Effect of sugar metabolite methylglyoxal on equine lamellar explants: An ex vivo model of laminitis.

Laminitis is one of the most devastating diseases in equine medicine, and although several etiopathogenetic mechanisms have been proposed, few clear answers have been identified to date. Several lines of evidence point towards its underlying pathology as being metabolism-related. In the carbonyl stress pathway, sugars are converted to methylglyoxal (MG)-a highly reactive α-oxoaldehyde, mainly derived during glycolysis in eukaryotic cells from the triose phosphates: D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. One common hypothesis is that MG could be synthesized during the digestive process in horses, and excessive levels absorbed into peripheral blood could be delivered to the foot and lead to alterations in the hoof lamellar structure. In the present study, employing an ex vivo experimental design, different concentrations of MG were applied to hoof explants (HE), which were then incubated and maintained in a specific medium for 24 and 48 h. Macroscopic and histological analyses and a separation force test were performed at 24 and 48 h post-MG application. Gene expression levels of matrix metalloproteinase (MMP)-2 and -14 and tissue inhibitor of metalloproteinase (TIMP)-2 were also measured at each time point for all experimental conditions. High concentrations of MG induced macroscopic and histological changes mimicking laminitis. The separation force test revealed that hoof tissue samples incubated for 24 h in a high concentration of MG, or with lower doses but for a longer period (48 h), demonstrated significant weaknesses, and samples were easily separated. All results support that high levels of MG could induce irreversible damage in HEs, mimicking laminitis in an ex vivo model.

Angelica polysaccharide attenuates LPS-induced inflammation response of primary dairy cow claw dermal cells via NF-κB and MAPK signaling pathways.

BACKGROUND: Laminitis, an inflammation of the claw laminae, is one of the major causes of bovine lameness, which can lead to enormous economic losses and animal welfare problems in dairy farms. Angelica polysaccharide (AP) is proved to possess anti-inflammatory properties. But the role of AP on inflammatory response of the claw dermal cells has not been reported. The aim of this study was to investigate the anti-inflammatory effects of AP on lipopolysaccharide (LPS)-induced primary claw dermal cells of dairy cow and clarify the potential mechanisms. In the current research, the primary claw dermal cells were exposed to gradient concentrations of AP (10, 50, 100 µg/mL) in the presence of 10 µg/mL LPS. The levels of cytokines and nitric oxide (NO) were detected with ELISA and Griess colorimetric method. The mRNA expressions of TLR4, MyD88 and chemokines were measured with qPCR. The activation of NF-κB and MAPK signaling pathways was detected with western blotting. RESULTS: The results indicated that AP reduced the production of inflammatory mediators (TNF-α, IL-1ß, IL-6 and NO), downregulated the mRNA expression of TLR4, MyD88 and some pro-inflammatory chemokines (CCL2, CCL20, CXCL2, CXCL8, CXCL10), and suppressed the NF-κB and MAPK signaling pathways evidenced by inhibition of the phosphorylation of IκBα, p65 and ERK, JNK, p38. CONCLUSIONS: Our results demonstrated that AP may exert its anti-inflammatory effects on claw dermal cells of dairy cow by regulating the NF-κB and MAPK signaling pathways.

Equine Hoof Stem Progenitor Cells (HPC) CD29 + /Nestin + /K15 + - a Novel Dermal/epidermal Stem Cell Population With a Potential Critical Role for Laminitis Treatment.

Laminitis is a life threating, extremely painful and frequently recurrent disease of horses which affects hoof structure. It results from the disruption of blood flow to the laminae, contributing to laminitis and in severe separation of bone from the hoof capsule. Still, the pathophysiology of the disease remains unclear, mainly due to its complexity. In the light of the presented data, in the extremally difficult process of tissue structure restoration after disruption, a novel type of progenitor cells may be involved. Herein, we isolated and performed the initial characterization of stem progenitor cells isolated from the coronary corium of the equine feet (HPC). Phenotype of the cells was investigated with flow cytometry and RT-qPCR revealing the presence of nestin, CD29, and expression of progenitor cell markers including SOX2, OCT4, NANOG and K14. Morphology of HPC was investigated with light, confocal and SEM microscopes. Cultured cells were characterised by spindle shaped morphology, eccentric nuclei, elongated mitochondria, and high proliferation rate. Plasticity and multilineage differentiation potential was confirmed by specific staining and gene expression analysis. We conclude that HPC exhibit in vitro expansion and plasticity similar to mesenchymal stem cells, which can be isolated from the equine foot, and may be directly involved in the pathogenesis and recovery of laminitis. Obtained results are of importance to the field of laminitis treatment as determining the repairing cell populations could contribute to the discovery of novel therapeutic targets and agents including and cell-based therapies for affected animals.

