Results of the 2nd scientific workshop of the ECCO (III): basic mechanisms of intestinal healing.

The second scientific workshop of the European Crohn's and Colitis Organization (ECCO) focused on the relevance of intestinal healing for the disease course of inflammatory bowel disease (IBD). The objective was to better understand basic mechanisms, markers for disease prediction, detection and monitoring of intestinal healing, impact of intestinal healing on the disease course of IBD as well as therapeutic strategies. The results of this workshop are presented in four separate manuscripts. This section describes basic mechanisms of intestinal healing, identifies open questions in the field and provides a framework for future studies.

Peroxisome proliferator-activated receptors (PPARs): from metabolic control to epidermal wound healing.

Peroxisome proliferator-activated receptors control many cellular and metabolic processes. They are transcription factors belonging to the family of ligand-inducible nuclear receptors. Three isotypes called PPARalpha, PPARbeta/delta and PPARgamma have been identified in lower vertebrates and mammals. They display differential tissue distribution and each of the three isotypes fulfills specific functions. PPARalpha and PPARgamma control energy homoeostasis and inflammatory responses. Their activity can be modulated by drugs such as the hypolipidaemic fibrates and the insulin sensitising thiazolidinediones (pioglitazone and rosiglitazone). Thus, these receptors are involved in the control of chronic diseases such as diabetes, obesity, and atherosclerosis. Little is known about the main function of PPARbeta, but it has been implicated in embryo implantation, tumorigenesis in the colon, reverse cholesterol transport, and recently in skin wound healing. Here, we present recent developments in the PPAR field with particular emphasis on both the function of PPARs in lipid metabolism and energy homoeostasis (PPARalpha and PPARgamma), and their role in epidermal maturation and skin wound repair (PPARalpha and PPARbeta).

Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing : a prospective randomized clinical trial on skin graft donor sites

La cicatrisation cutanée requiert de nombreux mécanismes et un nombre toujours croissant de molécules participant à ces réactions sont identifiées. La compréhension de cette cinétique et des différents facteurs impliqués dans ce processus permet d'accélérer la guérison des plaies. Certains facteurs de croissance (PDGF, FGF, EGF) ont déjà été utilisés localement sur des plaies mais leurs effets relatés sont inconsistants et contradictoires, probablement en raison de l'absence de cinétique ou de concentration physiologique. Les plaquettes jouent un rôle fondamental dans la cicatrisation cutanée, principalement par la formation du clou plaquettaire et le relargage de divers facteurs de croissance et de cytokines. De même, les kératinocytes ont un rôle similaire dans l'épithélialisation des plaies. Ainsi, l'apport de plaquettes et de kératinocytes sur une plaie pourrait accélérer sa cicatrisation. L'étude rapportée ici tente de vérifier si une solution de kératinocytes autologues en suspension dans un concentré plaquettaire ou un concentré plaquettaire seul pourrait stimuler la cicatrisation cutanée. Pour ce faire, nous avons comparé trois groupes de 15 patients bénéficiant, sur une prise de greffe de profondeur similaire, d'un traitement standard fait de pansement simple, d'un concentré plaquettaire seul ou de kératinocytes autologues suspendus dans un concentré plaquettaire. Sur ces plaies, la durée de cicatrisation ainsi que la douleur au cours de la guérison ont été étudiés. Nos résultats montrent une réduction significative de la durée de cicatrisation dans le groupe traité avec un concentré plaquettaire, passant de 13.9 +/- 0.5 à 7.2 +/- 0.2 jours. Cet effet est encore plus marqué dans le groupe traité avec des kératinocytes en suspension dans un concentré plaquettaire passant de 13.9 +/- 0.5 à 5.7 +/- 0.2 jours. De même, la douleur évaluée au cinquième jour montre une nette diminution dans les deux groupes traités avec des cellules. En conclusion, notre travail montre que l'application de plaquettes autologues ou de kératinocytes en suspension dans un concentré plaquettaire permet d'accélérer la cicatrisation cutanée et de diminuer les douleurs, sans aucun effet indésirable constaté. Notre étude montre également qu'un concentré plaquettaire peut être utilisé comme vecteur pour une suspension de kératinocytes. L'identification de la cinétique d'apparition et de la concentration de chacun des facteurs de croissance lors de la cicatrisation cutanée permettrait dans le futur d'analyser plus finement et de traiter physiologiquement des plaies chroniques.

LDLs stimulate p38 MAPKs and wound healing through SR-BI independently of Ras and PI3 kinase.

