Dermal-epidermal Interface Research Articles

Revealing the Therapeutic Potential of Muscle-Derived Mesenchymal Stem/Stromal Cells: An In Vitro Model for Equine Laminitis Based on Activated Neutrophils, Anoxia-Reoxygenation, and Myeloperoxidase.

Laminitis in horses is a crippling condition marked by the deterioration of the dermal-epidermal interface, leading to intense lameness and discomfort, often necessitating euthanasia. This study aimed to establish an in vitro model of laminitis using a continuous keratinocyte cell line exposed to anoxia-reoxygenation and an activated neutrophil supernatant. A significant decrease in the keratinocytes' metabolism was noted during the reoxygenation period, indicative of cellular stress. Adding muscle-derived mesenchymal stem/stromal cells during the reoxygenation demonstrated a protective effect, restoring the keratinocytes' metabolic activity. Moreover, the incubation of the keratinocytes with either an activated neutrophil supernatant or myeloperoxidase alone induced increased keratinocyte myeloperoxidase activity, which was modulated by stem cells. These findings underscore the potential of muscle-derived mesenchymal stem/stromal cells in mitigating inflammation and restoring keratinocyte metabolism, offering insights for future cell therapy research in laminitis treatment.

Artificial SA-I and RA-I afferents for tactile sensing of ridges and gratings.

For robot touch to reach the capabilities of human touch, artificial tactile sensors may require transduction principles like those of natural tactile afferents. Here we propose that a biomimetic tactile sensor (the TacTip) could provide suitable artificial analogues of the tactile skin dynamics, afferent responses and population encoding. Our three-dimensionally printed sensor skin is based on the physiology of the dermal-epidermal interface with an underlying mesh of biomimetic intermediate ridges and dermal papillae, comprising inner pins tipped with markers. Slowly adapting SA-I activity is modelled by marker displacements and rapidly adapting RA-I activity by marker speeds. We test the biological plausibility of these artificial population codes with three classic experiments used for natural touch: (1a) responses to normal pressure to test adaptation of single afferents and spatial modulation across the population; (1b) responses to bars, edges and gratings to compare with measurements from monkey primary afferents; and (2) discrimination of grating orientation to compare with human perceptual performance. Our results show a match between artificial and natural touch at single afferent, population and perceptual levels. As expected, natural skin is more sensitive, which raises a challenge to fabricate a biomimetic fingertip that demonstrates human sensitivity using the transduction principles of human touch.

Effects of Juglone on Neutrophil Degranulation and Myeloperoxidase Activity Related to Equine Laminitis.

Experimental laminitis, characterized by a failure of the dermal–epidermal interface of the foot, can be induced in horses by the oral administration of a black walnut extract (BWE). In the early phase of this severe and painful disease, an activation of neutrophil occurs, with the release of myeloperoxidase (MPO), a pro-oxidant enzyme of neutrophils, in plasma, skin, and laminar tissue. Juglone, a naphthoquinone derivative endowed with redox properties, is found in walnuts and has been incriminated in this neutrophil activation. We report for the first time the inhibitory activity of juglone on the degranulation of neutrophils induced by cytochalasin B and formyl-methionyl-leucyl-phenylalanine as monitored by the MPO release (>90% inhibition for 25 and 50 μM). Moreover, it also acts on the peroxidase activity of MPO by interacting with the intermediate “π cation radical,” as evidenced by the classical and specific immunological extraction followed by enzymatic detection (SIEFED) assays. These results are confirmed by a docking study showing the perfect positioning of juglone in the MPO enzyme active site and its interaction with one of the amino acids (Arg-239) of MPO apoprotein. By chemiluminescence and electron paramagnetic resonance techniques, we demonstrated that juglone inhibited reactive oxygen species (ROS) and superoxide anion free radical produced from phorbol myristate acetate (PMA)-activated polymorphonuclear neutrophils (PMNs). These results indicate that juglone is not the trigger for equine laminitis, at least if we focus on the modulation of neutrophil activation.

