Proteomics changes after negative pressure wound therapy in diabetic foot ulcers.

Label­free quantitative mass spectrometry was used to analyze the differences in the granulation tissue protein expression profiles of patients with diabetic foot ulcers (DFUs) before and after negative­pressure wound therapy (NPWT) to understand how NPWT promotes the healing of diabetic foot wounds. A total of three patients with DFUs hospitalized for Wagner grade 3 were enrolled. The patients received NPWT for one week. The granulation tissue samples of the patients prior to and following NPWT for one week were collected. The protein expression profiles were analyzed with label­free quantitative mass spectrometry and the differentially expressed proteins (DEPs) in the DFU patients prior to and following NPWT for one week were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to annotate the DEPs and DEP­associated signaling pathways. Western blotting and ELISA were performed to validate the results. By comparing the differences in the protein profiles of granulation tissue samples prior to and following NPWT for one week, 36 proteins with significant differences were identified (P<0.05); 33 of these proteins were upregulated and three proteins were downregulated. NPWT altered proteins mainly associated with antioxidation and detoxification, the cytoskeleton, regulation of the inflammatory response, complement and coagulation cascades and lipid metabolism. The functional validation of the DEPs demonstrated that the levels of cathepsin S in peripheral blood and granulation tissue were significantly lower than those prior to NPWT (P<0.05), while the levels of protein S isoform 1, inter α­trypsin inhibitor heavy chain H4 and peroxiredoxin­2 in peripheral blood and granulation tissue were significantly higher than those prior to NPWT (P<0.05). The present study identified multiple novel proteins altered by NPWT and laid a foundation for further studies investigating the mechanism of action of NPWT.

Micronutrient Status in Diabetic Patients with Foot Ulcers.

Objective: To explore the prevalence of micronutrient deficiencies in patients with diabetic foot ulcers and correlate this with foot disease severity and other clinical factors. Approach: Prospective cohort study of diabetic patients with foot ulcers seen in multidisciplinary foot clinics across Adelaide or admitted to the Vascular Surgery Unit at the Royal Adelaide Hospital between February 2017 and September 2018. A total of 131 patients were included in the study. Plasma serum levels of vitamins A, C, D, and E, copper, zinc, and ferritin were measured. Demographic and clinical data, including BMI, smoking status, duration of diabetes, HbA1c, and WIfI score, were obtained. Results: The most prevalent nutritional deficiency found was vitamin D affecting 55.7% of patients. Suboptimal levels of vitamin C affected 73% of patients, comprising marginal levels in 22.2% and deficient levels in 50.8%. Zinc deficiency, vitamin A deficiency, and low ferritin levels were present in 26.9%, 10.9%, and 5.9% of patients, respectively. There was no correlation between BMI, grip strength, duration of diabetes, HbA1c, or smoking status with micronutrient deficiency. Increased severity of diabetic foot disease was associated with lower vitamin C levels (p = 0.02). Innovation: This study has demonstrated that the deficiency of micronutrients, especially vitamin D, vitamin C, zinc, and vitamin A, is common in diabetic patients with foot ulcers. Conclusions: The prevalence of micronutrient deficiency is high in a diabetic population with foot ulcers/wounds. Special concerns regarding the high prevalence of vitamin C and zinc deficiency, given their roles in wound healing. Although further research needs to be performed to determine the clinical implications of our findings, micronutrient deficiency should be considered in diabetic patients with foot wounds.

Protein profile of leprosy patients with plantar ulcers from the Eastern Amazon region.

