No / This review focuses on treatment targets for the most common inflammatory skin diseases, eczema and psoriasis with an emphasis on cytokines expressed in the uppermost layer of the skin which is easily accessible for diagnostic and therapeutic approaches. Recently, a significant body of research has highlighted the influence of the skin barrier and the patients’ microbiome on skin inflammatory responses and we will comment on their impact on mediator regulation. Itch is a prominent dermatology symptom which is influenced by cytokines and can via itch–scratch cycle impact on the skin barrier and mediator expression associated with damage. Taking the contribution of pruritus and superficial skin damage into account, we address cytokines as targets for stratified treatment approaches in subgroups of eczema and psoriasis.

京都大学 / 新制・論文博士 / 博士(社会健康医学) / 乙第13612号 / 論社医博第18号 / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 森田 智視, 教授 古川 壽亮, 教授 今中 雄一 / 学位規則第4条第2項該当 / Doctor of Public Health / Kyoto University / DFAM

Le besoin de trouver de nouveaux pansements pour les personnes touchées par des plaies de la peau (brûlures, ulcères), et la nécessité de développer des modèles de peau adéquats pour tester de nouvelles formulations médicamenteuses développées in vitro, ont motivé la recherche dans le domaine des substituts de peau produits par génie tissulaire. Il est possible de produire des substituts de peau normale humaine (SPNH), constitués d’un derme et d’un épiderme stratifié (épiderme vivant, EV, et couche cornée, CC), en utilisant la méthode d’auto-assemblage développée par le Laboratoire d’Organogénèse Expérimentale (LOEX). Dans cette étude, des analyses par microspectroscopie vibrationnelle (infrarouge, IR, et Raman) ont été effectuées afin d’obtenir une caractérisation morpho-spectrale des trois couches caractéristiques des SPNH, qui ont été comparés à la peau normale humaine (PNH). Concernant la distribution et l’organisation des lipides, les résultats de microspectroscopie IR ont montré que les lipides de la CC étaient plus ordonnés que ceux de l’EV. En microspectroscopie Raman, les résultats confirment que la CC est une couche riche en lipides qui sont ordonnés dans la PNH et les SPNH. La quantité de lipides diminue et davantage de désordre apparait dans l’EV pour la PNH et les SPNH. Cependant les résultats montrent également qu’il y a moins de lipides dans les SPNH et que les lipides sont plus ordonnés dans la PNH. Concernant la structure secondaire des protéines et le contenu en protéines, les données montrent qu’ils sont similaires dans les SPNH et la PNH (kératine dans l’épiderme et collagène dans le derme). Finalement, l’organisation des lipides ainsi que le contenu en protéines des différentes couches sont similaires pour les SPNH et la PNH, confirmant que les SPNH reproduisent les propriétés essentielles de la peau native. Cette étude caractérise également la peau psoriasique humaine (PPH) et fournit une compréhension détaillée de son organisation et de sa composition moléculaire. Les microspectroscopies IR et Raman montrent une distribution similaire des lipides et du collagène pour la PNH et la PPH. Cependant, la PPH présente plusieurs caractéristiques montrant une perte globale d’organisation structurale qui pourrait expliquer la réduction de ses propriétés barrières. Il s’agit de la première caractérisation de la structure moléculaire de ces SPNH qui ont d’ores-et-déjà une application prometteuse dans le domaine clinique. La caractérisation de la PPH pourrait être le point de départ de la caractérisation des substituts pathologiques. / Research in the field of bioengineered skin substitutes is motivated by the need to find new dressings for people affected by skin injuries (burns, diabetic ulcers), and to develop adequate skin models to test new drug formulations developed in vitro. It is possible to produce human skin substitutes (HSS) consisting in a dermis and a stratified epidermis (living epidermis, LE, and stratum corneum, SC), using the self-assembly method developed by the Laboratoire d’Organogénèse Expérimentale (LOEX). In the present work, vibrational microspectroscopy analyses (infrared, IR, and Raman) were performed to obtain in-depth morpho-spectral characterization of the three characteristic layers of HSS as compared with normal human skin (NHS). Concerning the lipid distribution and organization, IR microspectroscopy results suggest that lipids in the SC are more ordered than those in the LE. Raman microspectroscopy results confirm that the SC is a layer rich in lipids which are well-ordered in both NHS and HSS. The amount of lipids decreases and more disorder appears in the LE for both NHS and HSS. However, the results also show that there are fewer lipids in the HSS and that the lipids are more organized in the NHS. Concerning the secondary structure of proteins and protein content, the data show that they are similar in the HSS and in NHS (keratin in the epidermis and collagen in the dermis). Finally, the lipid organization as well as the protein composition in the different layers are similar for HSS and NHS, confirming that the HSS reproduce essential features of real skin. This study also investigates psoriatic human skin (PHS) and provides a deep understanding of its molecular organization and composition. IR and Raman microspectroscopies reveal a similar distribution of lipids and collagen for NHS and PHS. However, PHS exhibits various characteristics showing a global decrease of the structural organization that is compatible with a reduction in its barrier properties. It is the first characterization of the molecular structure of these HSS, which are already considered as a promising biological wound dressing for clinical applications. The characterization of PHS could be the starting point of the characterization of the pathological substitutes.

