International survey on the use and welfare of zebrafish Danio rerio in research.

A survey was conducted regarding zebrafish Danio rerio use for scientific research with a focus on: anaesthesia and euthanasia; housing and husbandry; breeding and production; refinement opportunities. A total of 98 survey responses were received from laboratories in 22 countries in Europe, North America, South America, Asia and Australia. There appears a clear and urgent need to identify the most humane methods of anaesthesia and euthanasia. Aversive responses to MS-222 were widely observed raising concerns about the use of this anaesthetic for D. rerio. The use of anaesthesia in fin clipping for genetic identification is widely practised and there appears to be an opportunity to further develop less invasive methods and refine this process. Optimization (and potentially standardization) of feeding is an area for further investigation. Given that diet and body condition can have such profound effects on results of experiments, differences in practice could have significant scientific implications. Further research into transition between dark and light phases in the laboratory appears to represent an opportunity to establish best practice. Plants and gravel were not considered practical by many laboratories. The true value and benefits need to be established and communicated. Overproduction is a concern both from ethical and financial viewpoints. There is an opportunity to further reduce wastage of D. rerio. There are clear concerns and opportunities for the scientific community to work together to further improve the welfare of these important laboratory models.

Refinements in husbandry, care and common procedures for non-human primates: Ninth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement.

Preface Whenever animals are used in research, minimizing pain and distress and promoting good welfare should be as important an objective as achieving the experimental results. This is important for humanitarian reasons, for good science, for economic reasons and in order to satisfy the broad legal principles in international legislation. It is possible to refine both husbandry and procedures to minimize suffering and improve welfare in a number of ways, and this can be greatly facilitated by ensuring that up-to-date information is readily available. The need to provide such information led the British Veterinary Association Animal Welfare Foundation (BVAAWF), the Fund for the Replacement of Animals in Medical Experiments (FRAME), the Royal Society for the Prevention of Cruelty to Animals (RSPCA) and the Universities Federation for Animal Welfare (UFAW) to establish a Joint Working Group on Refinement (JWGR) in the UK. The chair is Professor David Morton and the secretariat is provided by the RSPCA. This report is the ninth in the JWGR series. The RSPCA is opposed to the use of animals in experiments that cause pain, suffering, distress or lasting harm and together with FRAME has particular concerns about the continued use of non-human primates. The replacement of primate experiments is a primary goal for the RSPCA and FRAME. However, both organizations share with others in the Working Group, the common aim of replacing primate experiments wherever possible, reducing suffering and improving welfare while primate use continues. The reports of the refinement workshops are intended to help achieve these aims. This report produced by the British Veterinary Association Animal Welfare Foundation (BVAAWF)/Fund for the Replacement of Animals in Medical Experiments (FRAME)/Royal Society for the Prevention of Cruelty to Animals (RSPCA)/Universities Federation for Animal Welfare (UFAW) Joint Working Group on Refinement (JWGR) sets out practical guidance on refining the husbandry and care of non-human primates (hereinafter primates) and on minimizing the adverse effects of some common procedures. It provides a valuable resource to help understand the physical, social and behavioural characteristics and needs of individual primates, and is intended to develop and complement the existing literature and legislative guidelines. Topics covered include refinements in housing, husbandry and common procedures such as restraint, identification and sampling, with comprehensive advice on issues such as primate communication, assessing and facilitating primate wellbeing, establishing and maintaining social groups, environmental and nutritional enrichment and animal passports. The most commonly used species are the key focus of this resource, but its information and recommendations are generally applicable to other species, provided that relevant individual species characteristics are taken into account.

Exhaled breath condensate hydrogen peroxide, pH and leukotriene B4 are associated with lower airway inflammation and airway cytology in the horse.