BACKGROUND: Studies investigating the nutritional status of patients with leprosy and plantar ulcers are sparse. Therefore, the objective of this study was to describe the protein profile of leprosy patients with plantar ulcers from the Eastern Amazon region. METHODS: A case record form was created for 75 patients with leprosy (31 with plantar ulcers and 44 without plantar ulcers) with the following data: sociodemographic characteristics, clinical form of leprosy, presence or absence of plantar ulcers, and nutritional assessment using anthropometry consisting of the measurement of body mass index, arm circumference, arm muscle circumference, and triceps skinfold. Levels of blood albumin, transferrin, and C-reactive protein (CRP) were also measured. Data regarding protein intake were obtained using a Food Frequency Questionnaire. RESULTS: Plantar ulcers occurred more frequently in male patients (67.7%), patients aged 40-49 years (mean ± SD: 47.3 ± 8.0 years), and patients receiving 300 or 600 USD (71.0%). The mean weight and height of patients were 71.6 ± 11.4 kg and 1.62 ± 0.1 m, respectively. High levels of CRP were detected in 51.6% of leprosy patients with plantar ulcers and only 9.1% of patients without plantar ulcers (P < 0.001). Nutritional depletion of transferrin was observed in 14.3% of patients with paucibacillary leprosy and 44.3% of patients with multibacillary leprosy (P = 0.0447). Most patients had normal levels of serum albumin (74.2% with plantar ulcers and 77.3% without plantar ulcers). CONCLUSIONS: Most leprosy patients with plantar ulcers have normal levels of serum albumin and transferrin and high CRP levels, which indicates the presence of an inflammatory process. Our findings suggest the need to monitor patients with leprosy to prevent the occurrence of plantar ulcers and to provide adequate treatment for patients with existing plantar ulcers.

Pathogenesis and Treatment of Callus in the Diabetic Foot.

Diabetic foot is one of the most common long term complications of diabetes. The risk of developing a foot ulcer is significantly increased when a patient presents with a callus. Callus develops due to various reasons, of which, the most important in people with diabetes is peripheral neuropathy. Motor neuropathy leads to deformity and sensory neuropathy causes lack of sensation, which results in persistent abnormal pressure on the foot. The cells of skin react to it by increasing keratinization and turns into a callus, which predisposes to foot ulceration. However, there is a lack of research in the field of callus. The link between hyperkeratosis, insulin and hyperglycaemia is not fully explored. There is also a lack of research on the relationship between genetic defects of hyperkeratosis, and the risk of developing a diabetic foot ulcer. There is scope for further research in this area, such as exploring whether development of callus is an individual risk factor, and whether glycaemic control or its treatment has any relationship with callus formation. The research around the genetic defects of hyperkeratosis may lead to identification of those, with diabetes, who may have increased risk of developing a foot ulcer.

Differences in leukocyte profile, gene expression, and metabolite status of dairy cows with or without sole ulcers.

Sole ulcers are one of the most severe pathologies causing lameness in dairy cows and are associated with abnormal behavior and impaired production performance. However, little is known about how or whether lameness caused by sole ulcers affects the cow systemically. This study compared hematology profile, leukocyte gene expression, and physiological responses [metabolite, cortisol, the endogenous steroid hormone dehydroepiandrosterone (DHEA), and haptoglobin concentrations] of cows with sole ulcers and healthy cows. Twelve clinically lame cows (lame) were identified as having at least one sole ulcer and no other disorder, and matched with a cow that had good locomotion and no disorders (sound), using days in milk, liveweight, body condition score, and diet. Blood samples were taken from all 24 cows within 24h of sole ulcer diagnosis. Leukocyte counts were obtained using an automated cell counter, cortisol and DHEA concentration by ELISA, and plasma haptoglobin, urea, total protein, creatine kinase, and glucose were analyzed on an Olympus analyzer. Expression of 16 genes associated with lameness or stress were estimated using reverse transcription-PCR. Data were analyzed using the MIXED procedure in SAS software (version 9.3; SAS Institute Inc., Cary, NC). Lame cows had a higher neutrophil percentage, a numerically lower lymphocyte percentage, and tended to have a higher neutrophil:lymphocyte ratio than sound cows. Serum cortisol and DHEA concentrations were higher in lame than in sound cows. Lame cows also tended to have higher haptoglobin and glucose levels than sound, as well as higher protein yet lower urea levels. Sound cows tended to have higher relative expression of the gene coding for colony-stimulating factor 2 than lame, but in all other cases where differences were detected in cytokine gene expression (IL-1α, IL-1ß, CXCL8, and IL-10), relative gene expression in sound cows tended to be, or was, lower than in lame. Relative expression of MMP-13, GR-α, Fas, haptoglobin, and CD62L were, or tended to be, higher in lame than sound cows. A high neutrophil:lymphocyte ratio in combination with higher cortisol levels in cows with ulcers is indicative of physiological stress. Moreover, increased DHEA and a higher cortisol:DHEA ratio, as well as a tendency for higher haptoglobin levels and increased haptoglobin mRNA expression, are indicative of systemic inflammation. Increased cytokine mRNA expression indicates activation of the immune system compared with healthy cows. Increased expression of MMP-13 mRNA has been found in cows with impaired locomotion and thus could be implicated in development of claw horn disorders.

