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Loss of perfusion in the burn wound might cause wound deepening and impaired healing. We previously showed persistent microvascular thrombosis coinciding with intraluminal neutrophils extracellular traps in human burned skin. This study investigates the presence of intraluminal citrullinated histone 3 (H3cit) from different cellular origins (neutrophils, monocytes, and lymphocytes) in relation to microvascular thrombosis of burn wounds. Eschar was obtained from burn patients (n = 18) 6–40 days postburn with a mean total burned body surface area of 23%. Microvascular presence of tissue factor (TF), factor XII (FXII) and thrombi was assessed by immunohistochemistry. Intramicrovascular cell death was analyzed via immunofluorescent microscopy, combining antibodies for neutrophils (MPO), monocytes (CD14), and lymphocytes (CD45) with endothelial cell markers CD31 and H3cit. Significantly increased microvascular expression of TF, FXII, and thrombi (CD31+) was found in all eschar samples compared with control uninjured skin. Release of H3cit from different cellular origins was observed in the lumen of the dermal microvasculature in the eschar tissue 7–40 days postburn, with release from neutrophilic origin being 2.7 times more abundant. Intraluminal presence of extracellular H3cit colocalizing with either MPO, CD14, or CD45 is correlated to increased microvascular thrombosis in eschar of burn patients. [ABSTRACT FROM AUTHOR]

Keloid tissues contain inflammatory cells and upregulated pro‐inflammatory cytokines. The Janus kinase (JAK)‐signal transducer and activator of transcription (STAT) pathway mediate cellular responses to these cytokines. We performed a systematic review on the role of the JAK–STAT pathway in keloid pathogenesis and the evidence for JAK–STAT inhibitors in keloid treatment. The search combined the terms (1) keloid and (2) JAK or TYK or STAT and included MeSH terms and synonyms. Two reviewers screened the articles and assessed the full texts on eligibility. Data were collected on the tested drugs and molecules, the type of cells and tissues used in the experiments, and study findings on the association between the JAK–STAT pathway and keloid cells and tissues. A total of twenty preclinical studies were included. Eleven preclinical studies proved that STAT3 expression and phosphorylation are enhanced in keloid tissue and keloid fibroblasts. Thirteen different JAK and/or STAT inhibitors were investigated. Tested drugs inhibited keloid progression as demonstrated by different processes, including reduced collagen production, cell proliferation and migration, increased cell cycle arrest and apoptosis, enhanced antioxidant responses, decreased (paracrine) signalling, and decreased profibrotic gene expression. No clinical studies have been published to date. Preclinical studies indicate a role for the JAK–STAT pathway in keloid pathogenesis and a potential role for JAK–STAT inhibitors in keloid treatment. The effect of these drugs should be further investigated on relevant biomarkers in a human keloid skin model, preferably including immune cells besides keloid fibroblasts and keratinocytes and in clinical studies. [ABSTRACT FROM AUTHOR]

Background: To date, it is unknown why some individuals develop late‐onset inflammatory adverse events after treatment with fillers. These events may result from various factors, including an immunological response of the adaptive immune system. Objective: In a pilot study, we looked for evidence that is there a relation between late‐onset inflammatory adverse events and the presence of immune cells surrounding the injected filler. Methods and Materials: We included 47 patients, of whom 20 experienced late‐onset inflammatory adverse events to different fillers (inflammatory group) and 27 who did not (reference group). A biopsy was taken from the area of the adverse event. Hematoxylin–eosin staining and immunohistochemistry analysis with CD3 (T‐cells) and CD68 (macrophages) on paraffin tissue sections was used to assess the biopsies. Results: Immune cells were found in biopsies obtained from 18 of 47 patients: Nine biopsies from the inflammation group and nine from the reference group. All these 18 cases showed CD68‐positive immune cells. Virtually no CD3‐positive immune cells were found. Conclusion: Our results indicate that there is no T‐cell activity in biopsies from areas with late‐onset adverse events after filler injections. The macrophages found in the biopsies are probably not responsible for the inflammatory response. [ABSTRACT FROM AUTHOR]

Background: Nickel‐induced proliferation or cytokine release by peripheral blood mononuclear cells may be used for in vitro diagnosis of nickel allergy. Objectives: Aim of this study was to explore the nickel‐specific cytokine profile to further elucidate the pathogenesis of nickel allergic contact dermatitis (ACD) and to identify potential new biomarkers for nickel ACD. Methods: Peripheral blood mononuclear cells from patients and controls were cultured with T‐cell skewing cytokine cocktails and/or nickel. Cytokine and chemokine concentrations were assessed in culture supernatants using validated multiplex assays. Specific cytokine production was related to history of nickel allergy and patch‐test results. Results: Twenty‐one of the 33 analytes included in the analysis were associated with nickel allergy and included type1 (TNF‐α, IFN‐γ, TNF‐β), type 2 (IL‐3, IL‐4, IL‐5, IL‐13), type 1/2 (IL‐2, IL‐10), type 9 (IL‐9), type 17/1 (IL‐17A[F], GM‐CSF, IL‐21) and type 22 (IL‐22) derived cytokines as well as the T‐cell/antigen presentation cell derived factors Thymus and activation regulated chemokine (TARC), IL‐27 and IP‐10. Receiver operator characteristics (ROC) analysis showed that IL‐5 was the strongest biomarker for nickel allergy. Conclusions: A broad spectrum of 33 cytokines and chemokines is involved in the allergen‐specific immune response in nickel allergic patients. IL‐5 remains, next to the lymphocyte proliferation test, the strongest biomarker for nickel allergy. [ABSTRACT FROM AUTHOR]