The Trichohyalin-Like Protein Scaffoldin Is Expressed in the Multilayered Periderm during Development of Avian Beak and Egg Tooth.

Scaffoldin, an S100 fused-type protein (SFTP) with high amino acid sequence similarity to the mammalian hair follicle protein trichohyalin, has been identified in reptiles and birds, but its functions are not yet fully understood. Here, we investigated the expression pattern of scaffoldin and cornulin, a related SFTP, in the developing beaks of birds. We determined the mRNA levels of both SFTPs by reverse transcription polymerase chain reaction (RT-PCR) in the beak and other ectodermal tissues of chicken (Gallus gallus) and quail (Coturnix japonica) embryos. Immunohistochemical staining was performed to localize scaffoldin in tissues. Scaffoldin and cornulin were expressed in the beak and, at lower levels, in other embryonic tissues of both chickens and quails. Immunohistochemistry revealed scaffoldin in the peridermal compartment of the egg tooth, a transitory cornified protuberance (caruncle) on the upper beak which breaks the eggshell during hatching. Furthermore, scaffoldin marked a multilayered peridermal structure on the lower beak. The results of this study suggest that scaffoldin plays an evolutionarily conserved role in the development of the avian beak with a particular function in the morphogenesis of the egg tooth.

Concentração de minerais no tecido queratinizado podal de equinos jovens da raça Crioula em diferentes fases de crescimento / [Mineral concentrations in the keratinized tissue on the feet of young Crioulo horses at different stages of growth]

O presente estudo avaliou o perfil mineral de Cálcio (Ca), Cobre (Cu) e Zinco (Zn) no estojo córneo pré e pós-desmame e sua associação com pigmentação, gênero e idade de potros da raça Crioula criados em pastagens nativas no Rio Grande do Sul. Foram coletadas amostras do casco de potros da raça Crioula 41 dias pré e 28 dias pós-desmame. Os teores de Ca, Cu e Zn variaram no casco fetal (571,0±39,4; 14,5±7,6 e 130,0±30,5mg/kg, respectivamente; P<0,05) e no definitivo (653,0±169,2; 33,8±11,5 e 69,3±36,8mg/kg, respectivamente; P<0,05), no pré (620,0±184; 17,2±21,2 e 103,0±75,5mg/kg, respectivamente; P<0,05) e no pós-desmame (517,5±181; 0 e 79,0±41,7mg/kg, respectivamente; P<0,05). Houve associação positiva (P<0,05) com a faixa etária no pré-desmame para Ca (r=0,5) e Cu (r=0,57), e negativa para Zn (r=-0,69; P<0,05). No pós-desmame, houve associação positiva (P<0,05) para Ca (r=0,36) e Zn (r=0,64) e negativa para Cu (r=-0,39; P<0,05). Tanto a pigmentação quanto o gênero não afetaram o perfil mineral. Conclui-se que há diferenças nas concentrações de Ca, Cu e Zn na epiderme do casco de potros da raça Crioula no pré e pós-desmame, independentemente da pigmentação e do gênero, onde as concentrações de Ca e Cu aumentam com a idade, enquanto as de Zn diminuem.(AU)

The aim of this study was to evaluate the mineral profile of Ca, Cu and Zn in the hoof horny capsule at pre and post-weaning and its association with pigmentation, gender and age range of Crioulo foals raised in native pastures in RS. Samples were collected from the epidermis of the Crioulo foal's hoof at two times, 41 pre and 28 post-weaning. The contents of Ca, Cu, Zn Varied in the fetal hoof ( 571.0±139.4, 14.5±7.6 and 130.0±30.5mg/kg, respectively, P<0.05) and definitive (653.0±169.2, 33.8±11.5 and 69.3±36.8mg/kg, respectively, P<0.05), in the pre (620.0±184, 17.2±21.2 and103.0±75.5mg/kg, respectively, P<0.05) and post-weaning (517.5±181, 0.1 and 79.0±41.7mg/kg, respectively, P<0.05).There was a positive association (P<0.05) with a preweaning age for Ca (r=0.5) and Cu (r=0.57) and negative for Zn (r=-0.69, P<0.05). In the post weaning there was a positive association (P<0.05) for Ca (r=0.36) and Zn (r=0.64) and negative for Cu (r=-0.39; P<0.05). Neither pigmentation nor gender affected the mineral profile. It is concluded that there are differences in Ca, Cu, Zn concentrations in the epidermis of foals of the crioulo breed at the pre and post weaning, regardless of pigmentation and sex, where Ca and Cu concentrations increase with age, while Zn concentrations decrease.(AU)

Nail matrix regenerative engineering: in vitro evaluation of poly(lactide-co-glycolide)/gelatin fibrous substrates.

