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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 vivonative 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-1β, 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.

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 vitrolymphoid models remain limited. Methods:: Here, we established an in vitrothree-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 vitroHuLN.

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.

A burn wound is a complex systemic disease at multiple levels. Current knowledge of scar formation after burn injury has come from traditional biological and clinical studies. These are normally focused on just a small part of the entire process, which has limited our ability to sufficiently understand the underlying mechanisms and to predict systems behaviour. Scar formation after burn injury is a result of a complex biological system—wound healing. It is a part of a larger whole. In this self-organising system, many components form networks of interactions with each other. These networks of interactions are typically non-linear and change their states dynamically, responding to the environment and showing emergent long-term behaviour. How molecular and cellular data relate to clinical phenomena, especially regarding effective therapies of burn wounds to achieve minimal scarring, is difficult to unravel and comprehend. Complexity science can help bridge this gap by integrating small parts into a larger whole, such that relevant biological mechanisms and data are combined in a computational model to better understand the complexity of the entire biological system. A better understanding of the complex biological system of post-burn scar formation could bring research and treatment regimens to the next level. The aim of this review/position paper is to create more awareness of complexity in scar formation after burn injury by describing the basic principles of complexity science and its potential for burn care professionals.

Organotypic models to investigate host–microbiome interactions are still a challenge for the field of tissue engineering. This is particularly the case for organs such as the urethra. Several cell line, animal, and tissue models are available to study Chlamydia trachomatisinfections, but none fully reflects natural infection in native human tissue. Therefore, we developed an organotypic reconstructed human urethral model (RhU) to study invasive and noninvasive strains of C. trachomatis. Primary urethra cells were used to reconstruct epithelium on a fibroblast populated collagen-fibrin hydrogel, yielding a RhU. Immunohistochemistry was used to compare RhU with native urethral tissue and to visualize the location of C. trachomatisbacteria in RhU after 10-day exposure. RhU closely resembled native urethral tissue with respect to proliferation and differentiation markers (keratins 6, 10, 13, 17, involucrin, SKALP [skin-derived antileucoproteinase], vimentin, and CD31). Exposure of RhU to noninvasive and invasive C. trachomatisstrains revealed relevant differences in infection ability because inclusions were observed (indicating active infection) in the epithelial layer after 10 days exposure only to the invasive strain. The noninvasive strain remained localized on the surface of the epithelial layer. Human primary urethral fibroblasts and keratinocytes can be used to construct RhU that closely resembles native tissue and can be used to investigate active C. trachomatisinfections. RhU provides a promising model to investigate host–microbiome interactions such as, but not limited to, the human pathogenesis of C. trachomatis.

To understand scar pathology, develop new drugs, and provide a platform for personalized medicine, physiologically relevant human scar models are required, which are characteristic of different scar pathologies. Hypertrophic scars and keloids are two types of abnormal scar resulting from unknown abnormalities in the wound healing process. While they display different clinical behavior, differentiation between the two can be difficult—which in turn means that it is difficult to develop optimal therapeutic strategies. The aim of this study was to develop in vitroreconstructed human hypertrophic and keloid scar models and compare these to normotrophic scar and normal skin models to identify distinguishing biomarkers. Keratinocytes and fibroblasts from normal skin and scar types (normotrophic, hypertrophic, keloid) were used to reconstruct skin models. All skin models showed a reconstructed differentiated epidermis on a fibroblast populated collagen–elastin matrix. Both abnormal scar types showed increased contraction, dermal thickness, and myofibroblast staining compared to normal skin and normotrophic scar. Notably, the expression of extracellular matrix associated genes showed distinguishing profiles between all scar types and normal skin (hyaluronan synthase-1, matrix-metalloprotease-3), between keloid and normal skin (collagen type IV), between normal scar and keloid (laminin α1), and between keloid and hypertrophic scar (matrix-metalloprotease-1, integrin α5). Also, inflammatory cytokine and growth factor secretion (CCL5, CXCL1, CXCL8, CCL27, IL-6, HGF) showed differential secretion between scar types. Our results strongly suggest that abnormal scars arise from different pathologies rather than simply being on different ends of the scarring spectrum. Furthermore, such normal skin and scar models together with biomarkers, which distinguish the different scar types, would provide an animal free, physiologically relevant scar diagnostic and drug testing platform for the future.

Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitrowound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and CXCL8) was observed followed by complete reepithelialization. Seven days after wounding, tissue integrity, metabolic activity, and cytokine levels had returned to the prewounded state. In conclusion, immortalized human gingiva KC and fibroblasts can be used to make physiologically relevant GE, which resemble either the healthy gingiva or a neoplastic disease model. These organotypic models will provide valuable tools to investigate oral mucosa biology and can also be used as an animal alternative for drug targeting, vaccination studies, microbial biofilm studies, and testing new therapeutics.

Salivary agglutinin (SAG), also known as gp340 or SALSA, is a glycoprotein encoded by the Deleted in Malignant Brain Tumours 1gene and is abundantly present in human saliva. SAG aggregates bacteria and viruses, thereby promoting their clearance from the oral cavity. The mucosa lining the oral cavity contains dendritic cells (DC) and Langerhans cells (LC), which express the C-type lectin receptors (CLR) DC-SIGN and Langerin, respectively. Both DC-SIGN and Langerin recognise mannose and fucose carbohydrate structures on pathogens and self-glycoproteins to regulate immunity and homeostasis. The purpose of this study was to investigate whether SAG interacts with these CLR and whether this interferes with the binding to oral pathogens. We show that whole parotid saliva and SAG, when coated to microplates, strongly interact with DC-SIGN and Langerin, probably via mannose and fucose structures. Also, primary human DC and LC bind parotid saliva and SAG via DC-SIGN and Langerin, respectively. Furthermore, SAG binding to DC-SIGN or Langerin prevented binding to the micro-organisms Candida albicansand Escherichia coliwhich express mannose and fucose-containing glycan structures. Thus, binding of saliva glycoprotein SAG to DC-SIGN and Langerin may inhibit pathogen-DC/LC interactions, and could prove to be a new immunomodulatory mechanism of SAG.

Currently, human skin equivalents (HSEs) used for in vitroassays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitrowound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin–eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely from human cell lines, provides an excellent opportunity to study in vitroskin biology and can also be used for drug targeting and testing new therapeutics, and ultimately, for incorporating into skin-on-a chip in the future.

Adipose tissue–derived stromal cells augment wound healing and skin regeneration. It is unknown whether and how they can also influence dermal scarring. The authors hypothesized that adipose tissue–derived stromal cells inhibit adverse differentiation of dermal fibroblasts induced by the pivotal factor in scarring, namely, transforming growth factor (TGF)-.

Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell–cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006–9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation factors. Our findings have implications for the choice of cell type (ASC or dermal fibroblast) to be used in regenerative medicine strategies and indicate the importance of taking into account interactions with the wound bed when developing advanced therapies for difficult-to-close cutaneous wounds.

New method to develop a human skin‐equivalent containing LC‐like cells in vitro, under conditions that mimic the natural tissue composition. In this report, the construction of a functional, immunocompetent, full‐thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ‐3 cell line (MUTZ‐LC) and a fibroblast‐populated dermal compartment. The CD1a+Langerin+HLA‐DR+MUTZ‐LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO4and resorcinol resulted in LC migration out of the epidermis toward the fibroblast‐populated dermal compartment. A significant dose‐dependent up‐regulation of the DC maturation‐related CCR7 and IL‐1β transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full‐thickness SE model containing epidermal MUTZ‐LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin‐based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

Upon implantation of tissue-engineered scaffolds, hypoxia will occur until neovascularization takes place. In vivo, the temporary fibrin matrix forms a suitable matrix for this process and fibrin variants can influence the extent of neovascularization. In this study, the influence of oxygen tension and naturally occurring fibrinogen variants on adipose tissue-derived mesenchymal stem cell (ASC) expansion and differentiation were determined. ASC proliferated 1.7-fold faster in 1% oxygen and showed reduced cell aging, and their stemness was preserved. The stem cell surface marker expression was similar in 1% and 20% oxygen. The various fibrinogen coatings did not influence ASC expansion and differentiation. Differentiation of ASC toward adipogenic and osteogenic lineages was improved in 20% oxygen, whereas 1% oxygen improved chondrogenic differentiation. In conclusion, optimal oxygen concentrations vary for the intended ASC application, and fibrinogen variants, which can be used to influence neovascularization, do not alter ASC behavior. These data emphasize the importance of oxygen concentrations during stem cell growth and differentiation.

The article examines the challenges faced by Catholic educators in terms of incorporating social media into their teaching environments. Catholic educators have more considerations than traditional educators when it comes to using social media, primary of which is the use of these channels as instruments of evangelization. The expected role of Catholic educators is to facilitate the proper use of social media in learning and to cooperate with parents to develop policies in using social media.