Surveillance of risk factors for diabetic foot ulceration with particular concern to local practice.

INTRODUCTION: Diabetes mellitus is a global health problem with rising prevalence worldwide. Diabetes mellitus is a multisystem disease affects many systems and tissues. Foot problems are not uncommon with diabetes and foot ulceration is one of theses problems. Risk factors for foot ulcerations may differ from community to community based on many factors. OBJECTIVES: To determine the risk factors for diabetic foot ulceration among Saudi diabetic patients with type 2 diabetes attending primary care center. METHODOLOGY: Cross sectional study was designed. Four hundred subjects were selected randomly. Inclusion criteria were settled. Three hundred and fifty subjects (350) were participated. Especial assessment form was designed. Data was collected and analyzed using SPPS ver 14. RESULTS: Three hundred and fifty subjects were participated (57% male and 43% female). The prevalence of peripheral vascular disease was 15%, hulux vulgus 22.5%, inappropriate foot wear 41%, peripheral neuropathy 47.5%. Peripheral neuropathy and inappropriate foot wear were the commonest risk factors for foot ulceration. CONCLUSION: Peripheral neuropathy and inappropriate foot wear were the commonest risk factors for foot ulceration.

[transcutaneous oximetry--between theory and practice]. / Transkutana oksimetrija (TcPO2)--izmedu teorije i prakse.

Transcutaneous oximetry is a procedure used to measure the pressure of oxygen in tissue and to determine oxygenation level. It is essential to determine the state of microcirculation and is used to assess the necessity and level of amputation and the effect of revascularization procedures, as a predictor of wound healing and hyperbaric oxygen therapy (HBOT) effectiveness tool. The measurement is done by the application electrode measuring point and the result is measured in mm Hg. Tissue with adequate oxygen level has a value greater than 50 mm Hg. Values between 20 and 40 mm Hg are considered hypoxic, while those below 20 mm Hg indicate extreme hypoxia. In Croatia, TcPO2 is commonly used for HBOT assessment but there is the need of broader application to objectify and facilitate procedures in the care of persons with impaired microcirculation.

Increased growth factors play a role in wound healing promoted by noninvasive oxygen-ozone therapy in diabetic patients with foot ulcers.

Management of diabetic foot ulcers (DFUs) is a great challenge for clinicians. Although the oxygen-ozone treatment improves the diabetic outcome, there are few clinical trials to verify the efficacy and illuminate the underlying mechanisms of oxygen-ozone treatment on DFUs. In the present study, a total of 50 type 2 diabetic patients complicated with DFUs, Wagner stage 2~4, were randomized into control group treated by standard therapy only and ozone group treated by standard therapy plus oxygen-ozone treatment. The therapeutic effects were graded into 4 levels from grade 0 (no change) to grade 3 (wound healing). The wound sizes were measured at baseline and day 20, respectively. Tissue biopsies were performed at baseline and day 11. The expressions of vascular endothelial growth factor (VEGF), transforming growth factor-ß (TGF-ß), and platelet-derived growth factor (PDGF) proteins in the pathologic specimens were determined by immunohistochemical examinations. The effective rate of ozone group was significantly higher than that of control group (92% versus 64%, P < 0.05). The wound size reduction was significantly more in ozone group than in control group (P < 0.001). After treatment, the expressions of VEGF, TGF-ß, and PDGF proteins at day 11 were significantly higher in ozone group than in control group. Ozone therapy promotes the wound healing of DFUs via potential induction of VEGF, TGF-ß, and PDGF at early stage of the treatment. (Clinical trial registry number is ChiCTR-TRC-14004415).

