Applying refinement to the use of mice and rats in rheumatoid arthritis research

Rheumatoid arthritis (RA) is a painful, chronic disorder and there is currently an unmet need for effective therapies that will benefit a wide range of patients. The research and development process for therapies and treatments currently involves in vivo studies, which have the potential to cause discomfort, pain or distress. This Working Group report focuses on identifying causes of suffering within commonly used mouse and rat ‘models’ of RA, describing practical refinements to help reduce suffering and improve welfare without compromising the scientific objectives. The report also discusses other, relevant topics including identifying and minimising sources of variation within in vivo RA studies, the potential to provide pain relief including analgesia, welfare assessment, humane endpoints, reporting standards and the potential to replace animals in RA research

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PMCID: PMC379391

The Mitochondrial Ca(2+) Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca(2+) uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca(2+) uptake and our current understanding of mitochondrial Ca(2+) homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca(2+) uniporter complex

Understanding emotionally relevant situations in primary dental practice. 3. Emerging narratives

Background and aims. Dentists experience considerable occupational stress. Stressful clinical situations can provoke high levels of negative emotions, and situations which are associated with positive emotions tend to be overlooked by practitioners. Reflection regarding difficult situations is encouraged to facilitate learning. Cognitive behavioural therapy (CBT) formulations may be applied to situations appraised both positively and negatively. Analysis and interpretation of the dentist's coping behaviour and the consequent outcomes facilitate learning and reflection upon individual interactions with patients. Method. Twenty primary care dental practitioners in the greater Lincoln area participated in a semi-structured interview which explored their stressful and positive clinical experiences. Some of the episodes were analysed to create CBT formulations. Results and discussion. CBT formulations are presented and the learning points highlighted by this structured presentation are discussed. In particular, it is suggested that this structured reconstruction of events, which highlights dentists' emotions, responses and the transactional effects of coping responses, might well facilitate objective reflective learning either individually or as part of peer to peer support. It should facilitate dentists' emotional processing of events and may thus contribute to stress reduction. Conclusion. CBT formulations of positive and negative dental scenarios may be constructed. It is proposed that this is a useful technique to foster reflection and learning in clinical situations and should lead to improved communication skills and shared decision-making, resulting in fewer complaints and thereby reduced stress. It should also improve dentists' emotional processing

Instability of Plastid DNA in the Nuclear Genome

Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes

Interdigital cell death in the embryonic limb is associated with depletion of Reelin in the extracellular matrix

Interdigital cell death is a physiological regression process responsible for sculpturing the digits in the embryonic vertebrate limb. Changes in the intensity of this degenerative process account for the different patterns of interdigital webbing among vertebrate species. Here, we show that Reelin is present in the extracellular matrix of the interdigital mesoderm of chick and mouse embryos during the developmental stages of digit formation. Reelin is a large extracellular glycoprotein which has important functions in the developing nervous system, including neuronal survival; however, the significance of Reelin in other systems has received very little attention. We show that reelin expression becomes intensely downregulated in both the chick and mouse interdigits preceding the establishment of the areas of interdigital cell death. Furthermore, fibroblast growth factors, which are cell survival signals for the interdigital mesoderm, intensely upregulated reelin expression, while BMPs, which are proapototic signals, downregulate its expression in the interdigit. Gene silencing experiments of reelin gene or its intracellular effector Dab-1 confirmed the implication of Reelin signaling as a survival factor for the limb undifferentiated mesoderm. We found that Reelin activates canonical survival pathways in the limb mesoderm involving protein kinase B and focal adhesion kinase. Our findings support that Reelin plays a role in interdigital cell death, and suggests that anoikis (apoptosis secondary to loss of cell adhesion) may be involved in this process

Bortezomib in combination with celecoxib in patients with advanced solid tumors: a phase I trial

