Loss-of-function mutations in the filaggrin gene(FLG) have recently been shown to be strongly associated with atopic dermatitis (AD). The overall aim of this study was to explore the role of filaggrin in skin barrier function and AD. There were two main focuses in this study. The first was a functional study whose primary objective was to determine if FLG mutations correlated with skin barrier dysfunction in AD. Fifty-five mild to moderate AD individuals were recruited, genotyped and had their skin barrier assessed using three different measures - transepidermal water loss (TEWL), skin capacitance and the number of tape strips required to abrogate skin barrier. A secondary aim of this functional study was to test the hypothesis that corneocytes were less adherent to one another in filaggrin-related AD compared to wild-type AD skin. The second main focus of this thesis was a structural study aimed at interrogating the structure-function relationship of filaggrin. Filaggrin protein was extracted and purified from a total of 21 AD and non-AD subjects and analysed using mass spectrometric techniques. Specifically, matrix assisted laser desorption/ionisation time-of-flight(MALDI-TOF)mass spectrometry (MS) and nano liquid chromatography tandem MS(LC-MS/MS) were utilised. Part of this structural study also involved developing and optimising the extraction and purification of filaggrin protein, including a novel way of extracting filaggrin from skin using tape stripping. In addition, novel filaggrin-specific enzyme-linked immunosorbant assay (ELISA) was also developed, which could serve as a useful screening test for the protein. In this study, FLG mutations were found to correlate with higher TEWL and fewer number of tape strips required to abrogate skin barrier, but not with skin capacitance. FLG mutations were also not shown to correlate with AD severity. The mean amount of protein extracted from filaggrin-related AD skin was also significantly higher compared to wild-type AD skin, supporting the hypothesis that corneocytes were less adherent to one another (and therefore, densely packed) in filaggrin-related AD skin. MS analysis of filaggrin confirmed the heterogeneic nature of filaggrin protein, even within a single individual. Interestingly, this structural study also showed that filaggrin was only minimally expressed in the skin of all the AD individuals studied, whether or not they possessed any FLG mutation. Due to the limited amount of filaggrin extracted from AD skin, it was not possible to conduct comparative structural analysis between filaggrin from AD and non-AD skin.

One of the most important roles of the skin is the formation of an effective barrier to prevent desiccation as well as to keep out foreign pathogens and allergens. This is a tightly regulated process and involves many structural proteins, lipids, enzymes and biochemical components. One of the proteins that has an indispensable role in barrier formation is filaggrin, which is encoded by the filaggrin gene (FLG) that lies within a cluster of epidermal genes known as the epidermal differentiation complex (EDC) on chromosome 1q21. Recent studies in Europe have shown that null mutations in FLG lead to the loss of the filaggrin protein; this is the underlying genetic cause of ichthyosis vulgaris (IV) and is a significant predisposing factor for atopic dermatitis (AD) and other atopic conditions such as asthma, allergic rhinitis and food allergy. In this thesis, the critical role of FLG-null mutations was examined and confirmed as a strong predisposing factor for AD in Singaporean Chinese patients. In addition, AD patients with FLG mutations also showed an increased susceptibility for recurrent skin infections. Interestingly, a diverse and wide spectrum of FLG-null mutations was identified in the Singaporean Chinese population, as opposed to the dominance of a few common FLG mutations in Europe. This result highlighted discrete genetic variations between different ethnic groups. FLG-null mutations were also shown to have significant gene modifying effects on other skin barrier genes such as steroid sulphatase gene (STS) to exacerbate the phenotype of X-linked ichthyosis (XLI). Next, the effect of FLG¬-null mutations on other complex conditions such as acne vulgaris and childhood peanut sensitisation was investigated but no significant association of FLG mutations with these diseases were observed in the Singaporean Chinese population. Lastly, a study was attempted to search for a candidate gene for psoriasis within the EDC, through the use of fine mapping techniques. With the advent of faster and cheaper next generation sequencing (NGS) in the near future, the quest for susceptibility factors in complex traits will increase in effectiveness and speed.

No description available.