BACKGROUND: Exhaled breath condensate (EBC) analysis is a noninvasive method to assess the lower respiratory tract. In human subjects, EBC hydrogen peroxide (H2 O2 ), pH and leukotriene B4 (LTB4 ) are useful for detection and monitoring of inflammatory lung diseases, including asthma. OBJECTIVES: To determine associations between EBC biomarkers and cytological and endoscopic definitions of lower airway inflammation (LAI) while controlling for sampling and environmental variables. STUDY DESIGN: Prospective, cross-sectional study. METHODS: Clinical, endoscopic and airway cytological findings from 47 horses were compared with EBC pH and concentrations of H2 O2 and LTB4 by univariate and multivariable analyses. Dichotomous (presence/absence of airway inflammation) and continuous outcome variables (differential cell counts in tracheal aspirate and bronchoalveolar lavage fluid, BALF) were evaluated and potential effects of collection and methodological factors were included. RESULTS: EBC pH and H2 O2 concentrations were higher in horses with LAI and both were positively associated with the percentage of neutrophils in BALF (P<0.05). Mast cell percentage in BALF was negatively associated with EBC pH, and BALF eosinophil percentage was positively associated with EBC LTB4 (P<0.05). Ambient temperature, relative humidity and assay methodology significantly impacted some analytes. MAIN LIMITATIONS: LAI is challenging to categorise due to a variety of clinical and cytological phenotypes. Although the study was designed to overcome this limitation, numbers of horses were small in some categories. CONCLUSIONS: EBC pH and H2 O2 concentrations are altered by airway inflammation, suggesting a role for these biomarkers in the diagnosis and monitoring of airway disease. Environmental and methodological factors can influence these biomarkers and should be considered in the interpretation of results.

Charcot neuroarthropathy triggered by osteomyelitis and/or surgery.

INTRODUCTION: Charcot neuroarthropathy (CN) is a rare but devastating complication of diabetic neuropathy. Osteomyelitis is also a complication of the diabetic foot and it may be difficult to differentiate from CN. PATIENTS AND METHODS: A patient with Type 1 diabetes and peripheral neuropathy developed a foot ulcer complicated by osteomyelitis of the first proximal phalanx. He was successfully treated with antibiotics and surgical excision of the infected bone. Six months later, he developed a hot, swollen, red foot and X-ray showed destruction of the second and third metatarsal heads. At the second presentation, it was difficult to determine whether this was a recurrence of osteomyelitis or a new onset of CN. Thus, to obtain a definitive diagnosis, recourse was made to more sophisticated imaging techniques. RESULTS: 99mTc methylenediphosphonate (MDP) bone scans and magnetic resonance imaging proved inconclusive to differentiate between osteomyelitis and CN. Subsequently, an indium-labelled white cell scan confirmed the absence of osteomyelitis and the patient was successfully treated for CN. DISCUSSION: Infection and/or surgery may be predisposing factors in the development of diabetic CN but the combination of the two could accelerate the onset of the Charcot process in people with diabetes and neuropathy.

Evaluation of Pharmacokinetic and Pharmacodynamic Drug-Drug Interaction of Sacubitril/Valsartan (LCZ696) and Sildenafil in Patients With Mild-to-Moderate Hypertension.

Sacubitril/valsartan (LCZ696) is indicated for the treatment of patients with heart failure and reduced ejection fraction (HFrEF). Since patients with HFrEF may receive sacubitril/valsartan and sildenafil, both increasing cyclic guanosine monophosphate, the present study evaluated the pharmacokinetic and pharmacodynamic drug interaction potential between sacubitril/valsartan and sildenafil. In this open-label, three-period, single sequence study, patients with mild-to-moderate hypertension (153.8 ± 8.2 mmHg mean systolic blood pressure (SBP)) received a single dose of sildenafil 50 mg, sacubitril/valsartan 400 mg once daily for 5 days, and sacubitril/valsartan and sildenafil coadministration. When coadministered with sildenafil, the AUC and Cmax of valsartan decreased by 29% and 39%, respectively. Coadministration of sacubitril/valsartan and sildenafil resulted in a greater decrease in BP (-5/-4/-4 mmHg mean ambulatory SBP/DBP/MAP (mean arterial pressure)) than with sacubitril/valsartan alone. Both treatments were generally safe and well tolerated in this study; however, the additional BP reduction suggests that sildenafil should be administered cautiously in patients receiving sacubitril/valsartan. Unique identifier: NCT01601470.

Training of staff for the delivery of PET/CT services in the UK.