This in vitro study describes a novel cell culture, transport, and transfer protocol that may be highly suitable for delivering cultured proliferating keratinocytes and melanocytes to large open skin wounds (e.g., burns). We have taken into account previous limitations identified using other keratinocyte transfer techniques, such as regulatory issues, stability of keratinocytes during transport (single cell suspensions undergo terminal differentiation), ease of handling during application, and the degree of epidermal blistering resulting after transplantation (both related to transplanting keratinocyte sheets). Large numbers of proliferating epidermal cells (EC) (keratinocytes and melanocytes) were generated within 10–14 days and seeded onto a three-dimensional matrix composed of elastin and collagen types I, III, and V (Matriderm®), which enabled easy and stable transport of the EC for up to 24 h under ambient conditions. All culture conditions were in accordance with the regulations set by the Dutch Central Committee on Research Involving Human Subjects (CCMO). As an in vitro model system for clinical in vivo transfer, the EC were then transferred from Matriderm onto human acellular dermis during a period of 3 days. After transfer the EC maintained the ability to regenerate into a fully differentiated epidermis containing melanocytes on the human dermis. Proliferating keratinocytes were located in the basal layer and keratin-10 expression was located in differentiating suprabasal layers similar to that found in human epidermis. No blistering was observed (separation of the epidermis from the basement membrane). Keratin-6 expression was strongly upregulated in the regenerating epidermis similar to normal wound healing. In summary, we show that EC-Matriderm contains viable, metabolically active keratinocytes and melanocytes cultured in a manner that permits easy transportation and contains epidermal cells with the potential to form a pigmented reconstructed epidermis. This in vitro study has produced a robust protocol that is ready for clinical studies in the future.

This study evaluated the impact of a curriculum called the Early Literacy Skills Builder on the language and early literacy skills of students with significant developmental disabilities. Students in the control group received the ongoing sight word and picture instruction prescribed by their individualized education programs. Results indicate statistically significant interaction effects for the treatment group for two research team-designed measures of early literacy (the Nonverbal Literacy Assessment and a pretest/posttest for the experimental curriculum). Significant interaction effects were also found for two standardized measures (Peabody Picture Vocabulary Test III and Memory for Sentences of the Woodcock Language Proficiency Battery). Implications and future research needs are provided.

Ideally tissue-engineered products should maintain the characteristics of the original tissue. For example, skin represents orthokeratinized epithelium and oral gingiva represents parakeratinized epithelium. The aim of this study was to develop an autologous full-thickness gingiva substitute suitable for clinical applications and to compare it with our autologous full-thickness skin substitute that is routinely used for healing chronic wounds. Autologous full-thickness skin and gingiva substitutes were constructed under identical culture conditions from 3-mm punch biopsies isolated from the upper leg or gingiva tissue, respectively. Both consisted of reconstructed epithelia on acellular dermis repopulated with fibroblasts. To compare the characteristics of the original and reconstructed tissue, differential morphological observations and expression of differentiation markers (keratins 6, 10, and 17 and stratum corneum precursors involucrin, loricrin, and SKALP) were determined. Skin and gingiva substitutes were transplanted onto therapy-resistant leg ulcers or tooth extraction sites in order to determine their effects on wound healing. The tissue-engineered constructs maintained many of the differential histological and immunohistochemical characteristics of the original tissues from which they were derived. The skin substitute was orthokeratinized, and the gingiva substitute was parakeratinized. Transplantation of skin (n = 19) and gingiva substitutes (n = 3) resulted in accelerated wound healing with no adverse effects. As identical culture systems were used to generate both the skin and gingiva substitutes, the differences observed in tissue (immuno)histology can be attributed to intrinsic properties of the tissues rather than to environmental factors (e.g., air or saliva). This study emphasizes the importance of closely matching donor sites with the area to be transplanted. Our results represent a large step forward in the area of clinical applications in oral tissue engineering, which have until now greatly lagged behind skin tissue engineering.