Despite the recent impressive clinical success of immunotherapy against melanoma, development of primary and adaptive resistance against immune checkpoint inhibitors remains a major issue in a large number of treated patients. This highlights the need for melanoma models that replicate the tumor's intricate dynamics in the tumor microenvironment (TME) and associated immune suppression to study possible resistance mechanisms in order to improve current and test novel therapeutics. While two‐dimensional melanoma cell cultures have been widely used to perform functional genomics screens in a high‐throughput fashion, they are not suitable to answer more complex scientific questions. Melanoma models have also been established in a variety of experimental (humanized) animals. However, due to differences in physiology, such models do not fully represent human melanoma development. Therefore, fully human three‐dimensional in vitro models mimicking melanoma cell interactions with the TME are being developed to address this need for more physiologically relevant models. Such models include melanoma organoids, spheroids, and reconstructed human melanoma‐in‐skin cultures. Still, while major advances have been made to complement and replace animals, these in vitro systems have yet to fully recapitulate human tumor complexity. Lastly, technical advancements have been made in the organ‐on‐chip field to replicate functions and microstructures of in vivo human tissues and organs. This review summarizes advancements made in understanding and treating melanoma and specifically aims to discuss the progress made towards developing melanoma models, their applications, limitations, and the advances still needed to further facilitate the development of therapeutics. [ABSTRACT FROM AUTHOR]

Background: Metal alloys containing contact sensitizers (nickel, palladium, titanium) are extensively used in medical devices, in particular dentistry and orthopaedic surgery. The skin patch test is used to test for metal allergy. Objective: To determine whether metal salts, when applied to freshly excised skin at patch test–relevant concentrations and using a method which mimics skin patch testing, cause in changes in the epidermis and dermis. Methods: Tissue histology, apoptosis, metabolic activity, and inflammatory cytokine release were determined for two nickel salts, two palladium salts, and four titanium salts. Results: Patch test–relevant concentrations of all metal salts caused localized cytotoxicity. This was observed as epidermis separation at the basement membrane zone, formation of vacuoles, apoptotic nuclei, decreased metabolic activity, and (pro)inflammatory cytokine release. Nickel(II) sulfate hexahydrate, nickel(II) chloride hexahydrate, titanium(IV) bis(ammonium lactato)dihydroxide, and calcium titanate were highly cytotoxic. Palladium(II) chloride, sodium tetrachloropalladate(II), titanium(IV) isopropoxide, and titanium(IV) dioxide showed mild cytotoxicity. Conclusion: The patch test in itself may be damaging to the skin of the patient being tested. These results need further verification with biopsies obtained during clinical patch testing. The future challenge is to remain above the elicitation threshold at noncytotoxic metal concentrations. [ABSTRACT FROM AUTHOR]

Background: The number of people within the European population having at least one tattoo has increased notably, and with it the number of tattoo‐associated clinical complications. Despite this, safety information and testing regarding tattoo inks remain limited. Objective: To assess cytotoxicity and sensitization potential of 16 tattoo inks after intradermal injection into reconstructed human skin (RHS). Methods: Commercially available tattoo inks were injected intradermally into RHS (reconstructed epidermis on a fibroblast‐populated collagen hydrogel) using a permanent makeup device. RHS biopsies, tissue sections, and culture medium were assessed for cytotoxicity (thiazolyl blue tetrazolium bromide assay [MTT assay]), detrimental histological changes (haematoxylin and eosin staining), and the presence of inflammatory and sensitization cytokines (interleukin [IL]‐1α, IL‐8, IL‐18; enzyme‐linked immunosorbent assay). Results: Varying degrees of reduced metabolic activity and histopathological cytotoxic effects were observed in RHS after ink injection. Five inks showed significantly reduced metabolic activity and enhanced sensitization potential compared with negative controls. Discussion: Using the RHS model system, four tattoo inks were identified as highly cytotoxic and classified as potential sensitizers, suggesting that allergic contact dermatitis could emerge in individuals carrying these inks. These results indicate that an RHS‐based assessment of cytotoxicity and sensitization potential by intradermal tattoo ink injection is a useful analytical tool to determine ink‐induced deleterious effects. [ABSTRACT FROM AUTHOR]

Unpredictable hypertrophic scarring (HS) occurs after approximately 35% of all surgical procedures and causes significant physical and psychological complaints. Parallel to the need to understanding the mechanisms underlying HS formation, a prognostic tool is needed. The objective was to determine whether (systemic) immunological differences exist between patients who develop HS and those who develop normotrophic scars (NS) and to assess whether those differences can be used to identify patients prone to developing HS. A prospective cohort study with NS and HS groups in which (a) cytokine release by peripheral blood mononuclear cells (PBMC) and (b) the irritation threshold (IT) after an irritant (sodium lauryl sulphate) patch test was evaluated. Univariate regression analysis of PBMC cytokine secretion showed that low MCP‐1, IL‐8, IL‐18 and IL‐23 levels have a strong correlation with HS (P <.010‐0.004; AUC = 0.790‐0.883). Notably, combinations of two or three cytokines (TNF‐a, MCP‐1 and IL‐23; AUC: 0.942, Nagelkerke R2: 0.727) showed an improved AUC indicating a better correlation with HS than single cytokine analysis. These combination models produce good prognostic results over a broad probability range (sensitivity: 93.8%, specificity 86.7%, accuracy 90,25% between probability 0.3 and 0.7). Furthermore, the HS group had a lower IT than the NS group and an accuracy of 68%. In conclusion, very fundamental immunological differences exist between individuals who develop HS and those who do not, whereas the cytokine assay forms the basis of a predictive prognostic test for HS formation, the less invasive, easily performed irritant skin patch test is more accessible for daily practice. [ABSTRACT FROM AUTHOR]

