An investigation of the equine epidermal growth factor system during hyperinsulinemic laminitis.

Equine laminitis is a disease of the digital epidermal lamellae typified by epidermal cell proliferation and structural collapse. Most commonly the disease is caused by hyperinsulinemia, although the pathogenesis is incompletely understood. Insulin can activate the epidermal growth factor (EGF) system in other species and the present study tested the hypothesis that upregulation of EGF receptor (EGFR) signalling is a key factor in laminitis pathophysiology. First, we examined lamellar tissue from healthy Standardbred horses and those with induced hyperinsulinemia and laminitis for EGFR distribution and quantity using immunostaining and gene expression, respectively. Phosphorylation of EGFR was also quantified. Next, plasma EGF concentrations were compared in healthy and insulin-infused horses, and in healthy and insulin-dysregulated ponies before and after feeding. The EGFR were localised to the secondary epidermal lamellae, with stronger staining in parabasal, rather than basal, cells. No change in EGFR gene expression occurred with laminitis, although the receptor showed some phosphorylation. No difference was seen in EGF concentrations in horses, but in insulin-dysregulated ponies mean, post-prandial EGF concentrations were almost three times higher than in healthy ponies (274 ± 90 vs. 97.4 ± 20.9 pg/mL, P = 0.05). Although the EGFR does not appear to play a major pathogenic role in hyperinsulinemic laminitis, the significance of increased EGF in insulin-dysregulated ponies deserves further investigation.

Plasma concentrations of inflammatory markers in previously laminitic ponies.

REASONS FOR PERFORMING STUDY: The mechanisms underlying individual animal predisposition to pasture-associated laminitis remain unclear; however, chronic inflammation is implicated. OBJECTIVES: To identify differences in the inflammatory profile of a group of previously laminitic ponies compared with control animals at pasture in late spring and winter. METHODS: Previously laminitic (PL; n = 38 and 42) and nonlaminitic control ponies (NL; n = 41 and 39) were sampled in late spring and winter. Body condition score, height, weight and crest height and thickness were measured. Plasma concentrations of tumour necrosis factor-α, serum amyloid A, haptoglobin, insulin, adiponectin, triglyceride, fibrinogen, interleukin-17, interleukin-4 and interferon-γ were assayed by validated/standard methods. Factors independently associated with each cytokine were determined by multivariate analysis. RESULTS: Plasma [adiponectin] was significantly influenced by laminitis status, being lower in PL (median [interquartile range] 2.1[1.4-3.2] µg/l) than in NL ponies (3.4 [2.6-4.1] µg/l; P<0.0001). No other cytokines or inflammatory markers were associated with laminitis status. Plasma fibrinogen and serum amyloid A concentrations were significantly (P = 0.04 and P = 0.01) higher in geldings (3.5 [3.0-4.0] g/l; 2.2 [0.5-3.6] mg/l) than in mares (3.0 [3.0-4.0] g/l; 1.5 [0.4-2.1] mg/l) and significantly (P = 0.04 and P<0.001) higher in winter (3.5 [3.0-4.0] g/l; 2.5 [0.9-3.6] mg/l) than in late spring (3.0 [3.0-3.5] g/l; 1.1 [0.3-1.9] mg/l). Serum haptoglobin concentration showed the same significant (P<0.001) seasonal difference (winter 2.1 [1.6-2.6 g/l; late spring 1.8 1.4-2.4 g/l) and was significantly (P = 0.01) inversely associated with weight. Serum interleukin-4 concentration was significantly (P<0.0001) higher in winter (2.0 [1.2-3.0] ng/l) than in late spring (0.0 [0.0-0.0] ng/l). Serum insulin concentration was significantly (P = 0.02) influenced by season (winter 31.7 [9.6-43.5] miu/l; late spring 84.0 [7.0-131.0] miu/l). Plasma triglyceride concentration was significantly (P = 0.02) higher in PL (0.5 [0.3-0.7] mmol/l) than in NL ponies (0.4 [0.2-0.5] mmol/l). CONCLUSIONS: There were significant effects of season, gender and bodyweight on a number of proinflammatory mediators or markers of inflammation. The only marker influenced by laminitis status was adiponectin, and concentrations of this anti-inflammatory marker were lower in previously laminitic animals. POTENTIAL RELEVANCE: Recurrent laminitis may be associated with reduced anti-inflammatory capacity rather than a proinflammatory state.