Atopische Dermatitis (Atopisches Ekzem) ist die häufigste Erkrankung der Haut, und sie ist mit der Entwicklung anderer schwerwiegenden atopischen Erkrankungen wie Lebensmittelallergien, allergischer Rhinitis, Heuschnupfen und Asthma vergesellschaftet. Inaktivierende Mutationen im Filaggrin (FLG) Gen zeigten die stärkste Assoziation mit dem Krankheitsbild der atopischen Dermatitis. FLG wird fast ausschließlich in der Epidermis der Haut ausgeprägt und trägt maßgeblich zur normalen Differenzierung von Keratinozyten und zur Integrität der Hautbarriere bei. Bi-allelische inaktivierende Mutationen in dem Gen verursachen die Hauterkrankung Ichthyosis vulgaris, bei es zu Ausbildung einer übermäßig trockenen und schuppigen Hautoberfläche kommt. Etwa die Hälfte aller Ichthyosis vulgaris Patienten entwickeln ebenfalls atopische Dermatitis im Laufe Ihres Lebens. Zusätzlich treten in Ichthyosis vulgaris Patienten häufiger atopische Krankheitsbilder auf als in Gesunden. Als Modell zur Erforschung molekularer Grundlagen der Pahtologie des atopischen Ekzemes und von Pathomechanismen der Atopie eignet sich die „flaky tail“ Mauslinie. Diese Mäuse entwickeln typische Merkmale einer systemischen Atopie wie z.B. einen Barriere Defekt der Haut, ein Hautekzem, eine Entzündung der Lunge, sowie erhöhte Mengen an Immunglobulin E im Serum. In diesem Modell wird die Krankheitsentwicklung durch zwei natürlich entstandene Mutationen in den Genen Flg und Tmem79 verursacht. Die das Gen Flg betreffende Mutation bewirkt eine reduzierte Expression, welche einen Ichthyosis vulgairs ähnlichen Phänotyp auslöst, der durch Schuppenartige („flaky“) Haut am Schwanz („tail“) gekennzeichnet ist (Flgft). Die Mutation im Tmem79 Gen bewirkt eine matte Fellfarbe und wird daher als „matted“ Mutation (Tmem79ma) bezeichnet. Obwohl die Mutationen in benachbarten Regionen des Maus Chromosoms 3 zu finden sind, konnten diese genetisch getrennt werden. Dadurch gelang es zu zeigen, dass allein die Tmem79ma Mutation ausreicht, um Ekzeme und systemische Atopie in Mäusen auszulösen. Nach Rückkreuzung der Flgft Mutation auf den pro-allergischen BALB/c genetischen Hintergrund entwickelten die Flgft/ft BALB/c Mäuse eine - Erkrankung ähnlich der atopischen Dermatitis, die ebenfalls von spontanem Asthma und hohen IgE Konzentrationen im Serum gekennzeichnet war. Beides sind Hauptmerkmale des sogenannten atopischen Marsches. Diese Versuche implizierten, dass Filaggrin eine wichtige Funktion im Schutz vor der Entwicklung von Atopie in Mäusen ausübt. Im Gegensatz zu diesen Ergebnissen wurden nach dem Rückkreuzen von Flg knockout (Flg-/-) Mäusen auf den BALB/c Hintergrund nur ein transienter neonataler Ichthyose Phänotyp, aber keine Hautentzündung oder Atopie beobachtet. Um diese Diskrepanz besser zu verstehen, generierten wir Flg knockout Mäuse direkt im BALB/c Hintergrund. Diese genetisch reinen Flg-/-/BALB/c Mäuse rekapitulierten den neonatalen Ichthyose Phänotyp, und sie zeigten einen Barriere Defekt, der die perkutane Sensibilisierung förderte. Spontane Entzündungen der Haut oder systemische Atopie wurden allerdings nicht beobachtet. Um die genetische Ursache der Atopie in Flgft/ft BALB/c Mäusen zu verstehen, sequenzierten wir das Genom mittels PacBio long read sequencing und verglichen es mit dem BALB/c Referenzgenom. Überraschenderweise zeigte die Analyse, dass die kongenen Flgft/ft BALB/c Mäuse ebenfalls die Atopie-verursachende Tmem79ma Mutation im homozygoten Zustand trugen. Zuvor wurde berichtet, dass die Flgft und Tmem79ma Mutationen vor dem Rückkreuzen des Flgft Allels auf den BALB/c Hintergrund voneinander getrennt werden konnten. Unsere Beobachtungen erklären nun die phänotypische Diskrepanz zwischen den Flgft/ft BALB/c und den Flg-/-/BALB/c Mäusen. Die Daten implizieren, dass der alleinige Funktionsverlust von FLG keine Atopie auslöst und werden durch Beobachtungen in einem Teil der FLG-defizienten Ichthyosis vulgaris Patienten gestützt, die ebenfalls keine Atopie zeigen. Es ist trotzdem wahrscheinlich, dass der Verlust von Filaggrin im Zusammenspiel mit weiteren genetischen Barriere Defekten, wie z.B. Mutationen im Tmem79 Gen