Evidence for the cost effectiveness of PET/CT imaging is now driving the widespread introduction of PET/CT services throughout the UK. The provision of PET/CT facilities will require a workforce of medical, scientific, technical and engineering staff who are adequately trained and fit for purpose. Suitably trained staff in this speciality are scarce. The development and accreditation of training courses and other educational resources for training programmes in all disciplines will therefore be required at a national and regional level. The implementation of PET/CT training can be achieved more cost-effectively by developing multi-professional learning resources whenever possible. It is intended that the recommendations would be implemented by close co-operation of both public and private healthcare providers together with educational establishments.

Identification and characterization of a 19q12 amplicon in esophageal adenocarcinomas reveals cyclin E as the best candidate gene for this amplicon.

Genomic DNA amplification in tumors is frequently associated with an increased gene copy number of oncogenes or other cancer-related genes. We have used a two-dimensional whole-genome scanning technique to identify gene amplification events in esophageal adenocarcinomas. A multicopy genomic fragment from a tumor two-dimensional gel was cloned, and genomic amplification encompassing this fragment was confirmed by Southern blot analysis. The corresponding DNA sequence was matched by BLAST to a BAC contig, which allowed the use of electronic-PCR to localize this amplicon to 19q12. Sequence tagged site-amplification mapping, an approach recently implemented in our laboratory (Lin, L. et al., Cancer Res., 60: 1341-1347,2000), was used to characterize the amplicon. Genomic DNA from 65 esophageal and 11 gastric cardia adenocarcinomas were investigated for 19q12 amplification using quantitative PCR at 11 sequence tagged site markers neighboring the cloned fragment. The amplicon was narrowed from >8 cM to a minimal critical region spanning <0.8 cM, between D19S919 and D19S882. This region includes the cyclin E gene. Fourteen expressed sequence tags (ESTs) covering the minimal region were then assayed for potential gene overexpression using quantitative reverse transcription-PCR. Seven of the selected ESTs were found to be both amplified and overexpressed. Among these seven ESTs, cyclin E showed the highest frequency of gene amplification and overexpression in the tumors examined, which allowed us to finalize the core-amplified region to <300 kb. These results indicate that cyclin E is the likely target gene selected by the amplification event at 19q12. The fact that cyclin E overexpression was found only in the amplified tumors examined indicates that gene amplification underlies the cyclin E gene overexpression. Our study represents the first extensive analysis of the 19q12 amplicon, and is the first to physically map the core-amplified domain to a region of <300 kb that includes cyclin E. Amplification of 19q12 was found neither in the 28 esophageal squamous cancers nor in the 39 lung adenocarcinomas examined but was observed in 13.8% of esophageal and 9.1% of gastric cardia adenocarcinomas.

The assembly of yeast mitochondrial ATP synthase: subunit depletion in vivo suggests ordered assembly of the stalk subunits b, OSCP and d.

The abundance in vivo of each of three subunits b, OSCP and d, components of the stalk region of the yeast mitochondrial ATP synthase complex, was manipulated by a controlled depletion strategy. Western blots of whole cell lysates were used to study the effect of depletion of each of these subunits on the cellular levels of other subunits of the enzyme complex. A hierarchy of subunit stability was determined and interpreted to indicate the order of assembly of these three subunits of the stalk region. Thus, subunit b is assembled first, followed by OSCP and then by subunit d.

Isoprenylation of polypeptides in the nematode Caenorhabditis elegans.

Covalent modification of eucaryotic proteins, involving addition of isoprenyl groups, is a widespread phenomenon. Here we provide direct evidence for this form of covalent modification in the free-living nematode, Caenorhabditis elegans. Following incubation in the presence of [3H]mevalonolactone, specific C. elegans polypeptides became labelled in both aqueous and detergent (Triton X-114)-enriched extracts. Chemical and GC-MS analysis of modifying groups, cleaved from C. elegans polypeptides, revealed that geranylgeranylation and, to a lesser extent, farnesylation of target polypeptides occurred. Immunoblot analysis provided preliminary evidence that the ras-like let-60 polypeptide was a target for isoprenylation in C. elegans.

Insights into ATP synthase assembly and function through the molecular genetic manipulation of subunits of the yeast mitochondrial enzyme complex.