Autologous G-CSF-mobilized peripheral blood CD34+ cell therapy for diabetic patients with chronic nonhealing ulcer.

Recently, animal studies have demonstrated the efficacy of endothelial progenitor cell (EPC) therapy for diabetic wound healing. Based on these preclinical studies, we performed a prospective clinical trial phase I/IIa study of autologous G-CSF-mobilized peripheral blood (PB) CD34(+) cell transplantation for nonhealing diabetic foot patients. Diabetic patients with nonhealing foot ulcers were treated with 2 × 10(7) cells of G-CSF-mobilized PB CD34(+) cells as EPC-enriched population. Safety and efficacy (wound closure and vascular perfusion) were evaluated 12 weeks posttherapy and further followed for complete wound closure and recurrence. A total of five patients were enrolled. Although minor amputation and recurrence were seen in three out of five patients, no death, other serious adverse events, or major amputation was seen following transplantation. Complete wound closure was observed at an average of 18 weeks with increased vascular perfusion in all patients. The outcomes of this prospective clinical study indicate the safety and feasibility of CD34(+) cell therapy in patients with diabetic nonhealing wounds.

Harnessing growth factors to influence wound healing.

Cutaneous wound healing is a dynamic process with the ultimate goal of restoring skin integrity. On injury to the skin, inflammatory cells, endothelial cells, fibroblasts, and keratinocytes undergo changes in gene expression and phenotype, leading to cell proliferation, migration, and differentiation. Cytokines and growth factors play an essential role in initiating and directing the phases of wound healing. These signaling peptides are produced by a variety of cells and lead to a concerted effort to restore the skin barrier function.

Oxygen tension assessment: an overlooked tool for prediction of delayed healing in a clinical setting.

Successful wound healing requires adequate transcutaneous oxygen tension (tcpO(2) ). TcpO(2) may not commonly be incorporated in clinical assessments because of variable measurement response at different sensory temperatures. This study aims to assess the relationship between changes in tcpO(2) , measured under basal (39°C) and stimulated (44°C) conditions and healing rate of chronic wounds over 4 weeks, to determine whether tcpO(2) measurement can predict delayed wound healing. TcpO(2) (Radiometer TCM400) measurements at sensor temperatures 39 and 44°C were recorded (twice, 4 weeks apart) adjacent to the ulcer site, and at a mirror image site on the contralateral leg. Ulcer outline was traced on clear acetate and perimeter and area measured (Visitrak™, Smith and Nephew). TcpO(2) measured at 44 and 39°C adjacent to all 13 wounds were lower compared to the contralateral site, significant at 44°C (P = 0·008). Significant correlation (r(2) = 0·8) occurred between wound healing rate and increased tcpO(2) at 44°C over 4 weeks. Importantly, the ratio of 39/44°C tcpO(2) , measured at the initial appointment, appeared to predict normal or delayed healing rate. TcpO(2) may provide clinicians with information regarding anticipated healing ability of wounds at the initial appointment, and hence identify wounds requiring early implementation of adjuvant therapies to accelerate healing.

Can procalcitonin predict bone infection in people with diabetes with infected foot ulcers? A pilot study.

