Prevalence and antimicrobial susceptibility of bacterial isolates from horses with synovial sepsis: A cross-sectional study of 95 cases.

Bacterial culture and antimicrobial susceptibility testing of septic synovial samples allows instigation of targeted antimicrobial therapy; however, bacterial culture takes more than 24 h and has low sensitivity. This study aimed to identify the most frequently cultured bacteria and their antimicrobial susceptibility profile from septic synovial samples in our referral equine hospital, to allow recommendations regarding appropriate initial antimicrobial therapy prior to culture results. Hospital records for all horses with synovial sepsis and a synovial sample submitted to the microbiology laboratory between 2004 and 2013 were retrieved (n= 379 samples). One horse had positive cultures from more than one synovial structure, and two horses had positive cultures obtained from repeat samples. Overall, 114 bacterial isolates were obtained. Gram-positive bacteria were isolated in 75% of cases, of which 22% were haemolytic Staphylococcus spp., and 52% were Staphylococcus aureus including two multidrug-resistant isolates. Gram-negative bacteria were isolated from 25% of cases. Anaerobic Clostridium spp. was isolated in 3% of cases. Of the first line antimicrobials, oxytetracycline and doxycycline were effective against 70-100% of the Gram-positive bacteria and 20-100% of the Gram-negative organisms, whilst trimethoprim-sulphamethoxazole and gentamicin efficacy ranged between 50% and 88% for both Gram-positive and Gram-negative bacteria. Of the equine protected antimicrobials, ceftiofur was effective against 70-90% of all bacterial isolates whilst 80% of isolates were susceptible to enrofloxacin. These results indicate that tetracyclines, trimethoprim-sulphamethoxazole or gentamicin may be suitable first-line antimicrobials for treatment of synovial sepsis cases while awaiting laboratory results, findings which support current recommendations for antimicrobial stewardship in equine medicine.

Exploring lay perceptions of the causes of crib-biting/windsucking behaviour in horses.

REASONS FOR PERFORMING STUDY: Crib-biting/windsucking behaviour has important consequences for equine health and welfare. Lay perceptions of health and illness are of interest to medical sociologists, providing important information to medical practitioners, but have infrequently been applied in veterinary research. OBJECTIVES: To demonstrate how lay epidemiology can be applied within veterinary research by exploring the lay perceptions regarding the causes of crib-biting/windsucking behaviour in horses. METHODS: Informants were recruited from professional and amateur horse owners who had or had not owned/cared for a horse that exhibited crib-biting/windsucking behaviour. In-depth interviews were used to examine perceptions about the development of this behaviour within each group until a 'saturation' of themes emerged. RESULTS: The main themes that emerged as causes of crib-biting/windsucking behaviour were 'boredom', 'stress' and 'habit/addiction'. In the group of owners/carers who did not have direct experience of this type of behaviour, 'copying' from other horses emerged as a strong theme and they stated that they would not wish to own a crib-biting/windsucking horse. In contrast, those who had direct experience of horses demonstrating this behaviour did not believe copying was a cause based on their own observations and would not be put off purchasing or caring for another horse displaying this behaviour. CONCLUSIONS: Perceptions about what causes crib-biting/windsucking was influenced by whether or not informants had personal experience of horses demonstrating this behaviour. The three main themes that emerged have some justification based on current research and highlight the need for further investigation into the underlying pathophysiology of crib-biting/windsucking behaviour. POTENTIAL RELEVANCE: Qualitative approaches to health, disease and behaviour have an important role in the medical field and are applicable to veterinary research.

Fluoxetine-induced change in rat brain expression of brain-derived neurotrophic factor varies depending on length of treatment.