Effects of continuous or intermittent lipopolysaccharide administration for 48 hours on the systemic inflammatory response in horses.

OBJECTIVE: To determine whether the method of lipopolysaccharide (LPS) administration (intermittent vs continuous) affects the magnitude and duration of the systemic inflammatory response in horses and whether prolonged (48 hours) endotoxemia induces laminitis. ANIMALS: 12 healthy adult horses (10 mares and 2 geldings). PROCEDURES: Horses were randomly assigned to receive LPS (total dose, 80 µg; n = 4) or saline (0.9% NaCl) solution (80 mL/h; 4) via constant rate infusion or 8 bolus IV injections of LPS (10 µg, q 6 h;4) during a 48-hour period. Physical examinations were performed every 4 hours, inflammatory cytokine gene expression was determined for blood samples obtained every 8 hours, and IV glucose tolerance tests were performed. RESULTS: All LPS-treated horses had signs of depression and mild colic; those signs abated as the study progressed. Administration of LPS increased expression of interleukin-1ß, interleukin-6, and interleukin-8, but results were not significantly different between LPS treatment groups. Cytokine expression was significantly higher on the first day versus the second day of LPS treatment. Interleukin-1ß expression was positively correlated with rectal temperature and expression of other cytokines. Glucose and insulin dynamics for both LPS groups combined did not differ significantly from those of the saline solution group. Signs of laminitis were not detected in any of the horses. CONCLUSIONS AND CLINICAL RELEVANCE: Horses developed LPS tolerance within approximately 24 hours after administration was started, and the method of LPS administration did not affect the magnitude or duration of systemic inflammation. Laminitis was not induced in horses.

Serum haptoglobin concentrations in dairy cattle with lameness due to claw disorders.

In cattle, elevated blood serum concentrations of haptoglobin, an acute phase protein, have been demonstrated in association with several diseases, but not with lameness. Serum haptoglobin was measured in 60 Holstein dairy cattle diagnosed with lameness due to four claw disorders, pododermatitis septica (PS; n=41), pododermatitis circumscripta (PC; n=8), interdigital necrobacillosis (IN; n=7), papillomatous digital dermatitis (PDD; n=4). Haptoglobin was measured on day 1 (0-3 days after lameness was observed but before treatment) and on days 3 and 5. A total of 10 healthy cows served as controls (haptoglobin values <1.0 mg/dL). Each of the claw disorders was associated with elevated haptoglobin on day 1 (PS, PC, IN and PDD: 65.9%, 37.5%, 71.4% and 25.0%, respectively). Trimming and antibiotic treatment led to a reduction in the number of PS and IN cows with increased haptoglobin concentrations, respectively (P<0.05), but trimming did not lead to any reduction in cows with PC. The study showed that lameness due to claw disorders can be associated with a systemic acute phase response and elevated serum haptoglobin in dairy cattle. Based on the course of haptoglobin, treatments seemed effective for all claw disorders except for PC.

Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis.