Le sérum ajouté aux milieux de culture est connu pour être une substance difficile à contrôler en raison de la présence de facteurs variables. Le but de cette étude était de faire varier les conditions de culture en retirant le sérum lors de la montée à l'interface air-liquide et de poursuivre les observations sur la qualité des substituts cutanés sains et psoriasiques produits par la méthode d'auto-assemblage. Les résultats histologique, macroscopique et immunohistochimique des substituts sains cultivés avec ou sans sérum ne montrent aucune différence significative. Les analyses infrarouges effectuées sur ces derniers permettent d'observer que ceux cultivés en absence de sérum possèdent une meilleure organisation lipidique au niveau de la couche cornée. Par contre, l'absorption percutanée démontre des différences au regard des propriétés physico-chimiques des molécules étudiées. De plus, les substituts conçus à partir de cellules psoriasiques affichent des résultats différents lorsque le sérum est retiré. Il semble que l'apport du sérum soit davantage important lors de l'utilisation de cellules pathologiques.

Various approaches are currently used to treat and manage psoriasis, and biological treatments are often the latest approaches. All biological treatments have major side effects as they are given systemically via injections. One of the latest biological treatments for psoriasis, one which has shown great efficacy with fewer side effects, is Secukinumab. Secukinumab is an anti-IL17 antibody that works by stopping the action of IL17, a cytokine that is known to have a major role in the pathogenesis of psoriasis. This work is based on the development of a new way to commence drug therapy to reduce the side effects of the treatment. Our work is based on the studies of the genotoxicity of the drug Secukinumab in its bulk and liposome form using comet and micronucleus assays on lymphocytes. The results from both assays have illustrated the safety of the drug and demonstrated the reduction of the DNA damage induced in both healthy individuals and patients with psoriasis. Secukinumab significantly decreases-H2O2 induced damage and efficiently attenuates its adverse effects both in the comet (p<0.0001) and micronucleus assays (p<0.01). The two concentrations of Secukinumab used (2.1 and 2.8μg/ml) efficiently decreased H2O2-induced DNA damage in both groups to nearly the level of the negative control. Overall, Secukinumab reveals protective and anti-genotoxic effects by demonstrating its potential in reducing DNA damage caused by oxidative stress and by not inducing any further damage in the lymphocytes of either healthy individuals or patients. Liposomes are highly versatile which have been proven efficient for therapy and research applications. The discovery of new therapies in the treatment of psoriasis is a considerable challenge and is now a necessity. Our study was the first one to determine the genotoxicity of various concentrations of the drug in the lymphocytes of psoriasis patients compared to healthy individuals. In the MTT assay, the data showed a decrease in % cell survival rates after exposure to different concentrations of Secukinumab. Also, the results demonstrated no statistically significant differences on confounding factors such as ethnicity, smoking, drinking habits, gender and age among psoriasis patient and healthy controls. The regulation of gene expression levels of IL-17, IL-22 and RORC were assessed after treatment with Secukinumab in the bulk and liposome form via RT-PCR analysis. Secukinumab bulk (2.1μg/ml) treatment significantly down-regulated gene expression of IL-17, IL22 and RORC to 0.46-fold, 0.47-fold and 0.5-fold, respectively. However, Secukinumab liposome (2.1μg/ml) only decreased the expression of IL-17 and IL-22 significantly, by 0.46-fold and 0.53-fold, respectively. On the other hand, studying the expression of P53 and P21 using qPCR revealed that Secukinumab bulk and liposome has no effect on the expression of these genes in lymphocytes from healthy individuals and psoriasis patients. Western blotting was used to investigate the effect of Secukinumab in both forms on protein expression levels IL-17, IL-22 and RORC. Analysis of the results showed that Secukinumab bulk and liposome had no significant effect on expression levels of any of these proteins in lymphocytes derived from healthy individuals. However, there was a statistically significant