In human skin, two main populations of dendritic cells (DC) can be discriminated: dermal DC (DDC) and epidermal Langerhans cells (LC). Although extensively studied, most of the knowledge about DDC and LC phenotype and function is obtained from studying DDC and LC cultured in vitro or DDC and LC migrated from skin explants. These studies have left the exact relationship between steady‐state human LC and DDC unclear: in particular, whether CD1a+DDC represent migrated LC or whether they constitute a separate subset. To gain further insight in the kinship between skin‐resident CD1a+DDC and LC, we analyzed CD1a+DDC and LC, isolated from steady‐state skin samples, by high‐density microarray analysis. Results show that the CD1a+DDC specifically express markers associated with DDC phenotype, such as the macrophage mannose receptor, DC‐specific ICAM‐grabbing nonintegrin, the scavenger receptor CD36, coagulation factor XIIIa, and chemokine receptor CCR5, whereas LC specifically express Langerin, membrane ATPase (CD39), and CCR6, all hallmarks of the LC lineage. In addition, under steady‐state conditions, both DC subsets display a strikingly different activation status, indicative of distinct functional properties. CD1a+DDC exhibit a more activated, proinflammatory, migratory, and T cell‐stimulatory profile, as compared with LC, whereas LC mainly express molecules involved in cell adhesion and DC retention in the epidermis. In conclusion, transcriptional profiling is consistent with the notion that CD1a+DDC and LC represent two distinct DC subsets but also that under steady‐state conditions, CD1a+DDC and epidermal LC represent opposites of the DC activation spectrum.

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.

SPRR genes (formerly SPR) encode a novel class of polypeptides (small proline rich proteins) that are strongly induced during differentiation of human epidermal keratinocytes in vitro and in vivo. Recently we found that the N- and C-terminal domains of these proteins show strong sequence homology to loricrin and involucrin, suggesting that SPRR proteins constitute a new class of cornified envelope precursor proteins. Here we show that SPRR proteins are encoded by closely related members of a gene family, consisting of two genes for SPRR1, approximately seven genes for SPRR2, and a single gene for SPRR3. All SPRR genes are closely linked within a 300-kb DNA segment on human chromosome 1 band q21-q22, a region where the related loricrin and involucrin genes have also been mapped. The most characteristic feature of the SPRR gene family resides in the structure of the central segments of the encoded polypeptides that are built up from tandemly repeated units of either eight (SPRR1 and SPRR3) or nine (SPRR2) amino acids with the general consensus *K*PEP**. Sequencing data of the different members, together with their clustered chromosomal organization, strongly suggest that this gene family has evolved from a single progenitor gene by multiple intra- and intergenic duplications. Analysis of the different SPRR subfamilies reveals a gene-specific bias to either intra- or intergenic duplication. We propose that a process of homogenization has acted on the different members of one subfamily, whereas the different subfamilies appear to have diverged from each other, at the levels of both protein structure and gene regulation. Copyright 1993, 1999 Academic Press

Abstract The limited life-span and irregularities in epidermal differentiation and barrier function that have restricted the utility of presently available skin culture models for pharmacological and toxicological studies indicate that further modifications of culture conditions are required for optimization of these models. In the present study epidermis reconstructed on de-epidermized dermis was used to investigate the effects of temperature and epidermal growth factor (EGF) on epidermal differentiation and lipogenesis. When cultured at 37° C, keratinocytes formed a well-differentiated epidermis whether EGF was present or not. However, the thickness of the epidermis, particularly of the stratum corneum, was higher in the presence of EGF. Both the differentiation-specific protein markers (keratins 1 and 10, involucrin and transglutaminase) and lipid markers (ceramides) were synthesized. EGF-induced increases in triglyceride content caused accumulation of lipid droplets within the stratum corneum which is indicative of a hyperproliferative effect of EGF. In the absence of EGF, a well-differentiated epidermis was generated at 33° C with a morphology showing a higher resemblance to native epidermis than cultures grown at 37° C. The stratum corneum was less compact and with practically no lipid droplets, irregularly shaped keratohyalin granules were abundant in the stratum granulosum, lamellar body extrusion was improved and the number of stratum corneum layers was reduced to normal levels. However, EGF supplementation had a deleterious effect on epidermal morphogenesis and differentiation of cultures grown at 33° C. The epidermis lacked a stratum granulosum and the stratum corneum contained a high number of nuclear remnants. The synthesis of the early specific protein differentiation markers (keratins 1 and 10) was suppressed on both the protein and mRNA levels without significant interference with the synthesis of late differentiation lipid markers, such as ceramides. From this observation it can be concluded that the synthesis of keratins associated with terminal differentiation is profoundly affected by the presence of EGF and is sensitive to temperature and that of ceramides is not. The finding that TGF α did not modulate the morphogenesis and synthesis of keratins 1 and 10 in cultures grown at 33° C indicates possible differences between the postreceptor binding processes of these EGF receptor ligands.:

SPRR3, a member of the SPRR family of cornified envelope precursor proteins, is expressed in oral and esophageal epithelia, where it is strictly linked to keratinocyte terminal differentiation. This gene is characterized by intragenic duplications that have created the characteristic proline-rich repeats in the coding sequence, an alternative noncoding exon, and a 200-bp polypyrimidine tract in the promoter region. Mutational analysis of the promoter region and transient transfection in normal human keratinocytes showed that in addition to the polypyrimidine tract, multiple regulatory elements are involved in differentiation-specific expression. These elements include a high-affinity Ets binding site bound by ESE-1, an AP-1 site (TRE) recognized by the Jun/Fos family of transcription factors, and an ATF/CRE bound by Jun/Fos and ATF factors. The repositioning of theSPRR3Ets binding site during evolution has a major effect on the relative contribution of this site to promoter activity.