AIMS: The diagnosis of osteomyelitis is a key step of diabetic foot management. Previous studies showed that procalcitonin (PCT), a novel infection marker, is superior to conventional infection markers in the diagnosis of diabetic foot infection. This study aimed to investigate the serum levels of PCT and other conventional infection markers in diabetic persons with and without osteomyelitis. METHODS: Twenty-four patients (18 male, mean age: 61.9±10.8 years) with infected foot ulcers were prospectively enrolled. Clinical characteristics of the wounds were noted. Blood samples were obtained for biochemical analysis. Magnetic resonance imaging of the foot was performed in all patients to diagnose osteomyelitis. RESULTS: Osteomyelitis was found in 13 of 24 (54%) patients. PCT levels were 66.7±43.5 pg/ml in patients with osteomyelitis and 58.6±35.5 pg/ml in patients without osteomyelitis. The difference did not reach statistical significance (p=0.627). Erythrocyte sedimentation rate, but not C-reactive protein and white blood cell count, was found significantly higher in patients with osteomyelitis. CONCLUSION: In this group of patients, PCT failed to discriminate patients with bone infection. Erythrocyte sedimentation rate can be used as a marker of osteomyelitis in diabetic persons.

Transfection of human HGF plasmid DNA improves limb salvage in Buerger's disease patients with critical limb ischemia.

AIM: Hepatocyte growth factor is a potent angiogenic agent. This study investigated the efficacy and safety of intramuscular injection of naked plasmid DNA encoding the human hepatocyte growth factor gene in Japanese patients with Buerger's disease and critical limb ischemia. METHODS: An open-label clinical study was performed at eight hospitals in Japan from May 2004 to April 2008. Ten patients were enrolled. They had Buerger's disease with ischemic ulcers, were not candidates for revascularization, and were unresponsive to conventional drug therapy. Treatment consisted of 8 injections (total dose: 4 mg) of hepatocyte growth factor plasmid, which were administered into the calf muscles and/or distal thigh muscles of the ischemic limbs under ultrasound guidance. Administration was done twice at an interval of 4 weeks. If there was no improvement after 2 doses, a 3rd dose could be administered. The response to treatment was evaluated from the reduction of ischemic ulcer size. RESULTS: The size of ischemic ulcers showed a decrease in 6/9 (66.7%) patients and the ulcers healed completely in 5/9 (55.6%) patients after gene therapy. Major amputation was not required. There were no deaths and no major safety concerns. CONCLUSION: Hepatocyte growth factor gene therapy is safe and effective for critical limb ischemia in patients with Buerger's disease.

Ulcerated dermatofibroma with osteoclast-like giant cells.

BACKGROUND: Ulceration and osteoclast-like giant cells are two pathological features uncommonly seen in dermatofibromas. To our knowledge, the presence of these features has not been previously described within the same lesion. METHODS: We report the clinical, histopathological and immunohistochemical findings of a 38-year-old man with an ulcerated dermatofibroma (DF) on the sole containing OLGC. COMMENTS: DF, or cutaneous fibrous histiocytoma, is a frequent dermatological lesion with many clinicopathological variants. Therefore, a correct diagnosis is not always straightforward, especially when two rare features co-exist in the same lesion. Differential diagnosis was performed with cutaneous and even non-cutaneous lesions. An explanation for the clinicopathological findings is also described.

Altered molecular mechanisms of diabetic foot ulcers.

The continuously increasing worldwide prevalence of diabetes will be accompanied by a greater incidence of diabetic foot ulcer, a complication in which many of the morphological processes involved in normal wound healing are disrupted. The highly complex and integrated process of wound healing is regulated by a large array of molecular factors. These often have overlapping functions, ensuring a certain degree of tolerance through redundancy. In diabetes, changes to the expression of a large number of molecular factors have been observed, overwhelming this inbuilt redundancy. This results in delayed healing or incomplete healing as in ulceration. Understanding the relationship between altered levels of molecular factors and the inhibited healing process in such ulcers will permit the development of targeted treatments aimed to greatly improve the quality of life of patients, at the same time helping to reduce the huge costs associated with treating this diabetic condition and its long-term consequences. This short review examines how changes in the expression of molecular factors are related to altered morphology in diabetic foot ulceration and very briefly considers treatment strategies at molecular level.

