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).

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.

Orientador: Luiz Henrique de Araújo Machado / Resumo: A dermatite atópica canina (DAC) apresenta uma complexa relação entre a inflamação e a barreira cutânea. O envolvimento de resposta imunológica do tipo Th2 e Th1, bem como produção de citocinas pró inflamatórias têm sido relacionadas com o padrão inflamatório, sinais clínicos e a gravidade da doença. As células-tronco mesenquimais (CTM) possuem características imunomoduladoras, sendo altamente sugeridas para reduzir a produção de citocinas inflamatórias e melhorar a qualidade de vida de animais atópicos. Visando comparar o efeito das CTM em cães atópicos, realizamos um estudo randomizado, simples-cego, com período inicial de placebo em seis cães. Foram comparados os perfis séricos inflamatórios por ELISA, além de resposta clínica por CADESI-04 e escala analítica de prurido. Ao final do estudo, os cães melhoraram significantemente o padrão clínico por CADESI-04 (p < 0,001) e pela escala de prurido (p < 0,05), contudo não houve mudança no perfil inflamatório das citocinas. Foi observado reações adversas em dois animais do estudo. Concluindo, o uso de CTM pode ser eficaz na melhora da sintomatologia relacionada a doença. / Abstract: Canine atopic dermatitis (CAD) presents a complex relationship between inflammation and cutaneous barrier. The involvement of Th2 and Th1 type immune responses, as well as the production of proinflammatory cytokines have been related to inflammatory pattern, clinical signs and severity of disease. Mesenchymal stem cells (MTCs) have immunomodulatory characteristics and are highly suggested to reduce the production of inflammatory cytokines and to improve quality of life of atopic animals. In order to compare effects of MTCs on atopic dogs, we performed a randomized, simple-blind, placebo-controlled study in 6 dogs. Inflammatory serum profiles were compared by ELISA, in addition to clinical response by CADESI-04 and analytical pruritus scale. At the end of the experiment, dogs significantly improved the clinical standard for CADESI-04 (p <0.001) and pruritus scale (p <0.05), however, there was no change in the inflammatory cytokine profile. Adverse reactions were observed in two study animals. Concluding that the use of CTM may be effective in improving symptoms related to disease / Mestre

1 Abstract Acylceramides belong to the subgroup of ultralong chain ceramides. They are essential components of the extracellular lipid matrix of stratum corneum, where they play a crucial role in proper function of skin barrier (they help preventing the excessive water loss and penetration of exogenous substances and pathogens to the organism). The 32-hydroxydotriacontanoic acid is one of the fatty acids forming the backbone of all the acylceramides. In the molecule of acylceramide, the carboxyl group of this acid is bound to a primary amino group of the sphingoid base and the ω-hydroxy group is esterified with linoleic acid. In the stratum corneum, 32-hydroxydotriacontanoic acid may remain as a part of free acylceramides or it can be covalently linked to the surface of corneocytes and form the "first lamela", which then serves as a basis for the orientation of other lipids in the matrix. The recent literature describes the synthesis of 32-hydroxydotriacontanoic acid but only with relatively small overall yields. The most problematic part of the synthesis seems to be the connection of two shorter fragments leading to the ultralong chain. The main aim of this research project was to optimalise the reaction conditions to increase the yield of formation of the utralong acid, focusing on the most complicated...