down-regulation observed in the protein expression levels of IL-17, IL-22 and RORC in lymphocytes obtained from the psoriasis patients, confirming the sensitivity of the compromised lymphocytes from patient group to Secukinumab treatment. With Secukinumab (bulk form) administration, a 0.5-fold decrease was observed in IL-17, 0.59-fold decrease in IL-22, and a 0.6-fold decrease in RORC expression. However, liposome form reduced their levels to 0.47–fold, 0.5-fold and 0.47–fold, respectively, when compared to the control group. While it had no significant effect on expression of P53 and P21 proteins in lymphocytes from healthy individuals and psoriasis patients and there was no difference observed in their regulation. In conclusion, the use of Secukinumab liposome as topical drug delivery system may be suitable replacement for improving the drug bioavailability and its side effects. / Libyan Cultural Attaché and Libyan embassy

Le métabolisme des lipides joue un rôle primordial au cœur de l'homéostasie cutanée. Précisément, les acides gras polyinsaturés (PUFAs) n-3 et n-6 sont impliqués dans des fonctions à la fois structurales et métaboliques, essentielles au développement et au maintien de la fonction barrière de la peau. Ces fonctions variables et complexes sont toujours partiellement incomprises à ce jour, et les conséquences qui en découlent sont évidentes et parfois néfastes. En effet, cette incompréhension limite notre capacité à reconstruire des modèles cutanés plus représentatifs de la peau normale humaine, qui sont nécessaires aux études pharmaceutiques. De plus, cette dernière fait partie intégrante de notre incapacité à déterminer les causes de certaines pathologies multifactorielles complexes telles que le psoriasis et, conséquemment, à développer des traitements efficaces pour les soigner. Les modèles de peau humaine sains et psoriasiques produits par génie tissulaire ont été exploités afin de mettre en lumière le rôle des acides gras polyinsaturés dans la fonction barrière de la peau. Au cours de cette thèse, plusieurs objectifs spécifiques ont été étudiés. Les deux premiers objectifs visaient l'optimisation du modèle de peau saine grâce à la supplémentation des milieux de culture avec, dans un premier temps, soit une supplémentation individuelle en acide alpha-linolénique (n-3 PUFAs) ou en acide linoléique (n-6 PUFAs). Dans un deuxième temps, une double supplémentation a été réalisée où l'acide alpha-linolénique et l'acide linoléique ont été ajoutés simultanément. Les résultats issus de ces deux études ont montré que la supplémentation des milieux de culture avec l'acide alpha-linolénique et celle avec les deux acides simultanément permettaient d'améliorer la fonctionnalité des substituts cutanés en diminuant l'absorption percutanée de la testostérone, alors que la supplémentation en acide linoléique seul n'avait pas d'effet. Ces résultats surprenants ont mis en évidence l'importance du ratio de n-3 et de n-6 PUFAs lors de la formation de la barrière cutanée in vitro. Le troisième objectif de cette thèse a été de caractériser l'endocannabinoïdome de la peau normale humaine et des substituts sains reconstruits par génie tissulaire. Cette étude a permis de mieux comprendre les différents acteurs du métabolisme des endocannabinoïdes au sein de la peau. Nos résultats ont aussi montré que les différents endocannabinoïdes présents dans la peau humaine étaient également retrouvés dans les substituts cutanés. De plus, une double supplémentation en acide alpha-linolénique et en acide linoléique a permis de moduler le profil d'endocannabinoïdes, confirmant que les peaux représentent un modèle adéquat pour l'étude du métabolisme des endocannabinoïdes cutanés. Finalement, le dernier objectif spécifique de cette thèse consistait à étudier le potentiel de l'acide alpha-linolénique (n-3 PUFAs) comme traitement pour soigner le psoriasis. Cette étude a permis de confirmer que les n-3 PUFAs pouvaient réguler la prolifération et la différenciation des kératinocytes psoriasiques in vitro afin qu'ils retrouvent un phénotype se rapprochant davantage de celui des cellules saines. Nos résultats montrent que cette régulation est induite par la modulation de la production des médiateurs lipidiques issus des acides gras polyinsaturés n-3 et n-6 ainsi que par l'activation et l'inhibition subséquentes des cascades de transduction du signal des kinases. En conclusion, le travail présenté dans