das Krankheitsbild der Atopie qualitativ beeinflusst.:Abbreviations 6 Summary 9 Zusammenfassung 11 Introduction 13 Skin 13 Epidermis 13 The immune system 15 Adaptive immune response effector mechanisms 17 Allergic diseases 19 Atopic march 19 Atopic dermatitis 19 Metabolics of AD skin 23 Filaggrin and AD 25 Current mouse models of barrier defect and AD 29 Induced AD mouse models 29 Transgenic and knock out (KO) AD mouse models 30 Inbred mouse strains spontaneously developing AD-like disease 31 Mouse models of FLG-deficiency 32 AD and microbiome 34 Aim 37 Material and methods 38 Generation of Flg-/- mice 38 Crispr targeting strategy 38 α-FLG Western Blot 39 Protein extract preparation 39 SDS Electrophoresis 39 α-FLG Immunofluorescence staining 40 PCR Typing 40 Isolation of genomic DNA from mouse tail tips 40 PCR typing strategy 41 H&E staining of neonatal mouse ear skin 42 Ear thickness measurement 44 Quantification of transepidermal water loss (TEWL) 44 Quantification of total and antigen-specific IgE 44 Whole back skin RNA Sequencing (RNA-Seq) 44 Sorting of basal ear skin keratinocytes for RNA extraction, by fluorescence-activated cell sorting (FACS) 45 Quantitative real time PCR (qRT-PCR) 47 Flow cytometric analysis of ear skin 48 Analysis of DO11.10+/4get transgenic T cell response to epicutaneous OVA immunization 49 Skin microbiome analysis 50 S.aureus colonization of Flg mutant mice 51 Long-term epicutaneous OVA treatment 52 Mapping of reads, assembly, annotation and variant calling 53 Statistical analysis 55 Results 56 Generation and validation of Flg-/-/ BALB/c mice 56 Large sequence deletions detected in Flg-mutant mice by PCR 56 Complete loss of FLG protein expression in Flg-/- mice 57 Flg-/-/BALB/c mice show a barrier-defective skin 59 FLG-deficient skin is devoid of inflammation 61 FLG-deficient skin shows age-related inside-out barrier defect 62 Mild T cell expansion and no other changes in the epidermal compartment of the Flg-/-/BALB/c mice 62 γδ T cell expansion in the whole ear skin suspensions of FLG-deficient mice 64 Lack of systemic atopy in FLG-deficient mice, as indicated by IgE quantification 67 S. aureus colonization does not trigger eczema or atopy in FLG-deficient mice 68 Sensitization with a protein Ag does not trigger inflammation in FLG-deficient mice 69 FLG-deficient skin shows age-related outside-in barrier defect 69 Mild inflammatory signature detected in Flg-/-/BALB/c back skin by RNA sequencing 71 Type 2 immune response signalling detected in Flg-/-/BALB/c ear skin keratinocytes by RNA sequencing 72 FLG-deficient ear skin shows elevated IL-1b expression 74 Reduced cutaneous commensal microbial diversity in FLG-deficient mice 75 Presence of atopy-causing Tmem79ma mutation in Flgft/ft BALB/c mouse genome account for phenotypic differences with our Flg-/-/BALB/c mice 76 Discussion 81 FLG-deficiency in mice does not trigger spontaneous atopic disease 81 FLG-deficiency in mice causes mild immunological perturbation 82 Atopic march, observed in Flgft/ft BALB/c congenic strain, can be explained by the presence of the matted mutation 88 Relevance of filaggrin deficiency for the pathogenesis of atopy in mouse and man 89 References 92 Acknowledgments 136 Erklärung zur Eröffnung des Promotionsverfahrens 137 Erklärung über die Einhaltung der gesetzlichen Vorgaben 138

No / IL-33 is a member of the IL-1 family of cytokines that is constitutively expressed in healthy skin and was found to be increased in the skin of patients with atopic dermatitis (AD). Because it can be released after tissue damage or physical stress including scratching of the skin,1 it has been classified as an alarmin concerned with alerting the immune system.2 It enhances TH2 responses by inducing IL-5 and IL-13 as well as TH1 responses via upregulation of IFN-γ. Keratinocytes are known producer cells of IL-33 and also express the receptor complex consisting of ST2 and IL-1RAcP on their surface. The aim of this study was to investigate the effect of IL-33 on keratinocytes, skin biopsies, and living skin equivalents with regard to the regulation of the skin barrier molecule filaggrin (FLG).

