Ageing is accompanied by a progressive decline in skeletal muscle mass and strength which may lead to impaired ability to perform activities of daily living in older adults. Although the exact cause of the gradual decline in muscle mass is unknown, identifying efficient strategies aiming to prevent age-related loss of muscle mass and strength is important in order to promote healthy ageing. The overall aim of this thesis was to explore the effects of resistance training alone or combined with a healthy diet on skeletal muscle mass and function of healthy recreationally active older women and to determine mechanisms by which elevated systemic inflammation may contribute to the age-related decline of muscle mass in older adults. The combination of resistance training and a healthy diet induced gains in leg lean mass as well as greater gains in dynamic explosive force than resistance training alone in healthy recreationally active older women. The observed gains in leg lean mass were accompanied by increases in the size of type IIA muscle fibres together with down-regulation in gene expression of a pro-inflammatory factor (IL-1β) and upregulation in gene expression of a regulator of cellular growth (mTOR) in skeletal muscle of older women. Additionally, reduced muscle protein synthesis and size of muscle cells may mediate the detrimental effects of elevated circulating markers of inflammation on muscle mass in older adults. In conclusion, the present thesis depicts mechanistic links between elevated systemic marker of inflammation and muscle mass and provides new information on the effects of combined resistance training and healthy diet on muscle mass and strength in a group of healthy recreationally active older women. This knowledge is instrumental for development of strategies aiming to prevent age-related loss of muscle mass and function.

The presented thesis discusses the issues of chronic inflammatory diseases of the digestive system and suggests the possibility of the alternative natural remedies improving the health conditions or prolonging the remission phase of IBD. The main goal is to design a probiotic supplement enriched by natural anti-inflammatory agents. Phytochemicals, concretely phenolic compounds, flavonoids, and carotenoids suppress harmful inflammatory processes by direct targeting the function of the immune cells or by inhibiting damaging oxidative stress in general. Therefore, there were several plants potentially rich for these biologically active substances selected. Concretely, blueberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), turmeric (Curcuma longa), peppermint (Mentha piperita), chamomile (Matricaria recutita), cinnamon (Cinnamomum zeylanicum), ginger (Zingiber officinale) and flax (Linum usitatissimum). The suitable parts of these plants were extracted by using a total of three different solvents – water, ethanol, and hexane for obtaining lipidic extracts. The concentration of total polyphenols, total flavonoids, total carotenoids, and the ascertainment of specific polyphenols was determined in the prepared extracts. These characteristics were enhanced by the measurement of antioxidant activity of the aqueous and ethanolic extracts. The interaction of individual samples with probiotic cultures (Bifidobacterium breve, Lactobacillus acidophilus) and the ability of extracts to influence the viability of probiotics in the model digestion process has been examined. The most interesting samples (aqueous extracts of blueberries, mint, chamomile, and cinnamon) were selected for further experiments. The possible cytotoxicity towards human intestinal epithelial cells was tested in vitro by the MTT tests utilizing the CaCo-2 cell line. Extracts showing the highest levels of beneficial phytochemicals and antioxidant activity, supporting the growth of probiotic cultures, and showing minimal cytotoxic effects on human intestinal cells were then co-encapsulated with probiotics into alginate particles of a diameter of 1 mm. Water extracts of mint, chamomile, and cinnamon have been chosen. The encapsulation efficiency of successfully entrapped probiotics and phenolic compounds was determined in prepared particles. Furthermore, the particles were studied during the process of model digestion, when the release of the desired substances in the various parts of the digestive system was observed and assessed whether the components would reach the crucial point of action – the colon. For the use of the proposed probiotic mixture as a dietary supplement, a recommended dose of 1 g has been determined. Lastly, possible adjustments such as lyophilization or sheathing by an extra protective polymerous layer, e.g. chitosan, were suggested to prolong the shelf life of the particles and volatile substances stability.