Although hypertrophic scars and keloids both generate excessive scar tissue, keloids are characterized by their extensive growth beyond the borders of the original wound, which is not observed in hypertrophic scars. Whether or not hypertrophic scars and keloids are two sides of the same coin or in fact distinct entities remains a topic of much debate. However, proper comparison between the two ideally occurs within the same study, but this is the exception rather than the rule. For this reason, the goal of this review was to summarize and evaluate all publications in which both hypertrophic scars and keloids were studied and compared to one another within the same study. The presence of horizontal growth is the mainstay of the keloid diagnosis and remains the strongest argument in support of keloids and hypertrophic scars being distinct entities, and the histopathological distinction is less straightforward. Keloidal collagen remains the strongest keloid parameter, but dermal nodules and α‐SMA immunoreactivity are not limited to hypertrophic scars alone. Ultimately, the current hypertrophic scars‐keloid differences are mostly quantitative in nature rather than qualitative, and many similar abnormalities exist in both lesions. Nonetheless, the presence of similarities does not equate the absence of fundamental differences, some of which may not yet have been uncovered given how much we still have to learn about the processes involved in normal wound healing. It therefore seems pertinent to continue treating hypertrophic scars and keloids as separate entities, until such a time as new findings more decisively convinces us otherwise. [ABSTRACT FROM AUTHOR]

Background: Skin and oral mucosa are continuously exposed to potential metal sensitizers while hosting abundant microbes, which may influence the host response to sensitizers. This host response may also be influenced by the route of exposure that is skin or oral mucosa, due to their different immune properties. Objective: Determine how commensal Streptococcus mitis influences the host response to nickel sulfate (sensitizer) and titanium(IV) bis(ammonium lactato)dihydroxide (questionable sensitizer) in reconstructed human skin (RHS) and gingiva (RHG). Methods: RHS/RHG was exposed to nickel or titanium, in the presence or absence of S. mitis for 24 hours. Histology, cytokine secretion, and Toll‐like receptors (TLRs) expression were assessed. Results: S. mitis increased interleukin (IL)‐6, CXCL8, CCL2, CCL5, and CCL20 secretion in RHS but not in RHG; co‐application with nickel further increased cytokine secretion. In contrast, titanium suppressed S. mitis–induced cytokine secretion in RHS and had no influence on RHG. S. mitis and metals differentially regulated TLR1 and TLR4 in RHS, and predominantly TLR4 in RHG. Conclusion: Co‐exposure of S. mitis and nickel resulted in a more potent innate immune response in RHS than in RHG, whereas titanium remained inert. These results indicate the important influence of commensal microbes and the route of exposure on the host's response to metals. [ABSTRACT FROM AUTHOR]

Background: The nature of clinically related adverse reactions to titanium is still unknown. Objective: To determine whether titanium salts have irritant or sensitizing potential in a reconstructed human skin (RHS) model with integrated Langerhans cells (LCs). Methods: RHS‐LCs (ie, reconstructed epidermis) containing primary differentiated keratinocytes and CFSE+CD1a+‐LCs generated from the MUTZ‐3 cell line on a primary fibroblast‐populated collagen hydrogel (dermis) were topically exposed to titanium(IV) bis(ammonium lactato)dihydroxide (TiALH). LC migration and plasticity were determined. Results: TiALH resulted in CFSE+CD1a+‐LC migration out of the epidermis. Neutralizing antibodies to CCL5 and CXCL12 showed that LC migration was CCL5 and not CXCL12 mediated. LCs accumulating within the dermis after TiALH exposure were CFSE+Lang+CD68+ which is characteristic of a phenotypic switch of MUTZ‐LC to a macrophage‐like cell. Furthermore, TiALH did not result in increased interleukin (IL)‐1β or CCR7 messenger RNA (mRNA) in the dermis, but did result in increased IL‐10 mRNA. In addition, monocultures of MUTZ‐LCs failed to increase LC maturation biomarkers CD83, CD86, and CXCL‐8 when exposed to noncytotoxic concentrations of four different titanium salts. Conclusion: These results classify titanium salts as irritants rather than sensitizers and indicate that titanium implant‐related complaints could be due to localized irritant‐mediated inflammation arising from leachable agents rather than a titanium metal allergy. [ABSTRACT FROM AUTHOR]

Millions of women worldwide have silicone breast implants. It has been reported that implant failure occurs in approximately a tenth of patients within 10 years, and the consequences of dissemination of silicone debris are poorly understood. Currently, silicone detection in histopathological slides is based on morphological features as no specific immunohistochemical technique is available. Here, we show the feasibility and sensitivity of stimulated Raman scattering (SRS) imaging to specifically detect silicone material in stained histopathological slides, without additional sample treatment. Histology slides of four periprosthetic capsules from different implant types were obtained after explantation, as well as an enlarged axillary lymph node from a patient with a ruptured implant. SRS images coregistered with bright‐field images revealed the distribution and quantity of silicone material in the tissue. Fast and high‐resolution imaging of histology slides with molecular specificity using SRS provides an opportunity to investigate the role of silicone debris in the pathophysiology of implant‐linked diseases. [ABSTRACT FROM AUTHOR]

Background: Abutment surfaces are being designed to promote gingival soft tissue attachment and integration. This forms a seal around prosthetics and consequently ensures long‐term implant survival. New scalable and reproducible models are necessary to evaluate and quantify the performance of these surfaces. Purpose: To evaluate a novel implantation model by histomorphometric and immunohistochemical characterization of the interactions between human oral gingival tissue and titanium abutments with either novel anodized or conventional machined surface. Materials and Methods: Abutments were inserted into an organotypic reconstructed human gingiva (RHG) model consisting of differentiated gingival epithelium cells on a fibroblast populated lamina propria hydrogel following a tissue punch. Epithelial attachment, down‐growth along the abutment surface, and phenotype were assessed via histomorphology, scanning electron microscopy, and immunohistochemistry 10 days after implantation. Results: The down‐growing epithelium transitioned from a gingival margin to a sulcular and junctional epithelium. The sulcus depth and junctional epithelial length were similar to previously reported pre‐clinical and clinical lengths. A collagen IV/laminin 5 basement membrane formed between the epithelium and the underlying connective tissue. The RHG expanded in thickness approximately 2‐fold at the abutment surface. The model allowed the evaluation of protein expression of adhering soft tissue cells for both tested abutments. Conclusions: The RHG model is the first in vitro 3D model to enable the assessment of not only human epithelial tissue attachment to dental abutments but also the expression of protein markers involved in soft tissue attachment and integration. The two abutments showed no noticeable difference in epithelial attachment. [ABSTRACT FROM AUTHOR]