The link between the fermentation of carbohydrate in the equine large intestine and the development of acute laminitis is poorly understood. Absorption of endotoxin (lipopolysaccharide; LPS) into the plasma has been observed in one experimental model of laminitis, but does not cause laminitis when administered alone. Thus, the potential role of endotoxin is unclear. Platelet activation has previously been demonstrated in the developmental stage of laminitis. Equine platelets are more sensitive than leukocytes to activation by endotoxin, and can be activated directly by LPS in the low pg/ml range, activating p38 MAP kinase and releasing serotonin (5-HT) and thromboxane. The objectives of this study were firstly to determine whether endotoxin and platelet activation could be measured in the plasma of horses in the developmental phase of laminitis induced with oligofructose. Secondly, the time course of events involving platelet activation and platelet-derived vasoactive mediator production was investigated. Laminitis was induced in six Standardbred horses by the administration of 10 g/kg bwt of oligofructose. Plasma samples were obtained every 4h, and platelet pellets were obtained by centrifugation. LPS was measured using a kinetic limulus amebocyte lysate assay, and platelet activation was assessed by Western blotting for the phosphorylated form of p38 MAP kinase. Plasma 5-HT was assayed by HPLC with electrochemical detection and thromboxane B(2) was measured by radioimmunoassay. Clinical signs of laminitis and histopathologic changes were observed in lamellar sections from five of the six horses. Onset of lameness was between 20 and 30 h after the administration of oligofructose. LPS increased above the limit of detection (0.6 pg/ml) to reach a peak of 2.4+/-1.0 pg/ml at 8 h. TNFalpha was also detectable in the plasma from 12 to 24 h. There was a time-dependent increase in platelet p38 MAPK phosphorylation, which peaked at approximately 12 h (3.8+/-1.3 fold increase); plasma 5-HT and thromboxane increased steadily after this time (2.9+/-0.6 and 11.3+/-5.0 fold increases, respectively). These data indicate that small quantities of endotoxin may move into the circulation from the large intestine after the sharp decrease in pH that occurs as a result of carbohydrate fermentation. Correlating these findings with in vitro studies suggests that LPS may primarily activate platelets, leading indirectly to the activation of leukocytes. Therefore, endotoxin may contribute in the initiation of the early inflammatory changes observed in experimental models of acute laminitis.

Effects of intramuscular administration of acepromazine on palmar digital blood flow, palmar digital arterial pressure, transverse facial arterial pressure, and packed cell volume in clinically healthy, conscious horses.

OBJECTIVE: To determine the magnitude and duration of effects of acepromazine administered intramuscularly (IM) on digital and systemic hemodynamic variables in clinically healthy horses. STUDY DESIGN: Experimental study. ANIMALS: Healthy adult horses (n=12). Methods- An ultrasonic Doppler flow probe was surgically implanted around the medial palmar digital artery before the study. Catheters were inserted in the transverse facial artery, lateral palmar digital artery, and jugular vein. A treatment group (n=6) was administered 0.04 mg/kg body weight of acepromazine IM; control horses (n=6) were administered an equivalent volume of saline IM. Palmar digital blood flow, and digital and facial arterial pressures were measured at baseline and for 6 hours after administration. Venous blood was collected for measurement of packed cell volume (PCV). RESULTS: Horses administered acepromazine had significantly lower facial arterial pressure compared with control horses administered saline. Palmar digital arterial blood flow in acepromazine-treated horses was not significantly different from that in control horses but increased significantly post-administration, compared with the respective baseline values for acepromazine-treated horses. PCV significantly decreased in horses administered acepromazine compared with their respective baseline value. CONCLUSION: IM acepromazine causes hypotension and increases palmar digital blood flow over time but the magnitude of the effect on digital blood flow was not sufficient to yield differences compared with saline-treated horses. CLINICAL RELEVANCE: IM acepromazine has a modest effect on palmar digital blood flow, facial arterial pressures and PCV in healthy horses with minimal sedation.

Effects of topical nitroglycerine patches and ointment on digital venous plasma nitric oxide concentrations and digital blood flow in healthy conscious horses.

OBJECTIVE: To measure and compare palmar digital venous plasma nitric oxide (NO) concentrations and digital arterial blood flow after application of topical nitroglycerine (NTG). STUDY DESIGN: Experimental study. ANIMALS: Healthy adult horses (n=8). METHODS: Digital blood flow was measured by an ultrasonic Doppler flow probe surgically implanted around the medial palmar digital artery. Blood was collected from a catheter placed in the medial palmar digital vein for quantification of NO. NTG patches, NTG ointment or control patches were placed over the palmar digital vessels at the level of the fetlock. Two horses had an intra-arterial infusion of an NTG solution into the medial palmar digital artery in a pilot study. RESULTS: Digital arterial blood flow did not change significantly with application of the NTG patches, NTG ointment, or control patches. There were no statistically significant or biologically important changes in digital venous NO concentrations across time or between treated and control horses. In the pilot study, digital arterial blood flow and palmar digital venous NO concentrations increased with intra-arterial infusion of NTG. CONCLUSIONS: In clinically healthy horses, digital arterial blood flow and digital venous plasma NO concentrations did not change significantly with application of the NTG patches/ointment. These treatments are unlikely to have an effect on the digital vasculature of laminitic horses, however, further investigation is warranted. CLINICAL RELEVANCE: Although NTG patches have been used as a method of decreasing vasomotor tone and improving digital blood flow in horses with laminitis, this study provides evidence in healthy conscious horses that this treatment is not effective in altering digital blood flow.