Les peptidyl-arginine désiminases (PADs) catalysent une modification post-traductionnelle, appelée désimination, qui correspond à la transformation des résidus arginine en résidus citrulline. À ce jour, leur rôle est encore mal compris, bien qu'elles aient été impliquées dans divers processus physiologiques et pathologiques. Dans l'épiderme et le follicule pileux, trois d'entre elles sont exprimées, les PAD1, 2 et 3. La désimination de la filaggrine (FLG), protéine essentielle de la différenciation épidermique, semble conduire à sa dissociation des filaments de kératines et permettre sa protéolyse totale. Les acides aminés libres ainsi générés sont nécessaires à la fonction de barrière que remplit la couche cornée, couche cellulaire la plus externe de l'épiderme. Dans le follicule pileux, la désimination de la trichohyaline (TCHH), protéine apparentée à la FLG, augmente sa solubilité et permet sa liaison covalente aux kératines par la transglutaminase 3 (TGase 3), ce qui participe à la formation de la tige pilaire. La description du rôle des PADs au cours de la différenciation des kératinocytes est cependant encore incomplète, et leurs implications dans les maladies de peau peu explorée. L'objectif de mon travail de thèse a été de mieux comprendre le rôle des PADs dans le métabolisme de la FLG et, plus généralement, dans l'homéostasie de l'épiderme et du follicule pileux. Tout d'abord, j'ai montré que la désimination de la FLG humaine par la PAD1 et/ou 3 est une étape majeure qui permet de réguler sa dégradation complète en fonction du taux d'humidité extérieure. Pour cela, j'ai utilisé comme modèle expérimental des épidermes reconstruits humains (ERHs). La diminution de l'humidité relative lors de leur production, de 95% à 30-50%, augmente la protéolyse de la FLG et la genèse des acides aminés correspondants. En parallèle, l'expression de la PAD1 et le taux de désimination de la FLG sont fortement accrus alors que ni l'expression ni l'activité des protéases impliquées dans cette protéolyse ne varient. De plus, le traitement d'ERHs pendant 24 heures avec un inhibiteur des PADs, le Cl-amidine, bloque en partie l'effet de la sécheresse sur le métabolisme de la FLG. J'ai ensuite recherché si la désimination joue un rôle plus global au cours de la différenciation des kératinocytes. J'ai traité des ERHs avec différentes concentrations de Cl-amidine pendant 48 heures et analysé l'effet du traitement sur leur morphologie. L'inhibition de la désimination est dose-dépendante et non toxique. À la plus forte concentration, le Cl-amidine entraîne un amincissement de la couche cornée, une augmentation importante du nombre de cellules transitionnelles, et l'accumulation de mitochondries et de vésicules dans le cytoplasme des kératinocytes granuleux. Ceci permet de proposer que la cornification, dernière étape de la différenciation kératinocytaire, est retardée. De plus, la protéine LC3B-II, marqueur des autophagosomes, étant plus fortement détectée dans les ERHs traités, les PAD1 et/ou 3 pourraient être impliquées dans le processus d'autophagie associé à la cornification.Enfin, j'ai participé à un nouveau projet collaboratif qui a permis de découvrir l'origine d'une maladie génétique rare, le syndrome des cheveux incoiffables. Des mutations des gènes de la TCHH, de la PAD3 ou de la TGase 3 ont été identifiées dans 11 familles et sont responsables de ce syndrome. La mutation non-sens du gène de la TCHH aboutit à la synthèse, si elle a lieu, d'une forme très courte, probablement incapable d'interagir avec les kératines. Les mutations des gènes de la PAD3 et de la TGase 3 induisent des changements structuraux et une quasi-absence de l'activité des enzymes correspondantes. De plus, les souris dont le gène de la Pad3 a été inactivé présentent des anomalies de la forme des poils. Ces résultats ont permis de comprendre la physiopathologie de cette maladie des cheveux et ont prouvé que la PAD3 est essentielle à leur morphogenèse. / Peptidylarginine deiminases (PADs) catalyze a post-translational modification, named deimination, corresponding to the transformation of arginine residues into citrulline. So far, their role is still poorly understood even if they have been associated with numerous physiological and pathological processes. In the epidermis and the hair follicle, three PADs are expressed, namely PAD1, 2 and 3. Deimination of filaggrin (FLG), a major protein of epidermal differentiation, would lead to its detachment from keratin filaments and allow its full proteolysis. The resulting free amino acids contribute to the barrier function of the cornified layer, the outmost cellular layer of the epidermis. In