Background: During the last decade, the number of people with ≥1 tattoo has increased noticeably within the European population. Despite this, limited safety information is available for tattoo inks. Objectives: To test the skin sensitization potential of 5 tattoo inks in vitro by using reconstructed human skin (RHS) and the contact sensitization biomarker interleukin (IL)‐18. Methods: Two red and 3 black tattoo inks, 1 additive (Hamamelis virginiana extract) and 1 irritant control (lactic acid) were tested. The culture medium of RHS (reconstructed epidermis on a fibroblast‐populated collagen hydrogel) was supplemented with test substances in a dose‐dependent manner for 24 hours, after which cytotoxicity (histology; thiazolyl blue tetrazolium bromide assay) and skin sensitization potential (IL‐18 secretion; enzyme‐linked immunosorbent assay) were assessed. Results: All but 1 ink showed cytotoxicity. Notably, 1 red ink and 1 black ink were able to cause an inflammatory response, indicated by substantial release of IL‐18, suggesting that these inks may be contact sensitizers. Conclusions: The in vitro RHS model showed that 4 tattoo inks were cytotoxic and 2 were able to cause an inflammatory IL‐18 response, indicating that an individual may develop allergic contact dermatitis when exposed to these tattoo inks, as they contain contact sensitizers. [ABSTRACT FROM AUTHOR]

Abnormal cutaneous wound healing can lead to formation of fibrotic hypertrophic scars. Although several clinical risk factors have been described, the cross‐talk between different cell types resulting in hypertrophic scar formation is still poorly understood. The aim of this in vitro study was to investigate whether endothelial cells (EC) may play a role in skin fibrosis, for example, hypertrophic scar formation after full‐thickness skin trauma. Using a collagen/elastin matrix, we developed an in vitro fibrosis model to study the interaction between EC and dermal fibroblasts or adipose tissue‐derived mesenchymal stromal cells (ASC). Tissue equivalents containing dermal fibroblasts and EC displayed a normal phenotype. In contrast, tissue equivalents containing ASC and EC displayed a fibrotic phenotype indicated by contraction of the matrix, higher gene expression of ACTA2, COL1A, COL3A, and less secretion of follistatin. The contraction was in part mediated via the TGF‐β pathway, as both inhibition of the ALK4/5/7 receptors and the addition of recombinant follistatin resulted in decreased matrix contraction (75 ± 11% and 24 ± 8%, respectively). In conclusion, our study shows that EC may play a critical role in fibrotic events, as seen in hypertrophic scars, by stimulating ASC‐mediated matrix contraction via regulation of fibrosis‐related proteins. [ABSTRACT FROM AUTHOR]

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL-6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP-1 secretion was reduced by CCL15/CCR3. In conclusion, this in-vitro study identifies chemokine receptorligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL-6 andHGF and reducing the secretion of TIMP-1. [ABSTRACT FROM AUTHOR]

Even though skin and oral mucosae are continuously in contact with commensal and opportunistic microorganisms, they generally remain healthy and uninflamed. Host defense peptides (HDPs) make up the body's first line of defense against many invading pathogens and are involved in the orchestration of innate immunity and the inflammatory response. In this study, we investigated the effect of two salivary HDPs, LL-37 and Hst1, on the inflammatory and antimicrobial response by skin and oral mucosa (gingiva) keratinocytes and fibroblasts. The potent antimicrobial chemokine CCL20 was investigated and compared with chemokines CCL2, CXCL1, CXCL8, and CCL27 and proinflammatory cytokines IL-1α and IL-6. Keratinocyte-fibroblast cocultures showed a synergistic increase in CCL20 secretion upon Hst1 and LL-37 exposure compared to monocultures. These cocultures also showed increased IL-6, CXCL1, CXCL8, and CCL2 secretion, which was IL-1α dependent. Secretion of the antimicrobial chemokine CCL20 was clearly IL-1α independent. These results indicate that salivary peptides can stimulate skin as well as gingiva cells to secrete antimicrobial chemokines as part of the hosts' defense to counteract infection. [ABSTRACT FROM AUTHOR]

Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis-, adipose-, and gingiva-derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin-derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation. [ABSTRACT FROM AUTHOR]

Both oral mucosa and skin have the capacity to maintain immune homeostasis or regulate immune responses upon environmental assault. Whereas much is known about key innate immune events in skin, little is known about oral mucosa. Comparative studies are limited due to the scarce supply of oral mucosa for ex vivo studies. Therefore, we used organotypic tissue equivalents (reconstructed epithelium on fibroblast-populated collagen hydrogel) to study cross talk between cells. Oral mucosa and skin equivalents were compared regarding secretion of cytokines and chemokines involved in LC migration and general inflammation. Basal secretion, representative of homeostasis, and also secretion after stimulation with TNFα, an allergen (cinnamaldehyde), or an irritant (SDS) were assessed. We found that proinflammatory IL-18 and chemokines CCL2, CCL20, and CXCL12, all involved in LC migration, were predominantly secreted by skin as compared to gingiva. Furthermore, CCL27 was predominantly secreted by skin whereas CCL28 was predominantly secreted by gingiva. In contrast, general inflammatory cytokines IL-6 and CXCL8 were secreted similarly by skin and gingiva. These results indicate that the cytokines and chemokines triggering innate immunity and LC migration are different in skin and gingiva. This differential regulation should be figured into novel therapy or vaccination strategies in the context of skin versus mucosa. [ABSTRACT FROM AUTHOR]