Intracellular signals elicited by LDLs are likely to play a role in the pathogenesis associated with increased LDL blood levels. We have previously determined that LDL stimulation of human skin fibroblasts, used as a model system for adventitial fibroblasts, activates p38 mitogen-activated protein kinases (MAPKs), followed by IL-8 production and increased wound-healing capacity of the cells. The proximal events triggering these responses had not been characterized, however. Here we show that MAPK kinases MKK3 and MKK6, but not MKK4, are the upstream kinases responsible for the activation of the p38 MAPKs and stimulation of wound closure in response to LDLs. Phosphoinositide 3 kinases (PI3Ks) and Ras have been suggested to participate in lipoprotein-induced MAPK activation. However, specific PI3K inhibitors or expression of a dominant-negative form of Ras failed to blunt LDL-induced p38 MAPK activation. The classical LDL receptor does not participate in LDL signaling, but the contribution of other candidate lipoprotein receptors has not been investigated. Using cells derived from scavenger receptor class B type I (SR-BI) knockout mice or the BLT-1 SR-BI inhibitor, we now show that this receptor is required for LDLs to stimulate p38 MAPKs and to promote wound healing. Identification of MKK3/6 and SR-BI as cellular relays in LDL-mediated p38 activation further defines the signaling events that could participate in LDL-mediated pathophysiological responses.

Development of a cost-effective method for platelet-rich plasma (PRP) preparation for topical wound healing.

Platelet-rich plasma (PRP) is a volume of plasma fraction of autologous blood having platelet concentrations above baseline whole-blood values due to processing and concentration. PRP is used in various surgical fields to enhance soft-tissue and bone healing by delivering supra-physiological concentrations of autologous platelets at the site of tissue damage. These preparations may provide a good cellular source of various growth factors and cytokines, and modulate tissue response to injury. Common clinically available materials for blood preparations combined with a two-step centrifugation protocol at 280g each, to ensure cellular component integrity, provided platelet preparations which were concentrated 2-3 fold over total blood values. Costs were shown to be lower than those of other methods which require specific equipment and high-cost disposables, while safety and traceability can be increased. PRP can be used for the treatment of wounds of all types including burns and also of split-thickness skin graft donor sites, which are frequently used in burn management. The procedure can be standardized and is easy to adapt in clinical settings with minimal infrastructure, thus enabling large numbers of patients to benefit from a form of cellular therapy.

Plasmacytoid dendritic cells sense skin injury and promote wound healing through type I interferons.

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-α/β)-producing cells that express intracellular toll-like receptor (TLR) 7 and TLR9 and recognize viral nucleic acids in the context of infections. We show that pDCs also have the ability to sense host-derived nucleic acids released in common skin wounds. pDCs were found to rapidly infiltrate both murine and human skin wounds and to transiently produce type I IFNs via TLR7- and TLR9-dependent recognition of nucleic acids. This process was critical for the induction of early inflammatory responses and reepithelization of injured skin. Cathelicidin peptides, which facilitate immune recognition of released nucleic acids by promoting their access to intracellular TLR compartments, were rapidly induced in skin wounds and were sufficient but not necessary to stimulate pDC activation and type I IFN production. These data uncover a new role of pDCs in sensing tissue damage and promoting wound repair at skin surfaces.

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells.

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing - a prospective randomized clinical trial on skin graft donor sites: platelet concentrate and keratinocytes on donor sites.

BACKGROUND: Wound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Lausanne University Hospital, Switzerland in 45 patients, randomized to three different topical treatment groups: standard treatment serving as control, autologous platelet concentrate (PC) and keratinocytes suspended in autologous platelet concentrate (PC + K). Split thickness skin graft donor sites were chosen on the anterolateral thighs of patients undergoing plastic surgery for a variety of defects. Wound healing was assessed by the duration and quality of the healing process. Pain intensity was evaluated at day five. RESULTS: Healing time was reduced from 13.9 ± 0.5 days (mean ± SEM) in the control group to 7.2 ± 0.2 days in the PC group (P < 0.01). An addition of keratinocytes in suspension further reduced the healing time to 5.7 ± 0.2 days. Pain was reduced in both the PC and PC + K groups. Data showed a statistically detectable advantage of using PC + K over PC alone (P < 0.01). CONCLUSION: The results demonstrate the positive contribution of autologous platelets combined with keratinocytes in stimulating wound healing and reducing pain. This strikingly simple approach could have a significant impact on patient care, especially critically burned victims for whom time is of the essence. CLINICAL TRIAL REGISTRY INFORMATION: Protocol Record Identification Number: 132/03Registry URL: http://www.clinicaltrials.gov.

