The biodistribution of triamcinolone acetonide injections in severe keloids: an exploratory three-dimensional fluorescent cryomicrotome study.

Intralesional corticosteroid injections are a first-line treatment for keloids; yet clinical treatment results are highly variable and often suboptimal. Variation in triamcinolone acetonide (TAC) biodistribution may be an important reason for the variable effects of TAC treatment in keloids. In this exploratory study we investigated the biodistribution of TAC in keloids and normal skin using different drug delivery techniques. Fluorescent-labeled TAC suspension was administered into keloids and normal skin with a hypodermic needle and an electronic pneumatic jet injector. TAC biodistribution was represented by the fluorescent TAC volume and 3D biodistribution shape of TAC, using a 3D-Fluorescence-Imaging Cryomicrotome System. Twenty-one keloid and nine normal skin samples were analyzed. With needle injections, the mean fluorescent TAC volumes were 990 µl ± 479 in keloids and 872 µl ± 227 in normal skin. With the jet injector, the mean fluorescent TAC volumes were 401 µl ± 252 in keloids and 249 µl ± 67 in normal skin. 3D biodistribution shapes of TAC were highly variable in keloids and normal skin. In conclusion, TAC biodistribution in keloids is highly variable for both needle and jet injection. This may partly explain the variable treatment effects of intralesional TAC in keloids. Future research is needed to confirm this preliminary finding and to optimize drug delivery in keloids.

In vitro immunity: an overview of immunocompetent organ-on-chip models.

Impressive advances have been made to replicate human physiology in vitro over the last few years due to the growth of the organ-on-chip (OoC) field in both industrial and academic settings. OoCs are a type of microphysiological system (MPS) that imitates functional and dynamic aspects of native human organ biology on a microfluidic device. Organoids and organotypic models, ranging in their complexity from simple single-cell to complex multi-cell type constructs, are being incorporated into OoC microfluidic devices to better mimic human physiology. OoC technology has now progressed to the stage at which it has received official recognition by the Food and Drug Administration (FDA) for use as an alternative to standard procedures in drug development, such as animal studies and traditional in vitro assays. However, an area that is still lagging behind is the incorporation of the immune system, which is a critical element required to investigate human health and disease. In this review, we summarise the progress made to integrate human immunology into various OoC systems, specifically focusing on models related to organ barriers and lymphoid organs. These models utilise microfluidic devices that are either commercially available or custom-made. This review explores the difference between the use of innate and adaptive immune cells and their role for modelling organ-specific diseases in OoCs. Immunocompetent multi-OoC models are also highlighted and the extent to which they recapitulate systemic physiology is discussed. Together, the aim of this review is to describe the current state of immune-OoCs, the limitations and the future perspectives needed to improve the field.

Intraluminal release of citrullinated histone 3 from various cellular origins coincides with microvascular thrombosis in burn wounds.

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.

Reconstructed Human Skin with Hypodermis Shows Essential Role of Adipose Tissue in Skin Metabolism.

BACKGROUND: Dysregulation of skin metabolism is associated with a plethora of diseases such as psoriasis and dermatitis. Until now, reconstructed human skin (RhS) models lack the metabolic potential of native human skin, thereby limiting their relevance to study human healthy and diseased skin. We aimed to determine whether incorporation of an adipocyte-containing hypodermis into RhS improves its metabolic potential and to identify major metabolic pathways up-regulated in adipose-RhS. METHODS: Primary human keratinocytes, fibroblasts and differentiated adipose-derived stromal cells were co-cultured in a collagen/fibrin scaffold to create an adipose-RhS. The model was extensively characterized structurally in two- and three-dimensions, by cytokine secretion and RNA-sequencing for metabolic enzyme expression. RESULTS: Adipose-RhS showed increased secretion of adipokines. Both RhS and adipose-RhS expressed 29 of 35 metabolic genes expressed in ex vivo native human skin. Addition of the adipose layer resulted in up-regulation of 286 genes in the dermal-adipose fraction of which 7 were involved in phase I (CYP19A1, CYP4F22, CYP3A5, ALDH3B2, EPHX3) and phase II (SULT2B1, GPX3) metabolism. Vitamin A, D and carotenoid metabolic pathways were enriched. Additionally, pro-inflammatory (IL-1ß, IL-18, IL-23, IL-33, IFN-α2, TNF-α) and anti-inflammatory cytokine (IL-10, IL-12p70) secretion was reduced in adipose-RhS. CONCLUSIONS: Adipose-RhS mimics healthy native human skin more closely than traditional RhS since it has a less inflamed phenotype and a higher metabolic activity, indicating the contribution of adipocytes to tissue homeostasis. Therefore it is better suited to study onset of skin diseases and the effect of xenobiotics.