Angiomatoid Spitz nevus with surrounding pagetoid melanocytic proliferation on the sole of the foot: An unusual case report with immunohistochemical studies for angiogenic factors

Angiomatoid Spitz nevus (ASN) is a rare histological variant of Spitz nevus (SN) that is characterized by prominent blood vessel proliferation around the intradermal melanocytes of SN. In contrast, SN may have pagetoid components, which are characterized by epidermal proliferation of single melanocytes. However, cases of ASN with predominant pagetoid melanocytic proliferation in the epidermis have not been reported. Here, we report a case of ASN with surrounding pagetoid melanocytic proliferation without formation of tumor nests in the epidermis in the plantar region. A 12-year-old girl presented with a bright red nodule surrounded by a brown macule on the sole of her right foot. Histologically, the nodule showed tumor nests in the dermis, composed of spindle or epithelioid melanocytes containing abundant cytoplasm and large nuclei. Around the nests, numerous blood vessels were seen. In the overlying epidermis of the nodule, numerous eosinophilic Kamino bodies were found along the dermal-epidermal interface. In the macule, proliferation of oval melanocytes was present as single-cell units in the epidermis. Theses melanocytes had abundant cytoplasm with large nuclei, which were larger than those of the surrounding keratinocytes. From these findings, a diagnosis of ASN with surrounding pagetoid melanocytic proliferation was made. Vascular endothelial growth factor and fibroblast growth factor 2 were strongly expressed in the melanocytes as well as in the endothelial cells in our case. Therefore, angiogenic factors produced by the melanocytes of SN might have played important roles in the surrounding angiogenesis of this case.

Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105+K14+ progenitor cells

The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning electron microscopy, immunocytochemistry, and CD105+K14+ cell trilineage plasticity. Cells from normal tissue had typical progenitor cell characteristics. Those from inflamed tissue had organelles and morphology consistent with catabolic activities including lysosomes, irregular rough endoplasmic reticulum, and fewer vacuoles and early endosomes than those from normal tissue. Cryopreserved tissue cells appeared apoptotic with an irregular cell membrane covered by cytoplasmic protrusions closely associated with endocytic and exocytic vesicles, chromatin aggregated on the nuclear envelop, abundant, poorly organized rough endoplasmic reticulum, and plentiful lysosomes. Cells that were CD105+K14+ were distinguishable from heterogenous cells by infrequent microvilli on the cell surface, sparse endosomes and vesicles, and desmosomes between cells. Cells expressed ectodermal (K15) and mesodermal (CD105) proteins in 2D and 3D cultures. Inflamed and cryopreserved tissue isolates attached poorly to tissue scaffold while normal tissue cells attached well, but only CD105+K14+ cells produced extracellular matrix after 4 d. The CD105+K14+ cells exhibited osteoblastic, adipocytic, and neurocytic differentiation. Ultrastructural information provided by this study contributes to understanding of equine hoof progenitor cells to predict their potential contributions to tissue maintenance, healing, and damage as well post-implantation behavior.

A multiscale hybrid mathematical model of epidermal-dermal interactions during skin wound healing.

Following injury, skin activates a complex wound healing programme. While cellular and signalling mechanisms of wound repair have been extensively studied, the principles of epidermal-dermal interactions and their effects on wound healing outcomes are only partially understood. To gain new insight into the effects of epidermal-dermal interactions, we developed a multiscale, hybrid mathematical model of skin wound healing. The model takes into consideration interactions between epidermis and dermis across the basement membrane via diffusible signals, defined as activator and inhibitor. Simulations revealed that epidermal-dermal interactions are critical for proper extracellular matrix deposition in the dermis, suggesting these signals may influence how wound scars form. Our model makes several theoretical predictions. First, basal levels of epidermal activator and inhibitor help to maintain dermis in a steady state, whereas their absence results in a raised, scar-like dermal phenotype. Second, wound-triggered increase in activator and inhibitor production by basal epidermal cells, coupled with fast re-epithelialization kinetics, reduces dermal scar size. Third, high-density fibrin clot leads to a raised, hypertrophic scar phenotype, whereas low-density fibrin clot leads to a hypotrophic phenotype. Fourth, shallow wounds, compared to deep wounds, result in overall reduced scarring. Taken together, our model predicts the important role of signalling across dermal-epidermal interface and the effect of fibrin clot density and wound geometry on scar formation. This hybrid modelling approach may be also applicable to other complex tissue systems, enabling the simulation of dynamic processes, otherwise computationally prohibitive with fully discrete models due to a large number of variables.