La persistance du VIH-1 est associée au maintien de l’inflammation chronique chez les patients infectés, malgré la mise en place de combinaison de traitements antirétroviraux. L’inflammation chronique est associée à un risque augmenté de développer des comorbidités, non associées au SIDA. Les polynucléaires neutrophiles (PNN) sont des cellules myéloïdes qui ont été impliqués dans de multiples maladies inflammatoires chroniques. Néanmoins, leur rôle dans l’infection par le VIH-1 est moins bien connue. Afin de pallier ce manque de connaissances, nous avons évalué l’hétérogénéité des PNNs dans le modèle macaque cynomolgus infecté par le SIVmac251. L’analyse phénotypique par cytométrie de masse a révélé la circulation de PNNs immatures en phase chronique de l’infection. En caractérisant l’hétérogénéité des PNNs au cours de l’infection par le SIV, nous avons observé une augmentation des fréquences des neutrophiles immatures et activés dans le sang dès la primo-infection. En phase chronique, les PNNs immatures et activés étaient toujours significativement augmentés dans le sang et la moelle osseuse. Au cours de l’infection, les PNNs avaient une fonction immunostimulatrice envers la prolifération et la sécrétion cytokinique des lymphocytaire T. L’initiation d’un traitement antirétroviral précoce a permis de restaurer le phénotype des PNNs. Les PNNs sont des cellules à fort potentiel pro-inflammatoire abondantes qui devraient être ainsi considérés comme de nouveaux effecteurs de l’inflammation chronique associée au VIH-1. / Even under combinational antiretroviral treatments (cART), HIV-1 persistence is associated with chronic inflammation in infected patients, leading to an increased risk of non-AIDS-related comorbidities. Polymorphonuclear neutrophils (PMN), have been less studied in HIV infection whereas they were associated with chronic inflammation diseases. To evaluate PMN heterogeneity in SIVmac251 nonhuman primate infection model, we first performed multiparameter single-cell phenotyping by mass cytometry giving a global vision of the immune system. This analysis demonstrated circulation of immature PMN with impaired during chronic infection. Then, we characterized neutrophils heterogeneity in the course of SIV infection. In primary infection, there was an increased frequency of CD10- immature and CD62L-low primed PMNs in peripheral blood. In chronic phase, CD10- immature PMNs were significantly higher in bone marrow and blood, maintaining a primed profile. During SIV infection, PMNs demonstrated variable immunomodulatory function against T cells proliferation and cytokine production. Early cART allowed to restore PMN phenotype. In this study, we provide unprecedented insight into PMN heterogeneity in the course of SIV infection. Since PMN represent 40-70% of circulating leukocytes and primed PMN are more potent to release pro-inflammatory cytokines and to transmigrate, they should be considered as a new player in HIV-1 chronic inflammation.

L'insulinorésistance, caractérisée par l'incapacité des organes impliqués dans le métabolisme énergétique (tissu adipeux, muscles squelettiques et foie) à répondre à l'insuline, tient une place centrale dans la physiopathologie des complications métaboliques de l'obésité. L'apparition d'une insulinorésistance chez un sujet obèse est plurifactorielle et les mécanismes moléculaires impliqués ne sont à ce jour pas complètement élucidés.L'expansion majoritairement hyperplasique du tissu adipeux conduit à une hypoxie et un stress des adipocytes, induisant un relargage accru de cytokines inflammatoires et d'acides gras libres (AGL). Les AGL se fixent eux-mêmes sur les récepteurs toll-like (TLRs) de l'immunité innée, dont l'activation aboutit également à la sécrétion de cytokines inflammatoires. Ces AGL et cytokines, véhiculés par la circulation systémique, contribuent, avec la coopération des macrophages infiltrant le tissu adipeux, au développement d'une inflammation chronique de bas grade. Ainsi, les perturbations de l'homéostasie énergétique, associées à une activation du système immunitaire sont à l'origine d'une atteinte globale de la sensibilité à l'insuline de l'organisme, particulièrement délétère au métabolisme musculaire.Cette étude porte sur le rôle d'un effecteur de l'immunité innée, l'endoribonucléase latente (RNase L), dont l'expression est régulée par les interférons de type I et l'activation, par un oligoadénylate, le 2-5A. La RNase L clive les ARNs cellulaires conduisant à l'inhibition spécifique de l'expression de certains gènes. Nous montrons par ce travail l'implication de la RNase L dans le contrôle de la