Wound closure and metabolic parameter variability in a db/db mouse model for diabetic ulcers.

BACKGROUND: Diabetic foot ulcers are a major cause of nontraumatic lower extremity amputations. Wound-healing researchers commonly use db/db mice as a model for diabetes, while the excisional wound correlates well with chronic foot ulcers. Recent clinical trials identified a correlation between glycemic control and cardiovascular complications in diabetic patients. The purpose of this study was to determine if the severity of diabetes was related to poor wound healing and the broad wound closure variability observed in diabetic db/db mice. MATERIALS AND METHODS: Adult female C57BLKS/J, db+/-, and db/db mice were anesthetized followed by creation of a 1.5 x 1.5 cm full-thickness excisional wound. Wound closure was measured on postoperative days (PODs) 1, 5, 7, 10, 14, and 21. Weight, fasting blood glucose, and fasting insulin were also measured during the study. RESULTS: By POD 21 both wild-type and db+/- mice demonstrated complete wound closure. In db/db mice open wounds were still present at POD 21. There was a broad range of percent wound closure from 24 to 81% with a mean of 55%. Despite strong correlations between diabetic parameters, there was no significant correlation between wound closure rate and severity of diabetes. CONCLUSIONS: Diabetic db/db mice exhibit a significant impairment of healing in the excisional wound model. The variability of wound closure for individual mice did not correlate with severity of obesity, hyperglycemia, hyperinsulinemia, or insulin resistance. An extensive evaluation of basic diabetes parameters does not provide significant insight into the wound-healing process in the db/db mouse model.

Basement membrane integrity and keratinization in healthy and ulcerated bovine hoof tissue.

Damage to, or deterioration of, the keratinized horn tissue of the bovine hoof claw culminates ultimately in the development of solear ulceration. We have observed abnormal keratin distribution at the site of solear ulceration in the bovine claw that may be due to alteration of the positional cues of the keratinocytes. In this study we have characterized key cell biological changes associated with ulceration in the claw that may precipitate abnormal keratinization. Loss of basement membrane at sites of ulceration was found by immunofluorescent detection of laminin and integrins. In other tissues, basement membrane breakdown results from degradation by matrix metalloproteinases (MMPs). Similarly, elevated levels of MMPs 2 and 9 were observed in ulcerated bovine claw tissue both by zymography and, quantitatively, by assay of enzyme activity. In the sole of claws that contained an ulcer, tissue distal to the ulcer site also had elevated MMP 2 when compared with healthy sole tissue from the same animals, as did sole tissue of claws recovering from ulceration. Tissue inhibitor of metalloproteinase 2 (TIMP 2) was detected by ELISA in healthy tissue. TIMP 2 tended to be lower in diseased tissue distal to ulcer sites, and was significantly lower in ulcerated tissue. MMP 2 was located by immunofluorescence in the dermal and basal epidermal region of sole tissue, in the region of the basement membrane. Increased punctate staining of material in the dermis was associated with ulcerated material. ELISA of TIMP 2 in tissue extracts enriched for dermis or epidermis confirmed that the inhibitor was located predominantly in the dermis. To investigate a possible causal relationship between basement membrane anchorage and epidermal keratinization, the effect of function-blocking antibodies to laminins and integrins was tested in tissue explant cultures prepared from healthy sole tissue. Anti-integrin antibody treatment had no effect on either protein or DNA synthesis. In contrast, in the presence of anti-laminin antibody, protein synthesis was decreased in a concentration-dependent manner, a significant effect being observed at the highest concentration after treatment for 24 h. At this concentration, DNA synthesis was also decreased after 48 h of culture, an effect that may be relevant to a hibernal reduction in claw cell turnover, and the associated seasonal vulnerability of cows to claw damage. The results provide evidence for basement membrane disruption at ulcer sites, and an increased potential for disruption in the diseased claw, and a causal link between this and abnormal epidermal keratinization. Basement membrane disruption is in turn associated with reciprocal changes in MMPs and their inhibitors, favouring extracellular proteolysis. Whether MMP activation is the primary cause of dermal-epidermal deterioration and, if so, how MMP activation is triggered, remains to be determined.