Recent studies indicate that brain-derived neurotrophic factor (BDNF) may be implicated in the clinical action of antidepressant drugs. Repeated (2-3 weeks) administration of antidepressant drugs increases BDNF gene expression. The onset of this response as well as concomitant effects on the corresponding BDNF protein is however, unclear. The present study investigated the effects of acute and chronic administration of the selective serotonin reuptake inhibitor, fluoxetine (10mg/kg p.o.), upon regional rat brain levels of BDNF mRNA and protein expression. To improve the clinical significance of the study, fluoxetine was administered orally and mRNA and protein levels were determined ex vivo using the techniques of in situ hybridisation histochemistry and immunocytochemistry respectively. Direct measurement of BDNF protein was also carried out using enzyme-linked immunosorbent assay (ELISA). Four days of once daily oral administration of fluoxetine induced decreases in BDNF mRNA (hippocampus, medial habenular and paraventricular thalamic nuclei). Whilst 7 days of treatment showed a non-significant increase in BDNF mRNA, there were marked and region-specific increases following 14 days of treatment. BDNF protein levels remained unaltered until 21 days of fluoxetine treatment, when the numbers of BDNF immunoreactive cells were increased, reaching significance in the pyramidal cell layer of CA1 and CA3 regions of Ammon's horn (CA1 and CA3) but not in the other sub-regions of the hippocampus. Indicative of the highly regional change within the hippocampus, the ELISA method failed to demonstrate significant up-regulation at 21 days, measuring levels of BDNF protein in the whole hippocampus. In contrast to the detected time dependent and biphasic response of the BDNF gene, activity-regulated, cytoskeletal-associated protein (Arc) mRNA showed a gradual increase during the 14-day course of treatment. The results presented here show that BDNF is expressed differentially depending on length of fluoxetine administration, which could contribute in explaining the slow onset of antidepressant activity observed with selective serotonin reuptake inhibitors.

A mutation in alpha-tropomyosin(slow) affects muscle strength, maturation and hypertrophy in a mouse model for nemaline myopathy.

Nemaline myopathy is a hereditary disease of skeletal muscle defined by a distinct pathology of electron-dense accumulations within the sarcomeric units called rods, muscle weakness and, in most cases, a slow oxidative (type 1) fiber predominance. We generated a transgenic mouse model to study this disorder by expressing an autosomal dominant mutant of alpha-tropomyosin(slow) previously identified in a human cohort. Rods were found in all muscles, but to varying extents which did not correlate with the amount of mutant protein present. In addition, a pathological feature not commonly associated with this disorder, cytoplasmic bodies, was found in the mouse and subsequently identified in human samples. Muscle weakness is a major feature of this disease and was examined with respect to fiber composition, degree of rod-containing fibers, fiber mechanics and fiber diameter. Hypertrophy of fast, glycolytic (type 2B) fibers was apparent at 2 months of age. Muscle weakness was apparent in mice at 5-6 months of age, mimicking the late onset observed in humans with this mutation. The late onset did not correlate with observed changes in fiber type and rod pathology. Rather, the onset of muscle weakness correlates with an age-related decrease in fiber diameter and suggests that early onset is prevented by hypertrophy of fast, glycolytic fibers. We suggest that the clinical phenotype is precipitated by a failure of the hypertrophy to persist and therefore compensate for muscle weakness.

Novel small molecule alpha v integrin antagonists: comparative anti-cancer efficacy with known angiogenesis inhibitors.