cette thèse a permis de faire avancer de manière importante l'état des connaissances en ce qui a trait au métabolisme des acides gras polyinsaturés n-3 et n-6 au sein des peaux saines et psoriasiques. Ce dernier a également permis l'optimisation de la reconstruction tissulaire d'un modèle de peau dans le but d'obtenir un tissu affichant une barrière cutanée similaire à celle d'une peau normale humaine et, par conséquent, de mener à un modèle répondant davantage aux besoins de l'industrie pharmaceutique. Finalement, cette thèse a contribué à supporter et à promouvoir l'utilisation des n-3 PUFAs en tant que traitement possible pour les patients atteints de psoriasis, ainsi qu'à démontrer la pertinence de poursuivre les recherches dans ce domaine puisqu'elles offrent un potentiel important quant à l'amélioration de la qualité de vie des patients. / Lipid metabolism plays a central role in the skin homeostasis. More specifically, omega-3 and omega-6 polyunsaturated fatty acids (n-3 and n-6 PUFAs) are involved in both structural and metabolic functions, which are essential for the development and maintenance of the skin barrier function. These highly variable and complex functions are still partially misunderstood to this day, and the resulting consequences are obvious. Indeed, these gaps in knowledge limit our ability to produce reconstructed skin models that are representative of normal human skin, which are necessary for pharmaceutical studies. In addition, improving our knowledge of skin lipids would help understand the causes of certain complex multifactorial pathologies such as psoriasis and would consequently lead to the development of more effective therapies. Tissue-engineered healthy and psoriatic human skin substitutes have been exploited to shed light on the role of polyunsaturated fatty acids regarding the barrier function of the skin. This thesis evaluated several specific objectives. The first two objectives aimed at the optimization of the healthy skin substitutes using first, individual supplementation of culture media with alpha-linolenic acid (n-3 PUFA) or linoleic acid (n-6 PUFA), as well as in a second step, a dual supplementation with both alpha-linolenic and linoleic acids. The results of these two studies showed that supplementation of culture media with alpha-linolenic acid and with the dual supplementation improved the functionality of skin substitutes by reducing the percutaneous absorption of testosterone, whereas the linoleic acid supplementation alone had no effect. These surprising results demonstrated the importance of the ratio of n-3 to n-6 PUFAs in the formation of the skin barrier in vitro. The third objective of this thesis was to characterize the endocannabinoidome of human skin compared to that of healthy tissue-engineered skin substitutes. This study provides a better understanding of the different actors in the metabolism of endocannabinoids within the skin. Our results show that the different endocannabinoids present in human skin were also found in the skin substitutes, and that they were modulated following a double supplementation with alpha-linolenic acid and linoleic acid, confirming that reconstructed skin equivalents are an adequate model for the study of the metabolism of cutaneous endocannabinoids. Finally, the last specific objective of this thesis was to study the potential of alpha-linolenic acid (n-3 PUFA) as a treatment for psoriasis. This study confirmed that n-3 polyunsaturated fatty acids can regulate the proliferation and differentiation of psoriatic keratinocytes so that they adopt a healthier behavior. This regulation is mediated by the modulation of the lipid mediators produced from n-3 and n-6 polyunsaturated fatty acids, as well as by the activation and inhibition of subsequent signal transduction cascades involving kinases. In conclusion, the results reported in this thesis significantly contribute to advancing the state of knowledge regarding the metabolism of n-3 and n-6 polyunsaturated fatty acids in healthy and psoriatic skin. This thesis also allowed the optimization of a skin model to be more representative of the functionality of normal human skin, and thus the generation of a more efficient model for the pharmaceutical industry. Ultimately, our work will help to support and promote the use of n-3 polyunsaturated fatty acids as a treatment for patients with psoriasis, which could greatly improve their quality of life.