Dermal-Epidermal Cross-Talk: Differential Interactions With Microvascular Endothelial Cells.

The biological importance of circulatory blood supply and angiogenesis for hair growth is now well recognized, but the their regulatory mechanisms require more mechanistic investigation. In vitro cocultures and tricultures can be successfully employed to greatly improve our knowledge on paracrine crosstalk between cell types that populate the dermal-epidermal interface and cutaneous vasculature. Here we report that human dermal fibroblasts (NHDF) promote viability and proliferation of microvascular endothelial cells (HMVEC), while HMVEC are not mitogenic for NHDF. In triculture setup, conditioned media (CM) obtained by cocultures (HMVEC/NHDF or HMVEC/follicle fibroblasts) differently modulate growth and proliferation of keratinocytes and alter the expression of metabolic and pro-inflammatory markers. In conclusion, tricultures were successfully employed to characterize in vitro dermal-epithelial and endothelial interactions and could integrate ex vivo and in vivo approaches by the use of high-throughput and standardized protocols in controlled conditions. J. Cell. Physiol. 232: 897-903, 2017. © 2016 Wiley Periodicals, Inc.

Oral lichen planus with antibodies to desmogleins 1 and 3

Lichen planus (LP) is a chronic inflammatory disorder involving the skin or mucous membranes. Previous studies have demonstrated that some LP patients showed positive enzyme-linked immunosorbent assay (ELISA) for desmoglein (DSG) antibodies. We report a case with intractable painful oral lesions. ELISA indices for DSG1 and 3 antibodies were increased by 49 and 36, respectively. Histopathological analysis revealed irregular acanthosis and band-like infiltration of lymphocytes at the dermal-epidermal interface. Direct immunofluorescence revealed negative deposits of immunoglobulin G and C3 in intracellular spaces of the epidermis. Indirect immunofluorescence of normal skin also did not detect any antibodies. Consequently, we made a final diagnosis of oral LP. The previous two LP cases with positive ELISA for DSG antibodies and our case manifested the erosive form, the most advanced oral LP. Therefore, it is a possibility that severe damage of keratinocytes may induce generation of DSG antibodies. However, negative results of immunofluorescence and no relation between disease severity and titers of antibodies make the possibility unlikely. We should measure titers of DSG antibodies in LP patients and accumulate data to establish a valid conclusion.

The Impact of Skin Type and Area on Skin Rejection in Limb Transplantation

Atypical manifestation of skin rejection observed in a series of hand transplant recipients suggests that appearance and mechanisms of rejection may vary and depend on skin type and location. We herein characterize skin rejection and inflammation at 3 defined areas of a rat hind-limb allograft. Allograft skin was harvested at the thigh, dorsum and planta pedis on postoperative day 5 and analyzed for histopathology, T-cell composition and expression of cytokines associated with skin inflammation. Isografts and naive animals served as controls. Histology of the allograft skin collected on day 5 showed a diffuse cell infiltrate in the dermis and at the dermal-epidermal interface in the thigh and dorsum, whereas in the planta pedis it was mainly located perivascularly in the dermis. At all areas, epidermal involvement such as apoptotic keratinocytes and infiltrating lymphocytes were observed. Isograft skin showed only very mild or no signs of inflammation at all 3 locations. No significant difference was obse...