différenciation cellulaire et la pathogenèse de l'insulinorésistance associée à l'obésité, via la régulation des voies de l'inflammation au niveau des tissus adipeux et musculaire. / Insulin resistance, which is characterized by the incapacity of organs involved in the energetic metabolism (adipose tissue, skeletal muscles and liver) to respond to insulin, has a central place in the pathophysiology of the metabolic complications associated to obesity. The onset of insulin resistance in obese subjects is multifactorial and the molecular mechanisms involved have not yet been completely elucidated.The mainly hyperplasic expansion of white adipose tissue leads to hypoxia and stress in adipocytes, inducing an increased release of inflammatory cytokines and free fatty acids (FFA). FFA bind and activate the toll-like receptors (TLR) of the innate immunity system, leading to the secretion of inflammatory cytokines. These FFA and cytokines, taken by the systemic circulation, contribute, with the cooperation of macrophages infiltrating the adipose tissue, to the development of a chronic low-grade inflammation. Thus, the disturbances of the energetic homeostasis, associated with an activation of the immune system cause a global impairment of insulin sensitivity of the body, with particularly deleterious effects on muscular metabolism.This study focuses on the role of an effector of innate immunity, the latent endoribonuclease (RNase L). RNase L expression is regulated by type I interferons and is activated by the 2-5A oligoadenylate. RNase L splits cellular RNA, thus leading to the specific inhibition of the expression of certain genes. In this study, we demonstrate the implication of RNase L in the control of cell differentiation and the pathogenesis of obesity-associated insulin resistance, via the regulation of inflammatory pathways in the adipose and muscular tissues.

Notre équipe a précédemment rapporté que l'utilisation d'un traitement d'induction lympho-déplétant [les globulines anti-lymphocytaires polyclonales (GALP)], peut entrainer une lymphopénie T CD4 persistante chez certains transplantés rénaux. Celle-ci est associée à certaines anomalies biologiques telles que la présence d'un syndrome inflammatoire chronique. Ce travail de thèse s'intéresse aux facteurs impliqués dans l'induction de ces anomalies puisqu'elles sont associées à une augmentation de l'incidence d'infections, de maladies cardiovasculaires et de décès, habituellement retrouvés chez les personnes âgées. Nous avons montré que les GALP entrainent une diminution de la fonction thymique et une expansion de lymphocytes T à un stade avancé de différenciation pouvant traduire une immunosénescence prématurée. Par ailleurs, une rupture de la barrière intestinale semble favoriser la translocation bactérienne digestive et l'activation d'une inflammation chronique observée en transplantation, via les lipopolysaccharides libérés dans la circulation périphérique. La composition du rnicrobiote intestinal pourrait jouer un rôle dans l'initiation, le maintien et la sévérité de l'inflammation. Nous avons observé des modifications du microbiote après transplantation et après l' antibioprophylaxie utilisée en transplantation. Les mécanismes à l'origine de toutes ces observations restent à élucider mais ce travail permet d'améliorer la compréhension des facteurs impliqués dans le développement de l'immunosénescence et de l'inflammation chronique en transplantation rénale, ouvrant potentiellement la voie à d'intéressantes perspectives thérapeutiques. / We bave previously described that polyclonal anti-lymphocytic globulins (GALP) may contribute to prolonged CD4 T­cell lymphopenia in some renal transplant recipients, associated with some biological abnormalities, such as a chronic inflammatory syndrome. Our work focuses on the factors involved in the induction of tbese abnormalities and susceptible to increase the incidence of infections, cardiovascular diseases and deatb, comparable to the incidence observed in the elderly. We showed that GALP are implicated in the decrease in thymie output and the expansion ofT cells at an advanced stage of differentiation. Both are hallmarks of premature immune senescence. Moreover, the dysfunction of the gut epithelial barrier is responsible for gut bacterial translocation (GBT) and the activation of chronic inflammation observed in chronic kidney disease as well as in renal transplant recipients. The composition of the intestinal microbiota may play a role in the initiation, maintenance and severity of GBT and systernic inflammation. We reported the existence of a dysbiosis a.fier transplantation. The mechanisms involved remain to be elucidated, yet, this work contributes to the understanding of the potential factors involved. in the progression of immune senescence and the persistence of chronic inflammation after kidney transplantation paving the way to new fields of therapeutic research in transplantation