In der vorliegenden Arbeit wurde die Expression von Angiotensinogen, Renin, Angiotensin-Converting-Enzym (ACE) und von den Agiotensin-Rezeptoren AT1 und AT2 in humaner Haut untersucht, um zu sehen, ob humane Haut ein lokales Gewebe Renin-Angiotensin-System (RAS) besitzt und fähig ist, Angiotensin II (Ang II) zu synthetisieren sowie welche physiologische Rolle Ang II in humaner Haut haben könnte. Außerdem wurde das Expressionsmuster von Angiotensinogen, Renin und ACE in gesunder humaner Haut mit dem in Psoriasis, Basaliom und Spinaliom (SCC) verglichen, um einen Einblick in pathophysiologische Funktionen des RAS zu gewinnen. Mit Hilfe von RT-PCR konnten alle Komponenten des RAS in vitro auf mRNA Ebene in kultivierten primären Keratinozyten, Melanozyten, dermalen Fibroblasten und dermalen mikrovaskulären Endothelzellen (MVEC´s) nachgewiesen werden, mit einer Ausnahme: Melanozyten scheinen keine AT2-Rezeptoren zu exprimieren. Immunhistochemische Untersuchungen zeigten die Expression aller Komponenten auf Proteinebene in Epidermis und dermalen Gefäßwänden in Gewebeschnitten humaner Haut. Zusätzlich erfolgte der Nachweis von Ang II in kultivierten Keratinozyten mittels enzymatischen immunometrischen Assays. Während Angiotensinogen, Renin und ACE bei immunhistochemischen Untersuchungen an Gewebeschnitten gesunder menschlicher Haut in allen Epidermalschichten gleichmäßig verteilt waren, zeigte sich bei der Psoriasis eine deutliche Betonung der unteren Epidermalschichten. Immunhistochemische Untersuchungen von Basaliomen erbrachten eine verminderte Expression von Angiotensinogen und Renin innerhalb der Tumornester. ACE wurde in den Tumorzellen noch weniger exprimiert. In immunhistochemischen Untersuchungen von Spinaliomen färbten sich die Tumorzellen deutlich homogen an. Die Experimente haben gezeigt, dass alle Komponenten des RAS in enger Lokalisation in menschlicher Haut vorkommen und dass folglich ein lokales Gewebe RAS in humaner Haut existiert sowie dass humane Haut fähig ist, Ang II ohne Zufuhr weiterer Komponenten und ohne regulatorische Einflüsse aus der Zirkulation zu synthetisieren. Eine mögliche physiologische Rolle von Ang II könnte die Regulation von Keratinozyten-Proliferation und –Differenzierung über seine Rezeptoren sein. Bezüglich der pathophysiologischen Rolle haben die Untersuchungen eine Fehlregulation des kutanen RAS in Epidermis psoriatisch veränderter Haut gezeigt, welches ein Hinweis auf eine pathogenetische Rolle des RAS bei der gestörten Keratinozyten-Proliferation und –Differenzierung sein könnte. Das Expressionsmuster in den untersuchten Tumoren war uneinheitlich, weshalb eine Interpretation der Rolle des RAS in kutanen Tumoren ohne weitere Untersuchungen kaum möglich erscheint. 1 / The present study was designed to elucidate whether a local tissue renin-angiotensin system (RAS) is expressed in human skin, whether cutaneous cells are able to autonomously synthesise angiotensin II (Ang II), and to get a first insight into a putative physiological role of Ang II in this location. For this purpose, the expression of angiotensinogen, renin, angiotensin-converting enzyme (ACE) and of the angiotensin receptors AT1 and AT2 was examined in human skin samples and in diverse cutaneous cells in primary culture on mRNA- and protein-level. Furthermore, the study compared the expression pattern of angiotensinogen, renin and ACE in healthy human skin with that in psoriasis, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) to look for possible differences between healthy and diseased skin. Using mRNA derived from cultured primary keratinocytes, melanocytes, dermal fibroblasts and dermal microvascular endothelial cells (MVECs), all components of the RAS could be demonstrated by RT-PCR except for AT2 receptors in melanocytes. Immunohistochemical stainings of cryostat sections of human skin revealed the expression of all components at protein level within the epidermis and in dermal vessel walls. In addition, the presence of Ang II in cultured keratinocytes and their supernatants could be proven by enzyme immunometric assay giving strong evidence for the ability of keratinocytes to autonomously synthesise Ang II. Regarding the comparison of RAS expression in healthy versus diseased skin, expression of angiotensinogen, renin and ACE was altered in all dermatoses examined. While in normal skin, RAS components were distributed equally and homogenously throughout all layers of the epidermis, in psoriatic skin their expression was more intense in the basal epidermal layers and less intense in the