Acute traumatic nail injury treatment repair procedures are commonly conducted in emergency departments and primary care offices. Current repair methods use nail splints that are inserted within the nail root to prevent the fusion of the proximal nail fold and the matrix tissue. Splints provide a protective barrier overlying the nail bed soft tissue during recovery periods, but uncertain prognoses (i.e., aesthetic and functional disadvantages) reveal a need for improved nail repair techniques. Nail splints are not specifically designed for nail organ restoration via biological mechanisms, thus, a clinical application that utilizes regenerative engineering techniques can prove useful in improving the nail injury prognoses. Using the coaxial electrospinning method, hybrid poly(lactide-co-glycolide) (PLGA) (85:15) and gelatin fibrous scaffolds (Hybrid1: PLGA shell, gelatin core and Hybrid2 : gelatin shell, PLGA core) with average fiber diameters of 540 ± 118 and 2,215 ± 1,135 nm, respectively, were produced and successful encapsulation of core fibers was observed. Furthermore, nail stem cells expressing stem cell characteristic markers CD90, CD29, and Lgr6 showed preferred attachment to Hybrid2 scaffolds after 24 hr. Overall, an in vitro regenerative engineered nail matrix may aid to improve the cosmetic appearance and function of injured nail organs post-traumatic injury.

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus.

The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three monoclonal antibodies generated against N- and C-terminal K124 peptides detected a band of the appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-K124 monoclonal antibodies.

Effects of silymarin on p65 NF-κB, p38 MAPK and CYP450 in LPS-induced hoof dermal inflammatory cells of dairy cows.

BACKGROUND: Laminitis is considered as one of the most important causes of hoof lameness in dairy cows, which can lead to enormous economic losses. However, the etiology and pathogenesis of laminitis have not been clarified yet. Besides, it is of great significant to find alternative herbs for the prevention and treatment of dairy hooves to avoid the antibiotic abuse. In this study, the primary hoof dermal cells of dairy cows were isolated, the inflammatory model was induced by LPS, and treated with silymarin to find whether silymarin has protective effect on the inflammatory dermal cells. The viability of dermal cells, the levels of IL-1ß and TNF-α, the degree of p65 NF-κB and p38 MAPK phosphorylation, the expressions of CYP3A4 and CYP1A1 were measured. RESULTS: Hoof dermal cells of dairy cows were successfully isolated and cultured by tissue adherent culture method. Certain concentrations of LPS can increase the levels of IL-1ß and TNF-α, promote the phosphorylation of p65 NF-κB and p38 MAPK, and inhibit the mRNA expressions of CYP3A4 and CYP1A1. The optimal concentration for LPS to establish a hoof dermal cells inflammatory model was 10 µg/mL. Certain concentrations of silymarin can markedly decrease the secretions of IL-1ß and TNF-α, inhibit the phosphorylation of p65 NF-κB and p38 MAPK, and promote the mRNA expressions of CYP3A4 and CYP1A1 in LPS-induced dermal inflammatory model. CONCLUSIONS: LPS can be used for inducing the hoof dermal cells inflammatory model of dairy cows. Silymarin has protective effects on the LPS-induced inflammatory model.

Equine Hoof Canker: Cell Proliferation and Morphology.