Endotoxin-induced digital vasoconstriction in horses: associated changes in plasma concentrations of vasoconstrictor mediators.

REASONS FOR PERFORMING STUDY: Lipopolysaccharide (LPS) infusion reduces digital perfusion, but the mediators responsible remain undetermined. OBJECTIVES: To identify vasoconstrictor mediators released following LPS infusion and relate their appearance in plasma to digital blood flow alterations. METHODS: Blood flow in the lateral digital vessels of 6 Thoroughbred horses, following a 30 min infusion of LPS (E. coli 055:B5; 30 ng/kg), was measured using Doppler ultrasonography. Concomitant measurements of hoof wall and coronary band surface temperatures (HWST and CBST) were made. Serial blood samples were collected and plasma LPS, tumour necrosis factor alpha (TNFalpha), 5-HT, thromboxane B2 (TxB2) and endothelin measured. RESULTS: Plasma LPS concentrations reached a maximum of 13.2 pg/ml during the infusion, followed by an increase in plasma TNFalpha concentration. Digital arterial and venous blood flow decreased by 43 and 63%, respectively; HWST and CBST similarly decreased. Systemic blood pressure remained unaltered. Plasma concentrations of TxB2 and 5-HT increased, coinciding with the onset of digital hypoperfusion. Plasma endothelin concentrations remained unchanged. CONCLUSIONS: The temporal relationship between the onset of digital hypoperfusion and increases in plasma 5-HT and TxB2 concentrations is consistent with these platelet-derived mediators being associated with LPS-induced laminitis. POTENTIAL RELEVANCE: These experimental data support the use of anti-platelet therapy in the prevention of laminitis associated with endotoxaemic conditions.

Pedal interdigital condylomata lata: a rare sign of secondary syphilis.

BACKGROUND: Condylomata lata of the toe webs are uncommon manifestations of secondary syphilis. Considering the recent decline in the incidence of syphilis in the United States, such lesions are likely to present infrequently. In some cases, this phenomenon may be the only physical sign of syphilis; therefore, it is important that a high index of suspicion is maintained when evaluating toe web lesions in patients at epidemiologic risk for syphilis. GOALS: A case of secondary syphilis presenting solely with interdigital condyloma lata in the toe web spaces is reported, and similar cases reported in the literature are reviewed. STUDY DESIGN: This article documents the diagnosis of secondary syphilis based on a positive serology in conjunction with the development of interdigital condyloma lata as the only physical finding suggestive of lues. RESULTS: The latter lesions resolved after appropriate, adequate antibiotic therapy. CONCLUSIONS: A case of condylomata lata of the toe webs without other pertinent physical findings is presented. Analogous to lesions typically seen in the anogenital region, moist exophytic toe web plaques may represent condyloma lata and thereby be a sign of secondary syphilis. The differential diagnosis includes tinea pedis, erythrasma, macerated corns, verrucae, and several tropical mycoses (chromomycosis, mycetoma).

Remitting seronegative symmetrical synovitis with pitting edema (RS3PE) syndrome: a review of the literature and a report of three cases.

