Influence of dietary zinc on the claw and interdigital skin of sheep.

Claw diseases like interdigital dermatitis and footrot threaten sheep health and are major welfare issues. Several studies mainly done in cattle suggested that zinc (Zn) supplementation may improve claw integrity. However, Zn supplements may differ markedly regarding Zn bioavailability. Zn bound to single amino acids has been shown to be more bioavailable than inorganic Zn sources. The aim of this study was to determine the effect of different Zn supplements on the integrity of the claw and interdigital skin of healthy sheep. At weaning 30 Merino lambs were randomly allocated to three different dietary treatments which were provided through the pelleted concentrates as follows: 1) no supplemental Zn (Zn0); 2) addition of 40 mg/kg Zn as Zn sulphate (ZnS); 3) addition of 40 mg/kg organic Zn as Zn amino acid complex (CZn). Barley straw and pelleted concentrates were given ad-libitum. The calculated Zn concentration of the total diet (roughage and concentrate) without supplemental Zn (Zn0) was 38 mg Zn/kg DM. The concentrates were formulated to meet the nutritional requirements for growing lambs and contained 207 g/kg DM crude protein and 12.4 MJ/kg DM metabolizable energy. After 8 weeks the lambs were slaughtered and the following specimens were collected: blood serum, liver, sole and coronary band of the claw, and interdigital skin. Serum and tissue Zn and copper (Cu) concentrations and claw hardness were determined. Routine pathohistology and electron microscopy were conducted. Franz diffusion cell system and Ki-67 immunostaining were used to determine the permeability of the interdigital skin and the keratinocyte proliferation in the basal layer of sole horn, coronary band and interdigital skin, respectively. The concentrations of Zn and Cu in serum and liver tissue as well as the Zn concentration in claw horn were not affected by dietary treatment. Zn0 lambs showed higher (p < 0.05) Cu concentrations in claw horn compared to both Zn supplemented groups. Routine pathohistology as well as electron microscopy did not show significant morphological differences between the three groups. Franz diffusion cell system proved to be a suitable method examining the interdigital skin permeability, but the group differences in this study were not significant. Dietary treatment did not affect keratinocyte proliferation in the coronary band. In the sole keratinocyte proliferation was significantly higher (p < 0.05) in the Zn0 group compared to CZn with ZnS being intermediate. Keratinocyte proliferation in the interdigital skin was significantly higher (p < 0.05) in the CZn group compared to the Zn0 with ZnS being intermediate. The results of the current experiment indicate that serum and tissue Zn concentrations and horn hardness are not affected by adding a moderate amount of Zn sulphate or Zn amino acid complex to a basal diet. However, supplemental Zn amino acid complex seems to affect keratinocyte proliferation of interdigital skin and sole horn of lambs. Effects on skin permeability should be retested using a higher number of animals prospectively.

Terbinafine hydrochloride nail lacquer for the management of onychomycosis: formulation, characterization and in vitro evaluation.

AIM: The present investigation's intention was to develop an optimized nail lacquer (NL) for the management of onychomycosis. MATERIALS & METHODS: The NL was optimized statistically adopting 32 full factorial design having different polymer ratios and solvent ratios. The formulations were assessed for drug permeation drying time and peak adhesive strength of the film. Characterization was done using techniques including attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), x-ray diffraction (XRD), etc. RESULTS & CONCLUSION: The formulation that had 1:1 polymer ratio and 80:20 solvent ratio was chosen as the optimized formulation. In vitro permeation studies showed better penetration (∼3.25-fold) as well as retention (∼11-fold) of the optimized NL formulation in the animal hoof as compared with the commercial formulation. The findings of in vitro and ex vivo studies elucidated the potential of the optimized formulation. [Formula: see text].

Corium molecular biomarkers reveal a beneficial effect on hoof transcriptomics in peripartal dairy cows supplemented with zinc, manganese, and copper from amino acid complexes and cobalt from cobalt glucoheptonate.