Porphyromonas gingivalis interaction with Candida albicans allows for aerobic escape, virulence and adherence.

In the oral cavity Candida albicans interacts with many oral bacteria, including Porphyromonas gingivalis, both physically and metabolically. The aim of this in vitro study was to characterize these interactions and study their effects on the survival of P. gingivalis. First, metabolic interactions were evaluated by counting the colony forming units (CFU) after co-culturing. The results indicated that the anaerobic bacterium P. gingivalis survives under aerobic conditions when co-cultured with C. albicans. This is due to the oxygen consumption by C. albicans as determined by a reduction in survival upon the addition of Antimycin A. By measuring the protease activity, it was found that the presence of C. albicans induced gingipain activity by P. gingivalis, which is an important virulence factor. Adherence of P. gingivalis to hyphae of C. albicans was observed with a dynamic flow system. Using various C. albicans mutants, it was shown that the mechanism of adhesion was mediated by the cell wall adhesins, members of the agglutinin-like sequence (Als) family: Als3 and Als1. Furthermore, the two microorganisms could be co-cultured into forming a biofilm in which P. gingivalis can survive under aerobic culturing conditions, which was imaged using scanning electron microscopy. This study has further elucidated mechanisms of interaction, virulence acquisition and survival of P. gingivalis when co-cultured with C. albicans. Such survival could be essential for the pathogenicity of P. gingivalis in the oxygen-rich niches of the oral cavity. This study has emphasized the importance of interaction between different microbes in promoting survival, virulence and attachment of pathogens, which could be essential in facilitating penetration into the environment of the host.

Towards Full Thickness Small Intestinal Models: Incorporation of Stromal Cells.

INTRODUCTION: Since small intestine is one of the major barriers of the human body, there is a need to develop reliable in vitro human small intestinal models. These models should incorporate both the epithelial and lamina propria compartments and have similar barrier properties compared to that of the human tissue. These properties are essential for various applications, such as studying cell-cell interaction, intestinal diseases and testing permeability and metabolism of drugs and other compounds. The small intestinal lamina propria contains multiple stromal cell populations with several important functions, such as secretion of extracellular matrix proteins and soluble mediators. In addition, stromal cells influence the intestinal epithelial barrier, support the intestinal stem cell niche and interact with immune cells. METHODS: In this review, we provide an extensive overview on the different types of lamina propria stromal cells found in small intestine and describe a combination of molecular markers that can be used to distinguish each different stromal cell type. We focus on studies that incorporated stromal cells into human representative small intestine models cultured on transwells. RESULTS AND CONCLUSION: These models display enhanced epithelial morphology, increased cell proliferation and human-like barrier properties, such as low transepithelial electrical resistance (TEER) and intermediate permeability, thus better mimicking the native human small intestine than models only consisting of an epithelium which generally show high TEER and low permeability.

An Organotypic Human Lymph Node Model Reveals the Importance of Fibroblastic Reticular Cells for Dendritic Cell Function.

BACKGROUND: Human lymph node (HuLN) models have emerged with invaluable potential for immunological research and therapeutic application given their fundamental role in human health and disease. While fibroblastic reticular cells (FRCs) are instrumental to HuLN functioning, their inclusion and recognition of importance for organotypic in vitro lymphoid models remain limited. METHODS: Here, we established an in vitro three-dimensional (3D) model in a collagen-fibrin hydrogel with primary FRCs and a dendritic cell (DC) cell line (MUTZ-3 DC). To study and characterise the cellular interactions seen in this 3D FRC-DC organotypic model compared to the native HuLN; flow cytometry, immunohistochemistry, immunofluorescence and cytokine/chemokine analysis were performed. RESULTS: FRCs were pivotal for survival, proliferation and localisation of MUTZ-3 DCs. Additionally, we found that CD1a expression was absent on MUTZ-3 DCs that developed in the presence of FRCs during cytokine-induced MUTZ-3 DC differentiation, which was also seen with primary monocyte-derived DCs (moDCs). This phenotype resembled HuLN-resident DCs, which we detected in primary HuLNs, and these CD1a- MUTZ-3 DCs induced T cell proliferation within a mixed leukocyte reaction (MLR), indicating a functional DC status. FRCs expressed podoplanin (PDPN), CD90 (Thy-1), CD146 (MCAM) and Gremlin-1, thereby resembling the DC supporting stromal cell subset identified in HuLNs. CONCLUSION: This 3D FRC-DC organotypic model highlights the influence and importance of FRCs for DC functioning in a more realistic HuLN microenvironment. As such, this work provides a starting point for the development of an in vitro HuLN.

