Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells.

Murine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.

The reliance of glycerol utilization by Cupriavidus necator on CO2 fixation and improved glycerol catabolism.

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization.

Digital histology of tissue with Mueller microscopy and FastDBSCAN.

We present the results of the automated post-processing of Mueller microscopy images of skin tissue models with a new fast version of the algorithm of density-based spatial clustering of applications with noise (FastDBSCAN) and discuss the advantages of its implementation for digital histology of tissue. We demonstrate that using the FastDBSCAN algorithm, one can produce the diagnostic segmentation of high resolution images of tissue by several orders of magnitude faster and with high accuracy (>97%) compared to the original version of the algorithm.

Pharmaceutical Development of Nanostructured Vesicular Hydrogel Formulations of Rifampicin for Wound Healing.

Chronic wounds exhibit elevated levels of inflammatory cytokines, resulting in the release of proteolytic enzymes which delay wound-healing processes. In recent years, rifampicin has gained significant attention in the treatment of chronic wounds due to an interesting combination of antibacterial and anti-inflammatory effects. Unfortunately, rifampicin is sensitive to hydrolysis and oxidation. As a result, no topical drug product for wound-healing applications has been approved. To address this medical need two nanostructured hydrogel formulations of rifampicin were developed. The liposomal vesicles were embedded into hydroxypropyl methylcellulose (HPMC) gel or a combination of hyaluronic acid and marine collagen. To protect rifampicin from degradation in aqueous environments, a freeze-drying method was developed. Before freeze-drying, two well-defined hydrogel preparations were obtained. After freeze-drying, the visual appearance, chemical stability, residual moisture content, and redispersion time of both preparations were within acceptable limits. However, the morphological characterization revealed an increase in the vesicle size for collagen-hyaluronic acid hydrogel. This was confirmed by subsequent release studies. Interactions of marine collagen with phosphatidylcholine were held responsible for this effect. The HPMC hydrogel formulation remained stable over 6 months of storage. Moving forward, this product fulfills all criteria to be evaluated in preclinical and clinical studies.

Epidermal Regeneration Induced by Comfrey Extract: A Study by Light and Electron Microscopy.

INTRODUCTION: An accelerated healing of superficial wounds was demonstrated in clinical trials with a topical comfrey preparation (Symphytum × uplandicum Nyman). The effect has previously not been examined in skin models. METHODS: An established in vitro model of epidermal cells with the typical strata was used for the observation of effects of applied substances on skin regeneration. Damage corresponding to a typical abrasion was created on day 1 by punching an opening into the epidermal fine structure down to the stratum basale. Samples were either untreated (controls) or exposed to comfrey cream on days 2, 3, 5, and 6. Tissue samples were taken for light and electron microscopy on days 1, 4, and 7. RESULTS AND CONCLUSIONS: Application of comfrey cream led to a quicker regeneration of skin cells and to an earlier differentiation of the cells towards a normal fine structure with a visible distinction of epidermal strata, keratin, and corneocyte formation within 4-7 days. The study covered the early days of skin regeneration and confirms the benefits observed in published clinical trials and non-interventional studies in patients with abrasions.

Vascularised human skin equivalents as a novel in vitro model of skin fibrosis and platform for testing of antifibrotic drugs.

OBJECTIVES: Fibrosis is a complex pathophysiological process involving interplay between multiple cell types. Experimental modelling of fibrosis is essential for the understanding of its pathogenesis and for testing of putative antifibrotic drugs. However, most current models employ either phylogenetically distant species or rely on human cells cultured in an artificial environment. Here we evaluated the potential of vascularised in vitro human skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs. METHODS: Skin equivalents were assembled on a three-dimensional extracellular matrix by sequential seeding of endothelial cells, fibroblasts and keratinocytes. Fibrotic transformation on exposure to transforming growth factor-ß (TGFß) and response to treatment with nintedanib as an established antifibrotic agent were evaluated by quantitative polymerase chain reaction (qPCR), capillary Western immunoassay, immunostaining and histology. RESULTS: Skin equivalents perfused at a physiological pressure formed a mature, polarised epidermis, a stratified dermis and a functional vessel system. Exposure of these models to TGFß recapitulated key features of SSc skin with activation of TGFß pathways, fibroblast to myofibroblast transition, increased release of collagen and excessive deposition of extracellular matrix. Treatment with the antifibrotic agent nintedanib ameliorated this fibrotic transformation. CONCLUSION: Our data provide evidence that vascularised skin equivalents can replicate key features of fibrotic skin and may serve as a platform for evaluation of antifibrotic drugs in a pathophysiologically relevant human setting.