Supplying trace minerals in more bioavailable forms such as amino acid complexes (AAC) could help ameliorate the incidence of hoof disorders in peripartal dairy cows. The aim of this study was to evaluate the effects of supplementing metal AAC during the peripartal period on expression of 28 genes in corium tissue related to claw composition, oxidative stress, inflammation, chemotaxis, and transcriptional regulation. Forty-four multiparous Holstein cows received a common diet from -30 to 30 d relative to parturition and were assigned to receive an oral bolus containing either inorganic trace minerals (INO) or AAC (i.e., organic) Zn, Mn, Cu, and Co to achieve supplemental levels of 75, 65, 11, and 1 ppm, respectively, in the total diet dry matter. Inorganic trace minerals were provided in sulfate form, and AAC were supplied via Availa Zn, Availa Mn, Availa Cu, and COPRO (Zinpro Corp., Eden Prairie, MN). Locomotion score was recorded before enrollment and weekly throughout the experiment. Incidence of hoof health problems at 30 d in milk was evaluated before a hoof biopsy in a subset of cows (INO=10; AAC=9). Locomotion score did not differ between treatments in the prepartum or postpartum period. The incidence of heel horn erosion was lower in AAC cows, but the incidence of sole ulcers did not differ. Downregulation of KRT5, CTH, CALML5, and CYBB, and upregulation of BTD in AAC cows indicated a decrease in the need for activation of cellular pathways to regenerate corium tissue and increase biotin availability in the sole claw. These molecular changes in the sole could have been triggered by the lower incidence of heel erosion in response to AAC. Among the genes associated with oxidative stress, the AAC cows had greater expression of NFE2L2, a transcription factor that regulates the antioxidant response, and the antioxidant enzyme SOD1. Among genes associated with inflammation, AAC cows had greater expression of TLR4, and lower expression of TLR2, IL1B, and TNF compared with INO cows. Supplementation with metal AAC during the peripartal period affected the expression of genes involved in composition, oxidative stress, and inflammation status in the corium. The hoof biopsy procedure used in the present study should be further perfected and implemented in future lameness research to expand our understanding of hoof biology in dairy cows.

Evaluation of the possible role of prostaglandin F(2 alpha) in laminitis induced in horses by nasogastric administration of black walnut heartwood extract.

OBJECTIVE: To provide insights into the role of prostaglandin F(2 alpha) (PGF(2 alpha)) in the developmental stages of laminitis induced in horses by ingestion of black walnut heartwood extract (BWHE). SAMPLE POPULATION: 10 adult mixed-breed horses. PROCEDURES: Horses were separated into 2 groups and were euthanatized at 12 hours after placebo (water) administration (control horses) or after BWHE administration and development of Obel grade 1 laminitis. Blood samples were obtained to determine plasma PGF(2 alpha) concentrations hourly for the first 4 hours and subsequently every 2 hours after substance administration. Laminar arteries and veins were isolated, and responses to increasing concentrations of PGF(2 alpha) were measured before and after preincubation of blood vessels with prostanoid and thromboxane receptor antagonists SQ 29,548, SC-19220, and AH 6809. RESULTS: Plasma PGF(2 alpha) concentrations increased in horses given BWHE; the WBC count decreased concurrently. In control horses, PGF(2 alpha) was a potent contractile agonist for laminar veins but not for laminar arteries. In horses given BWHE, PGF(2 alpha) was similarly selective for laminar veins; however, the magnitude of PGF(2 alpha)-induced venoconstriction was less than that in control horses. After preincubation with SQ 29,548, laminar veins from control horses responded to PGF(2 alpha) with a small degree of dilation, whereas laminar veins from horses given BWHE did not. CONCLUSIONS AND CLINICAL RELEVANCE: PGF(2 alpha) may play a role in the inflammatory and vascular dysfunction associated with the prodromal stages of laminitis. Prostanoids such as PGF(2 alpha) may be viable targets for the prevention of acute laminitis in horses.

Tissue concentrations of 4-HNE in the black walnut extract model of laminitis: indication of oxidant stress in affected laminae.

In the septic horse prone to laminitis, a similar activation of the innate immune system appears to occur as reported in the septic human prone to organ failure. Because oxidant injury plays a central role in organ failure occurring due to an overzealous innate immune response in human sepsis, this study was performed to determine whether there was evidence of oxidant stress in the laminar tissue in the early stages of laminitis. 4-Hydroxy-2-nonenal (4-HNE), a lipid aldehyde that forms due to lipid peroxidation occurring during episodes of oxidant stress, readily forms adducts with cellular proteins; these adducts can be assessed as a marker of oxidant stress in the form of lipid peroxidation. In this study, a slot blot technique was used to assess 4-HNE adduct concentrations in the laminae, lung, liver, and intestinal tract in the black walnut extract (BWE) model of laminitis. Significant increases in laminar 4-HNE adduct concentrations were identified at two early stages in the BWE model, in the absence of such changes in the other tissues. These data indicate that oxidant stress may play an important role in the laminar failure in laminitis, and further support the concept that a poor antioxidant response in the laminae relative to other equine tissues may be responsible for failure of the laminae in the septic horse. In contrast, tissues such as the lung and liver that undergo oxidant injury in human sepsis appear to be relatively protected in horses.

Effect of biotin supplementation on hoof health and ceramide composition in dairy cattle.