Development of an increasingly detailed understanding of the eucaryotic mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry, structure and function of F(0) sector subunits in the contexts of the proton channel and the stator stalk. Still to be resolved are the precise locations and roles of other supernumerary subunits present in mitochondrial ATP synthase complexes, but not found in the bacterial or chloroplast enzymes. The highly developed system of molecular genetic manipulation available in the yeast Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene function based on the effects of gene disruption or deletion. In addition, the genes encoding ATP synthase subunits can be manipulated to introduce specific amino acids at desired positions within a subunit, or to add epitope or affinity tags at the C-terminus, enabling questions of stoichiometry, structure and function to be addressed. Newly emerging technologies, such as fusions of subunits with GFP are being applied to probe the dynamic interactions within mitochondrial ATP synthase, between ATP synthase complexes, and between ATP synthase and other mitochondrial enzyme complexes.

Differential cell cycle progression patterns of infiltrating leukocytes and resident cells after balloon injury of the rat carotid artery.

The heterogeneous nature of the cell populations involved in vascular repair remains a major hurdle for the assessment of the cellular events that take place in injured arteries. The present experiments were designed to estimate the proportions and cell cycle progression of infiltrating leukocytes versus resident vascular cells after balloon injury of the rat common carotid artery. After tissue disaggregation, cell suspension samples from each artery were analyzed by flow cytometry. Cells were stained with anti-CD45 or anti-alpha-smooth muscle actin antibodies to identify leukocytes and smooth muscle cells, respectively. A day after injury, a 12-fold increase in CD45+ leukocytes was found. Double labeling with CD45 and CD-3, ED-1, or granulocyte markers revealed that most infiltrating cells were monocytes and granulocytes. Approximately 14% of infiltrating leukocytes were found to enter apoptosis at day 1, and 17% entered S phase at day 3. In contrast, the highest proliferation rate of resident alpha-smooth muscle actin-positive cells was observed at day 7 (19%). The present results demonstrate that infiltrating leukocytes and resident vascular smooth muscle cells have dissimilar cell cycle profiles. Furthermore, our study demonstrates the feasibility of using flow cytometry to quantitatively determine the cell types and their relative activation state in injured arteries.

Lesion-targeted injectable vectors for vascular restenosis.

Pathologic lesions caused by catheter-based revascularization procedures for occlusive artery disease include disruption of the endothelium, exposure of extracellular matrix (ECM) proteins, and proliferation of vascular smooth muscle cells, which lead to neointima formation and restenosis. We have developed matrix-collagen-targeted retroviral vectors that are able to accumulate at sites of vascular injury (Hall et al., Hum. Gene Ther. 1997;8:2183-2192; Hall et al., Hum. Gene Ther. 2000;11:983-993). The primary tissue-targeting motif, adapted from the physiological surveillance sequence found in von Willebrand factor, served to localize and concentrate the vector within vascular lesions. In the present study, we evaluated the efficiency of this vector-targeting system in rats with nonligated balloon-injured carotid arteries. Both intraarterial (by retrograde femoral artery catheterization) and intravenous (via femoral vein) injection of a matrix-targeted vector enhanced transduction of neointimal cells ( approximately 20%) at severely denuded areas when compared with the nontargeted vector (<1%). Further, intraarterial instillation of a matrix-targeted, but not a nontargeted, vector bearing an antisense cyclin G1 construct inhibited neointima lesion formation in the injured carotid arteries. Taken together, these data indicate that strategic targeting of retroviral vectors to vascular lesions would have therapeutic potential in the management of vascular restenosis and many other disorders of uncontrolled proliferation where endothelial disruption, ECM remodeling, and collagen deposition form the nexus for preferential vector localization and concentration in vivo.

Characterisation of a thermostable pepstatin-insensitive acid proteinase from a Bacillus sp.