<p>Abstract</p> <p>Background</p> <p>COX-2 inhibitors, such as celecoxib, and ubiquitin-proteasome pathway inhibitors, such as bortezomib, can down-regulate NF-κB, a transcription factor implicated in tumor growth. The objective of this study was to determine the maximum tolerated dose and dose-limiting toxicities of bortezomib in combination with celecoxib in patients with advanced solid tumors.</p> <p>Methods</p> <p>Patients received escalating doses of bortezomib either on a weekly schedule (days 1, 8, 15, 22, and 29 repeated every 42 days) or on a twice-weekly administration schedule (days 1, 4, 8, and 11 repeated every 21 days), in combination with escalating doses of celecoxib twice daily throughout the study period from 200 mg to 400 mg twice daily.</p> <p>Results</p> <p>No dose-limiting toxicity was observed during the study period. Two patients had stable disease lasting for four and five months each, and sixteen patients developed progressive disease.</p> <p>Conclusion</p> <p>The combination of bortezomib and celecoxib was well tolerated, without dose limiting toxicities observed throughout the dosing ranges tested, and will be studied further at the highest dose levels investigated.</p> <p>Trial registration number</p> <p>NCT00290680.</p

Use of functional feeding strategies to protect Atlantic salmon from virally-induced inflammatory diseases- mechanistic insights revealed by transcriptomic analysis

Over the past few years one of the major concerns in the Atlantic salmon (Salmo salar) farming industry has been the increasing incidence and severity of inflammatory viral diseases. Heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS) are currently two of the most prevalent viral diseases in commercial Atlantic salmon farms in Norway. Mortality levels in both diseases are generally low but morbidity can be very high with the associated chronic inflammatory response lasting for several months. The consequent reduced growth performance is causing considerable financial impact as HSMI has become increasingly widespread in recent years. The impact of CMS is further exacerbated as it generally affects large fish close to harvest. HSMI lesions occur in the atrium and ventricle in the heart including inflammation and necrosis in epi- endo- and myocardium along with myositis of red skeletal muscle. CMS lesions are commonly observed in the spongy myocardium in the atrium and ventricle of the heart with severe mononuclear inflammation and necrosis. Furthermore, circulatory disturbances associated with reduced cardiac function cause multifocal liver steatosis and necrosis in both diseases. Currently there are no vaccines or any other effective treatments for these diseases and so alternative therapies that could potentially modulate the intensity of the inflammatory response could be crucial to improve the clinical manifestation of the diseases. Therefore, the overall aim of the present study was to evaluate the concept of “clinical nutrition” to improve the clinical symptoms of both viral diseases, HSMI and CMS, through the use of functional feeds formulated with reduced lipid content and increased proportions of anti-inflammatory fatty acids to moderate the apparently uncontrolled inflammatory response in the heart tissue associated with both diseases and also alleviate the secondary hepatic lesions. The experimental work consisted of three major dietary trials in Atlantic salmon in seawater. Two large trials investigated the effects of functional feeds in Atlantic salmon challenged with Atlantic salmon piscine reovirus (ASRV) and piscine myocarditis virus (PMCV), the causal agents of HSMI and CMS, respectively. In both trials, heart transcriptome, heart and liver histopathology and tissue lipid and fatty acid compositions and metabolism were determined post-infection in fish fed with the functional feeds in comparison with fish fed with a standard commercial feed formulation considered as a reference diet. All the functional feeds were formulated to have reduced digestible energy through lower dietary lipid and higher protein contents, and increased levels and proportions of anti-inflammatory long-chain polyunsaturated fatty acids (LC-PUFA), particularly eicosapentaenoic acid (EPA) compared with the reference diets. Histopathology, fatty acid composition and gene expression of heart were assessed over a long time-period of 16 weeks and 14 weeks post-challenge with ASRV and PMCV, respectively. Viral load in heart tissue, hepatic histopathology and fatty acid composition of liver and head kidney along with expression of the genes involved in the eicosanoid and LC-PUFA and eicosanoid biosynthesis pathways were also determined in the HSMI trial. The third trial was a nutritional trial evaluating the effects of dietary digestible energy content on lipid and fatty acid metabolism in salmon fed diets containing graded amounts of lipid. Fatty acid composition of liver and heart were assessed over 12 weeks, along with the hepatic expression of genes of lipid and fatty acid metabolism. The results of this research are presented in four chapters (Chapters 2-5) as four paper manuscripts. The manuscripts/Papers are either published (Chapter 2), in review (Chapter 3 and 4) or drafted for submission (Chapter 5) in appropriate peer-reviewed international journals. Chapter 2 and 3 correspond to the HSMI trial, Chapter 4 to the nutritional trial, and Chapter 5 to the CMS trial. Chapter 2 showed that viral load and histopathology scores were lower in fish fed the functional feeds, especially diet FF1, which displayed better performance. Diet strongly influenced the expression of genes related with the immune and inflammatory responses, with delayed expression in fish fed the functional feeds. Up-regulation of pro-inflammatory genes was correlated with the higher viral load observed at early-mid stages of the disease in fish fed the reference diet (ST). Expression of genes related with the immune response at 16-weeks post challenge reflected the differences in immunomodulation between the functional feeds, with fish fed diet FF1 showing lower expression. Therefore, severity of the heart lesions was correlated with the intensity of the immune response and could be associated with tissue anti-inflammatory LC-PUFA levels. Chapter 3 was focused on liver histopathology, fatty acid composition and LC-PUFA biosynthesis, along with phospholipid fatty acid composition and eicosanoid production in head kidney and heart tissue at early and late stages of ASRV infection. Liver was severely affected by the virus at the beginning of the infection in fish fed the reference ST diet, but the level of lesions were similar in all dietary groups at the end of the trial. Hepatic expression of fatty acyl desaturases was significantly depressed in fish fed the ST diet compare with fish fed the functional feeds despite the lower levels of dietary LC-PUFA in that feed. Thus endogenous production and bioavailability of anti-inflammatory LC-PUFA was potentially enhanced in fish fed the functional feeds. Changes in tissue lipid content, mobilization of fatty acids involved in inflammatory responses and changes in expression of transcription factors and genes involved in eicosanoid biosynthesis were more prominent in head kidney, confirming the important role of this organ in dietary immunomodulation after viral infection. To a lesser extent similar changes were observed in heart tissue, suggesting in situ production of eicosanoids could also be important. The unexpected effects of diet on expression of genes of LC-PUFA biosynthesis were specifically investigated in the trial described in Chapter 4. One aim of this study was to clarify whether dietary lipid content or viral infection was the cause of altered expression of desaturase genes between the different diets. Hepatic expression of other genes of lipid and fatty acid metabolism were also determined to evaluate metabolic changes associated with dietary lipid/energy level. In general, reduction of dietary energy and lipid contents while maintaining similar proportions of dietary fatty acids, led to a general up-regulation of genes involved in lipid biosynthetic pathways. Thus salmon fed lower energy diet showed increased liver expression of fatty acyl desaturases in comparison with fish fed higher energy levels. Heart transcriptomic data in Chapter 5 showed a similar delay in the inflammatory response in fish fed the functional feeds after PCMV infection as observed in the HSMI study. Modulation of inflammatory responses, similar to that previously described after ASRV infection, was also observed in fish fed the functional feeds. However, the differences in the expression of immune related genes and the level of heart lesions were not as prominent at mid-late stages of the disease as in fish fed FF1 in the HSMI trial. The present study demonstrated the beneficial effects of a clinical nutrition approach via functional feeds in two viral inflammatory diseases, HSMI and CMS, currently affecting farmed Atlantic salmon. Dietary immunomodulation increased the availability of anti-inflammatory LC-PUFA and significantly influenced the expression of the genes related with the immune/inflammatory response reducing the level and severity of cardiac and liver lesions and therefore improving the performance of fish suffering the diseases

