in psoriasis. Besides the different cellular location of CCHCR1 protein in normal and psoriasis skins, a higher level of CCHCR1 expression was measured in normal skin than that in psoriatic skin by immunoblotting analysis, and immunofluorescence microscopy further revealed a co-localization of CCHCR1 protein with keratin 17 (K17), a proliferation marker protein and also putative autoantigen for psoriasis, to basal keratinocytes of normal skins, and extended further fro m the basal to granular keratinocytes in the epidermis of psoriasis patients, suggesting a close association of CCHCR1 protein to cell proliferation. During epidermal development in OTC, the CCHCR1 protein was expressed in all keratinocyte layers in a time-dependent manner from day 1 0 to day 21, reaching a maximal level at day 21 when cell proliferation decreased and differentiation to the corneum became active. Furthermore, immunofluorescence for K17 remained co-localized to CCHCR1-positive cells in OTC from day 10 to day 14, but the level of K17 was very weak in the keratinocytes of suprabasallayers on day 21. In HaCaT cell culture, CCHCR1 transcription level at 100% confluence was 25 folds higher than that in subconfluence. Taken all these results together, the up-regulation of CCHCR1 was closely related to cell growth inhibition and differentiation. Immunofluorescence microscopy revealed a co-localization of CCHCR1 protein with a Golgi marker protein, golgin-97, in the compact Golgi complex at the juxtanuclear region of HaCaT cells and HeLa cells. CCHCR1 gene transcribed in all phases of the cell cycle, but was slightly higher in the G2/M phase of synchronous HaCaT cells, and its translation reached its maximal level in the G 1 phase. In the G2/M phase, CCHCR1 protein was dispersed in the cytoplasm like the golgin. Such dispersal was also observed in HaCaT cells and HeLa cells treated with CPT for 24 h and 48 h, respectively. CCHCR1 expression increased in both transcriptional and translational levels as well as apoptotic changes following growth arrest in both HeLa cells and HaCaT cells. Depletion of the CCHCR1 protein by means of siRNAor shRNA-mediated knockdown induced HeLa cells proliferation, cell elongation and disassembly of the Golgi complex. / Conclusion: CCHCR1 protein was expressed in the cytoplasm of epidermal keratinocytes of both normal and psoriasis skins, with a higher level detected in the normal skins. It was also found especially abundant in the keratinocytes of skin organotypic cultures during their transition from proliferation to differentiation. CCHCR1 gene transcription was increased obviously in cultured HaCaT cells at confluence. CCHCR1 was also up-regulated at both transcriptional and translational levels in response to the antiproliferative drug, camptothecin, in HaCaT cell experiments, and was accompanied by G 1 arrest and subsequent apoptosis. When CCHCR1 was knockdowned in HeLa cells, the Golgi complex disassembled, implicating a role of CCHCRl protein in the maintenance of Golgi integrity and in the control of cell proliferation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Lijun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 107-133). / Abstracts also in Chinese. / Abstract --- p.i / Acknowledgements --- p.viii / List of Abbreviation --- p.ix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Psoriasis --- p.1 / Chapter 1.2 --- Skin --- p.9 / Chapter 1.3 --- An In Vitro Model for Human Skin Equivalent --- p.10 / Chapter 1.4 --- Aim of Study --- p.12 / Chapter Chapter2 --- Expression of CCHCRl Protein in Psoriasis skin --- p.13 / Chapter 2.1 --- Background of Skin and Psoriasis --- p.13 / Chapter 2.2 --- Materials and Methods --- p.18 / Chapter 2.3 --- Results --- p.21 / Chapter 2.4 --- Discussion and Conclusion --- p.25 / Chapter Chapter3 --- Expression of CCHCRl Protein in Skin Organotypic Culture --- p.37 / Chapter 3.1 --- Background about Skin Organotypic Culture --- p.37 / Chapter 3.2 --- Materials and Methods --- p.41 / Chapter 3.3 --- Results --- p.45 / Chapter 3.4 --- Discussion and Conclusion --- p.49 / Chapter Chapter4 --- Expression of CCHCRl Protein in HaCaT Cells Treated with Camptothecin --- p.60 / Chapter 4.1 --- Background --- p.60 / Chapter 4.2 --- Materials and Methods --- p.61 / Chapter 4.3 --- Results --- p.68 / Chapter 4.4 --- Discussion and Conclusion --- p.73 / Chapter Chapter 5 --- General Discussion --- p.95 / References --- p.107 / Publications --- p.172

Atopic Dermatitis (AD) is a common chronic relapsing inflammatory skin disease affecting 15 - 20% of children and 2 - 10% of adults worldwide, with significant morbidity. A hallmark of AD is disruption of the critical barrier function of upper epidermal layers, causatively linked to environmental stimuli, genetics and infections. Another typical feature of AD is skin infections, especially from Staphylococcus aureus (S. aureus), which closely relates with the disease severity. Although not a normal flora, S. aureus is found on 75-100% of AD lesions (< 30% on healthy skin). S. aureus secrete a range of virulence factors, including extracellular toxins and proteases which contribute to disease pathogenesis. S. aureus serine protease A (SspA/V8) is a well-characterised extracellular protease widely expressed among different S. aureus strains. The pathogenic effect of V8 protease has been demonstrated in vivo, damaging murine skin integrity via effects on the stratum corneum (SC), but the targets for this V8-mediated damage remains unclear. The capacity of proteases to induce barrier dysfunction has been proposed as a key driving force in the initiation and exacerbation of AD. Thus, understanding the host factors that maintain barrier function is a priority in developing novel therapeutic approaches. This thesis therefore aimed at detecting host factors which can combat the barrier dysfunction caused by pathogenic proteases, assessing their relevance in vitro and ex vivo and elucidating the underlying mechanisms. Firstly, an in vitro skin barrier integrity model was developed, using both immortalized and primary keratinocytes, to evaluate the barrier damage mediated by pathogenic proteases. The results revealed that S. aureus protease SspA/V8 is the dominant secreted factor (in laboratory and AD clinical strains of S. aureus) inducing barrier integrity impairment. In addition, studies demonstrated that V8 protease itself was sufficient to induce barrier disruption, and this phenotype was not dependent on cell death, but rather on breaking down of cell-cell junctions. Key tight junction proteins including claudin-1 and occludin were found to be degraded by V8 protease. Next, a wide range of host and bacterial factors were investigated to determine whether they could promote protection of keratinocytes against V8 damage. Several factors, including IL-1β, TNF-α, heat-killed Staphylococcus epidermidis (which is the main skin normal flora), were found to induce protection against V8 protease, with IL-1β having the strongest effect. In addition, data indicated that this IL-1β-mediated protection was independent of effects on claudin-1 but occurred via secretion of a transferrable host factor. Induction of keratinocyte expression of the antimicrobial/host defence peptide human beta-defensin 2 (hBD2) was found to be the mechanism underpinning this IL-1β- induced protective effect. Endogenous hBD2 expression was required and sufficient for protection against V8 protease-mediated integrity damage, and exogenous application of hBD2 was also protective. An ex vivo model using human skin tissue was also established to address this novel function of hBD2, and preliminary data indicated that exogenous hBD2 protected against V8-mediated damage in this system. Overall, my data reveal a novel function for the antimicrobial/host defence peptide hBD2. This modulatory property of hBD2, independent of its antibacterial effects, gives new significance to the defective induction of hBD2 in the barrier-defective skin lesions of AD and indicates therapeutic potential to prevent S. aureus-mediated aggravation of skin barrier dysfunction in AD.