The small proline rich protein (SPRR) genes constitute a family of conserved genes which are part of the human epidermal differentiation complex (EDC) on chromosome 1q21 and code for precursor proteins of the cornified cell envelope. The expression of these genes is strictly linked to keratinocyte terminal differentiation bothin vivoandin vitro.Here we show that cultured cell lines derived from squamous cell carcinoma (SCC) show significantly lower levels ofSPRRexpression than normal human keratinocytes. However, the residualSPRRexpression in SCC lines appears to be both gene and cell line specific. Expression ofSPRR2appears to correlate well with the residual ability of these cells to differentiate. However, the kinetics ofSPRR2expression, following treatment with calcium, an inducer of keratinocyte differentiation, are typical for each cell line and differ substantially from the ones found in normal cells. In most cell lines a rapid transient expression ofSPRR2contrasts with a slow induction leading to a high sustained level of expression in normal cells. This pattern of expression is typical forSPRR2and not observed for the otherSPRRgenes or involucrin. Our analysis indicates that the expression of various keratinocyte terminal differentiation markers, even when involved in the same biological process (cornification), can be differentially affected by carcinogenic transformation.

1. Twelve healthy subjects received high-voltage pulsed galvanic stimulation (115–475V d.c.) delivered in separate treatments of 2, 32 and 128 pulses/s for 10 min at the subject's maximum tolerable voltage while calf muscle blood flow was measured by non-invasive Whitney strain-gauge venous occlusion plethysmography. 2. The high-voltage pulsed galvanic stimulation was administered with negative polarity by an intermittent mode of 30 s on, 30 s off. Measurements of calf muscle blood flow were made during each 30 s period when the stimulus was off. The effect of one 30 s maximum isometric contraction of the calf muscles on blood flow was used as a standard for evaluating the effectiveness of high-voltage pulsed galvanic stimulation on calf muscle blood flow. 3. Significant (paired t-tests; P < 0.05) increases in calf muscle blood flow over the preceding baseline levels occurred for the isometric contraction (322%) and for frequencies of 2 pulses/s (33.5%) and 128 pulses/s (13.36%), but not for a frequency of 32 pulses at which calf muscle blood flow increased in only six of 12 subjects. The mean increases in calf muscle blood flow at 2 and 128 pulses/s represented 11.63% and 4.0%, respectively, of that resulting from the isometric contraction. 4. A clear positive correlation between voltage level and the magnitude of increase in calf muscle blood flow was demonstrated but differed for each frequency used. 5. It is concluded that high-voltage pulsed galvanic stimulation results in a measurable increase in calf muscle blood flow when it is applied at frequencies of 2 or 128 pulses/s on intermittent mode and at maximum tolerable voltages, but the magnitude of the increase in blood flow is small compared with that stimulated by a maximal isometric contraction.

Highlights the “Barn Again!” exhibition at the Calhoun County Museum and Cultural Center in Calhoun County, South Carolina. Examination of the regional style and roles of barns; List of antiques in the museum; Contact information.

Reports on the maintenance of several historic buildings by the Lexington County Museum in Lexington County, South Carolina. Demonstration of the life in the 18th and 19th centuries; Financial support given by the nonprofit group Friends of the Lexington County Museum to the museum; Contact information.

We have isolated genomic clones from several members of the UV and TPA inducible human spr2 gene-family in order to analyse the regulation of these genes at a molecular level. From one of these members, the spr2–1 gene, we have identified and sequenced the regulatory region. By using CAT fusion plasmids and a liposome mediated transfection procedure we show that the isolated promoter region contains all the cis-elements necessary for induced expression after UV irradiation or phorbolester treatment of cultured human keratinocytes. Additionally the spr2–1 promoter is shown to be regulated aswell during the normal process of keratinocyte differentiation. This makes the spr2–1 promoter sequence an ideal tool to study the molecular mechanisms by which environmental agents such as UV radiation and chemical tumor promoters interfere with normal gene expression during cell proliferation and differentiation.