Recent evidence supports the involvement of integrins in angiogenesis: blockade of alpha v beta 3 and alpha v beta 5 integrins disrupts angiogenesis leading to decreased blood vessel formation and hence decreased tumor growth. We hypothesized that av antagonists could inhibit tumor growth in tumor cells devoid of alpha v beta 3 integrins. We evaluated SM256 and SD983, novel small molecules that are specific av antagonists in mouse models of angiogenesis and tumorigenesis, and compared them with standards: TNP470, a fumagillin analog in the clinic, and flavopiridol, a cell cycle kinase inhibitor. In vitro SM256 was a selective alpha v beta 3 inhibitor with an IC50 = 4nM, and the affinity of SD983 against the mouse endothelial alpha v beta 3 integrin yielded an IC50 = 2nM and an IC50 = 54nM against alpha v beta 5. In the mouse Matrigel model of angiogenesis SM256 decreased blood vessel formation (hemoglobin content) with an ED50 = 0.055 ug/kg/day, tenfold more potent than TNP470. SG545, an ester of SD983, decreased blood vessel formation with an ED50 = 6 ug/kg/day, while flavopiridol ED50 = 18 ug/kg/day. In the mouse xenograft model, using human colon carcinoma RKO cells that do not express alpha v beta 3 but express alpha v beta 5, tumor growth was inhibited by SG545 (10 mg/kg/day) and flavopiridol (5 mg/kg/every other day) 40% and 70%, respectively (p < 0.05). Although the proliferative index (measured by BrdU incorporation) was not significantly changed with SM256, SG545 or flavopiridol (29-32%), the apoptotic index increased significantly (p < 0.05) in the SM256 and SG545-treated groups (2.3-2.7%) compared with controls (1.1%), suggesting increased cell death contributed to decreased tumor volumes. Neovascularization decreased with SM256 and SG545 treatment. The data demonstrate that potent selective av antagonist can target endothelial cells, tumor cells, inhibit angiogenesis and inhibit tumor growth.

Identification of a novel slow-muscle-fiber enhancer binding protein, MusTRD1.

The molecular mechanisms which are responsible for restricting skeletal muscle gene expression to specific fiber types, either slow or fast twitch, are unknown. As a first step toward defining the components which direct slow-fiber-specific gene expression, we identified the sequence elements of the human troponin I slow upstream enhancer (USE) that bind muscle nuclear proteins. These include an E-box, a MEF2 element, and two other elements, USE B1 and USE C1. In vivo analysis of a mutation that disrupts USE B1 binding activity suggested that the USE B1 element is essential for high-level expression in slow-twitch muscles. This mutation does not, however, abolish slow-fiber specificity. A similar analysis indicated that the USE C1 element may play only a minor role. We report the cloning of a novel human USE B1 binding protein, MusTRD1 (muscle TFII-I repeat domain-containing protein 1), which is expressed predominantly in skeletal muscle. Significantly, MusTRD1 contains two repeat domains which show remarkable homology to the six repeat domains of the recently cloned transcription factor TFII-I. Furthermore, both TFII-I and MusTRD1 bind to similar but distinct sequences, which happen to conform with the initiator (Inr) consensus sequence. Given the roles of MEF2 and basic helix-loop-helix (bHLH) proteins in muscle gene expression, the similarity of TFII-I and MusTRD1 is intriguing, as TFII-I is believed to coordinate the interaction of MADS-box proteins, bHLH proteins, and the general transcription machinery.

Transcription occurs in pulses in muscle fibers.

We report a novel mechanism of gene regulation in skeletal muscle fibers. Within an individual myofiber nucleus, not all muscle loci are transcriptionally active at a given time and loci are regulated independently. This phenomenon is particularly remarkable because the nuclei within a myofiber share a common cytoplasm. Both endogenous muscle-specific and housekeeping genes and transgenes are regulated in this manner. Therefore, despite the uniform protein composition of the contractile apparatus along the length of the fiber, the loci that encode this structure are not transcribed continuously. The total number of active loci for a particular gene is dynamic, changing during fetal development, regeneration, and in the adult, and potentially reflects the growth status of the fiber. The data reveal that transcription in particular stages of muscle fiber maturation occurs in pulses and is defined by a stochastic mechanism.

The characterization of potent novel warfarin analogs.

Racemic sodium warfarin, Coumadin, is widely used in the prevention of thromboembolic disease. The present study was undertaken to characterize three novel classes of warfarin analogs, and to compare them with the warfarin enantiomers. All three classes of compounds inhibit vitamin K epoxide reductase, the enzyme inhibited by racemic warfarin. The alcohol and the ester analogs have reduced protein binding compared with R-(+)-warfarin. The ester and the fluoro-derivatives have similar in vivo anticoagulant activity in the rat to that of S-(-)-warfarin. Thus, it is possible to synthesize novel warfarin analogs that differ from racemic warfarin or its enantiomers in certain selected properties.