upper layers. In BCC sections, expression of angiotensinogen and renin was down-regulated, and tumour cells stained negatively for ACE. In SCC cryostat sections, tumour cells stained positively for all RAS components with an intensity comparable to normal skin. Taken together, the experiments revealed that a local tissue RAS exists in human skin, and that human skin is able to autonomously synthesise Ang II without any supply of components from the circulation. The physiological role of Ang II in normal skin may comprise the regulation of keratinocyte proliferation and differentiation. Concerning a putative pathophysiological role of Ang II in skin, this study provides evidence for a deregulation of the RAS in psoriatic skin and in BCC pointing to an involvement of the RAS in the pathomechanisms of these dermatoses. 1

Background: Financial constraints challenge evidence of the effectiveness of dermatological inpatient management. Objective: To evaluate the effectiveness of hospitalization in atopic dermatitis and psoriasis regarding initial and sustained benefits. Methods: Prospective study on adults with psoriasis vulgaris (n = 22) and atopic dermatitis (n = 14). At admission, discharge, and 3 months after discharge, validated outcomes of objective and subjective disease severity were assessed by trained investigators. Results: Hospitalization resulted in substantial benefit in quality of life and clinical disease severity. Looking at mean scores, the observed benefit appeared stable until 3-month follow-up. The analysis of individual patient data revealed significant changes in disease severity between discharge and 3-month follow-up with some patients relapsing, others further improving. Reasons for hospitalization and treatment performed were not related to sustained benefit. Conclusions: In psoriasis vulgaris and atopic dermatitis, hospitalization effectively improved quality of life and clinical disease severity. Further research should focus on prognostic factors for sustained improvement. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

In order to convince, one must know which arguments are most suitable for the specific situation. But even if you think you know how others will react to your arguments, you may be wrong. Therefore, the purpose of this paper is to find out a way of examining to what extent informative text take account to the subjective views of their addressees. The theoretical framework is Blitzer's theory of the rhetorical situation, pragmatic theory of speech acts and their presuppositions, Burk´s theory of consubstantiality and phenomenographic theory of subjective perceptions. Methodologically, phenomenography has also been used as a guideline for selecting informants, collecting data and processing the interviews. The material analyzed consists interviews with four persons suffering from psoriasis, a brochure addressed to an unspecified audience with the same problem plus an interview with a spokesperson for the Swedish Psoriasis Association. The analysis reveals a clear difference in certain important respects between the subjective views of the intended reader and the implied addressees of the text, i.e. a lack of consubstantiality between sender and receiver. This points to a rhetorical problem when addressing an unknown audience without exploring their specific perceptions of concepts related to the transmitted information, a problem which may be grasped and perhaps to some extent even solved by applying a method similar to the one used in this study.

We present a system for data-driven therapy decision support based on techniques from the field of recommender systems. Two methods for therapy recommendation, namely, Collaborative Recommender and Demographic-based Recommender, are proposed. Both algorithms aim to predict the individual response to different therapy options using diverse patient data and recommend the therapy which is assumed to provide the best outcome for a specific patient and time, that is, consultation. The proposed methods are evaluated using a clinical database incorporating patients suffering from the autoimmune skin disease psoriasis. The Collaborative Recommender proves to generate both better outcome predictions and recommendation quality. However, due to sparsity in the data, this approach cannot provide recommendations for the entire database. In contrast, the Demographic-based Recommender performs worse on average but covers more consultations. Consequently, both methods profit from a combination into an overall recommender system.