Hypertrophic scar formation is a result of adverse cutaneous wound healing. The pathogenesis of hypertrophic scar formation is still poorly understood. A problem next to the lack of suitable animal models is that often normal skin is compared to hypertrophic scar ( HTscar) and not to normotrophic scar ( NTscar) tissue. Another drawback is that often only one time period after wounding is studied, while scar formation is a dynamic process over a period of several months. In this study, we compared the expression of genes involved in inflammation, angiogenesis and extracellular matrix ( ECM) formation and also macrophage infiltration in biopsies obtained before and up to 52 weeks after standard surgery in five patients who developed HTscar and six patients who developed NTscar. It was found that HTscar formation coincided with a prolonged decreased expression of inflammatory genes ( TNF α, IL-1 α, IL-1 RN, CCL2, CCL3, CXCL2, CXCR2, C3 and IL-10) and an extended increased expression of ECM-related genes ( PLAU, Col3A1, TGF β3). This coincided with a delayed but prolonged infiltration of macrophages (type 2) in HTscar tissue compared to NTscar tissue. These findings were supported by immunohistochemical localization of proteins coding for select genes named above. Our study emphasizes that human cutaneous wound healing is a dynamic process that is needed to be studied over a period of time rather than a single point of time. Taken together, our results suggest innate immune stimulatory therapies may be a better option for improving scar quality than the currently used anti-inflammatory scar therapies. [ABSTRACT FROM AUTHOR]

Most cutaneous wounds heal with scar formation. Ideally, an inconspicuous normotrophic scar is formed, but an abnormal scar (hypertrophic scar or keloid) can also develop. A major challenge to scientists and physicians is to prevent adverse scar formation after severe trauma (e.g. burn injury) and understand why some individuals will form adverse scars even after relatively minor injury. Currently, many different models exist to study scar formation, ranging from simple monolayer cell culture to 3D tissue-engineered models even to humanized mouse models. Currently, these high-/medium-throughput test models avoid the main questions referring to why an adverse scar forms instead of a normotrophic scar and what causes a hypertrophic scar to form rather than a keloid scar and also, how is the genetic predisposition of the individual and the immune system involved. This information is essential if we are to identify new drug targets and develop optimal strategies in the future to prevent adverse scar formation. This viewpoint review summarizes the progress on in vitro and animal scar models, stresses the limitations in the current models and identifies the future challenges if scar-free healing is to be achieved in the future. [ABSTRACT FROM AUTHOR]

Chronic ulcers ((arterio)venous, decubitus, or postoperative) have no tendency to heal within a period of at least 3 months despite optimal therapy according to internationally accepted guidelines. This retrospective study evaluates the safety and efficacy of an autologous, dermal-epidermal skin substitute (SS) for treating ulcers of various origins. Ulcers were treated within 7 Dutch centers over 5 years. Sixty-six ulcers (size: 0.75-150 cm2; duration: 0.25-32 years) with a follow-up time of 24 weeks after a single-skin substitute application were assessed. Wound-bed preparation consisted of vacuum-assisted-closure-therapy (5 days, hospitalized) or application of acellular dermis (5-7 days, outpatient). Time to heal, adverse events, and recurrence 1 year after complete healing were recorded. Complete ulcer healing occurred in 36 of 66 ulcers (55%) at 24 weeks. At that time point, a further 29% of ulcers showed decrease in ulcer size between 50 and 99%. No difference was observed between the hospitalized vs. outpatient treatment with complete healing. There were 32 of 36 healed ulcers that were available for follow-up 1 year after complete closure, of which 27 (84%) were still closed. Only two minor/moderate possibly related adverse events were recorded. This retrospective analysis shows that SS provides a safe and successful treatment for particularly chronic ulcers of various origins. [ABSTRACT FROM AUTHOR]

A major challenge for clinicians treating (arterio) venous leg ulcers is to decide between standard therapy and advanced interventions. Here, we developed a simple method to collect human material representative of the ulcer wound bed, which can be used to identify biomarkers for prognostic test development. Superficial surgical debridement was performed using a small vidal curette during the weekly visit to the outpatient clinic. Moist, easily removable debridement material essentially blood free (including necrotic and nonviable slough) was collected from the surface of the ulcer. The amount ranged from 5.5 mg to 78 mg material per ulcer. Seventeen cytokines, chemokines, and growth factors were extracted and analyzed by enzyme-linked immunosorbent assay (concentration range: 0.0005-78 ng/mg total protein). Notably, CXCL8 was by far the most abundant protein present. Inflammatory mediators were more abundant than anti-inflammatory mediators (e.g., interleukin ( IL)-10 and transforming growth factor-β1). Bioactivity assays showed chronic wound extracts to be capable of stimulating fibroblast migration in a chemokine-dependent manner and also capable of stimulating healthy cells within skin substitutes to secrete wound healing mediators ( CCL2, CXCL1, CXCL8, IL-6) in an IL-1α dependent manner. Collection of debridement tissue enables investigation of the ulcer environment in an easy noninvasive manner that may be suitable for prognostic test development. [ABSTRACT FROM AUTHOR]

Skin irritation is generally not considered to be an immunological event; however, alterations in the density of Langerhans cells (LC) in the epidermis do occur, which is indicative of LC migration. In this study, we investigated the migration of LC out of the epidermis after skin exposure to contact irritants and identified the chemokines involved. With the aid of ex vivo-intact human skin and epidermal sheets we show that dermal fibroblasts play a role in mediating LC migration towards the dermis. Exposure of ex vivo-intact human skin to a panel of seven irritants (SDS, salicylic acid, phenol, isopropanol, DMSO, TritonX, or benzalkonium chloride) resulted in decreased numbers of CD1a cells in the epidermis and the accumulation of CD1a cells in the dermis. In contrast to allergen exposure, neutralizing antibodies to either CXCL12 or CCL19/CCL21 did not inhibit LC migration out of the epidermis. Exposure of epidermal sheets to the prototypical irritant SDS resulted in a TNF-α-dependent LC migration towards dermal fibroblasts. This was a result of CCL2/MCP-1 and CCL5/RANTES chemokine secretion by fibroblasts: injection of CCL2- and CCL5-neutralizing antibodies into intact human skin totally inhibited LC migration into the dermis. We have thus identified a novel role for TNF-α-inducible dermis-derived CCL2 and CCL5 in initiating migration of irritant-exposed human LC out of the epidermis. [ABSTRACT FROM AUTHOR]