Hoof canker is described as progressive pododermatitis of the equine hoof with absent epidermal cornification and extensive proliferation of the dermal papillary body; however, in-depth research on the type of proliferative activity has not yet been reported. The aim of the present study was to determine cell-specific proliferation patterns together with morphological analysis of hoof canker tissue. Tissues removed during surgery from 19 horses presented for treatment of canker were compared with similar postmortem tissues of healthy hooves of 10 horses. Morphological alterations visible in light microscopy were assessed semiquantitatively and graded for severity. Proliferative activity was evaluated by means of anti-PCNA (proliferative cell nuclear antigen) and anti-Ki67 immunohistochemistry. Histologically, canker tissue showed 5 major morphological alterations-the presence of lacunae, vacuoles, giant cells, hemorrhage, and inflammation-not seen in control tissue. Also, there was a notable koilocytotic appearance of keratinocytes in canker tissue. Immunohistochemistry revealed increased levels of PCNA protein expression in keratinocytes and fibroblasts of canker tissue compared with control tissue. In control tissue, keratinocytes showed higher levels of Ki67 compared with canker tissue, while the dermal fibroblasts of both groups showed similar levels of Ki67, indicating similar proliferative activity of less than 3% of total dermal fibroblasts. These results demonstrate that, in contrast to previous reports, there is no evidence for increased proliferative activity of the dermal papillary body associated with hoof canker. Increased levels of PCNA protein expression and morphological alterations indicate that dysregulation of keratinocyte differentiation constitutes a key event in equine hoof canker development.

Fumonisin B1 (FB1) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants.

One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B1 (FB1) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB1 at any concentration on dermal or epidermal cells. However, FB1 significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB1 (2.5-10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB1 impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB1 might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB1 on the equine hoof in more detail.

Lgr6 marks nail stem cells and is required for digit tip regeneration.

The tips of the digits of some mammals, including human infants and mice, are capable of complete regeneration after injury. This process is reliant on the presence of the overlaying nail organ and is mediated by a proliferative blastema. Epithelial Wnt/ß-catenin signaling has been shown to be necessary for mouse digit tip regeneration. Here, we report on Lgr5 and Lgr6 (leucine-rich repeat-containing G protein-coupled receptor 5 and 6), two important agonists of the Wnt pathway that are known to be markers of several epithelial stem cell populations. We find that Lgr5 is expressed in a dermal population of cells adjacent to the specialized epithelia surrounding the keratinized nail plate. Moreover, Lgr5-expressing cells contribute to this dermis, but not the blastema, during digit tip regeneration. In contrast, we find that Lgr6 is expressed within cells of the nail matrix portion of the nail epithelium, as well as in a subset of cells in the bone and eccrine sweat glands. Genetic lineage analysis reveals that Lgr6-expressing cells give rise to the nail during homeostatic growth, demonstrating that Lgr6 is a marker of nail stem cells. Moreover, Lgr6-expressing cells contribute to the blastema, suggesting a potential direct role for Lgr6-expressing cells during digit tip regeneration. This role is confirmed by analysis of Lgr6-deficient mice, which have both a nail and bone regeneration defect.

Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof.

BACKGROUND: The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. HYPOTHESIS/OBJECTIVES: To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. METHODS: Indirect immunofluorescence microscopy and immunoblotting were used to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14 and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 [pp63; a marker of ESC transition to transit-amplifying (TA) cell] in epithelial tissues of the foot (haired skin, hoof coronet and hoof lamellae) and the eye (limbus and cornea). RESULTS: Expression of K14 was restricted to the basal layer of epidermal lamellae and to basal and adjacent suprabasal layers of the haired skin, coronet and corneal limbus. Coronary and lamellar epidermis was negative for both K3 and K10, which were expressed in the cornea/limbus epithelium and haired skin epidermis, respectively. Variable expression of p63 with relatively low to high levels of phosphorylation was detected in individual basal and suprabasal cells of all epithelial tissues examined. CONCLUSIONS: To the best of the author's knowledge, this is the first report of the characterization of tissue-specific keratin marker expression and the localization of putative epithelial progenitor cell populations, including ESCs (high p63 expression with low pp63 levels) and TA cells (high expression of both p63 and pp63), in the horse. These results will aid further investigation of epidermal and corneal epithelial biology and regenerative therapies in horses.

Bifunctional ectodermal stem cells around the nail display dual fate homeostasis and adaptive wounding response toward nail regeneration.