Heparin-like inhibitory activity to fibroblast growth factor-2 in wound fluids of patients with chronic skin ulcers and its modulation during wound healing.

Fibroblast growth factors are potent mitogens and angiogenic factors which play a critical role in wound healing. Fibroblast growth factors require heparan sulfates as cofactors in order to activate their cognate receptors and exert their cellular and biological effects. Heparan sulfates were extracted from wound fluids of 5 patients with chronic diabetic foot ulcers or chronic venous stasis ulcers and tested for their capacity to modulate fibroblast growth factor-receptor binding, during the course of the ulcers' resolution, until complete healing (3-8 months). Total heparan sulfates concentration measured as iduronic acid equivalents, decreased in wound fluids from 1.1 +/-0.3 microg/ml to 0.26 +/-0.1 microg/ml as wound healing progressed. These heparan sulfates exhibited a predominant inhibitory effect on fibroblast growth factor-2 binding to fibroblast growth factor receptor-1, when tested in cells deficient in cell surface heparan sulfates. During wound healing, there was a marked decrease in the relative inhibitory activity of the extracted heparan sulfates on fibroblast growth factor-2-receptor binding. Heparan sulfates extracted from chronic skin ulcers of different etiologies such as diabetic foot or chronic venous stasis ulcers showed the same pattern of alternating balance in heparan sulfates mediated activity. The presence of fibroblast growth factor inhibitory factors which possess heparin-like activity in fluids of chronic skin ulcers and their ability to modulate fibroblast growth factor-receptor activity throughout the process of wound healing, may significantly contribute to the mechanism of chronicity. Treatments to counter this inhibition may offer new possibilities for healing chronic wounds.

Synthesis and distribution of cytokeratins in healthy and ulcerated bovine claw epidermis.

Keratinization of the epidermal cells of the bovine claw generates the horn that gives the tissue its mechanical strength. Disruption of keratinization is likely to have a detrimental effect on the strength and integrity of the horn, and could lead to solar lesions and lameness. As part of a wider investigation of the cell biological causes of lameness in dairy animals, we have compared keratin synthesis and distribution in healthy bovine claw tissue with those in hooves with solar ulcers. Protein synthesis was measured by [35S]-labelled amino acid incorporation in claw tissue explant cultures. [35S]-labelled protein synthesis was higher in tissue from diseased claws than in healthy claws, and highest at the ulcer site. The identity of proteins synthesised in vitro did not differ between healthy and diseased tissue. DNA synthesis indicative of cell proliferation was also elevated in diseased tissue. Immunoblotting after one- or two-dimensional electrophoresis showed cytokeratins (CK) 4, 5/6, 10 and 14 to be amongst those expressed in healthy claw tissue. The relative abundance of these keratins was not altered in healthy regions of ulcerated hooves, nor at the ulcer site, but CK16, not usually found in healthy tissue, was detected in the sole of diseased claws. CK5/6 and CK14 were shown by immunohistochemistry to be present in the basal epidermis of healthy tissue, whereas CK10 was found in supra-basal layers. In healthy tissue from ulcerated claws, this distribution was unaltered, but at the site of solar ulcers, CK5/6 and CK14 were each found in both basal and supra-basal epidermis. The study suggests that solar ulceration of the bovine claw is not associated with gross alteration in the keratin composition of the tissue, but causes abnormal distribution of cytokeratins, perhaps as a result of loss of positional cues from the basement membrane. Ulceration did, however, stimulate cell repair involving epidermal protein synthesis (including keratins), and keratinocyte proliferation.