Remitting seronegative symmetrical synovitis with pitting edema (RS3PE) syndrome is a disease that commonly produces symmetrical synovitis and swelling of both the upper and lower extremities. It generally involves the wrists, hands, feet, and ankles of the affected individual. This syndrome most often resembles that of polymyalgia rheumatica and rheumatoid arthritis and usually affects elderly Caucasian males. Serological testing is typically negative except for a mild to moderate elevation of the erythrocyte sedimentation rate. The HLA-B7 phenotype is present in approximately 50% of patients with this syndrome. Treatment of RS3PE syndrome is heralded by the predictable response to low-dose corticosteroid or hydroxychloroquine therapy. There has been no previous mention of this condition in the podiatric literature. Presented below is a review of this syndrome and three case studies.

Paraneoplastic remitting seronegative symmetrical synovitis with pitting edema.

Remitting seronegative symmetrical synovitis with pitting edema (RS3PE) is a syndrome that may be associated with many conditions, including malignancy. Three further cases of paraneoplastic RS3PE are described and the literature is reviewed. Paraneoplastic RS3PE is more frequently associated with solid tumors, in particular adenocarcinoma. The two clinical characteristics suggestive of paraneoplastic RS3PE are systemic sign/symptoms and the poor response to corticosteroid therapy.

Review and consideration of coagulopathies.

Blood components bring nutrients to the tissues, remove wastes, provide defenses against microorganisms and foreign bodies, and retain sufficient fluidity to keep the system functioning properly. Coagulation factors are also present in the blood as part of a sensitive system that stems the flow of blood from a wound and maintains homeostasis. These factors include anuclear platelets that originate from megakaryocytes in the bone marrow, as well as soluble factors in precursor form, and calcium ion. Faults in single or multiple parts of this system, which can be determined in the patient's history, may result in bleeding problems that may be clinically relevant. Clarification of coagulation defects requires cooperation with a hematologist before medication that affects the coagulation system is prescribed or before any procedures that may result in bleeding are undertaken.

The foot and systemic disease. Differential diagnoses.

The discipline of medicine is truly a combination of both science and art, and nowhere is this more true than in the practice of differential diagnosis. From the initial clinic visit, when the patient presents with a nonspecific symptom, until the final treatment is rendered, the clinician must constantly and critically evaluate his or her diagnosis. This process can be expedited by using a consistent and comprehensive methodology that reminds the clinician to entertain the full spectrum of causes. Two such methodologies have been presented in this article. It is important, however, to note that the systems presented here are by no means the sole, or even best, methods. The clinician should experiment and discover what works best for him or her. Any system is ideal if it allows the clinician to consistently derive the correct diagnosis in a short amount of time. After all, the most important part of any treatment is the proper diagnosis.

Evaluation of platelet activation and platelet-neutrophil aggregates in ponies with alimentary laminitis.

OBJECTIVES: To determine whether platelets are hyperaggregable or form platelet-neutrophil aggregates during the prodromal stages of acute laminitis of ponies. ANIMALS: Healthy adult ponies: 8 experimental and 6 control. PROCEDURES: Acute laminitis was induced by oral administration of corn starch and wood flour to 8 ponies, and indices of platelet activation were evaluated. Blood samples were collected before and at 4, 8, 12, 24, 28, and 32 hours after carbohydrate administration, and PCV, total plasma protein concentration, platelet count, activated clotting time, whole blood recalcification time, spontaneous platelet aggregation, ex vivo platelet aggregation responses, and platelet-neutrophil aggregates were determined. When lameness was first detected, ponies were euthanatized and arteriography and histologic examination of hooves were performed. RESULTS: Carbohydrate overload was associated with hyperaggregability of platelets throughout the prodromal stages of laminitis and increased numbers of platelet-neutrophil aggregates. Reduction of blood supply to affected hooves was variable, and blood clots were found in 6 of 11 laminitis-affected hooves. CONCLUSIONS: Platelets were hyperaggregable throughout the prodromal stages of carbohydrate-induced laminitis and formed platelet-neutrophil aggregates. Platelet-neutrophil aggregates may initiate or contribute to development of acute laminitis. CLINICAL RELEVANCE: Anti-platelet therapy may be useful for treatment of acute alimentary laminitis in horses.

Unusual localisation of tophaceous gout. A report of four cases and review of the literature.