Role of PPARβ in keratinocyte adhesion and migration during skin wound healing

RESUME L'homéostasie du tissu cutané est assurée par des interactions étroites entre les cellules le composant et par l'équilibre entre la différenciation et la prolifération des kératinocytes devant permettre un renouvellement constant du tissu. Après une blessure, les kératinocytes environnant la zone blessée sont activés par des cytokines. Ils acquièrent alors un phénotype migratoire qui s'accompagne d'une modulation de l'activité protéolytique de la matrice extra cellulaire, d'une modulation de la dynamique du cytosquelette d'active, de la polarisation de la cellule, de l'affaiblissement des contacts entre cellules et de changements dans leurs contacts avec la matrice extra cellulaire. PPARβ est un facteur de transcription activé par les acides gras et leurs dérivés. Il appartient à la famille des récepteurs nucléaires aux hormones et son expression est avérée dans les kératinocytes des follicules pileux et dans les kératinocytes inter-folliculaires activés par la blessure cutanée. Le rôle de PPARβ dans la peau est principalement lié à son effet protecteur contre l'apoptose ainsi qu'à son implication dans l'équilibre dynamique entre la prolifération et la différentiation des kératinocytes. L'objet de ce travail fut de déterminer le rôle de PPARβ dans les processus d'adhésion et de migration des kératinocytes activés durant la régénération de l'épithélium blessé. Nous avons montré que les souris dépourvues du gène codant pour PPARβ ont de sévères imperfections affectant la morphologie de l'épithélium. Ce phénotype est corrélé à la modulation imparfaite du réseau d'active chez les souris dépourvues de PPARβ, à un défaut de localisation de l'intégrine α3 impliquée dans les complexes induisant la migration cellulaire, ainsi qu'à la modulation de l'expression d'acteurs majeurs affectant l'activité protéolytique de la matrice extra cellulaire. En conclusion, nos résultats montrent que PPARβ est impliqué dans le contrôle de la dynamique du cytosquelette d'active et la polarisation des kératinocytes activés. PPARβ étant impliqué dans l'acquisition d'un phénotype migratoire, il est légitime de se demander s'il intervient de même dans d'autres types cellulaires, par exemple dans la transition épithéliale-mésenchymateuse durant le développement, ou encore la progression de cellules tumorales. SUMMARY Highly coordinated intercellular interactions and single cell metabolism ensure cell and tissue maintenance of the skin. Healing of a skin wound involves keratinocyte activation by cytokines and growth factors. Activated keratinocytes acquire a motile phenotype that requires extracellular matrix remodeling and subsequent ligand activation through proteolytic activity, as well as cytoskeletal reorganisation induced by the release of cell-cell junctions and by the signalling relayed via integrin receptors and their cytoplasmic adaptors. PPARβ is a transcription factor activated by polyunsaturated fatty acids and fatty acid derivatives which belong to the nuclear hormone receptor superfamily. It is expressed in activated keratinocytes where it plays an essential role in protecting them from apoptosis. In addition, it plays an important function in hair follicle morphogenesis at the time of elongation, via the regulation of the balance between keratinocyte differentiation and proliferation. The aim of the present work was to determine if PPARβ is also involved in the regulation of migration and adhesion properties of keratinocytes during skin wound healing. We have shown that wounded PPARβ null mice display severe abnormalities of the keratinocyte migratory layer as shown at the histological level and using three-dimensional reconstruction. This altered migratory phenotype is correlated to altered dynamic of the actin cytoskeleton network, impaired α3 integrin localisation in migrating keratinocytes and changes in the expression of a key actor involved in extracellular matrix proteolytic activity. These results show that PPARβ is implicated in the fine tuning of the actin network organisation and the polarisation of activated keratinocytes following an epithelial wound. Whether these mechanisms are also controlled by PPARβ in other cell types during epithelial mesenchymal transition or tumour cell progression is an interesting question to rise.

Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells.

Engineering of fetal tissue has a high potential for the treatment of acute and chronic wounds of the skin in humans as these cells have high expansion capacity under simple culture conditions and one organ donation can produce Master Cell Banks which can fabricate over 900 million biological bandages (9 x 12cm). In a Phase 1 clinical safety study, cases are presented for the treatment of therapy resistant leg ulcers. All eight patients, representing 13 ulcers, tolerated multiple treatments with fetal biological bandages showing no negative secondary effects and repair processes similar to that seen in 3rd degree burns. Differential gene profiling using Affymetrix gene chips (analyzing 12,500 genes) were accomplished on these banked fetal dermal skin cells compared to banked dermal skin cells of an aged donor in order to point to potential indicators of wound healing. Families of genes involved in cell adhesion and extracellular matrix, cell cycle, cellular signaling, development and immune response show significant differences in regulation between banked fetal and those from banked old skin cells: with approximately 47.0% of genes over-expressed in fetal fibroblasts. It is perhaps these differences which contribute to efficient tissue repair seen in the clinic with fetal cell therapy.