Pancreatic cancer is the fourth most lethal cancer and ranks as the eighth most commonly diagnosed cancer worldwide. This is due to its rapid proliferation, strong metastatic potential and its delayed detection. One major risk factor for developing pancreatic cancer is the aggressive inflammatory disease chronic pancreatitis. Chronic inflammation frequently precedes the development of certain pancreatic cancers. Inflammation is a protective and necessary process by which the body can alert the immune system of the existence of a wound or infection and mount an immune response to remove the harmful stimuli and start wound healing. The cross-talking of cells of the immune system and infected cells happens through cytokines, soluble proteins that activate and recruit other immune cells to increase the system’s response to the pathogen. Failure to resolve the injury can result in persistent cytokine production that in turn allows a cell that is damaged or altered to survive when in normal conditions it would be killed. Inflammation is thought to create a microenvironment that facilitates the initiation and/or growth of pancreatic cancer cells. Cytokines use two important kinases for their signaling: Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). The JAKs are activated upon the binding of cytokines to their corresponding receptors. When activated, the JAKs activate STATs through tyrosine phosphorylation. The STATs transduce signals to the nucleus of the cells to induce expression of critical genes essential in normal physiological cellular events such as differentiation, proliferation, cell survival, apoptosis and angiogenesis. STAT3 (a member of the STAT family) is constitutively activated in some pancreatic cancers, promoting cell cycle progression, cellular transformations and preventing apoptosis. Therefore, STAT3 is a promising target for cancer treatment. Novel therapies that inhibit STAT3 activity in cancers are urgently needed. Natural products are a very good resource for the discovery of new drugs against pancreatic cancer. Covering more than 70% of the Earths surface, The Ocean is an excellent source of bioactive natural products. Harbor Branch Oceanographic Institute’s Center for Marine Biomedical and Biotechnology Research (HBOI-CMBBR) situated in Florida, aims to find new marine natural products useful in disease prevention and drug therapy. Their current focus is to look for novel treatments for preventing both the formation of new pancreatic tumors and the metastasis of existing tumors. The hypothesis of this degree project was that novel inhibitors of STAT3 useful in the treatment of pancreatitis and/or pancreatic cancer could be found from marine-natural products. The first specific aim of this degree project was to set up an assay to identify bioactive marine natural products as inhibitors of inflammation. Furthermore the assay was validated using a commercially available inhibitor of inflammation (Cucurbitacin I). The last aim was to further validate the assay by screening pure compounds and peak library material from the HBOI marine specimen collection. At the end of the experimentation time, the assay still was not set-up as there were difficulties in proper cell culture techniques and the cell line did not respond as advertised. While the results were not as expected, the work performed resulted in familiarization with research laboratory practices and increased laboratory skills. Moreover, the results from the assays point to future directions to accomplish this project. / Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer

Le muscle squelettique a la capacité de se régénérer suite à une lésion grâce aux cellules satellites qui sont les cellules souches du muscle. Après dommage musculaire, les cellules satellites s’activent, prolifèrent, se différencient et fusionnent afin de reformer le muscle lésé. Cependant les cellules myogéniques ne sont pas les seules cellules impliquées dans la régénération musculaire. Des études précédentes réalisées au laboratoire ont montré chez la souris que les macrophages sont des cellules essentielles à la régénération musculaire. En effet, peu de temps après un dommage musculaire, les monocytes infiltrent le tissu lésé et se différencient en macrophages pro-inflammatoires Ly6Cpos (M1). Ces macrophages stimulent la prolifération des myoblastes et inhibent leur fusion. Puis les macrophages pro-inflammatoires changent de phénotype et deviennent des macrophages anti-inflammatoires Ly6Cneg (M2) qui stimulent la différenciation des myoblastes et les protègent de l’apoptose. Ainsi, en fonction de leur phénotype, les macrophages exercent des rôles trophiques séquentiels sur les myoblastes tout au long du processus de régénération musculaire. Dans la première partie de notre étude, nous montrons in vitro que les macrophages humains soutiennent les différentes étapes de la myogenèse. Les macrophages M1 sont fortement attirés par les myoblastes. De plus ils stimulent la prolifération des myoblastes et inhibent leur fusion. Les macrophages M2 attirent les myoblastes et stimulent leur différenciation permettant ainsi la formation de larges myotubes. En utilisant des anticorps bloquants spécifiques, nous avons identifié plusieurs molécules sécrétées par les macrophages régulant la myogenèse chez l’homme. Nos analyses in vivo chez l'homme confirment nos résultats obtenus in vitro. En effet, les macrophages M1 sont préférentiellement associés aux aires de régénération contenant des myoblastes non différenciés alors que les macrophages M2 sont associés aux aires de régénération contenant des myoblastes en différenciation. Dans un contexte de myopathie dégénérative, nous avons montré que les macrophages adoptent des phénotypes et des fonctions totalement différents des macrophages présents au cours de la régénération musculaire. Nous avons observé chez la souris et chez l’homme, que les macrophages exprimant des marqueurs M1 sont associés à la fibrose et qu’un traitement anti-inflammatoire réduit leur nombre dans le muscle dystrophique murin. Par isolement spécifique et cocultures ex vivo, nous avons montré qu'au cours de la régénération musculaire, les macrophages Ly6Cneg stimulent la production de collagène par les fibroblastes. A l'inverse au cours des myopathies dégénératives, ce sont les macrophages Ly6Cpos qui stimulent fortement l’établissement de la fibrose en agissant directement sur les fibroblastes. De plus, ces macrophages Ly6Cpos, qui régulent négativement les fibroblastes au cours de la régénération musculaire, stimulent la différenciation des fibroblastes et myofibroblastes dans les myopathies. De plus, ils les protègent de l'apoptose, participant ainsi à la persistance de ces cellules fibrosantes. Ainsi, nous avons confirmé chez l’homme in vitro et in vivo, le rôle de support séquentiel des macrophages au cours de la régénération musculaire. De plus, nous avons identifié différents effecteurs sécrétés par les macrophages M1 et M2 impliqués dans la régulation du processus myogénique chez l'adulte. Nous avons également montré que lors des myopathies dégénératives et au cours de la régénération musculaire, les macrophages adoptent un phénotype et des fonctions totalement différents, avec notamment un rôle profibrotique des macrophages pro-inflammatoires. / Skeletal muscle has the ability to regenerate after a chemical or physical injury thanks to satellite cells, the muscle stem cells. After damage, satellite cells proliferate, differentiate and fused to reform muscle. Myogenic cells are not the only on cells involved. Previous studies in the laboratory showed that, in mice, macrophages are crucial for skeletal muscle regeneration. Soon after an injury, macrophages infiltrate damaged muscle and differentiate into Ly6Cpos pro-inflammatory (M1) macrophages. They stimulate proliferation of myoblasts and inhibit their fusion. Then, pro-inflammatory macrophages skew towards a Ly6Cneg anti-inflammatory phenotype (M2). Anti-inflammatory macrophages stimulate differentiation of myoblasts and protect them from apoptosis. Thus, depending on their phenotype, macrophages exert sequential trophic roles on myoblasts throughout muscle regeneration. Here, we showed in vitro that human macrophages also support different steps of myogenesis. M1 macrophages are strongly attracted by mpcs. Moreover, they secrete molecules, which stimulate proliferation of mpcs and inhibit their fusion. M2 macrophages attract mpcs and stimulate differentiation of mpcs in order to form large myotubes. Using specific blocking antibodies, we identified molecules involved in the regulation of myogenesis by M1 and M2 macrophages in a human in vitro system. In vivo analysis of regenerating human muscle sections confirmed our results obtained in vitro. M1 macrophages are preferentially associated with proliferating myogenic cells while M2 macrophages are associated with