We report four cases with very unusual manifestations of tophaceous gout. All patients were male, aged between 29 and 67 years. Only one patient had an acute gouty arthritis in his medical history, whereas the other three had never developed clinical symptoms from their hyperuricemia. Two patients had gout tophi in the patella, while the others showed gout tophi in the space of bipartite bones, one in a bipartite patella and one in a tripartite sesamoid bone of the first toe. To our knowledge, gout tophi in the space of bi- or tripartite bones has never been described before.

HPV-1 capsids expressed in vitro detect human serum antibodies associated with foot warts.

Seventy-eight human serum samples were screened for their ability to immunoprecipitate the major (L1) and minor (L2) capsid proteins of HPV1. The L1 and L2 proteins expressed from a recombinant vaccinia virus were able to self assemble into capsids in the nuclei of infected cells. Twenty-eight of the sera precipitated the L1 protein. The L1 protein was only precipitated when the protein was native, denatured protein was not precipitated by the human sera. None of the sera precipitated the L2 protein. The assay demonstrated a significant association between the ability of sera to precipitate the L1 protein and a clinical history of foot warts (P = 0.001). The same serum samples were tested by immunoblots using L1 and L2-trpE bacterial fusion proteins. It was found that almost half of the sera reacted with the L2 fusion protein and few reacted with the L1 protein. Immunoblot results did not correlate well with a clinical history of foot warts (P = 0.7), suggesting that immune precipitation of capsid proteins may be superior to immunoblotting for serodiagnosis of HPV infections.

Transcutaneous oxygen tension in the dysvascular foot with infection.

Eight adult insulin-requiring diabetics with peripheral vascular disease were admitted with foot infection and signs of systemic sepsis. Transcutaneous oxygen tension was measured at the foot and ankle prior to surgery. None of the values were sufficient to support wound healing. Four of the patients underwent open ray resection and four open midfoot amputation. After resolution of the local infections, transcutaneous oxygen tensions were repeated. Seven of the eight patients exhibited an appreciable increase in the value following decompression of the foot infection, sufficient to support wound healing.

Erythromelalgia in a patient with thrombotic thrombocytopenic purpura.

Erythromelalgia is characterized by paroxysmal burning pain and redness of the distal extremities; primary and secondary forms exist. Secondary erythromelalgia is most commonly associated with essential thrombocythemia and polycythemia vera, and the pathogenic mechanism is thought to be platelet aggregation. Thrombotic thrombocytopenic purpura is characterized by thrombocytopenia, microangiopathic anemia, renal failure, and fluctuating neurologic signs and symptoms. The pathogenic mechanism suggested for thrombotic thrombocytopenic purpura is diffuse platelet aggregation. We describe a female patient in whom thrombotic thrombocytopenic purpura was associated with erythromelalgia. This has not been previously reported.

Comparison of serum and tissue antibiotic levels in diabetes-related foot infections.

Parenteral antibiotics are used as an adjunct to amputation or operative debridement for patients with diabetes who require emergency surgery for a septic foot. In 26 patients with a diabetes-related foot infection, one dose of various intravenous antibiotic regimens (gentamicin and clindamycin, ticarcillin/clavulanate, ampicillin/sulbactam) was administered during the hour before the procedure, and assays were performed to measure the antibiotic serum and tissue levels at the time of surgical debridement. Aerobic and anaerobic cultures were performed on infected tissue. The 172 bacterial isolates, including 95 aerobes and 77 anaerobes, (6.6 isolates per patient) underwent antibiotic susceptibility testing. Antibiotic levels were calculated by biologic assay from serum and tissue biopsies from the viable margins of the surgical site, which subsequently healed primarily or supported a split-thickness skin graft. Sixteen of the patients achieved therapeutic serum levels, and therapeutic tissue levels were reached in six patients at the time of surgery. A significantly lower number of patients had therapeutic tissue levels compared to serum levels (p less than 0.01, chi square). Initial intravenous antibiotic administration provides inadequate tissue concentrations for treating foot infections in patients with diabetes. Adequate serum antibiotic levels do not reflect therapeutic tissue antibiotic levels at the surgical margins in this group of patients.