Muscle Mitochondrial Dysfunction in Horses Affected by Acute Laminitis

Laminitis is a common and debilitating disease affecting horses and ponies. It often leads to the demise of the animal. Energy deficiency is suspected to entrain the disruption of the hemidesmosomes leading to the failure of the dermal-epidermal interface. The aim of this study was to measure the muscle mitochondrial function by high resolution respirometry. Muscle micro-biopsies were obtained from 11 horses affected by acute metabolic laminitis, 6 horses affected by acute laminitis resulting from a systemic inflammation response syndrome and 28 healthy horses distributed in 2 control groups: 17 horses with a body condition score [BSC, ranging from 0 (emaciated) to 5 (obese)] of 2 to 3 and 11 horses with a BSC of 4 to 5. During the acute phase of laminitis, a significant reduction of the muscle mitochondrial respiration was observed. The muscle mitochondrial dysfunction occurred independently of the etiology (metabolic disorder or systemic inflammation) leading to laminitis. The reduction of the oxidative phosphorylation and of the maximal respiratory capacity (after uncoupling) may induce depletion of the cell’s ATP content. If the same mitochondrial alteration occurs in the foot lamina, mitochondria targeting should be considered for the future, not only to better understand the physiopathology of the disease but also to maintain and to support the mitochondrial function before reaching the « mitochondrial dysfunction threshold » that may lead to the failure of the dermal-epidermal interface.

The role of activated neutrophils in the early stage of equine laminitis

Despite ongoing research and a widening range of treatment options, laminitis remains a severely damaging condition with poorly understood pathophysiology. Results obtained from cytokine regulation studies during the last decade have highlighted the inflammatory nature of laminitis. This review will describe the role of systemic activation and local infiltration of neutrophils in laminar tissues in the induction of laminitis. Particular emphasis is placed on the role of neutrophil activation in subsequent vascular dysfunction and oxidative and proteolysis imbalances that are pathways previously implicated in laminitis. Neutrophils, by the way of their interdependent relationship with endothelial cells and keratinocytes, dramatically increase the inflammatory response culminating in the failure of the laminar dermal-epidermal interface.

Temporal Changes in Basement Membrane Proteolysis and Protease Expression During Laminitis Development

s Vol 30, No 2 (2010) 101 (range)] and terminal elimination half-life was 8.5 (7.113.3) h. After PO gabapentin Cmax was 3.75 (1.9-5.8) mg/ml, and terminal elimination half-life was 7.7 (6.711.9) h. The fractional absorption was 16.2 2.8%. There was no significant effect on cardiovascular parameters. Sedation scores were increased during the first hour after IV infusion (P < 0.05). DISCUSSION There were no major adverse effects noted after single dose IV or PO gabapentin. Bioavailability was only16% following PO administration. CLINICAL RELEVANCE Inability to control pain and subsequent euthanasia is the most common cause of treatment failure in laminitis cases. Gabapentin may be a useful analgesic agent in the treatment of laminitis. CONCLUSION Gabapentin was safe and well tolerated. Further research is required to establish a dosage that will provide effective analgesia in horses with laminitis and to determine if combinations with other agents create an enhanced effect. 1. Jones E, et al. Neuropathic changes in equine laminitis pain. Pain 2007;132(3):321–331. The Laminitis Discovery Database Hannah Galantino-Homer, Rebecca Carter, Susan Megee, Julie Engiles, James Orsini, and Christopher Pollitt, Laminitis Institute, Department of Clinical Studies/New Bolton Center and Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, Australian Equine Laminitis Research Unit, School of Veterinary Science, The University of Queensland, St. Lucia, Queensland, Australia TAKE HOME MESSAGE The Laminitis Discovery Database is a collaborative tissue bank and information resource. INTRODUCTION Establishment of a centralized laminitis tissue bank was identified as a top priority at the first AAEP Foundation Equine Laminitis Research Workshop. Initiated in 2008, the purpose of the Laminitis Discovery Database is to collect and provide well-documented tissue samples from non-laminitic and experimental and natural laminitic horses for collaborative research. MATERIALS AND METHODS Samples collected: ante-mortem serum and plasma, gross specimen images and lamellar length measurements, liquid nitrogen snap frozen, formalin-fixed/paraffin-embedded, and paraformaldehyde-fixed/OCT-embedded frozen tissue collected from five regions along the dorsal dermal-epidermal interface: skin, coronary, proximal, mid, and distal lamellar tissues, and frozen mixed cells. Tissue and horse information stored in a master catalog and freezer log. A mid-lamellar sample from each foot is submitted for histopathological evaluation. RESULTS Currently, samples have been banked from 78 horses, including 48h hyperinsulinemia model (4, plus matched controls), 24h oligofructose model (5), fetuses (6), foals (3), non-laminitic horses (32), and laminitic horses (16) including supporting limb (3), retained placenta (1), PPID (4), chronic (6), and acute (2) laminitis. Various ages (non-laminitic: 9.2 5.7 (mean S.D.) years; and laminitic: 14 10 years) and breeds are represented. DISCUSSION Major challenges of tissue banking include case recruitment, timing and technical difficulty of tissue retrieval, and proper archiving. More samples are needed for age/ breed/sex-matched case-control studies. CLINICAL RELEVANCE Understanding laminitis pathophysiology is essential for the rational development of an evidence-based approach to this disease. CONCLUSION Banked tissue from clinical cases of laminitis is needed to validate research based on experimental models of laminitis.