Polymorphic light eruption (PLE) is the most common photodermatosis, affecting ~17% of the population. PLE is a delayed-type hypersensitivity response to an antigen induced by solar ultra-violet radiation (UVR). Its effects vary between patients, but the main symptom is a non-scarring, red papular rash in areas exposed to UVR. An effective therapy is low dose ultra-violet B (NBUVB) phototherapy. It is thought that NBUVB phototherapy desensitises the skin to further UVR exposure, but the mechanism by which this happens is unknown. Current immune based studies have been unable to clarify a mechanism as to how PLE arises. However, research in other skin diseases, such as psoriasis and atopic dermatitis, has shown that the barrier function of the skin is compromised by these disorders. Furthermore, research in lesional PLE skin showed an increase in barrier permeability of the skin. Recent research has specifically linked claudin proteins of tight junctions to the barrier dysfunction. Therefore, this study used quantitative immunofluorescent staining to measure tight junction (TJ) proteins and other barrier proteins of interest. Barrier function was also measured by transepidermal water loss (TEWL); a tracer dye penetration assay was used to measure TJ barrier function specifically. All measurements were made in non-lesional PLE skin, as compared to skin from healthy human volunteers. In photoprotected PLE skin the TJ protein claudin-1 was significantly reduced compared to healthy skin. The use of a tracer dye highlighted there was a reduction in TJ barrier function in PLE skin compared to healthy individuals. PLE and healthy skin were then exposed to ultra-violet B (UVB) and 24h later TJ proteins and TJ barrier function were measured. There was no change to claudin-1 after UVB exposure in PLE skin, but claudin-7 was reduced and claudin-12 increased. In contrast, in UVB-irradiated skin in normal controls after UVB exposure claudin-7 and claudin-12 were both increased, whilst claudin-1 was reduced. In PLE patients there was no further change to TJ barrier function, however, in normal controls, skin TJ barrier function was reduced post UVB. Both in healthy and PLE skin TEWL was unchanged before and after UVB exposure. Lastly TJ proteins were investigated after NBUVB in PLE patients. There was a further reduction in claudin-1 in PLE patients as well as a reduction in the TJ protein occludin, however the stratum corneum was significantly thickened. It could be suggested that this is a compensatory measure for the reduction seen in TJ barrier proteins, however further studies are needed to understand this. These data show significant differences in the TJ skin barrier in patients with PLE as compared to healthy human volunteers before and after UVB exposure. Furthermore, in PLE skin there is a significant change to the epidermis after NBUVB phototherapy. These data demonstrate that TJ protein expression and function is altered in PLE skin and may contribute to aetiology of the disorder, however the role of TJ barrier in aetiology is yet to be firmly established.

Des formes galéniques spécialement étudiées permettent l'administration topique d'un médicament ou d'un actif cosmétique afin d'obtenir un effet local ou une action systémique. Les excipients présents dans la formulation, comme les tensioactifs dans les solutions micellaires, émulsions, microémulsions, nanoémulsions, nanostructures, peuvent être alors des éléments influençant la pénétration et la perméation de l'actif. L'étude de leurs effets sur la barrière cutanée peut être utile afin de choisir au mieux les différents composés de la formulation. L'objectif de cette thèse a été d'étudier l'effet de différents tensioactifs (alkylpolyglucosides, lipoaminoacides, alcools gras éthoxylés et copolymères à blocs de type Poloxamer) en solution aqueuse sur la structure et la fonction barrière de la peau, grâce à des techniques d'infrarouge à transformée de Fourier, de calorimétrie différentielle à balayage, de mesure de la perte insensible en eau et de microscopie électronique à transmission. L'objectif spécifique de ce travail a porté sur l'étude de l'absorption cutanée ex vivo de trois principes actifs de lipophilie différente (caféine, kétoprofène et progestérone) à travers un modèle de peau animale co-traitée par différents tensioactifs. Plus spécifiquement, la pénétration de la progestérone au sein des différentes couches de la peau a été étudiée compte tenu de sa lipophilie élevée limitant son passage dans les couches cutanées plus hydrophiles. A l'issue de ces travaux, nous montrons que les tensioactifs peuvent exercer non seulement des effets sur l'organisation lamellaire des lipides intercornéocytaires mais aussi sur les cornéocytes du stratum corneum (SC). Par ailleurs, ces différents résultats ont permis de définir les structures chimiques des tensioactifs favorisant l'absorption cutanée de principes actifs. Les tensioactifs anioniques comportant une chaîne aliphatique de 12 carbones augmentent sélectivement la perméation cutanée de principes actifs hydrophiles et la pénétration de principes actifs lipophiles dans les tissus cutanés. La pénétration cutanée de principes actifs est étroitement corrélée à la taille des micelles de tensioactif susceptible de s'incorporer dans les espaces intercornéocytaires du SC. Enfin, la valeur de concentration micellaire critique est une propriété physico-chimique permettant d'expliquer en partie l'effet des tensioactifs sur leur perméabilité cutanée / Dosage forms specially designed allow the topical administration of drugs or cosmetic active ingredients to obtain a local effect or a systemic action. Excipients, such as surfactants in micellar solutions, emulsions, microemulsions, nanoemulsions, nanostructures can influence the drug penetration and permeation. Understanding their effects on skin barrier is helpful in the choice of surfactant. The aim of this thesis was to study the effect of different surfactants (alkylpolyglucosides, lipoaminoacids, ethoxylated fatty alcohol and copolymers blocks) in aqueous solution on the skin barrier structure and function, using techniques like infrared Fourier transform, differential scanning calorimetry, transepidermal water loss measurement and transmission electron microscopy. The specific aim of this work focused on the ex vivo cutaneous absorption of three drugs showing different lipophilicity (caffeine, ketoprofen and progesterone) through animal skin co-treated by different surfactants. More specifically, the penetration of progesterone in the different skin layers has been studied due to its high lipophilicity limiting its permeation in the most hydrophilic layers (viable epidermis and dermis). At the outset, we showed that surfactants could provide not only an effect on lamellar organization of intercorneocyte lipids but also on the corneocytes into the stratum corneum (SC). Moreover, these different results allowed to define surfactant chemical structure increasing drug cutaneous absorption. Anionic surfactant with a C12 chain length increased significantly cutaneous permeation of hydrophilic drug and penetration of lipophilic drug into cutaneous tissue. Drug penetration is correlated with micelle size allowed its incorporation into SC intercorneocyte spaces. Finally, the value of its critical micellar concentration is a physico-chemical properties allowed to partially explain the surfactant effect on their cutaneous permeability