Involvement of endothelial PECAM-1/CD31 in angiogenesis.

The adhesive interactions of endothelial cells with each other and the adhesion receptors that mediate these interactions are probably of fundamental importance to the process of angiogenesis. We therefore studied the effect of inhibiting the function of the endothelial cell-cell adhesion molecule, PECAM-1/ CD31, in rat and murine models of angiogenesis. A polyclonal antibody to human PECAM-1, which cross-reacts with rat PECAM-1, was found to block in vitro tube formation by rat capillary endothelial cells and cytokine-induced rat corneal neovascularization. In mice, two monoclonal antibodies against murine PECAM-1 prevented vessel growth into subcutaneously implanted gels supplemented with basic fibroblast growth factor (bFGF). Taken together these findings provide evidence that PECAM-1 is involved in angiogenesis and suggest that the interactions of endothelial cell-cell adhesion molecules are important in the formation of new vessels.

Cryo-immunogold ultrastructural localization of laminin in adult rat peripheral nerve.

Elucidation of the functional roles of the extracellular matrix component laminin in adult peripheral nerve has been hindered by differing accounts of its ultrastructural localization. This is the first report applying the advantages of the cryo-immunogold technique to laminin localization in peripheral nerve. Laminin labelling was found over the basal lamina and possibly over the immediately subjacent Schwann cell plasma membrane, but specific labelling appeared to be absent from other membranes (including those of non-myelinated axon/Schwann cell clusters) and from endoneurial collagen fibrils. It would appear that the functional roles played by laminin in normal adult peripheral nerve are likely to be mediated via its localization in the basal lamina, rather than through a more widespread distribution within the endoneurium.

Acyl CoA:cholesterol acyltransferase (ACAT) inhibitors: synthesis and structure-activity relationship studies of a new series of trisubstituted imidazoles.

A series of 4,5-diaryl-2-(substituted thio)-1H-imidazoles has been synthesized and demonstrated to be potent inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). The design, synthesis, and structure-activity relationships for this series are reported herein. One of the compounds from this series, N'-(2,4-difluorophenyl)-N-[5-[(4,5-diaryl-1H-imidazol-2- yl)thio]pentyl]-N-heptylurea (DuP 128), was selected for development as an intestinally active ACAT inhibitor. DuP 128 is a potent ACAT inhibitor in vitro and in vivo, inhibiting ACAT in rat hepatic microsomes with an IC50 = 10 nM and possessing potent antihypercholesterolemic activity in vivo.

Whole blood and plasma concentrations of 4-hydroxy-2-nonenal in Watanabe heritable hyperlipidemic versus New Zealand White rabbits.

Both plasma and whole blood concentrations of 4-hydroxy-2-nonenal (4HNE) were significantly elevated in a population (n = 6) of 2 year old Watanabe heritable hyperlipidemic rabbits relative to a population (n = 6) of New Zealand White rabbits. The plasma concentrations were 74 +/- 10 nmol/L for the Watanabe group and 47 +/- 6 nmol/L for the New Zealand White group. The whole blood concentrations were 364 +/- 55 nmol/L for the Watanabe group and 188 +/- 64 nmol/L for the New Zealand White group. These results indicate that 4HNE concentrations in blood can be elevated in individuals with atherosclerosis and demonstrate the potential link between the formation of 4HNE and the progression of atherosclerosis.

Regulation of ACAT activity by a cholesterol substrate pool during the progression and regression phases of atherosclerosis: implications for drug discovery.