An acid proteinase, Wai 21a, produced by a thermophilic Bacillus species (strain Wai 21a) has been purified to homogeneity by cation-exchange chromatography, phenyl-Sepharose chromatography and anion-exchange chromatography. A pI of 3.8 was determined by isoelectric focussing. The protein contained some associated carbohydrate (20 mol hexose equiv/mol proteinase). Optimal proteolytic activity was observed at pH 3.0 (at 60 degrees C). The Leu15-Tyr16 bond was the major site of hydrolysis for the oxidized B chain of insulin. Enzyme activity was not affected by inhibitors of the cysteine, metallo or serine class of proteinases. The aspartate proteinase inhibitor, pepstatin, did not inhibit enzyme activity. Inhibition of enzyme activity by 1,2-epoxy-3-(p-nitrophenoxy)-propane indicated the presence of at least one carboxyl group essential to the catalytic mechanism of the enzyme. Proteinase activity was inhibited by diazoacetyl-DL-norleucine methyl ester in a slow and non-specific manner atypical of pepstatin-sensitive aspartate proteinases. Wai 21a proteinase may be classified as member of the pepstatin-insensitive group of aspartate proteinases. The thermal stability at pH 3.0 and 60 degrees C increased 2.1-fold (t1/2, 4.5-9.7 hr) in the presence of 5 mM Ca++. An increase in both pH (3.0-4.5) and Ca++ concentration (0-30 mM) resulted in a 15-fold increase (t1/2, 15-230 min) in thermal stability at 75 degrees C. The amino acid composition of Wai 21a proteinase was found to be similar to other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin.

Effects of hypertension and its reversal on aortic intima lesions of the rat.

A combined transmission (TEM) and scanning (SEM) electron microscopic study was performed on aortae of deoxycorticosterone-salt (DOC-salt)-treated rats and spontaneously hypertensive rats (SHR) to compare the effects of hypertension as well as its reversal on the aortic intima. To best reproduce the in vivo state of the vasculature, rats were perfusion-fixed at pressures corrected for each individual animal (30 mm Hg below measured systolic pressure). The intimal alterations were focal and thus were best appreciated with the combined use of SEM and TEM. Qualitatively, both models of hypertension showed similar intimal changes, which consisted of subintimal thickening due to an accumulation of both extracellular material and cells. Subendothelial cells with a morphology indicating a blood-borne origin were present simultaneously with cells derived from the vessel wall. The increased subendothelial extracellular material included precipitated plasma proteins, reticulated basement membrane, collagen fibers, and fragments of elastin. Increase in the height of endothelial cells with distortion of nuclear shape was prominent. Withdrawal of DOC-salt combined with low-salt diet for 11 weeks did not result in a discernible regression of these intimal changes despite normalization of blood pressure. We conclude that vascular injury, once induced, may be difficult to reverse and suggest that areas of prior damage may serve as foci for later vascular complications.

Aortic endothelial and subendothelial cells in experimental hypertension and aging.

The endothelial and subendothelial cellular changes occurring as a result of hypertension and aging were characterized in deoxycorticosterone/salt-treated (D/S), spontaneously hypertensive (SHR), and Wistar-Kyoto (WKY) rats. An increase in the number of subendothelial cells occurred with both hypertension and aging and was most dramatic with D/S hypertension. Many of the cells found in the widened subendothelium showed morphological characteristics of mononuclear cells (both macrophages and lymphocytes), and of smooth muscle cells. Normalization of blood pressure by withdrawal of D/S and maintenance of rats on a low salt diet reversed the number of subendothelial cells to levels of control animals of comparable age. Significant alterations were seen in the aortic endothelial cells of D/S animals. Within 2 to 4 weeks of D/S administration, the endothelial cells doubled in number and often assumed bizarre shapes with nuclear folding and bulging toward the lumen. Some similar abnormalities in endothelial cell shape and appearance occurred with increasing age in the SHR and control WKY, although the number of endothelial cells increased only slightly in these groups. These results suggest that profound cellular changes in the aortic intima occur with an increase in blood pressure. These changes are pronounced in the D/S model of hypertension, while virtually absent in SHR. Comparable alterations also may be seen in aged normotensive animals, but to a lesser extent and with slower progression.

The use of [99mTc]-HM-PAO in the diagnosis of primary degenerative dementia.

The clinical value of single photon emission computed tomography (SPECT) in the differential diagnosis of dementia due to cerebral atrophy was evaluated by comparing the pattern of distribution [99mTc]-HM-PAO in three dementing conditions. Imaging was carried out in 26 patients with suspected Alzheimer's disease, 14 with dementia of the frontal-lobe type, and 13 with progressive supranuclear palsy. Images were evaluated and reported without knowledge of clinical diagnosis with respect to regions of reduced uptake of tracer. Reduced uptake in the posterior cerebral hemispheres was characteristic of Alzheimer's disease, while selective anterior hemisphere abnormalities characterized both dementia of the frontal-lobe type and progressive supranuclear palsy. The latter conditions could be distinguished on the basis of the appearance of integrity of the rim of the frontal cortex. The technique has an important role in the differentiation of degenerative dementias.