the hair follicle, deimination of trichohyalin (TCHH), a FLG-related protein, increases its solubility and improves its crosslinking by transglutaminase 3 (TGase 3) to keratins, contributing to the hair shaft formation. However, the function of PADs during keratinocyte differentiation is incompletely described and their involvements in skin diseases have been poorly investigated. The objective of my work was to better understand the role of PADs in the metabolism of FLG, and more generally in the epidermis and hair follicle homeostasis. First of all, I demonstrated that a major step in the metabolism of human FLG is its deimination by PAD1 and/or PAD3. This regulates its complete degradation according to the external humidity level. For this purpose, I used reconstructed human epidermis (RHEs) as an experimental model. Lowering relative humidity from 95 to 30-50% during RHE generation enhanced FLG proteolysis as well as the resulting amino acid amount. In parallel, PAD1 expression and FLG deimination were highly increased while the expression or activity of proteases known to target FLG did not vary. Moreover, treatment of RHEs during 24 hours with Cl-amidine, a PAD inhibitor, partially blocked the effect of dryness on FLG metabolism. Then, I investigated whether deimination could play a more general role during keratinocyte differentiation. I treated RHEs with various concentrations of Cl-amidine during 48 hours and I analyzed the effect of treatments on the epidermis morphology. The inhibition of deimination was dose-dependent and not cytotoxic. At the strongest concentration, Cl-amidine was shown to cause thinning of the cornified layer, to highly increase the number of transitional cells and to induce accumulation of mitochondria and vesicles in the cytoplasm of granular keratinocytes. This suggested that cornification, the ultimate stage of keratinocyte differentiation, is slowed down by Cl-amidine treatment. Besides, as the autophagosome marker LC3B-II was up-regulated in Cl-amidine treated RHEs, PAD1 and/or PAD3 could be involved in the cornification-associated process of autophagy. Finally, I participated in a new collaborative work that led to discover the cause of uncombable hair syndrome, a rare genetic disorder. Mutations of the genes encoding TCHH, PAD3 and TGase 3 were identified in 11 families and were shown to be responsible for the disease. The nonsense mutation in TCHH gene results in the synthesis, if any, of a very short protein, probably not able to interact with keratins. Mutations in PAD3 and TGase3 genes were shown to induce structural changes and almost total absence of activity of the corresponding enzymes. Moreover, examination of Pad3 deficient mice revealed alterations in the hair morphology. These results allowed a better understanding of this hair disease physiopathology and proved that PAD3 is essential for the hair shaft morphogenesis.

Introdução: A dermatite atópica (DA) é uma doença cutânea inflamatória crônica que cursa em surtos. Possui manifestação clínica variável, mas o prurido e a xerose são características frequentes, e pode estar associada a outras manifestações extra-cutâneas de atopia. Pacientes com DA apresentam maior risco de infecções por bactérias e vírus, destacando-se a erupção variceliforme de Kaposi, causada por herpes simples. A DA mostra-se como exemplo de dermatose com comprometimento da barreira cutânea, aliado a disfunção imunológica. São descritas alterações das proteínas da barreira cutânea na DA (filagrina e claudinas ), relacionadas ao maior risco de infecção . Objetivo: Avaliar a expressão de proteínas relacionadas à barreira cutânea como a filagrina, e as claudinas -1 e -4 na pele de pacientes adultos com dermatite atópica, acompanhados no Departamento de Dermatologia da Faculdade de Medicina da Universidade de São Paulo. Métodos: 32 indivíduos com diagnóstico de DA (estabelecido pelos critérios de Hanifin & Rajka) e 23 controles (indivíduos sem DA), maiores de 18 anos, foram submetidos a biópsias cutâneas. Os indivíduos com DA foram biopsiados em dois pontos, tanto na pele lesada, quanto na pele não-lesada. O material obtido foi analisado por imuno-histoquímica, através de marcadores específicos para filagrina, claudina-1 e claudina-4. As lâminas foram digitalizadas pelo Panoramic Scan - 3DHistech - Hungria, e as imagens analisadas pelo software Image Pro Plus 4,5, quanto à intensidade da expressão do marcador. A espessura média da epiderme do local estudado foi também avaliada. O grupo com DA foi também analisado quanto à gravidade da doença (EASI), níveis séricos de IgE e grau de eosinofilia. Resultados: Houve redução da expressão