The effect of biotin supplementation on various foot lesions and hoof ceramide composition of toe (wall) and sole portions of hooves was studied in crossbred dairy cattle. Biotin supplementation was done for five months in 14 cattle at a farm and the other 14 animals kept as control. A significant decline was observed in heel erosions and sole avulsions along with total disappearance of white line fissures and double soles in the biotin supplemented cattle resulting in decrease in the overall disease score. Thin layer chromatographs of the hoof lipids revealed 11 types of ceramides in sole lipids and 6 types of ceramides in toe (wall) lipids. The ceramides were typed and identified according to their Rf values. A qualitative increase in the density of thin layer chromatographs of sole lipids was observed in biotin supplemented cattle whereas a non-significant difference in density of thin layer chromatographs of toe lipids was observed after supplementation of biotin.

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.

Nail swelling as a pre-formulation screen for the selection and optimisation of ungual penetration enhancers.

INTRODUCTION: Targeting drug treatment to fungal infections that reside within or below the nail plate is problematic due to the highly restrictive barrier of the human nail. To optimise topical formulations for ungual drug delivery, inclusion of an effective penetration enhancer (PE) is imperative. At present, in vitro nail permeation studies can take weeks or months in order to obtain any meaningful data because the lack of a simple in vitro model to identify and develop nail PEs makes the selection and optimisation of novel PEs an empirical and inefficient process. The aim of this study was to compare three methods for pre-formulation screening of putative ungual PEs and then to select the most suitable technique for screening candidates that may enhance the permeation of therapeutic agents through the human nail. METHODS: Three screening techniques were evaluated; nail swelling (weight increase of human nail clippings), horse hoof swelling (weight increase of horse hoof clippings) and nail penetration of a radiolabelled permeability probe. Four test PEs were evaluated using each screening method and nail swelling was identified as a simple, rapid, economic, relevant and reliable technique. This screen was then used to evaluate 20 potential PEs. Thioglycolic acid (TA), hydrogen peroxide (H(2)O(2)) and urea H(2)O(2) produced the greatest nail weight increases; 71.0+/-4.6%, 69.2+/-6.6%, and 69.0+/-9.9 respectively. To confirm the relationship between human nail swelling and altered ungual barrier function, a permeation study was performed in human nails using caffeine as a model penetrant. RESULTS AND DISCUSSION: Human nails pre-treated with TA in vitro had a 3.8-fold increase in caffeine flux compared to the control (TA-free solution). This study illustrated the potential to use human nail clipping swelling as a surrogate marker of PE activity for topical ungual drug delivery.

Bioadhesive patch-based delivery of 5-aminolevulinic acid to the nail for photodynamic therapy of onychomycosis.

The in vitro penetration of 5-aminolevulinic acid (ALA) across human nail and into neonate porcine hoof when released from a novel bioadhesive patch containing 50 mg cm(-2) ALA is described. ALA is a naturally occurring precursor of the photosensitiser protoporphyrin IX (PpIX). Topical application of excess ALA bypasses negative feedback inhibition and yields photosensitising concentrations of PpIX at the application site. ALA-based photodynamic therapy (PDT) has been extensively investigated in the topical treatment of various skin neoplasias. Recently, its use has been extended to the microbiological field. If sufficient concentrations of ALA could be achieved within the nail matrix, and at the nail bed, PDT may prove to be a useful treatment for onychomycosis. Patch application for 24 h allowed an ALA concentration of 2.8 mM to be achieved on the ventral side of excised human nail. Application for 48 h induced a concentration of 6.9 mM. Application time had no significant effect on the ALA concentration at mean depths of 2.375 mm in neonate porcine, with application times of 24, 48 and 72 h all producing concentrations of 0.1 mM. Incubation of Candida albicans and Trichophyton interdigitale with ALA concentrations of 10.0 mM for 30 min and 6 h, respectively, caused reductions in viability of 87% and 42%, respectively, following irradiation with red light. Incubation with 0.1 mM ALA for 30 min and 6 h, respectively, caused reductions in viability of 32% for Candida albicans and 6% for Trichophyton interdigitale, following irradiation. Drug penetration across nail may be improved using penetration enhancers, or by filing of the impenetrable dorsal surface of the nail. Moreover, iron chelators can be used to increase PpIX production for a given ALA dose. Therefore, with suitable modifications, ALA-PDT may prove to be a viable alternative in the treatment of onychomycosis.

Effects of biotin supplementation on serum biotin levels and physical properties of samples of solar horn of Holstein cows.