Improved metal allergen reactivity of artificial skin models by integration of Toll-like receptor 4-positive cells.

BACKGROUND: Reconstructed human epidermis (RhE) is widely used to replace animal models in order to assess the proinflammatory and allergenic effects of chemicals. Unfortunately, RhE lacks proinflammatory responsiveness for metal haptens, which are the most prevalent human contact allergens, raising concerns about its reliability for predicting skin allergens. OBJECTIVES: To investigate whether this limitation of RhE might be attributable to a lack of functional expression of Toll-like receptor 4 (TLR4), which governs proinflammatory sensitivity to nickel and cobalt. MATERIALS AND METHODS: RhE, dendritic cell (DC)-containing RhE and full-thickness skin equivalent (FTSE) were compared regarding their proinflammatory responsiveness to metal allergens. RESULTS: The incorporation of dermal fibroblasts was sufficient to confer metal sensitivity to RhE. Unlike keratinocytes, normal human fibroblasts expressed high levels of TLR4 mRNA and induced interleukin-8 expression upon stimulation with nickel or cobalt. Consistently, dermal isolates from FTSE expressed considerable amounts of TLR4 mRNA, whereas RhE or epidermis isolated from FTSE, normal human epidermis or inflamed human epidermis failed to express TLR4. Similarly, co-culture with TLR4-positive DCs bestowed RhE with proinflammatory responsiveness to metals. CONCLUSION: Our data suggest that FTSE or DC/RhE co-culture models can circumvent the shortcomings of RhE assays, and combine the benefits of complex and monoculture-based test systems in a single assay.

Noncanonical WNT5A controls the activation of latent TGF-ß to drive fibroblast activation and tissue fibrosis.

Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Conformational regulation and target-myristoyl switch of calcineurin B homologous protein 3.

Calcineurin B homologous protein 3 (CHP3) is an EF-hand Ca2+-binding protein involved in regulation of cancerogenesis, cardiac hypertrophy, and neuronal development through interactions with sodium/proton exchangers (NHEs) and signalling proteins. While the importance of Ca2+ binding and myristoylation for CHP3 function has been recognized, the underlying molecular mechanism remained elusive. In this study, we demonstrate that Ca2+ binding and myristoylation independently affect the conformation and functions of human CHP3. Ca2+ binding increased local flexibility and hydrophobicity of CHP3 indicative of an open conformation. The Ca2+-bound CHP3 exhibited a higher affinity for NHE1 and associated stronger with lipid membranes compared to the Mg2+-bound CHP3, which adopted a closed conformation. Myristoylation enhanced the local flexibility of CHP3 and decreased its affinity to NHE1 independently of the bound ion, but did not affect its binding to lipid membranes. The data exclude the proposed Ca2+-myristoyl switch for CHP3. Instead, a Ca2+-independent exposure of the myristoyl moiety is induced by binding of the target peptide to CHP3 enhancing its association to lipid membranes. We name this novel regulatory mechanism 'target-myristoyl switch'. Collectively, the interplay of Ca2+ binding, myristoylation, and target binding allows for a context-specific regulation of CHP3 functions.

Biomimetic Connection of Transcutaneous Implants with Skin.

Bacterial infection is a crucial complication in implant restoration, in particular in permanent skin-penetrating implants. Therein, the resulting gap between transcutaneous implant and skin represents a permanent infection risk, limiting the field of application and the duration of application. To overcome this limitation, a tight physiological connection is required to achieve a biological and mechanical welding for a long-term stable closure including self-healing probabilities. This study describes a new approach, wherein the implant is connected covalently to a highly porous electrospun fleece featuring physiological dermal integration potential. The integrative potential of the scaffold is shown in vitro and confirmed in vivo, further demonstrating tissue integration by neovascularization, extracellular matrix formation, and prevention of encapsulation. To achieve a covalent connection between fleece and implant surface, self-initiated photografting and photopolymerization of hydroxyethylmethacrylate is combined with a new crosslinker (methacrylic acid coordinated titanium-oxo clusters) on proton-abstractable implant surfaces. For implant modification, the attached fleece is directed perpendicular from the implant surface into the surrounding dermal tissue. First in vitro skin implantations demonstrate the implants' dermal integration capability as well as wound closure potential on top of the fleece by epithelialization, establishing a bacteria-proof and self-healing connection of skin and transcutaneous implant.