da filagrina na pele de doentes de DA em relação aos controles, tanto na pele com lesão quanto na pele sem lesão. Demonstrou-se correlação inversa na expressão da filagrina, tanto com relação à gravidade da doença quanto à espessura da epiderme. A análise das claudinas -1 e -4 demonstrou redução de ambas na pele dos doentes de DA, mas não houve correlação com a gravidade, espessura da epiderme, níveis de IgE sérica ou eosinofilia. Conclusão: No adulto com dermatite atópica, existe redução da expressão das proteínas relacionadas à barreira cutânea, como a filagrina e as claudinas -1 e -4. A redução da expressão da filagrina relacionou-se inversamente com a gravidade da doença, e com a espessura da epiderme, sugerindo cronicidade das lesões. Houve redução da expressão das claudinas -1 e -4, sem relação com a gravidade da doença, espessura da epiderme, eosinofilia ou com os níveis séricos de IgE / Introduction: Atopic dermatitis (AD) is a chronic, inflammatory dermatosis with ocasional flares. Its clinical features are variable, but pruritus and xerosis are frequent, and the disease may be associated to extracutaneous atopy. Patients with AD have increased risk for bacterial or viral infection, with emphasis on eczema herpeticum due to herpes simplex. AD is an example of a compromised skin barrier, allied to na imune dysfunction. There are reports on efective proteins of the skin barrier (filaggrin and claudins), related to increased risk for infection. Objectives: To evaluate the expression of proteins related to the skin barrier, such filaggrin and claudins-1 and-4 in the skin of adults with AD, followed at the Department of Dermatology, University of Sao Paulo Medical School. Methods: 32 individuals diagnosed as AD, according to Hanifin & Rajka\'s criteria, and 23 non-atopic controls, above the age of 18, were biopsied. Individuals with AD were biopsied in two different sites (lesional and nonlesional skin). The specimens were analyzed by immunohistochemistry through specific markers for filaggrin, claudins 1 and 4. The slides were scanned utilizing Panoramic Scan - 3DHistech - Hungary, and images analyzed by Image Pro Plus 4,5 for the intensity of each marker. The mean epidermal thickness was also evaluated. AD patients were also analyzed for disease severity (EASI), circulating IgE levels and eosinophilia. Results: In lesional and nonlesional skin of AD patients there was a reduced expression of filaggrin, when compared to nonatopic controls. There was an inverse correlation of filaggrin expression with disease severity and epidermal thickness. In the skin of AD individuals, there was reduced expression of claudins 1-and-4, which did not correlate with disease severity, epidermal thickness or eosinophilia. Conclusion: In adults with AD, there is reduced expression of skin barrier proteins, such as filaggrin, claudins 1 and 4. The reduction of filaggrin expression had an inverse correlation with disease severity and epidermal thickness, suggesting disease chronicity. There was reduction of claudins 1 and 4, with no relation with disease severity, epidermal thickness, circulating IgE levels or eosinophilia

Introdução: A dermatite atópica (DA) é uma doença cutânea inflamatória crônica que cursa em surtos. Possui manifestação clínica variável, mas o prurido e a xerose são características frequentes, e pode estar associada a outras manifestações extra-cutâneas de atopia. Pacientes com DA apresentam maior risco de infecções por bactérias e vírus, destacando-se a erupção variceliforme de Kaposi, causada por herpes simples. A DA mostra-se como exemplo de dermatose com comprometimento da barreira cutânea, aliado a disfunção imunológica. São descritas alterações das proteínas da barreira cutânea na DA (filagrina e claudinas ), relacionadas ao maior risco de infecção . Objetivo: Avaliar a expressão de proteínas relacionadas à barreira cutânea como a filagrina, e as claudinas -1 e -4 na pele de pacientes adultos com dermatite atópica, acompanhados no Departamento de Dermatologia da Faculdade de Medicina da Universidade de São Paulo. Métodos: 32 indivíduos com diagnóstico de DA (estabelecido pelos critérios de Hanifin & Rajka) e 23 controles (indivíduos sem DA), maiores de 18 anos, foram submetidos a biópsias cutâneas. Os indivíduos com DA foram biopsiados em dois pontos, tanto na pele lesada, quanto na pele não-lesada. O material obtido foi analisado por imuno-histoquímica, através de marcadores específicos para filagrina, claudina-1 e claudina-4. As lâminas foram digitalizadas pelo Panoramic Scan - 3DHistech - Hungria, e as imagens analisadas pelo software Image Pro Plus 4,5, quanto à intensidade da