Background Head and neck squamous cell carcinomas develop in preneoplastic mucosal fields that can extend over several centimeters in diameter. Most of these fields are microscopically recognized as dysplasias. These fields are often not adequately treated and might cause local relapse. Previous investigations demonstrated that mouthwash therapy with oncolytic adenoviruses appears to be a good option for the treatment of these fields, although, at present, with limited efficacy. Methods Immunohistochemistry on normal and preneoplastic mucosa was applied to determine the expression levels of the coxsackie adenoviral receptor (CAR) and a few surface antigens that might allow retargeting: Ly-6D, CD44v6 and K928. Monoclonal antibodies directed against these surface antigens were used for retargeting of adenoviruses in model experiments with organotypic cultures of mucosal epithelium. A bispecific single chain antibody was constructed against both the adenoviral knob and Ly-6D. Results Immunohistochemical staining revealed that CAR is present only at a low level in the basal layers of the oral mucosa of both normal and dysplastic lesions. By contrast, Ly-6D, CD44v6 and K928 were abundantly expressed and Ly-6D even on the most superficial layers. Monoclonal antibodies against Ly-6D and CD44v6 were shown to enhance infection in an organotypic cell culture by one log. Based on these observations, we constructed a bispecific single chain antibody against Ly-6D and adenovirus fiber knob, and showed that this engineered molecule allows efficient CAR-independent infection. Conclusions Retargeting of oncolytic adenovirus to other surface molecules might improve the efficacy of virotherapy of preneoplastic fields in the oral mucosa. Copyright © 2010 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Wounds in the mouth heal faster and with less scarification and inflammation than those in the skin. Saliva is thought to be essential for the superior oral wound healing, but the involved mechanism is still unclear. We have previously discovered that a human-specific peptide, histatin, might be implicated in the wound-healing properties of saliva. Here we report that histatin enhances reepithelialization in a human full-skin wound model closely resembling normal skin. The peptide does not stimulate proliferation but induces cell spreading and migration, two key initiating steps in reepithelialization. Activation of cells by histafin requires a C-protein-coupled receptor that activates the ERK1/2 pathway. Using a stepwise-truncation method, we determined the minimal domain (SHREFPFYGDYGS) of the 38-mer-parent peptide that is required for activity. Strikingly, N- to C-terminal cyclization of histatin-I potentiates the molar activity ∼1000-fold, indicating that the recognition of histatin by its cognate receptor requires a specific spatial conformation of the peptide. Our results emphasize the importance of histatin in human saliva for tissue protection and recovery and establish the experimental basis for the development of synthetic histatins as novel skin wound-healing agents. [ABSTRACT FROM AUTHOR]

Allergic contact dermatitis results from a T-cell-mediated, delayed-type hypersensitivity immune response induced by allergens. Skin dendritic cells (DCs) play a central role in the initiation of allergic skin responses. Following encounter with an allergen, DCs become activated and undergo maturation and differentiate into immunostimulatory DCs and are able to present antigens effectively to T cells. The frequency of allergic skin disorders has increased in the past decades. Therefore, the identification of potential sensitizing chemicals is important for skin safety. Traditionally, predictive testing for allergenicity has been conducted in animal models. For regulatory reasons, animal use for sensitization testing of compounds for cosmetic purposes is shortly to be prohibited in Europe. Therefore, new non-animal-based test methods need to be developed. Several DC-based assays have been described to discriminate allergens from irritants. Unfortunately, current in vitro methods are not sufficiently resilient to identify allergens and therefore need refinement. Here, we review the immunobiology of skin DCs (Langerhans’ cells and dermal dendritic cells) and their role in allergic and irritant contact dermatitis and then explore the possible use of DC-based models for discriminating between allergens and irritants. [ABSTRACT FROM AUTHOR]

Background/purpose: Cytokines play an important role in inflammatory and repair processes occurring in the skin. The objectives of this study were to determine the amounts of cytokines and protein isolated by tape stripping in the different layers of the stratum corneum (SC), and to compare normal skin with skin exposed in vivo to the irritant sodium lauryl sulphate (SLS). Methods: In eight volunteers, we determined the amount of total and soluble protein and also interleukin-1α (IL-1α) in pooled tape strips obtained from the upper, intermediate and lower parts of the SC. Three different types of tape were compared (Diamond®, D-squame® or Sentega® tape). In a separate study, 20 volunteers were repeatedly exposed to 0.1% SLS over a 3-week period. The amounts of IL-1α, IL-1RA and IL-8 in strips obtained from the three different SC levels of SLS-exposed skin were compared with an unexposed site. Results: For normal skin, the amounts of soluble protein and IL-1α were similar for the three tapes. Diamond® tape showed the highest yield of total protein. The total protein yield per strip decreased to lower SC levels, whereas soluble protein and IL-1α normalized by soluble protein did not change across the SC. After SLS induced skin irritation, IL-1α decreased and IL-1RA and IL-8 increased at increasing depth into the SC. Conclusions: Tape stripping is a suitable method to determine SC cytokine concentrations in human skin. With this technique, it is possible to study changes in cytokine concentrations at different SC layers after skin irritation. [ABSTRACT FROM AUTHOR]