differentiating myogenic cells. In degenerative myopathies, we showed that macrophages are completely different from those present during skeletal muscle regeneration. We observed in mouse and human that M1 marker-expressing macrophages are associated with fibrosis while anti-inflammatory treatment reduced this population, in association with an improvement of the dystrophic muscle. Isolated Ly6Cneg macrophages exhibit a mixed M1/M2 phenotype. In ex-vivo coculture experiments, we showed that Ly6Cpos macrophages strongly favor establishment of fibrosis by directly acting on fibroblasts while in regenerating muscle, these Ly6Cpos macrophages negatively regulate fibrosis. To resume, we confirm in human the supportive sequential roles of macrophages during skeletal muscle regeneration in vitro and in vivo. Moreover, we identified effectors secreted by M1 and M2 macrophages involved in the regulation of the myogenic process. We also highlight that during muscle regeneration and in degenerative myopathies, macrophages exhibit different phenotype associated with opposite functions, with a pro-fibrotic role for pro-inflammatory macrophages.

T-Helfer(Th)-Lymphozyten spielen aufgrund ihrer Expression von Zytokinen eine zentrale Rolle in der Regulation von Immunantworten. Durch die Zytokine fördern die Th-Zellen die Rekrutierung und Aktivierung von Zellen des angeborenen Immunsystems, den Immunglobulin-Klassenwechsel von B-Zellen und ihre Differenzierung zu antikörper-sezernierenden Plasmazellen. Welche Zytokine eine aktivierte Th-Zelle exprimiert hängt von instruktiven Signalen ab, die sie in der Regel von antigen-präsentierenden Signalen bekommt. Mit den instruktiven Signalen wird ein ganzes Differenzierungsprogramm in den Th-Zellen initiiert, welches am Ende zu einer epigenetischen Prägung des Effektorprogramms führt, sodass die Th-Zelle auch bei nachfolgenden Aktivierungen die gleichen Funktionen ausführt. In dieser Arbeit wurde untersucht wie diese Differenzierungsprogramme zur Pathogenese von chronischen Entzündungen beitragen und wie eine chronische Entzündung wiederum die Differenzierung der Th-Zellen beeinflusst. Des Weiteren haben wir molekulare Adaptationen identifiziert, die selektiv in chronisch aktivierten entzündungsfördernden Th Typ 1 (Th1)-Zellen hochgeregelt werden. Diese Adaptationen, wie die Expression von Twist1 und Hopx, fördern das Überleben der Th1-Zellen am Ort der Entzündung und könnten zur Perpetuation der Entzündung entscheidend beitragen. Zusammengenommen zeigen unsere Daten, dass Th-Zellen durch ihre pro-inflammatorische Prägung zwar gut gerüstet sind uns gegen Pathogene zu schützen, im Falle einer Autoreaktivität aber auch Immunpathologie und chronische Entzündungen auslösen können. Allerdings weisen unsere Arbeiten auch daraufhin, dass selektive Anpassungen der Th-Zellen an eine chronische Entzündung auch gleichzeitig Ansatzpunkte für ein therapeutisches Eingreifen bei chronisch-entzündlichen Krankheiten darstellen. / T helper (Th) lymhocytes play a central role in the regulation of immune responses. Through the expression of cytokines, Th cells orchestrate the recruitment and activation of cells of the innate immune system, and induce antibody class switch recombination in B lymphocytes and their differentiation into antibody-secreting plasma cells. Which cytokines an activated Th cell expresses is dependent on instructive signals, the Th cells receives from antigen-presenting cells. Such instructive signals initiate a differentiation program, which in the end leads to the epigenetic imprinting of the effector program, ensuring that the Th cell execute the same functions in subsequent activations. In this thesis, we investigated how such differentiation programs contribute to the pathogenesis of chronic inflammation and how chronic inflammation, in turn, affects the differentiation of the Th cells. Furthermore, we have identified molecular adaptations, which are selectively upregulated in chronically activated pro-inflammatory Th type 1 (Th1) cells. Such adaptations, like the upregulation of Twist1 and Hopx, promote the survival of Th1 cells in the inflamed tissue and could thereby contribute to the perpetuation of the inflammation. Taken together, our data show that Th cells with a pro-inflammatory imprint are well equipped to protect us against pathogens, in case of autoimmunity, however, cause immune pathology and chronic inflammation. At the same time, our work also shows that the selective adaptations of Th cells to chronic inflammation could also be promising targets for novel therapies for the treatment of chronic inflammatory diseases.