The Laminitis Discovery Database

s Vol 30, No 2 (2010) 101 (range)] and terminal elimination half-life was 8.5 (7.113.3) h. After PO gabapentin Cmax was 3.75 (1.9-5.8) mg/ml, and terminal elimination half-life was 7.7 (6.711.9) h. The fractional absorption was 16.2 2.8%. There was no significant effect on cardiovascular parameters. Sedation scores were increased during the first hour after IV infusion (P < 0.05). DISCUSSION There were no major adverse effects noted after single dose IV or PO gabapentin. Bioavailability was only16% following PO administration. CLINICAL RELEVANCE Inability to control pain and subsequent euthanasia is the most common cause of treatment failure in laminitis cases. Gabapentin may be a useful analgesic agent in the treatment of laminitis. CONCLUSION Gabapentin was safe and well tolerated. Further research is required to establish a dosage that will provide effective analgesia in horses with laminitis and to determine if combinations with other agents create an enhanced effect. 1. Jones E, et al. Neuropathic changes in equine laminitis pain. Pain 2007;132(3):321–331. The Laminitis Discovery Database Hannah Galantino-Homer, Rebecca Carter, Susan Megee, Julie Engiles, James Orsini, and Christopher Pollitt, Laminitis Institute, Department of Clinical Studies/New Bolton Center and Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, Australian Equine Laminitis Research Unit, School of Veterinary Science, The University of Queensland, St. Lucia, Queensland, Australia TAKE HOME MESSAGE The Laminitis Discovery Database is a collaborative tissue bank and information resource. INTRODUCTION Establishment of a centralized laminitis tissue bank was identified as a top priority at the first AAEP Foundation Equine Laminitis Research Workshop. Initiated in 2008, the purpose of the Laminitis Discovery Database is to collect and provide well-documented tissue samples from non-laminitic and experimental and natural laminitic horses for collaborative research. MATERIALS AND METHODS Samples collected: ante-mortem serum and plasma, gross specimen images and lamellar length measurements, liquid nitrogen snap frozen, formalin-fixed/paraffin-embedded, and paraformaldehyde-fixed/OCT-embedded frozen tissue collected from five regions along the dorsal dermal-epidermal interface: skin, coronary, proximal, mid, and distal lamellar tissues, and frozen mixed cells. Tissue and horse information stored in a master catalog and freezer log. A mid-lamellar sample from each foot is submitted for histopathological evaluation. RESULTS Currently, samples have been banked from 78 horses, including 48h hyperinsulinemia model (4, plus matched controls), 24h oligofructose model (5), fetuses (6), foals (3), non-laminitic horses (32), and laminitic horses (16) including supporting limb (3), retained placenta (1), PPID (4), chronic (6), and acute (2) laminitis. Various ages (non-laminitic: 9.2 5.7 (mean S.D.) years; and laminitic: 14 10 years) and breeds are represented. DISCUSSION Major challenges of tissue banking include case recruitment, timing and technical difficulty of tissue retrieval, and proper archiving. More samples are needed for age/ breed/sex-matched case-control studies. CLINICAL RELEVANCE Understanding laminitis pathophysiology is essential for the rational development of an evidence-based approach to this disease. CONCLUSION Banked tissue from clinical cases of laminitis is needed to validate research based on experimental models of laminitis.