Full-skin substitutes, epidermal substitutes, and dermal substitutes are currently being used to heal deep burns and chronic ulcers. In this study, we investigated which wound-healing mediators are released from these constructs and whether keratinocyte–fibroblast interactions are involved. Autologous skin substitutes were constructed from human keratinocytes, fibroblasts, and acellular donor dermis. Full-thickness skin was used to represent an autograft. Secretion of wound-healing mediators was investigated by means of protein array, enzyme-linked immunosorbent assay, neutralizing antibodies, and conditioned culture supernatants. Full-skin substitutes and autografts produce high amounts of inflammatory/angiogenic mediators (IL-6, CCL2, CXCL1, CXCL8, and sST2). Epidermal and dermal substitutes produced less of these proteins. Epidermal-derived proinflammatory cytokines interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α) were found to mediate synergistically the secretion of these wound-healing mediators (with the exception of sST2) from fibroblasts in dermal substitutes. The secretion of proinflammatory cytokines (IL-1α, TNF-α), chemokine/mitogen (CCL5) and angiogenic factor (vascular endothelial growth factor) by epidermal substitutes and tissue remodeling factors (tissue inhibitor of metalloproteinase-2, hepatocyte growth factor) by dermal substitutes was not influenced by keratinocyte–fibroblast interactions. The full-skin substitute has a greater potential to stimulate wound healing than epidermal or dermal substitutes. Both epidermal-derived IL-1α and TNF-α are required to trigger the release of dermal-derived inflammatory/angiogenic mediators from skin substitutes. [ABSTRACT FROM AUTHOR]

At this moment, pressure ulcer risk assessment is dominated by subjective measures and does not predict pressure ulcer development satisfactorily. Objective measures are, therefore, needed for an early detection of these ulcers. The current in vitro study evaluates cytokines and chemokines [interleukin 1 α (IL-1 α), interleukin 1 receptor antagonist (IL-1RA), tumor necrosis factor α (TNF- α) and interleukin 8 (CXCL8/IL-8)] as early markers for mechanically-induced epidermal damage. Various degrees of epidermal damage were induced by subjecting commercially available epidermal equivalents (EpiDerm) to increasing pressures (0, 50, 75, 100, 150, and 200 mmHg) for 24 h, using a loading device. At the end of the loading experiment, tissue damage was assessed by histological examination and by evaluation of the cell membrane integrity. Cytokines and chemokines were determined in the culture supernatant. Sustained epidermal loading resulted in an increased release of IL-1 α, IL-1RA, TNF- α and CXCL8/IL-8. This was first observed at 75 mmHg, when the tissue was only slightly damaged. Swollen cells, vacuoles, necrosis and affected cell membranes were observed at pressures higher than 75 mmHg. Furthermore, at 150 and 200 mmHg, the cells in the lower part of the epidermis were severely compressed. In conclusion, IL-1 α, IL-1RA, TNF- α and CXCL8/IL-8 are released in vitro as a result of sustained mechanical loading of the epidermis. The first increase in cytokines and chemokines was observed when the epidermal tissue was only slightly damaged. Therefore, these cytokines and chemokines are potential markers for the objective, early detection of mechanically-induced skin damage, such as pressure ulcers. [ABSTRACT FROM AUTHOR]

To date, no in vitro test is suitable for routine diagnosis of contact allergy. The aim of our study was to establish improved in vitro test protocol for the detection of antigen-specific responses of lymphocytes from patients with allergic contact dermatitis to nickel (Ni-ACD). Blood leucocytes from 14 Ni-ACD patients and 14 controls were cultured in the presence of ‘cytokine cocktails’ skewing lymphocytes towards ‘type 1’ [interferon-γ (IFN- γ)-secreting] or ‘type 2’ [interleukin (IL)-5 and IL-13-secreting] phenotypes. The cocktails consisted of IL-7 and, respectively, either IL-12 or IL-4. Cell responses to nickel were measured with enzyme-linked immunospot assay (ELISpot), enzyme-linked immunosorbent assay (ELISA), and lymphocyte proliferation test (LPT). Significant differences between patients with Ni-ACD and controls were found for the ‘type 2’ cytokines IL-13 and IL-5, with further increase of allergen-specific responses occurring when cultures were supplemented with IL-7 and IL-4. No significant differences were found for IFN- γ. The best correlate to clinical diagnosis was LPT with ‘type 2’ skewing ( r= 0.739, P < 0.001), followed by IL-13 ELISpot with ‘type 2’ skewing ( r= 0.654, P < 0.001). The non-radioactive method that correlated best with LPT was IL-2 ELISpot ( r= 0.809, P < 0.001). Overall, we conclude that combining ELISpot assay with proposed modifications of culture conditions improves detection of specific lymphocyte responses in contact allergy to nickel. [ABSTRACT FROM AUTHOR]

Type 1 and type 2 cytokines are primary mediators in contact allergy and aeroallergen-mediated disorders, respectively. For both types of disease, dendritic cells (DCs) are pivotal in initiating immune hyperresponsiveness. We studied whether contact and respiratory allergens possess intrinsic capacities to polarize DC towards DC1 and DC2 functions, independent of environmental factors. Human monocyte-derived DCs were exposed to the positive controls [type 1: lipopolysaccharide (LPS) + interferon-γ; type 2: LPS + prostaglandin E2], contact allergens [2,4-dinitrochlorobenzene (DNCB), oxazolone (OXA), and nickel sulfate (NiSO4)], and respiratory allergens [trimellitic anhydride (TMA) and the protein allergen derived from Dermatophagoides pteronyssinus (Der p1)]. The polarizing potentials of the allergens on DCs were determined by the secretion of type 1 [tumour necrosis factor-α (TNF-α), CXCL10, and interleukin (IL)-12p70] and type 2 (IL-10) cytokines. The contact allergens, DNCB and OXA, induced strict type 1 DC polarization, whereas the respiratory allergens, TMA and Der p1, showed strict type 2 DC polarization. The contact allergen, NiSO4, induced both DC1 (TNF-α and CXCL10 production) and DC2 (decreased IL-12p70/IL-10 ratio) features. These results support the view that allergens have an intrinsic capacity to skew immune responses at the DC level, irrespective of local factors such as those determined by cutaneous or mucosal epithelial microenvironments. [ABSTRACT FROM AUTHOR]