Regulation of adult stem cells (SCs) is fundamental for organ maintenance and tissue regeneration. On the body surface, different ectodermal organs exhibit distinctive modes of regeneration and the dynamics of their SC homeostasis remain to be unraveled. A slow cycling characteristic has been used to identify SCs in hair follicles and sweat glands; however, whether a quiescent population exists in continuously growing nails remains unknown. Using an in vivo label retaining cells (LRCs) system, we detected an unreported population of quiescent cells within the basal layer of the nail proximal fold, organized in a ring-like configuration around the nail root. These nail LRCs express the hair stem cell marker, keratin 15 (K15), and lineage tracing show that these K15-derived cells can contribute to both the nail structure and peri-nail epidermis, and more toward the latter. Thus, this stem cell population is bifunctional. Upon nail plucking injury, the homeostasis is tilted with these SCs dominantly delivering progeny to the nail matrix and differentiated nail plate, demonstrating their plasticity to adapt to wounding stimuli. Moreover, in vivo engraftment experiments established that transplanted nail LRCs can actively participate in functional nail regeneration. Transcriptional profiling of isolated nail LRCs revealed bone morphogenetic protein signaling favors nail differentiation over epidermal fate. Taken together, we have found a previously unidentified ring-configured population of bifunctional SCs, located at the interface between the nail appendage organ and adjacent epidermis, which physiologically display coordinated homeostatic dynamics but are capable of rediverting stem cell flow in response to injury.

Tooth, hair and claw: comparing epithelial stem cell niches of ectodermal appendages.

The vertebrate ectoderm gives rise to organs that produce mineralized or keratinized substances, including teeth, hair, and claws. Most of these ectodermal derivatives grow continuously throughout the animal׳s life and have active pools of adult stem cells that generate all the necessary cell types. These organs provide powerful systems for understanding the mechanisms that enable stem cells to regenerate or renew ectodermally derived tissues, and remarkable progress in our understanding of these systems has been made in recent years using mouse models. We briefly compare what is known about stem cells and their niches in incisors, hair follicles, and claws, and we examine expression of Gli1 as a potential example of a shared stem cell marker. We summarize some of the features, structures, and functions of the stem cell niches in these ectodermal derivatives; definition of the basic elements of the stem cell niches in these organs will provide guiding principles for identification and characterization of the niche in similar systems.

Wnt activation in nail epithelium couples nail growth to digit regeneration.

The tips of mammalian digits can regenerate after amputation, like those of amphibians. It is unknown why this capacity is limited to the area associated with the nail. Here we show that nail stem cells (NSCs) reside in the proximal nail matrix and that the mechanisms governing NSC differentiation are coupled directly with their ability to orchestrate digit regeneration. Early nail progenitors undergo Wnt-dependent differentiation into the nail. After amputation, this Wnt activation is required for nail regeneration and also for attracting nerves that promote mesenchymal blastema growth, leading to the regeneration of the digit. Amputations proximal to the Wnt-active nail progenitors result in failure to regenerate the nail or digit. Nevertheless, ß-catenin stabilization in the NSC region induced their regeneration. These results establish a link between NSC differentiation and digit regeneration, and suggest that NSCs may have the potential to contribute to the development of novel treatments for amputees.

Effects of long-term extracorporeal blood perfusion of the distal portion of isolated equine forelimbs on metabolic variables and morphology of laminar tissue.

OBJECTIVE: To establish an ex vivo model of blood perfusion in the distal portion of isolated equine forelimbs that closely represents the in vivo situation in the laminar tissue of the hoof. SAMPLE POPULATION: 18 forelimbs collected from 9 healthy adult horses following slaughter at a licensed abattoir. PROCEDURES: The distal portion of isolated equine forelimbs from 9 horses were perfused under physiologic conditions over a period of 6, 8, and 10 hours with autologous blood. To determine cell viability in perfused tissues, indicators for metabolism (lactate generation and glucose and oxygen consumption) as well as indicators for cell damage (potassium concentration and lactate dehydrogenase activity) were examined at 1-hour intervals from samples of the perfusate. Weight gain in the forelimb was used to determine the edema index. After perfusion, light and electron microscopic examinations of laminar tissue specimens were performed. RESULTS: During hemoperfusion of the isolated forelimbs, mean +/- SD glucose consumption was 197.4 +/- 65.1 mg/h, lactate generation was 1.84 +/- 0.79 mmol/h, and oxygen consumption was 6.4 x 10(-6) +/- 8.9 x 10(-5) mL.g(-1).min(-1). Neither an efflux of potassium into the perfusate nor a relevant increase of the lactate dehydrogenase activity was detected, indicating low amounts of cellular damage in the perfused tissues. Weight gain of forelimbs was 1.02 +/- 0.95%. Histologic and ultrastructural appearance of the laminar tissue revealed no signs of tissue damage. CONCLUSIONS AND CLINICAL RELEVANCE: Isolated equine limbs were perfused under physiologic conditions over a period of < or = 10 hours without structural damage to the laminar tissue.