expressão do marcador. A espessura média da epiderme do local estudado foi também avaliada. O grupo com DA foi também analisado quanto à gravidade da doença (EASI), níveis séricos de IgE e grau de eosinofilia. Resultados: Houve redução da expressão da filagrina na pele de doentes de DA em relação aos controles, tanto na pele com lesão quanto na pele sem lesão. Demonstrou-se correlação inversa na expressão da filagrina, tanto com relação à gravidade da doença quanto à espessura da epiderme. A análise das claudinas -1 e -4 demonstrou redução de ambas na pele dos doentes de DA, mas não houve correlação com a gravidade, espessura da epiderme, níveis de IgE sérica ou eosinofilia. Conclusão: No adulto com dermatite atópica, existe redução da expressão das proteínas relacionadas à barreira cutânea, como a filagrina e as claudinas -1 e -4. A redução da expressão da filagrina relacionou-se inversamente com a gravidade da doença, e com a espessura da epiderme, sugerindo cronicidade das lesões. Houve redução da expressão das claudinas -1 e -4, sem relação com a gravidade da doença, espessura da epiderme, eosinofilia ou com os níveis séricos de IgE / Introduction: Atopic dermatitis (AD) is a chronic, inflammatory dermatosis with ocasional flares. Its clinical features are variable, but pruritus and xerosis are frequent, and the disease may be associated to extracutaneous atopy. Patients with AD have increased risk for bacterial or viral infection, with emphasis on eczema herpeticum due to herpes simplex. AD is an example of a compromised skin barrier, allied to na imune dysfunction. There are reports on efective proteins of the skin barrier (filaggrin and claudins), related to increased risk for infection. Objectives: To evaluate the expression of proteins related to the skin barrier, such filaggrin and claudins-1 and-4 in the skin of adults with AD, followed at the Department of Dermatology, University of Sao Paulo Medical School. Methods: 32 individuals diagnosed as AD, according to Hanifin & Rajka\'s criteria, and 23 non-atopic controls, above the age of 18, were biopsied. Individuals with AD were biopsied in two different sites (lesional and nonlesional skin). The specimens were analyzed by immunohistochemistry through specific markers for filaggrin, claudins 1 and 4. The slides were scanned utilizing Panoramic Scan - 3DHistech - Hungary, and images analyzed by Image Pro Plus 4,5 for the intensity of each marker. The mean epidermal thickness was also evaluated. AD patients were also analyzed for disease severity (EASI), circulating IgE levels and eosinophilia. Results: In lesional and nonlesional skin of AD patients there was a reduced expression of filaggrin, when compared to nonatopic controls. There was an inverse correlation of filaggrin expression with disease severity and epidermal thickness. In the skin of AD individuals, there was reduced expression of claudins 1-and-4, which did not correlate with disease severity, epidermal thickness or eosinophilia. Conclusion: In adults with AD, there is reduced expression of skin barrier proteins, such as filaggrin, claudins 1 and 4. The reduction of filaggrin expression had an inverse correlation with disease severity and epidermal thickness, suggesting disease chronicity. There was reduction of claudins 1 and 4, with no relation with disease severity, epidermal thickness, circulating IgE levels or eosinophilia

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-04-26T15:54:34Z No. of bitstreams: 1 klausrubackbertges.pdf: 2812002 bytes, checksum: 85312f3aa11372affe21388fe45dce43 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-04-27T11:52:01Z (GMT) No. of bitstreams: 1 klausrubackbertges.pdf: 2812002 bytes, checksum: 85312f3aa11372affe21388fe45dce43 (MD5) / Made available in DSpace on 2018-04-27T11:52:01Z (GMT). No. of bitstreams: 1 klausrubackbertges.pdf: 2812002 bytes, checksum: 85312f3aa11372affe21388fe45dce43 (MD5) Previous issue date: 2018-03-21 / A esofagite eosinofílica vem tendo uma importância crescente na literatura médica. É uma doença inflamatória crônica, imunoantígeno mediada, caracterizada por sintomas de disfunção esofágica e uma infiltração predominantemente eosinofílica na mucosa do esôfago. O diagnóstico histopatológico é definido pela presença de 15 ou mais eosinófilos por campo de grande aumento e o tratamento de cada paciente deve ser individualizado. O processo de remodelamento do esôfago na esofagite eosinofílica ainda não está completamente esclarecido, mas parece envolver a presença de fibrose na lâmina própria, sendo a grande responsável pelos sintomas de disfagia e impactação alimentar. A IL-13 contrarregula a expressão