Subcutaneous Panniculitis-Like T-Cell Lymphoma With Overlapping Clinicopathologic Features of Lupus Erythematosus: Coexistence of 2 Entities?

We observed 5 patients with subcutaneous panniculitis-like T-cell lymphoma (SPTCL) who were unusual, in that they also exhibited features of lupus erythematosus (LE). This observation is in keeping with a recent study that reported an increased rate of autoimmune disease, including systemic lupus erythematosus (SLE), among patients with SPTCL. In all cases, attributes indicating SPTCL included an infiltrate of lymphocytes with pleomorphic nuclei involving subcutaneous lobules exhibiting a cytotoxic T-cell (CD3/CD8/betaF1) immunophenotype. Additionally, a high proliferation rate and a monoclonal T-cell receptor-gamma gene rearrangement were observed in most cases. The manifestations of LE in these patients included a spectrum of clinical and histopathological abnormalities. Clinical manifestations of LE included, in some patients, morphologic evidence of lupus erythematosus panniculitis (LEP) with subcutaneous nodules that healed with lipoatrophy on the face. In addition, all the patients exhibited serologic and/or extracutaneous end-organ abnormalities seen in patients with SLE, with 2 patients having sufficient findings to meet American College of Rheumatology criteria for SLE. Histopathological evidence of LE included vacuolar change at the dermal-epidermal interface in 3 patients, 2 of whom also showed interstitial deposition of mucin in the reticular dermis. One of these patients also had findings of LEP in the subcutaneous lobules with clusters of CD20 B cells partially arranged within germinal centers. In 2 patients in which neither the epidermis nor dermis was available for review, histopathological features of LE included, in one patient, a few small clusters of CD123 plasmacytoid dendritic cells within the adipose tissue and, in the other patient, a positive direct immunofluorescence test (lupus band) on clinically uninvolved and lesional skin. Our study shows that some patients show overlap between SPTCL and LE. We suspect that these patients may suffer from both diseases concomitantly. Furthermore, patients with LE, particularly LEP, should be monitored for evolution into SPTCL with biopsy of any subcutaneous lesion that is not typical of LEP. Additionally, screening for cutaneous LE and SLE could be considered in patients with SPTCL.

Conversion of the Nipple to Hair-Bearing Epithelia by Lowering Bone Morphogenetic Protein Pathway Activity at the Dermal-Epidermal Interface

Epithelial appendages, such as mammary glands and hair, arise as a result of epithelial-mesenchymal interactions. Bone morphogenetic proteins (BMPs) are important for hair follicle morphogenesis and cycling and are known to regulate a wide variety of developmental processes. For example, overexpression of BMPs inhibits hair follicle formation. We hypothesized that the down-regulation of the BMP signaling pathway in the basal epidermis expands regions that are competent to form hair follicles and could alter the fate of the epithelium in the mouse nipple to a hair-covered epidermal phenotype. To test our hypothesis, we used a transgenic mouse model in which keratin 14 (KRT14) promoter-mediated overexpression of Noggin, a BMP antagonist, modulates BMP activity. We observed the conversion of nipple epithelium into pilosebaceous units. During normal mammary gland organogenesis, BMPs are likely used by the nipple epithelium to suppress keratinocyte differentiation, thus preventing the formation of pilosebaceous units. In this report, we characterize the morphology and processes that influence the development of hairs within the nipple of the KRT14-Noggin mouse. We demonstrate that Noggin acts, in part, by reducing the BMP signal in the epithelium. Reduction of the BMP signal in turn leads to a reduction in the levels of parathyroid hormone-related protein. We propose that during evolution of the nipple, the BMP pathway was co-opted to suppress hair follicle formation and create a more functional milk delivery apparatus.