Effects of dietary biotin supplementation on serum biotin levels and physical properties of sole horn of 40 Holstein cows were evaluated. The mean serum biotin level in biotin-supplemented cows after 10 mo of biotin supplementation (1163.2 +/- 76.2 pg/mL) was significantly higher (P = 0.007) than that in control cows (382.0 +/- 76.2 pg/mL). The sole horn of biotin-supplemented cows was significantly harder (P = 0.026) and had a significantly lower moisture content (P = 0.021) than that of control cows. No morphologic differences in horn tubules or intertubular horn were found between the biotin-supplemented and control cows. The total lipid content of sole horn was significantly higher (P = 0.030) in the biotin-supplemented cows than in the control cows. These results suggest that dietary biotin supplementation causes increases in serum biotin levels and changes in physical properties and fat content of sole horn.

Dermatopharmacology of ciclopirox nail lacquer topical solution 8% in the treatment of onychomycosis.

Ciclopirox is a synthetic hydroxypyridone antifungal agent. In contrast to the azoles, glucuronidation is the main metabolic pathway of ciclopirox; therefore interactions with drugs metabolized via the cytochrome P450 system are unlikely Ciclopirox is also distinct from the common systemic agents, which interfere with sterol biosynthesis. In fact, ciclopirox chelates trivalent cations (such as Fe3+), inhibits metal-dependent enzymes that are responsible for degradation of toxic metabolites in the fungal cells, and targets diverse metabolic (eg, respiratory) and energy producing processes in microbial cells. Ciclopirox is a broad spectrum antimicrobial with activity against all the usual dermatophytes as well as yeast and nondermatophyte molds. It has demonstrated activity against gram positive and negative bacteria, including resistant strains of Staphlococcus aureus. Ciclopirox exhibits fungal inhibitory activity (minimum inhibitory concentration < 4 microg/mL for dermatophytes) as well as fungicidal activity; to date resistance to the drug has not been identified. Ciclopirox has been formulated in a nail lacquer delivery system. After evaporation of volatile solvents in the lacquer, the concentration of ciclopirox in the remaining lacquer film reaches approximately 35%, providing a high concentration gradient for penetration into the nail. Radiolabel data demonstrate penetration into infected nails after only 1 application of the lacquer. Ciclopirox nail lacquer is a topical product that provides an active fungicidal agent in a delivery system capable of promoting nail penetration. With repeated applications, the antifungal agent is homogeneously distributed through all layers of the toenail achieving concentrations of ciclopirox in excess of inhibitory and fungicidal concentrations for most pathogens. Although ciclopirox readily penetrates nails, very low levels of ciclopirox are recoverable systemically, even after chronic use. Ciclopirox nail lacquer 8% is a topical product that provides an active fungicidal agent in a delivery system capable of penetrating nails.

HLA-B27 heavy chains contribute to spontaneous inflammatory disease in B27/human beta2-microglobulin (beta2m) double transgenic mice with disrupted mouse beta2m.

MHC class I allele, HLA-B27, is strongly associated with a group of human diseases called spondyloarthropathies. Some of these diseases have an onset after an enteric or genitourinary infection. In the present study, we describe spontaneous disease in HLA-B27 transgenic mice where endogenous beta2-microglobulin (beta2m) gene was replaced with transgenic human beta2m gene. These mice showed cell surface expression of HLA-B27 similar to that of human peripheral blood mononuclear cells. In addition, free heavy chains (HCs) of HLA-B27 were also expressed on thymic epithelium and on a subpopulation of B27-expressing PBLs. These mice developed spontaneous arthritis and nail changes in the rear paws. Arthritis occurred primarily in male animals and only when mice were transferred from the pathogen-free barrier facility to the conventional area. Transgenic mice expressing HLA-B27 with mouse beta2m have undetectable levels of free HCs on the cell surface and do not develop arthritis. In vivo treatment with anti-HC-specific antibody delayed the onset of disease. Our data demonstrate specific involvement of HLA-B27 'free' HCs in the disease process.

Incorporation of L-75Se-cystine in tissue fragments from the matrix of the hoof and the claw--a tool for studying the pathogenesis of laminitis?

An in vitro method has been designed and used to study the incorporation of 75Se-cystine into matrix fragments from hooves and claws of healthy horses and cattle. Tissue fragments from the zone of keratinisation were incubated with L-75Se-cystine in a tissue culture medium for 4 to 6 h, during which time there was continuous incorporation of the labelled selenocystine. The incorporation was greatly decreased by adding L-cystine to the incubation mixture. It is concluded that the incorporation of 75Se-cystine depends on the presence of a specific receptor for cystine in the tissue fragments studied. The possible application of the method to studies of the pathogenesis of laminitis is discussed.