de filagrina nas células epiteliais, promovendo um mecanismo pelo qual antígenos alimentares ativam o sistema imunológico. Este estudo objetivou avaliar a prevalência das características histopatológicas de remodelamento tecidual e a expressão da filagrina em biópsias esofágicas de pacientes com esofagite eosinofílica. Foram avaliadas, retrospectivamente, 50 pacientes com suspeita clínica e/ou endoscópica de esofagite eosinofílica. Deste total, 27 foram selecionados e distribuídos em dois grupos: GI, com 15 a 24 eosinófilos por campo de grande aumento e GII, contendo mais de 24. Após a histomorfometria e imuno-histoquímica para filagrina, comparou-se os dois grupos. Nos parâmetros de fibrose, houve diferença estatisticamente significante, com maior prevalência no GII (p < 0,05). Também houve mais espessamento da camada basal no GII (p < 0,001). No estudo de correlação entre o número de eosinófilos e o percentual de fibrose, encontrou-se correlação positiva: 50,8% (p = 0,016), ou seja, mais fibrose nos casos do GII. Na correlação entre o número de eosinófilos e a espessura da camada basal, o resultado também foi positivo: 52,1% (p = 0,08), ou seja, membrana basal mais espessa no GII. A filagrina mostrou-se reduzida nos pacientes com esofagite eosinofílica. / Eosinophilic esophagitis is of increasing importance in the medical literature. It is a chronic inflammatory disease, mediated immunoantigen, characterized by symptoms of esophageal dysfunction and a predominantly eosinophilic infiltration in the esophagic mucosa. The histopathological diagnosis is defined by the presence of 15 or more eosinophils per large increase field and the treatment of each patient should be individualized. The process of esophageal remodeling into eosinophilic esophagitis is still not fully understood, but it seems to involve a presence of fibrosis in the lamina propria, being a major cause of the symptoms of dysphagia and food impaction. IL-13 counteracts the expression of filaggrin in epithelial cells, promoting a mechanism by which food antigens activate the immune system. This study aimed to evaluate a prevalence of the histopathological characteristics of tissue remodeling and the filaggrin expression in esophageal biopsies of patients with eosinophilic esophagitis. Fifty patients with clinical and/or endoscopic suspicion of eosinophilic esophagitis were retrospectively evaluated. Of these, 27 were selected and distributed in two groups: GI, with 15 to 24 eosinophils per large increase field and GII, containing more than 24. After a histomorphometry and immunohistochemistry for filaggrin, the two groups were compared. In the fibrosis parameters, there was a statistically significant difference, with a higher prevalence in GII (p < 0.05). There was also more thickening of the basal layer in GII (p < 0.001). The correlation study between the number of eosinophils and the percentage of fibrosis found a positive correlation: 50.8% (p = 0.016), that means, more fibrosis in cases of GII. In the correlation between the number of eosinophils and the thickness of the basal layer, the result was also positive: 52.1% (p = 0.08), that means, thicker basal layer in GII. Filaggrin was reduced in patients with eosinophilic esophagitis.

Erhöhte Hautoberflächen-pH-Werte werden bei atopischer Dermatitis beobachtet.Störungen der pH-Regulation, insbesondere eine unzureichende Azidifizierug des Stratum corneum, werden mit der Störung der epidermalen Barrierefunktion bei atopischer Dermatitis als pathogenetischer Faktor diskutiert.Zielsetzung der vorliegenden Arbeit war die Klärung der Frage, ob Störungen der pH-Regulation schon unmittelbar im Verlauf der postpartalen Phase bei Neugeborenen nachweisbar sind. Ferner sollte geklärt werden, ob der Hautoberflächen-pH-Wert als Prädiktor der atopischen Dermatitis herangezogen werden kann.Es wurden 108 Säuglinge der Entbindungsstation des Städtischen Klinikums Gütersloh im postpartalen Verlauf (4 bis 7 Tage,sowie 6, 12 und 24 Wochen nach der Geburt) mittels Hautoberflächen-pH-Messung und Corneometrie (24 Woche postpartum) untersucht. Es wird gezeigt, dass der Hautoberflächen-pH-Wert atopisch disponierter Säuglinge und von Säuglingen ohne atopische Disposition keinen signifikanten Unterschied im postpartalen Verlauf aufweist.Dagegen zeigten atopisch disponierte Säuglinge einen deutlich niedrigeren Wert der Stratum-corneum-Hydratation (Corneometrie). Die Ergebnise lassen den Schluß zu, dass der Barrierefunktionsstörung der atopischen Dermatitis keine primäre pH-Regulationsstörung zugrunde liegt. Die Messung der Hautoberflächen-pH eignet sich nicht zur Früherkennung der atopischen Dermatitis.