Early TRAIL-engagement elicits potent multimodal targeting of melanoma by CD34+ progenitor cell-derived NK cells.

Umbilical cord blood (UCB) CD34+ progenitor cell-derived natural killer (NK) cells exert efficient cytotoxicity against various melanoma cell lines. Of interest, the relative cytotoxic performance of individual UCB donors was consistent throughout the melanoma panel and correlated with IFNγ, TNF, perforin and granzyme B levels. Importantly, intrinsic perforin and Granzyme B load predicts NK cell cytotoxic capacity. Exploring the mode of action revealed involvement of the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46 and most importantly of TRAIL. Strikingly, combinatorial receptor blocking led to more pronounced inhibition of cytotoxicity (up to 95%) than individual receptor blocking, especially in combination with TRAIL-blocking, suggesting synergistic cytotoxic NK cell activity via engagement of multiple receptors which was also confirmed in a spheroid model. Importantly, lack of NK cell-related gene signature in metastatic melanomas correlates with poor survival highlighting the clinical significance of NK cell therapies as a promising treatment for high-risk melanoma patients.

Current Practice in Keloid Treatment: a Survey of Dutch Dermatologists and Plastic Surgeons.

BACKGROUND: Several therapeutic options are available for the treatment of keloids, but it remains unclear which treatment options are most commonly used by practitioners. OBJECTIVE: To explore the prevailing treatment for different keloid phenotypes among dermatologists and plastic surgeons in the Netherlands. METHODS: Members of the Dutch society for Plastic surgery and the Dutch society for Dermatology and Venereology were asked to participate. Questions elaborated on the treatment for a small and a large keloid on the mandibula and multiple keloids on the chest. RESULTS: One hundred forty-three responses were obtained. Heterogeneity in treatment was extremely high for the small, large, and multiple keloids with 27, 35, and 33 various first choices, respectively. Intralesional corticosteroids were most often chosen for all 3 different keloid phenotypes. These were mostly (61%) administered as monotherapy for the small keloid and mostly combined with other treatments for the large keloid (19%) and multiple keloids (43%). Surgery was chosen regularly (22%) for the large keloid, mostly combined with intralesional corticosteroids (10%) or brachytherapy (8.4%). CONCLUSION: Keloid treatment is very heterogeneous among dermatologists and plastic surgeons, even in a relatively small country as the Netherlands. Moreover, the treatment choice depends on the keloid phenotype.

A Reconstructed Human Melanoma-in-Skin Model to Study Immune Modulatory and Angiogenic Mechanisms Facilitating Initial Melanoma Growth and Invasion.

Invasion, immune modulation, and angiogenesis are crucial in melanoma progression. Studies based on animals or two-dimensional cultures poorly recapitulate the tumor-microenvironmental cross-talk found in humans. This highlights a need for more physiological human models to better study melanoma features. Here, six melanoma cell lines (A375, COLO829, G361, MeWo, RPMI-7951, and SK-MEL-28) were used to generate an in vitro three-dimensional human melanoma-in-skin (Mel-RhS) model and were compared in terms of dermal invasion and immune modulatory and pro-angiogenic capabilities. A375 displayed the most invasive phenotype by clearly expanding into the dermal compartment, whereas COLO829, G361, MeWo, and SK-MEL-28 recapitulated to different extent the initial stages of melanoma invasion. No nest formation was observed for RPMI-7951. Notably, the integration of A375 and SK-MEL-28 cells into the model resulted in an increased secretion of immune modulatory factors (e.g., M-CSF, IL-10, and TGFß) and pro-angiogenic factors (e.g., Flt-1 and VEGF). Mel-RhS-derived supernatants induced endothelial cell sprouting in vitro. In addition, observed A375-RhS tissue contraction was correlated to increased TGFß release and α-SMA expression, all indicative of differentiation of fibroblasts into cancer-associated fibroblast-like cells and reminiscent of epithelial-to-mesenchymal transition, consistent with A375's most prominent invasive behavior. In conclusion, we successfully generated several Mel-RhS models mimicking different stages of melanoma progression, which can be further tailored for future studies to investigate individual aspects of the disease and serve as three-dimensional models to assess efficacy of therapeutic strategies.

Mechanisms of Porphyromonas gingivalis to translocate over the oral mucosa and other tissue barriers.