Equine laminitis: Membrane type matrix metalloproteinase‐1 (MMP‐14) is involved in acute phase onset

Enzymatic separation at the hoof lamellar dermal-epidermal interface may play a role in the development of laminitis and characterising and locating matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMPs or TIMPs) in lamellar tissues may further understanding of pathogenesis. To clone and sequence the cDNA encoding lamellar MMP-14 and TIMP-2, and quantify their transcription in normal and laminitic tissue; and to develop antibody to locate MMP-14 in lamellar tissues. Tissue samples were obtained from an oligofructose induced model of laminitis. Total RNA was isolated, amplified by RT-PCR, cloned into a vector and sequenced. Real-time PCR was used to quantify MMP-14 and TIMP-2 expression. Rabbit anti-equine MMP-14 antibody was developed to analyse MMP-14 proteins from hoof tissues. Immunohistochemistry detected MMP-14 in the cytoplasm of normal lamellar basal and parabasal cells in close proximity to the lamellar basement membrane. In laminitis affected tissue MMP-14 immunostaining was depleted in lamellar basal cells. Quantitative real-time PCR showed MMP-14 and TIMP-2 expression significantly (P<0.05) elevated and lowered respectively in laminitis affected tissues. MMP-14, located in the cytoplasm of normal lamellar basal cells, disappears during laminitis development. The pathology of laminitis is associated with increased and lowered transcription of MMP-14 and TIMP-2, respectively. Enzymes have a role in laminitis pathology and inhibition of their activity may prevent laminitis.

Evaluation of architectural changes along the proximal to distal regions of the dorsal laminar interface in the equine hoof

To describe architectural changes along the dorsal laminar interface of the equine foot. 6 macroscopically normal forefeet obtained from 6 equine cadavers. Histologic sections of 8 evenly spaced, proximal to distal, samples of the dorsal laminar interface were photographed, digitized, and examined for differences in architecture. Laminar depth; secondary laminar density; number and consistency of bifurcations occurring within the secondary laminae, and areas composed of primary dermal lamina, primary epidermal lamina, and secondary laminar interface were recorded. Data were examined to test for differences in architecture associated with the proximal to distal positioning of the sample. With exception of the areas of the primary epidermal and primary dermal lamina, all measured variables were significantly different between the proximal and distal regions of the dorsal laminar interface. Changes included increases in laminar depth and the secondary laminar density. Bifurcation of secondary laminae principally occurred proximally and had an increased depth of bifurcation distally. The secondary laminar dermal-epidermal interface had a 109% increase in area between the most proximal and distal sections. Results of this study indicate that the interface normally contributes a substantial volume of dermal components to the internal surface of the wall. These data also indicate that 2 distinct mechanisms (i.e., bifurcation of secondary laminae and an increase in the length of secondary laminae) contribute to changes in the architecture of the laminar interface, which allows for the hypothesis that the normal laminar interface is capable of responding to mechanical load.