A cytochrome c-GFP fusion is not released from mitochondria into the cytoplasm upon expression of Bax in yeast cells.

To study Bax-induced release of cytochrome c in vivo, we have expressed a cytochrome c-GFP (green fluorescent protein) fusion in Saccharomyces cerevisiae cells null for the expression of the endogenous cytochrome. We show here that cytochrome c-GFP is efficiently localised to mitochondria and able to function as an electron carrier between complexes III and IV of the respiratory chain. Strikingly, while natural cytochrome c is released into the cytoplasm upon expression of Bax, the cytochrome c-GFP fusion is not. Nevertheless, cells co-expressing Bax and the cytochrome c-GFP fusion die, indicating that mitochondrial release of cytochrome c is not essential for cell death to occur in yeast. The failure to release cytochrome c-GFP is presumed to arise from increased bulk due to the GFP moiety. We propose that in intact yeast cells, Bax-induced release of cytochrome c into the cytoplasm occurs through a selective pore and not as a consequence of the non-specific breakage of the mitochondrial outer membrane.

A novel fluorescent marker for assembled mitochondria ATP synthase of yeast. OSCP subunit fused to green fluorescent protein is assembled into the complex in vivo.

We have shown that OSCP, a subunit of yeast mitochondrial ATP synthase, can be incorporated into the intact enzyme as a fusion protein representing OSCP fused at its C-terminus to the green fluorescent protein (GFP) of Aequorea victoria. The relevant fusion OSCP-GFP-h6 additionally contains a hexahistidine tag at the C-terminus. Expression of OSCP-GFP-h6 in yeast cells lacking endogenous OSCP led to the efficient restoration of growth of cells on the non-fermentable substrate, ethanol. Confocal laser scanning microscopy revealed fluorescence due to GFP in mitochondria of cells expressing OSCP-GFP-h6. Use of immobilised metal ion affinity chromatography enabled the recovery of assembled ATP synthase complexes which contained OSCP-GFP-h6 identified by its mobility on SDS-PAGE and immunoreactivity to anti-OSCP and anti-GFP antibodies. The successful isolation of the assembled multisubunit ATP synthase containing GFP fused to one of the essential subunits of the complex widely expands the potential applications of GFP. In principle, these include the spatial and temporal monitoring of ATP synthase complexes in vivo, and the exploration of interactions involving ATP synthase subunits by fluorescence resonance energy transfer (FRET).

CGP 43371 paradoxically inhibits development of rabbit atherosclerotic lesions while inducing extra-arterial foam cell formation.

Ansamycins are hypolipidemic compounds which, when administered to various animal species, dramatically lower high density lipoprotein (HDL) cholesterol levels, in addition to reducing the levels of other lipoprotein classes. The current study tested one of these ansamycins (CGP 43371) for its hypolipidemic and anti-atherosclerotic activity in cholesterol-fed rabbits. Rabbits were fed a 0.25% cholesterol-enriched diet with or without admixed CGP 43371, equivalent to 30 mg/kg per day for 16 weeks. Compared with control rabbits, CGP 43371 treatment lowered total cholesterol levels (46%, P<0.05) and lipoprotein cholesterol levels (HDL, 58%; VLDL, 49% [both P<0.05]; LDL, 28% [not significant]). Despite the dramatic lowering of HDL cholesterol levels, aortic atherosclerosis, assessed by grossly visible sudanophilia, was significantly inhibited versus controls (total aorta=38%; aortic arch=32%; thoracic aorta=60%). Of particular note in CGP 43371-treated rabbits was a striking splenomegaly, which correlated with the presence of massive accumulations of macrophage foam cells in the splenic red pulp. We speculate that CGP 43371 inhibits the development of atheroselerotic lesions in rabbits by both a hypolipidemic mechanism, and by a mechanism(s) in which macrophage foam cells accumulate in the spleen.