Introduction: The oral pathogen Porphyromonas gingivalis is not only associated with periodontitis but also with systemic diseases elsewhere in the body. The mechanisms by which P. gingivalis travels from the oral cavity to other organs in the body are largely unknown. This review describes the four putative mechanisms supported by experimental evidence, which enable translocation of P. gingivalis over the oral mucosa, endothelial barriers and subsequent dissemination into the bloodstream. Mechanisms: The first mechanism: proteolytic enzymes secreted by P. gingivalis degrade adhesion molecules between tissue cells, and the extracellular matrix. This weakens the structural integrity of the mucosa and allows P. gingivalis to penetrate the tissue. The second is transcytosis: bacteria actively enter tissue cells and transfer to the next layer or the extracellular space. By travelling from cell to cell, P. gingivalis reaches deeper structures. Thirdly, professional phagocytes take up P. gingivalis and travel to the bloodstream where P. gingivalis is released. Lastly, P. gingivalis can adhere to the hyphae forming Candida albicans. These hyphae can penetrate the mucosal tissue, which may allow P. gingivalis to reach deeper structures. Conclusion: More research could elucidate targets to inhibit P. gingivalis dissemination and prevent the onset of various systemic diseases.

Oral host-microbe interactions investigated in 3D organotypic models.

The oral cavity is inhabited by abundant microbes which continuously interact with the host and influence the host's health. Such host-microbe interactions (HMI) are dynamic and complex processes involving e.g. oral tissues, microbial communities and saliva. Due to difficulties in mimicking the in vivo complexity, it is still unclear how exactly HMI influence the transition between healthy status and disease conditions in the oral cavity. As an advanced approach, three-dimensional (3D) organotypic oral tissues (epithelium and mucosa/gingiva) are being increasingly used to study underlying mechanisms. These in vitro models were designed with different complexity depending on the research questions to be answered. In this review, we summarised the existing 3D oral HMI models, comparing designs and readouts, discussing applications as well as future perspectives.

Baker Grade IV Capsular Contracture Is Correlated with an Increased Amount of Silicone Material: An Intrapatient Study.

BACKGROUND: Breast implant surgery is one of the most frequently performed procedures by plastic surgeons worldwide. However, the relationship between silicone leakage and the most common complication, capsular contracture, is far from understood. This study aimed to compare Baker grade I with Baker grade IV capsules regarding their silicone content in an intradonor setting, using two previously validated imaging techniques. METHODS: Twenty-two donor-matched capsules from 11 patients experiencing unilateral complaints were included after bilateral explantation surgery. All capsules were examined using both stimulated Raman scattering (SRS) imaging and staining with modified oil red O (MORO). Evaluation was done visually for qualitative and semiquantitative assessment and automated for quantitative analysis. RESULTS: Using both SRS and MORO techniques, silicone was found in more Baker grade IV capsules (eight of 11 and 11 of 11, respectively) than in Baker grade I capsules (three of 11 and five of 11, respectively). Baker grade IV capsules also showed significantly more silicone content compared with the Baker grade I capsules. This was true for semiquantitative assessment for both SRS and MORO techniques ( P = 0.019 and P = 0.006, respectively), whereas quantitative analysis proved to be significant for MORO alone ( P = 0.026 versus P = 0.248 for SRS, respectively). CONCLUSIONS: In this study, a significant correlation between capsule silicone content and capsular contracture is shown. An extensive and continued foreign body response to silicone particles is likely to be responsible. Considering the widespread use of silicone breast implants, these results affect many women worldwide and warrant a more focused research effort. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

The Complexity of the Post-Burn Immune Response: An Overview of the Associated Local and Systemic Complications.

Burn injury induces a complex inflammatory response, both locally and systemically, and is not yet completely unravelled and understood. In order to enable the development of accurate treatment options, it is of paramount importance to fully understand post-burn immunology. Research in the last decades describes insights into the prolonged and excessive inflammatory response that could exist after both severe and milder burn trauma and that this response differs from that of none-burn acute trauma. Persistent activity of complement, acute phase proteins and pro- and anti-inflammatory mediators, changes in lymphocyte activity, activation of the stress response and infiltration of immune cells have all been related to post-burn local and systemic pathology. This "narrative" review explores the current state of knowledge, focusing on both the local and systemic immunology post-burn, and further questions how it is linked to the clinical outcome. Moreover, it illustrates the complexity of post-burn immunology and the existing gaps in knowledge on underlying mechanisms of burn pathology.