Moed H, von Blomberg M, Bruynzeel DP, Scheper R, Gibbs S, Rustemeyer T. Improved detection of allergen-specific T-cell responses in allergic contact dermatitis through the addition of ‘cytokine cocktails’. The gold standard for the diagnosis of allergic hypersensitivity is skin patch testing with the suspected allergens. This diagnostic tool, however, has distinct disadvantages, and therefore the development of alternative or complementary in vitro tests is of great importance. In this study, we evaluate the applicability of an in vitro test method, as developed earlier for nickel allergy, to detect allergen-specific T cells in the blood of patients allergic to frequent sensitizers (chromate, cobalt, paraphenylenediamine, fragrances and chloromethyl-isothiazolinone). Peripheral blood mononuclear cells (PBMCs) of allergic patients and healthy controls were cultured in the absence or presence of allergen. Additionally, type 1 (IL-7 and IL-12) or type 2 (IL-7 and IL-4) stimulating cytokines were added; after 6-day proliferation, IFN-γ and IL-5 secretions were determined. Without the addition of cytokines, consistent allergen-induced proliferation was observed in PBMCs of nickel-allergic patients only. By contrast, the addition of type 1 or type 2 stimulating cytokines resulted in a significantly enhanced allergen-specific proliferation for all allergens tested (sensitivity increased from 26 to 43% or 38%, respectively, P < 0.05). In these cultures, allergen-induced IFN-γ and IL-5 secretion was also significantly increased, compared to healthy controls ( P < 0.05, for IFN-γ sensitivity 79%, specificity 93%; for IL-5 sensitivity 74%, specificity 81%). In conclusion, these results demonstrate an increased proliferative capacity and cytokine production by allergen-specific T cells from allergic patients, but not of healthy individuals upon stimulation with allergens in combination with type 1 or 2 skewing cytokines. The present data warrant further exploration of the application of this test to a broader set of allergens. [ABSTRACT FROM AUTHOR]

Abstract: For screening of a potential irritant it is essential that an early marker for irritation should be chosen which could be detected before the physiological signs of irritation occur. Interleukin 1 alpha (IL-1α) is widely accepted as such a marker in both in vivo and in vitro test systems. In this study, we have determined the mRNA levels of IL-1α in the epidermis after topical application of sodium dodecyl sulphate (SLS) in both a commercially available epidermal kit (EpiDerm) and in excised skin. Furthermore, we have determined the effect of water, the vehicle for SLS, on IL-1α mRNA levels. Topical application of water to excised skin increases IL-1α mRNA levels sixfold in the epidermis whereas topical application of water to EpiDerm cultures did not alter IL-1α mRNA levels. This is explained by the finding that EpiDerm cultures have a sub-optimal barrier function when compared with excised skin – topical application of SLS was clearly toxic at much lower concentrations in EpiDerm cultures (0.2% SLS) than in excised skin (5% SLS). Also caffeine penetration was 10-fold higher through EpiDerm cultures than through the excised skin. Therefore, incubation of control EpiDerm cultures at 100% humidity effectively mimics topical exposure to water. An additional increase in IL-1α mRNA levels observed between topical application of water and SLS is similar (about threefold) in both experimental systems. In conclusion, in vitro reconstructed epidermis models, such as EpiDerm, can be used as a predictive model for irritancy screening. However, great care should be taken when interpreting the results due to the fact that EpiDerm cultures do not have a competent barrier function and therefore lower irritant concentrations are required than in in vivo or ex vivo studies in order to induce cytotoxic effects. Furthermore, the irritant effects of the vehicle should not be neglected. Our results show clearly that the topical... [ABSTRACT FROM AUTHOR]

Various growth factors such as epidermal growth factor and keratinocyte growth factor have been reported to promote wound closure and epidermal regeneration. In the present study epidermis reconstructed on de-epidermized dermis was used to investigate the effects of epidermal growth factor and keratinocyte growth factor on keratinocyte proliferation, migration and differentiation. Our results show that epidermal growth factor supplemented cultures share many of the features which are observed during regeneration of wounded epidermis: a thickening of the entire epidermis, an enhanced rate of proliferation and migration, and an increase in keratin 6, keratin 16, skin-derived antileukoproteinase, involucrin and transglutaminase 1 expression. The increase in transglutaminase 1 protein is accompanied by an increase in the amount of active transglutaminase 1 enzyme. Surprisingly no increase in keratin 17 is observed. Prolonging the culture period for more than two weeks results in rapid senescence and aging of the cultures. In contrast, keratinocyte growth factor supplemented cultures have a tissue architecture that is similar to healthy native epidermis and remains unchanged for at least 4 weeks of air-exposure. The rate of proliferation and the expression of keratins 6, 16 and 17, skin-derived antileukoproteinase, involucrin and transglutaminase 1 is similar to that found in healthy epidermis and furthermore keratinocyte migration does not occur. When the culture medium is supplemented with a combination of keratinocyte growth factor and a low concentration of epidermal growth factor, skin-derived antileukoproteinase, involucrin and keratins 6, 16 and 17 expression is similar to that found in cultures supplemented with keratinocyte growth factor alone and in healthy epidermis. Only high transglutaminase 1 expression remains similar to that observed in cultures supplemented with epidermal growth factor alone. Our results show that the regulation of keratinocyte growth, migration and differentiation depends on the availability of these growth factors. Epidermal growth factor may play a dominant early role in wound healing by stimulating keratinocyte proliferation and migration while keratinocyte growth factor may play a role later in the repair process by stabilizing epidermal turnover and barrier function. [ABSTRACT FROM AUTHOR]