Bovine coronary region keratinocyte colony formation is supported by epidermal-dermal interactions.

Delineating the factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronary region keratinocytes and dermal fibroblasts, determine the colony-forming capacity of epidermal keratinocytes in the coronary region, and characterize transcriptional changes in specific cytokine, growth factor, and receptor genes during colony formation in coculture. Fibroblasts and keratinocytes from the coronary region of the lateral, hind limb claw were collected, and 5.0 x 10(3) and 7.5 x 10(3) keratinocytes were cultured in the presence or absence of fibroblast monolayers, respectively. The 2 densities of keratinocytes formed 144 +/- 15.8 and 183 +/- 26.9 colonies, respectively, in the presence of dermal fibroblasts, whereas no colonies developed in the absence of dermal fibroblasts. Keratinocytes with the ability to show colony formation comprised 1.09% +/- 0.16 to 1.77% +/- 0.28 of the keratinocyte population isolated from the coronary region. Keratinocyte-fibroblast cocultures developed a time-dependent increased expression of several growth factors, cytokines, and receptors. These findings demonstrated that keratinocytes from the bovine coronary region formed colonies in vitro and that colony formation occurred with an absolute dependence on dermal fibroblasts. Colony growth was associated with increased transcriptional expression of cytokine, growth factor, and receptor expression known to drive keratinocyte colony formation in other species. The results indicate that horn-producing keratinocytes must interact with dermal fibroblasts during normal tissue homeostasis in the bovine claw.

Quantitative morphology of the equine laminar junction in relation to capsule shape in the forehoof of Standardbreds and Thoroughbreds.

REASONS FOR PERFORMING STUDY: Differences in hoof morphology have largely been underappreciated in the literature until recently, and it is these that hold the key to interpreting functional adaptation in the hoof. HYPOTHESES: Primary laminar morphology correlates with hoof capsule shape; and breeds with different hoof shapes and loadings show different patterns of correlation. METHODS: Seventeen measurements of capsule shape and 3 of primary epidermal laminae (PEL) morphology (spacing, orientation and curvature) were made on right and left front hooves from 27 Standardbred and 25 Thoroughbred horses, and tested for breed differences. Three laminar variables (spacing, orientation and curvature) were measured on each hoof for samples of 25 PEL in 5 circumferential and 4 proximodistal locations. Pairwise correlations of capsular and laminar measurements were compared within breeds. Significant correlations were mapped onto the 20 sampling sites. RESULTS: Capsule shape differed significantly between breeds in 7 measurements and in a multivariate test. Between breeds, PEL differed in orientation and spacing primarily at the medial quarters and heels, and in curvature at both quarters (P<0.05). Significant correlations between several pairs of capsule and laminar variables were found at sample locations that differed markedly between breeds. CONCLUSIONS: Laminar morphology, hoof capsule shape and correlations between them differ between Standardbreds and Thoroughbreds. These results support the concept that remodelling of PEL is, at least in part, stimulated and directed by varying stress or strain levels in the laminar junction. POTENTIAL RELEVANCE: Understanding the biological responses of hoof tissues to stress should add to the ability to prevent lameness involving the hoof and maintain its health.

Muscle-specific calpain is localized in regions near motor endplates in differentiating lobster claw muscles.

Calpains are Ca2+-dependent proteinases that mediate protein turnover in crustacean skeletal muscles. We used an antibody directed against lobster muscle-specific calpain (Ha-CalpM) to examine its distribution in differentiating juvenile lobster claw muscles. These muscles are comprised of both fast and slow fibers early in development, but become specialized into predominantly fast or exclusively slow muscles in adults. The transition into adult muscle types requires that myofibrillar proteins specific for fast or slow muscles to be selectively removed and replaced by the appropriate proteins. Using immunohistochemistry, we observed a distinct staining pattern where staining was preferentially localized in the fiber periphery along one side of the fiber. Immunolabeling with an antibody directed against synaptotagmin revealed that the calpain staining was greatest in the cytoplasm adjacent to synaptic terminals. In complementary analyses, we used sequence-specific primers with real-time PCR to quantify the levels of Ha-CalpM in whole juvenile claw muscles. These expression levels were not significantly different between cutter and crusher claws, but were positively correlated with the expression of fast myosin heavy chain. The anatomical localization of Ha-CalpM near motor endplates, coupled with the correlation with fast myofibrillar gene expression, suggests a role for this intracellular proteinase in fiber type switching.