The influence of microtextured basal lamina analog topography on keratinocyte function and epidermal organization

The rational design of future bioengineered skin substitutes requires an understanding of the mechanisms by which the three-dimensional microarchitecture of tissue scaffolds modulates keratinocyte function. Microtextured basal lamina analogs were developed to investigate the relationship between the characteristic topography at the dermal-epidermal interface of native skin and keratinocyte function. Microfabrication techniques were used to create master patterns, negative replicates, and collagen membranes with ridges and channels of length scales (e.g., grooves of 50-200 microm in depth and width) similar to the invaginations found in basal lamina at the dermal-epidermal junction of native skin. Keratinocytes were seeded on the surfaces of basal lamina analogs, and histological analyses were performed after 7 days of tissue culture at the air-liquid interface. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microtextured membranes. Morphometric analyses of immunostained skin equivalents suggest that keratinocyte stratification and differentiation increases as channel depth increases and channel width decreases. This trend was most pronounced in channels with the highest depth-to-width ratios (i.e., 200 microm deep, 50 microm wide). It is anticipated that the findings from these studies will elucidate design parameters to enhance the performance of future bioengineered skin substitutes.

Glutamine and glutamine synthetase immunoreactivities in Schwann cells of the rat glabrous skin

We have shown recently that glial cells of the peripheral nervous system contain substrates and enzymes related to the glutamine cycle, e.g. glutamine, glutamine synthetase, and glutamate dehydrogenase (Miller et al., Brain Res 2002: 945: 202–11). The present study used immunohistochemistry to determine the cellular distribution of glutamine and glutamine synthetase in the rat glabrous skin. Normal adult Sprague–Dawley rats were used for these studies. Glutamine and glutamine synthetase were localized with both immunoperoxidase and immunofluoresent techniques. Glutamine and glutamine synthetase immunoreactivities were abundant in Schwann cells of large dermal nerve bundles. An enrichment of glutamine and glutamine synthetase immunoreactivies occurred in terminal and near‐terminal Schwann cells in dermal papilla and at the dermal–epidermal interface. We hypothesize that the peripheral glial glutamine cycle is important for supplying neuronal energy demands in dermal nerve fibers. Furthermore, the peripheral glial glutamine cycle in terminal Schwann cells most likely produces glutamine for sensory terminal uptake. This glutamine would be converted to glutamate for release from sensory afferents. Glutamate released from sensory afferents may be important for the development of hyperalgesia and allodynia. Terminal Schwann cells, therefore, are potential targets for modulating sensory afferent sensitivity during acute and chronic pain.Supported by NIH AR47410 (KEM).

Immunopathology of vesicular cutaneous lupus erythematosus in the rough collie and Shetland sheepdog: a canine homologue of subacute cutaneous lupus erythematosus in humans.

Clinical and histological features of an erosive disease in the rough collie and Shetland sheepdog are most consistent with a vesicular variant of cutaneous lupus erythematosus (VCLE). This paper reports the immunopathological findings of canine VCLE using samples from 17 affected dogs. Lesional skin sections were stained with monoclonal antibodies specific for CD3 (11 dogs) or a panel of monoclonal antibodies specific for leukocyte antigens (two dogs). Apoptotic cells were detected using the TUNEL method in 12 cases. Direct (14 dogs) and indirect immunofluorescence tests (five dogs) were also performed. Circulating antibodies to extractable nuclear antigens (ENA) were surveyed in 11 dogs by immunoblotting and ELISA. The predominant cells at the dermal-epidermal interface were identified as CD3(+) T lymphocytes expressing CD4 or CD8 and CD1(+) dendritic antigen presenting cells. In 7/12 dogs (58%), apoptosis of basal keratinocyte nuclei was present. Up-regulation of MHCII and ICAM-1 was observed on basal keratinocytes from the two dogs examined. Direct immunofluorescence revealed deposition of immunoglobulins bound to the cytoplasm of keratinocytes (6/14 dogs; 43%), to the dermal-epidermal junction (7/14 dogs; 50%), or to superficial dermal venules (13/14 dogs; 93%). Circulating IgG auto-antibodies targeting one or more ENA were detected in nine (82%) and eight (73%) of 11 dogs by immunoblotting and ELISA, respectively. These auto-antibodies recognized Ro/SSA and/or La/SSB in four (36%) and six (55%) of 11 dogs respectively by these two methods. Altogether, results of these studies provide evidence supporting the hypothesis that canine VCLE is an immunological homologue of subacute cutaneous lupus erythematosus in humans.