The regulation of aortic ACAT by a cholesterol substrate pool (CSP) was investigated in a rabbit progression/regression model of dietary-induced atherosclerosis. ACAT activity increased 25-fold during the 10-week progression phase of the study. ACAT activity decreased 8-fold during the 24-week regression phase of the study, however, it was still 14-fold greater than in normal aortas. ACAT activity assayed in the absence vs. the presence of exogenous cholesterol was used as a qualitative measure of the amount of cholesterol in the CSP. The CSP was filled to 28% of capacity in normal aortas, this increased to 75% during the progression phase. By the end of the regression phase, the CSP was filled to 100% of capacity even though serum cholesterol levels had returned to normal. The data are discussed in terms of emerging concepts of intracellular cholesterol trafficking, ACAT inhibitors, and the types of atherosclerotic lesions which may be subject to amelioration by ACAT inhibitors.

Cloning and characterization of Treponema hyodysenteriae antigens and protection in a CF-1 mouse model by immunization with a cloned endoflagellar antigen.

We cloned genes that code for Treponema hyodysenteriae antigens into Escherichia coli with the purpose of identifying protective antigens for vaccine development. Three different genomic libraries were screened with various antisera reactive with T. hyodysenteriae antigens. The cloned antigens and corresponding native T. hyodysenteriae antigens were analyzed for molecular size, serum reactivity, solubility in sarcosine, and segregation during phase partitioning with the nonionic detergent Triton X-114. The results from these analyses suggested that the gene products were components of either the cytoplasmic membrane, periplasm, or endoflagella of T. hyodysenteriae. The cloned antigens were tested as vaccine candidates in a CF-1 mouse model of T. hyodysenteriae infection and immunity. Intraperitoneal injection of crude E. coli extracts containing cloned antigens did not protect mice from challenge. However, serum from mice injected with a crude extract of an E. coli clone which expressed an endoflagellar antigen killed T. hyodysenteriae in vitro. Partially purified preparations of this cloned endoflagellar antigen protected mice against oral challenge with both the homologous serotype (B204) and a heterologous serotype (B234) of T. hyodysenteriae. These results suggest that the endoflagellar proteins could be used as an effective subunit vaccine against T. hyodysenteriae.

Effect of lanthanum chloride on established atherosclerosis in the cholesterol-fed rabbit. Mitral valve as a site for assessment of treatment effects.

The ability of lanthanum chloride (LaCl3) to retard the progression of established atherosclerosis was investigated in cholesterol-fed rabbits. Rabbits were initially maintained on a high-fat plus cholesterol-supplemented diet for 10 weeks to induce lesions and were then changed to a low-fat diet or a low-fat diet supplemented with LaCl3 for an additional 24 weeks to permit their serum cholesterol levels to normalize. LaCl3 did not affect the rate at which serum cholesterol levels returned to normal. The dose of LaCl3 was approximately 30 mg/kg body weight/day. In comparison with controls, LaCl3-treated rabbits exhibited histologically less severe coronary artery and mitral valve atherosclerosis. Lesion severity in the carotid arteries was unaffected by LaCl3 treatment. Although statistically significant, the salutary effects of LaCl3 were relatively small. The data support the hypothesis that calcium antagonists can retard the progression of established atherosclerotic lesions. The data also illustrate the value of the mitral valve as a site to assess treatment effects on monocyte/macrophages in vivo.

Regulation of acyl-CoA:cholesterol acyltransferase activity in normal and atherosclerotic rabbit aortas: role of a cholesterol substrate pool.

A new substrate optimized assay for acyl-CoA:cholesterol acyltransferase (ACAT) was developed that permits the accurate measurement of ACAT activity in normal arterial microsomes. The apparent Km and Vmax of ACAT with respect to oleoyl-CoA were determined to be 3 microM and 17.7 pmole min-1 mg-1. While the Km value is similar to other values reported in the literature, the Vmax is 5- to 8-fold higher. The higher Vmax is attributable to the saturation of ACAT with not only oleoyl-CoA, but also cholesterol. The observation that exogenous cholesterol was necessary for the determination of maximal ACAT activity indicates that under normal conditions the endogenous level of microsomal cholesterol does not saturate ACAT. Assay of ACAT in the presence and absence of exogenous cholesterol permits a qualitative assessment of the amount of cholesterol in the cholesterol substrate pool of ACAT. Using this approach, it was found that hypercholesterolemia results in the expansion of the cholesterol substrate pool of ACAT. Of the 21-fold increase in ACAT activity in atherosclerotic aortas observed in this study. 80% of the increase was attributable to expansion of the cholesterol substrate pool, while 20% was attributable to more enzyme. Notably, the increase in the amount of ACAT was observed after only 2 weeks of hypercholesterolemia.