No description available.

Keloid disease (KD) is a benign fibroproliferative dermal disease of unknown aetiopathogenesis that occurs in genetically susceptible individuals. KD shows high heterogeneity within the lesion, harbouring different immune cell profiles, which are poorly characterised in KD at different lesional sites. Although, it has long been appreciated that chronic inflammation and dermal fibrosis is associated with other fibrotic diseases (e.g. scleroderma), this link has not, yet, been established in KD through direct evidence. Additionally, the limited availability of a simple KD animal model has hindered our understanding of the underlying pathogenesis of KD. Therefore, the main objectives were a) to identify and profile different immune cells at defined KD lesional and histological sites, b) to further characterize the potential contribution of viral particles in KD by investigating the gene and protein expression profile of toll like receptors that recognise viral particles in KD, and c) to develop an optimized long-term serum-free organ culture (OC) model for KD research as a tool for probing novel hypotheses in KD pathobiology deduced from a) and b) and to also validate the reliability and instructiveness of this novel ex vivo KD model with conventional (e.g. dexamethasone) and potential future anti-KD compounds [(-)-epigallocatechin-3-gallate (EGCG) and plasminogen activator inhibitor-1 (PAI-1) knock-down by siRNA]. To achieve above objectives, different cellular and molecular techniques were applied. Immune profiling of KD (chapter 2) at defined lesional and histological sites generated the first comprehensive analysis of KD-associated inflammatory cell infiltrates. This work demonstrated for the first time the presence of specific type of chronic inflammation in KD that resembles the formation of tertiary lymphoid tissues (TLTs) (in 14.7%, out of 68 KD cases). Although, these TLTs are not strictly linked to defined lesional sites within the KD, they are similar in structure to mucosa-associated lymphoid tissue (MALT). Therefore, we named this phenomenon as keloid-associated lymphoid tissue (KALT). Immunophenotyping of KD lesional sites also showed a predominance of T-cells, B-cells, M2 macrophages and OX40L+ degranulated mast cells in intralesional and perilesional sites of KD compared to normal skin and normal scar tissue. In the epidermis, Langerhans cells showed no changes, whereas the intra-epidermal T-cells were significantly increased in both the intralesional and perilesional sites of KD with an increased CD4:CD8 ratio. Intra-epidermal B-cells were only rarely found in KD. Interestingly, there was no significant statistical difference between intralesional and perilesional sites of KD immunophenotyping. These abnormal immune profiles suggest the persistence of non-resolving inflammation presence towards unknown stimuli, which require further investigation. The chronic inflammation could be followed by a reparative phase in a repetitive manner leading to KD formation. Evaluation of toll-like receptor (TLR) gene and protein expression in KD showed a significant increase in the expression of intra-epidermal TLR-6, -7 and dermal TLR-8. Since these TLRs are typically up regulated during anti-viral responses, these results further support the hypothesis that certain viruses or yet unidentified ligand may play a role in KD pathogenesis (chapter 3). A successful long-term, serum-free keloid OC model was established using a 4 mm sized punch biopsy embedded in collage matrix as air liquid interface in supplemented William’s E medium for up to 6 weeks (Chapter 4).