Vector-borne Trypanosoma brucei parasites develop in artificial human skin and persist as skin tissue forms.

Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly's bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites' development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.

[Regenerative medicine for the corneal epithelium : Cell therapy from bench to bedside]. / Regenerative Medizin für das Hornhautepithel : Zelltherapie in Wissenschaft und Klinik.

In the case of thermal or caustic burns of the ocular surface, loss of limbal epithelial stem cells leads to compromised self-renewal of the corneal epithelium. This results in permanent loss of vision. In these situations, transplantation of cultured limbal epithelial cells on an amniotic membrane or fibrin gel as substrate (Holoclar®) can help to regenerate the corneal surface. The required cells are obtained from the healthy partner eye, if available. Adult stem cells from other parts of the body potentially serve as alternative cell sources: hair follicles, oral mucosa, mesenchymal stromal cells, or induced pluripotent stem cells (originally, e.g., skin fibroblasts). The reprogramming of such cells can be achieved with the help of transcription factors. In addition, work is being done on biosynthetic or synthetic matrices, which not only serve as substrate material for the transplantation but also support the functional properties of these cells (self-renewal, corneal epithelial-typical phenotype).

Fully Synthetic 3D Fibrous Scaffolds for Stromal Tissues-Replacement of Animal-Derived Scaffold Materials Demonstrated by Multilayered Skin.

The extracellular matrix (ECM) of soft tissues in vivo has remarkable biological and structural properties. Thereby, the ECM provides mechanical stability while it still can be rearranged via cellular remodeling during tissue maturation or healing processes. However, modern synthetic alternatives fail to provide these key features among basic properties. Synthetic matrices are usually completely degraded or are inert regarding cellular remodeling. Based on a refined electrospinning process, a method is developed to generate synthetic scaffolds with highly porous fibrous structures and enhanced fiber-to-fiber distances. Since this approach allows for cell migration, matrix remodeling, and ECM synthesis, the scaffold provides an ideal platform for the generation of soft tissue equivalents. Using this matrix, an electrospun-based multilayered skin equivalent composed of a stratified epidermis, a dermal compartment, and a subcutis is able to be generated without the use of animal matrix components. The extension of classical dense electrospun scaffolds with high porosities and motile fibers generates a fully synthetic and defined alternative to collagen-gel-based tissue models and is a promising system for the construction of tissue equivalents as in vitro models or in vivo implants.

In Model, In Vitro and In Vivo Killing Efficacy of Antitumor Peptide RDP22 on MUG-Mel2, a Patient Derived Cell Line of an Aggressive Melanoma Metastasis.

The host defense derived peptide was assessed in different model systems with increasing complexity employing the highly aggressive NRAS mutated melanoma metastases cell line MUG-Mel2. Amongst others, fluorescence microscopy and spectroscopy, as well as cell death studies were applied for liposomal, 2D and 3D in vitro models including tumor spheroids without or within skin models and in vivo mouse xenografts. Summarized, MUG-Mel2 cells were shown to significantly expose the negatively charged lipid phosphatidylserine on their plasma membranes, showing they are successfully targeted by RDP22. The peptide was able to induce cell death in MUG-Mel2 2D and 3D cultures, where it was able to kill tumor cells even inside the core of tumor spheroids or inside a melanoma organotypic model. In vitro studies indicated cell death by apoptosis upon peptide treatment with an LC50 of 8.5 µM and seven-fold specificity for the melanoma cell line MUG-Mel2 over normal dermal fibroblasts. In vivo studies in mice xenografts revealed effective tumor regression upon intratumoral peptide injection, indicated by the strong clearance of pigmented tumor cells and tremendous reduction in tumor size and proliferation, which was determined histologically. The peptide RDP22 has clearly shown high potential against the melanoma cell line MUG-Mel2 in vitro and in vivo.

Orchestrating the Dermal/Epidermal Tissue Ratio during Wound Healing by Controlling the Moisture Content.