Poly-N-acetyl glucosamine fibers are synergistic with vacuum-assisted closure in augmenting the healing response of diabetic mice.

BACKGROUND: Vacuum-assisted closure (VAC) has become the preferred modality to treat many complex wounds but could be further improved by methods that minimize bleeding and facilitate wound epithelialization. Short fiber poly-N-acetyl glucosamine nanofibers (sNAG) are effective hemostatic agents that activate platelets and facilitate wound epithelialization. We hypothesized that sNAG used in combination with the VAC device could be synergistic in promoting wound healing while minimizing the risk of bleeding. METHODS: Membranes consisting entirely of sNAG nanofibers were applied immediately to dorsal excisional wounds of db/db mice followed by application of the VAC device. Wound healing kinetics, angiogenesis, and wound-related growth factor expression were measured. RESULTS: The application of sNAG membranes to wounds 24 hours before application of the VAC device was associated with a significant activation of wounds (expression of PDGF, TGFβ, EGF), superior granulation tissue formation rich in Collagen I as well as superior wound epithelialization (8.6% ± 0.3% vs. 1.8% ± 1.1% of initial wound size) and wound contraction. CONCLUSIONS: The application of sNAG fiber-containing membranes before the application of the polyurethane foam interface of VAC devices leads to superior healing in db/db mice and represents a promising wound healing adjunct that can also reduce the risk of bleeding complications.

New perspective on the study of the authority relationship: Integrating individual and societal level research.

The concept of authority crosses many social sciences, but there is a lack of common taxonomy and definitions on this topic. The aims of this review are: (1) to define the basic characteristics of the authority relationship, reaching a definition suitable for the different domains of social psychology and social sciences; (2) to bridge the gap between individual and societal levels of explanation concerning the authority relationship, by proposing an interpretation within the framework of social representations. The authority relationship can be conceived as a negotiation of meanings and it is closely linked to shared value orientation and the attribution of meanings negotiated within a society. We assume that the authority relationship is socially constructed and represents both a shared representation of society and a normative principle of social life. A multidisciplinary approach is adopted, crossing definitions and studies provided in sociology, political science, law and social psychology.

Deletion of delta-opioid receptor in mice alters skin differentiation and delays wound healing.

In addition to their well-known antinociceptive action, opioids can modulate non-neuronal functions, such as immune activity and physiology of different cell types. Several findings suggest that the delta-opioid receptor (DOR) and its endogenous ligands (enkephalins) are important players in cell differentiation and proliferation. Here we show the expression of DOR in mouse skin and human skin cultured fibroblasts and keratinocytes using RT-PCR. In DOR knock-out (KO) mice, a phenotype of thinner epidermis and higher expression of cell differentiation marker cytokeratin 10 (CK 10) were observed compared with wild type (WT). Using a burn wound model, significant wound healing delay (about 2 days) and severe epidermal hypertrophy were shown at the wound margin of DOR KO mice. This wound healing delay was further investigated by immunohistochemistry using markers for proliferation, differentiation, re-epithelialization, and dermal repair (CK 6, CK 10, and collagen IV). The levels of all these markers were increased in wounds of KO mice compared with WT. During the wound healing, the epidermal thickness in KO mice augments faster and exceeds that of the WT by day 3. These results suggest an essential role of DOR in skin differentiation, proliferation, and migration, factors that are important for wound healing.

Housewife or working mum--each to her own? The relevance of societal factors in the association between social roles and alcohol use among mothers in 16 industrialized countries.

Aims To investigate whether differences in gender-income equity at country level explain national differences in the links between alcohol use, and the combination of motherhood and paid labour. Design Cross-sectional data in 16 established market economies participating in the Gender, Alcohol and Culture: An International Study (GenACIS) study. Setting Population surveys. Participants A total of 12 454 mothers (aged 25-49 years). Measurements Alcohol use was assessed as the quantity per drinking day. Paid labour, having a partner, gender-income ratio at country level and the interaction between individual and country characteristics were regressed on alcohol consumed per drinking day using multi-level modelling. Findings Mothers with a partner who were in paid labour reported consuming more alcohol on drinking days than partnered housewives. In countries with high gender-income equity, mothers with a partner who were in paid labour drank less alcohol per occasion, while alcohol use was higher among working partnered mothers living in countries with lower income equity. Conclusion In countries which facilitate working mothers, daily alcohol use decreases as female social roles increase; in contrast, in countries where there are fewer incentives for mothers to remain in work, the protective effect of being a working mother (with partner) on alcohol use is weaker. These data suggest that a country's investment in measures to improve the compatibility of motherhood and paid labour may reduce women's alcohol use.