Intralesional corticosteroid administration in the treatment of keloids: a survey among Dutch dermatologists and plastic surgeons.

BACKGROUND: Intralesional corticosteroid administration (ICA) is a first-line therapy in keloid treatment. However, its clinical results are still highly variable and often suboptimal. Treatment results may strongly be influenced by various ways of ICA. OBJECTIVE: To explore the prevailing practice of ICA in keloid treatment among dermatologists and plastic surgeons in the Netherlands. METHODS: The survey was constructed based on a scoping review on ICA in keloid treatment. Members of the Dutch Society for Plastic surgery and the Dutch Society for Dermatology and Venereology were asked to participate. RESULTS: One hundred and thirty-six responses were obtained. One hundred and thirty (95.6%) participants used triamcinolone acetonide. The majority (54.7%) did not use local anesthesia for pain reduction. Reported corticosteroid dosing that one would inject in one specific keloid differed by a factor of 40. Treatment intervals varied from 1 week to more than 8 weeks. The keloid center was most often injected (46.9%), followed by subepidermal (18.0%). CONCLUSIONS: A wide variety in ICA for keloids is noted among dermatologists and plastic surgeons, even in a limited geographic region and when evidence points toward an optimal way of treatment. Future studies and better implementation of existing evidence may reduce variation in ICA and optimize its treatment results.

The JAK-STAT pathway in keloid pathogenesis: a systematic review with qualitative synthesis.

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.

Intralesional Corticosteroid Administration in the Treatment of Keloids: A Scoping Review on Injection Methods.

BACKGROUND: Intralesional corticosteroid administration (ICA) is a first-line treatment for keloids. However, its clinical results are still highly variable and often suboptimal. Treatment results may strongly be influenced by various operator-dependent factors. The aim of this study was to map the details of ICA in keloids described in randomized controlled trials (RCTs), hence presenting the scientific practice of a first-line treatment for keloids in the best available evidence. SUMMARY: A systematic search was performed on PubMed, Ovid MEDLINE, Ovid EMBASE, and CENTRAL. Eligible studies were RCTs including patients with keloids treated with intralesional corticosteroids. Treatment and study design-related data were charted on a predefined form. Thirty-eight RCTs were included for data extraction. Triamcinolone acetonide was used in 37 (97.4%) studies. Dosing per cm2 could only be compared among ten (26%) studies and varied from 1 to 20 mg. The maximum dose per session varied from 20 to 80 mg. Local anesthetics were administered in seven (20%) RCTs. Treatment intervals varied from weekly to monthly, with 4 weeks most frequently (50%) used. Needle size was reported in eleven (29%) studies and varied from 26 to 30-gauge. Syringe size was specified in four (11%) studies, being 1 mL. The injection level was described in eleven (29%) studies. Blanching as endpoint was reported in ten (26%) studies. Outcome measures varied widely, from height, surface area, or volume, to Vancouver Scar Scale, Patient and Observer Scar Assessment Scale, pain and itch scores, patient satisfaction, and different efficacy rates. Only six studies had a follow-up of ≥6 months. Recurrence was identified in two studies with 18 weeks and 1 year of follow-up. Adverse events were reported in 23 (61%) studies.

No association found between late-onset inflammatory adverse events after soft tissue filler injections and the adaptive immune system.

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.

A Humanized In Vitro Model of Innervated Skin for Transdermal Analgesic Testing.

Sensory innervation of the skin is essential for its function, homeostasis, and wound healing mechanisms. Thus, to adequately model the cellular microenvironment and function of native skin, in vitro human skin equivalents (hSE) containing a sensory neuron population began to be researched. In this work, a fully human 3D platform of hSE innervated by induced pluripotent stem cell-derived nociceptor neurospheres (hNNs), mimicking the native mode of innervation, is established. Both the hSE and nociceptor population exhibit morphological and phenotypical characteristics resembling their native counterparts, such as epidermal and dermal layer formation and nociceptor marker exhibition, respectively. In the co-culture platform, neurites develop from the hNNs and navigate in 3D to innervate the hSE from a distance. To probe both skin and nociceptor functionality, a clinically available capsaicin patch (Qutenza) is applied directly over the hSE section and neuron reaction is analyzed. Application of the patch causes an exposure time-dependent neurite regression and degeneration. In platforms absent of hSE, axonal degeneration is further increased, highlighting the role of the skin construct as a barrier. In sum, an in vitro tool of functional innervated skin with high interest for preclinical research is established.

Nickel allergy is associated with a broad spectrum cytokine response.

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-ß), 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.