Background: The differential effects of using topical oils for the prevention or treatment of baby dry skin on skin barrier function may contribute to the development of childhood atopic eczema. Prevalence of atopic eczema has increased from 5% of children aged 2 to 15 years in the 1940s, to approaching 30% more recently. This increase cannot be attributed to genetic changes. It is likely that increases stem from environmental factors, including the increased use of some inappropriately formulated commercial and natural baby skincare products. Midwives, health visitors and other maternity service health professionals, in the UK, routinely recommend the use of olive oil and sunflower oil for baby dry skin or massage, but the effect of these oils on newborn baby skin has not been studied. Aim: The aim of this research was to assess the feasibility and acceptability of testing the hypothesis that the regular application of sunflower oil, when compared to no oil or olive oil, had an effect on skin barrier function of newborn term babies. Study Design: A pilot, assessor-blinded, single centre, three-arm, randomised controlled trial, with nested qualitative component, underpinned by post-positivism. Methods: Quantitative methods were used to establish proof of concept that the use of topical oils had some effect on newborn baby skin barrier function, and to assess the feasibility of trial processes and parameters. Qualitative methods were used to explore the acceptability to parents of having a newborn baby participating in a randomised controlled trial, and trial design and procedures. The study was conducted in St. Mary’s hospital, a large teaching hospital in North West England. Data were collected between September 2013 and August 2014.The randomised controlled trial included 115 babies who were randomised to three groups: sunflower oil, olive oil and no oil, using a computer-generated varied size block randomisation with concealed allocation. Parents of babies randomised to the oil groups were blinded to which oil they were allocated. Data were collected using standardised case report forms for demographic and clinical observation data, weekly telephone questionnaires and a follow-up questionnaire, informed by previous baby skincare trials. The qualitative study encompassed semi-structured interviews, conducted within six months of birth. The sample was a subset of the trial participants, purposively sampled to incorporate a mix of treatment groups and positive and negative experiences derived from the follow-up questionnaire. Data also included two open-text questions from the follow-up questionnaire. Quantitative data were managed using IBM SPSS Statistics versions 20 and 22 and analysed descriptively. Qualitative data were managed in NVivo 10 and analysed using Framework Analysis. Results: The pilot study found that a definitive randomised controlled trial is not the optimal next step. A longitudinal observational study and further mechanistic work is recommended. Recruitment was challenging and loss to follow-up was higher than anticipated. Protocol adherence was reasonable and the study was acceptable to parents. Some statistically significant results were obtained, which must be interpreted with caution as the study was not powered to detect such a difference. These results showed that both oils may impede the development of the skin barrier function from birth; clinical importance of the results is not known. Conclusion: A longitudinal observational study is required, which maps the diagnosis of atopic eczema with environmental factors such as the use of baby skincare products from birth. Mechanistic work is also required to consider the optimal skincare formulation. As any intervention should do more good than harm, it would be wrong to support the recommendation of topical olive oil or sunflower oil for newborn baby dry skin or massage, based on the study data.

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京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21804号 / 農博第2317号 / 新制||農||1065(附属図書館) / 学位論文||H31||N5176(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 菅原 達也, 教授 佐藤 健司, 教授 松井 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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