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal

Candidate Vaccine Sequences to Represent Intra- and Inter-Clade HIV-1 Variation

A likely key factor in the failure of a HIV-1 vaccine based on cytotoxic T lymphocytes (CTL) is the natural immunodominance of epitopes that fall in variable regions of the proteome, which both increases the chance of epitope sequence mismatch with the incoming challenge strain and replicates the pathogenesis of early CTL failure due to epitope escape mutation during natural infection. To identify potential vaccine sequences to focus the CTL response on highly conserved epitopes, the whole proteomes of HIV-1 clades A1, B, C, and D were assessed for Shannon entropy at each amino acid position. Highly conserved regions in Gag (cGag-1, Gag 148–214, and cGag-2, Gag 253–331), Env (cEnv, Env 521–606), and Nef (cNef, Nef 106–148) were identified across clades. Inter- and intra-clade variability of amino acids within the regions tended to overlap, suggesting that polyvalent representation of consensus sequences for the four clades would allow broad HIV-1 strain representation. These four conserved regions were rich in both known and predicted CTL epitopes presented by a breadth of HLA types, and screening of 54 persons with chronic HIV-1 infection revealed that these regions are commonly immunogenic in the context of natural infection. These data suggest that vaccine delivery of a 16-valent mixture of these regions could focus the CTL response against conserved epitopes that are broadly representative of circulating HIV-1 strains