Water environment microwave thawing.

A rapid, controlled microwave oven thawing technic for fresh frozen plasma (FFP) that minimizes flocculent formation while maintaining excellent coagulation factor, total protein, and albumin levels is presented. This method, using an unmodified microwave oven and a tap-water environment, decreases thawing time from 37.5 minutes for the standard 37 degrees C water bath to approximately 11 minutes for either one or two units of FFP.

Differential endocytosis of lipoproteins by capillary endothelial vesicles.

Plasma levels of lipoproteins are believed to be controlled largely by lipoprotein receptors on the surfaces of tissue cells which facilitate their internalization and degradation. This presupposes transit of lipoproteins across the walls of blood vessels in order to gain access to the receptor sites. The endothelium of tissue capillaries may therefore constitute an additional regulatory barrier for lipoproteins. In order to test this hypothesis, we have measured the uptake of fluorescent-labeled lipoproteins into endothelial vesicles of capillaries isolated from fat tissue. HDLs are ingested at more than twice the efficiency of LDLs and VLDLs are excluded from vesicular ingestion. This represents a decreased efficiency of ingestion with an increase in molecular diameter of lipoproteins. This phenomenon correlates well with the dimensions of endothelial vesicles and caveolae which may restrict entry of very large serum lipoproteins. Selective transport of lipoproteins by capillary endothelial vesicles on the basis of molecular size may therefore serve to regulate blood-tissue interchanges of lipid.

Diagnostic exercise testing in 104 patients over 65 years of age.

Since 1979 we have carried out symptom limited exercise stress tests for the diagnosis of chest pain in 104 patients, 61 male, 43 female, over 65 years of age; mean age 68 +/- 3 years. An upright bicycle ergometer was used for 64 tests, a treadmill for 38 tests and a supervised walk for 2 patients unable to undergo formal exercise testing. A positive result of greater than or equal to 1 mm of ST depression was recorded in 45% of patients; males 57%, females 28% (P less than 0.01). Bicycle and treadmill tests were equally likely to produce a positive result; bicycle 43%, treadmill 50% (NS). The limiting symptom was chest pain in 43%, dyspnoea in 26% and fatigue in 30% of patients. No serious arrhythmias or collapses occurred. During a mean follow up to 24 +/- 18 months 13 patients died. A positive exercise test was associated with a significantly increased risk of cardiac death; 8 of 47 patients with positive tests died compared with 1 of 57 patients with negative or equivocal tests (P less than 0.02). The remaining 4 deaths were due to malignancy. Exercise testing can thus be safely performed in elderly subjects with the expectation of a high diagnostic yield. A positive result confers a poor prognosis.

High-voltage electron microscopy of capillary endothelial vesicles.

Conventional electron microscopy of thin sections through capillary walls is inadequate to discern the relationships between endothelial vesicles and their association with the cell surface. High-voltage electron microscopy of thick sections through diaphragm muscle capillaries has been employed to visualize the three-dimensional structure of the vesicular system. Stereopairs of thick sections provide for direct three-dimensional observations of samples several vesicle diameters deep. A variety of simple and compound vesicular forms are present but not all are conjoined or maintain connections with the endothelial cell surface. This contributes to the concept of a dynamic interaction between free and attached vesicular structures where fission and fusion events compartmentalize and reconnect repeatedly. Such interactions would provide for a discontinuous pathway across the capillary wall but with a higher degree of complexity than a simple shuttling mechanism.