A balanced and moist wound environment and surface increases the effect of various growth factors, cytokines, and chemokines, stimulating cell growth and wound healing. Considering this fact, we tested in vitro and in vivo water evaporation rates from the cellulose dressing epicitehydro when combined with different secondary dressings as well as the resulting wound healing efficacy in a porcine donor site model. The aim of this study was to evaluate how the different rates of water evaporation affected wound healing efficacy. To this end, epicitehydro primary dressing, in combination with different secondary dressing materials (cotton gauze, JELONET◊, AQUACEL® Extra ™, and OPSITE◊ Flexifix), was placed on 3 × 3 cm-sized dermatome wounds with a depth of 1.2 mm on the flanks of domestic pigs. The healing process was analyzed histologically and quantified by morphometry. High water evaporation rates by using the correct secondary dressing, such as cotton gauze, favored a better re-epithelialization in comparison with the low water evaporation resulting from an occlusive secondary dressing, which favored the formation of a new and intact dermal tissue that nearly fully replaced all the dermis that was removed during wounding. This newly available evidence may be of great benefit to clinical wound management.

A preclinical model of cutaneous melanoma based on reconstructed human epidermis.

Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery.

Handheld Spectral Sensing Devices Should Not Mislead Consumers as Far as Non-Authentic Food Is Concerned: A Case Study with Adulteration of Milk Powder.

With the rising trend of consumers being offered by start-up companies portable devices and applications for checking quality of purchased products, it appears of paramount importance to assess the reliability of miniaturized sensors embedded in such devices. Here, eight sensors were assessed for food fraud applications in skimmed milk powder. The performance was evaluated with dry- and wet-blended powders mimicking adulterated materials by addition of either ammonium sulfate, semicarbazide, or cornstarch in the range 0.5-10% of profit. The quality of the spectra was assessed for an adequate identification of the outliers prior to a deep assessment of performance for both non-targeted (soft independent modelling of class analogy, SIMCA) and targeted analyses (partial least square regression with orthogonal signal correction, OPLS). Here, we show that the sensors have generally difficulties in detecting adulterants at ca. 5% supplementation, and often fail in achieving adequate specificity and detection capability. This is a concern as they may mislead future users, particularly consumers, if they are intended to be developed for handheld devices available publicly in smartphone-based applications.

In vitro skin culture media influence the viability and inflammatory response of primary macrophages.

The replacement of animal models for investigation of inflammation and wound healing has been advancing by means of in vitro skin equivalents with increasing levels of complexity. However, the current in vitro skin models still have a limited pre-clinical relevance due to their lack of immune cells. So far, few steps have been made towards the incorporation of immune cells into in vitro skin and the requirements for immunocompetent co-cultures remain unexplored. To establish suitable conditions for incorporating macrophages into skin models, we evaluated the effects of different media on primary keratinocytes, fibroblasts and macrophages. Skin maturation was affected by culture in macrophage medium, while macrophages showed reduced viability, altered cell morphology and decreased response to pro- and anti-inflammatory stimuli in skin differentiation media, both in 2D and 3D. The results indicate that immunocompetent skin models have specific, complex requirements for supporting an accurate detection of immune responses, which point at the identification of a suitable culture medium as a crucial pre-requisite for the development of physiologically relevant models.

A 3D In Vitro Model for Burn Wounds: Monitoring of Regeneration on the Epidermal Level.

Burns affect millions every year and a model to mimic the pathophysiology of such injuries in detail is required to better understand regeneration. The current gold standard for studying burn wounds are animal models, which are under criticism due to ethical considerations and a limited predictiveness. Here, we present a three-dimensional burn model, based on an open-source model, to monitor wound healing on the epidermal level. Skin equivalents were burned, using a preheated metal cylinder. The healing process was monitored regarding histomorphology, metabolic changes, inflammatory response and reepithelialization for 14 days. During this time, the wound size decreased from 25% to 5% of the model area and the inflammatory response (IL-1ß, IL-6 and IL-8) showed a comparable course to wounding and healing in vivo. Additionally, the topical application of 5% dexpanthenol enhanced tissue morphology and the number of proliferative keratinocytes in the newly formed epidermis, but did not influence the overall reepithelialization rate. In summary, the model showed a comparable healing process to in vivo, and thus, offers the opportunity to better understand the physiology of thermal burn wound healing on the keratinocyte level.

Analysis of tissue microstructure with Mueller microscopy: logarithmic decomposition and Monte Carlo modeling.

Significance: Definitive diagnostics of many diseases is based on the histological analysis of thin tissue cuts with optical white light microscopy. Extra information on tissue structural properties obtained with polarized light would help the pathologist to improve the accuracy of his diagnosis.

Aim: We report on using Mueller matrix microscopy data, logarithmic decomposition, and polarized Monte Carlo (MC) modeling for qualitative and quantitative analysis of thin tissue cuts to extract the information on tissue microstructure that is not available with a conventional white light microscopy.