Laboratory Animal Science Associations Research Articles

Transfer of HBV Genomes Using Low Doses of Adenovirus Vectors Leads to Persistent Infection in Immune Competent Mice

Studies of mechanisms responsible for the persistence of hepatitis B virus (HBV) infection have been hindered by a lack of appropriate animal models. HBV genomes can be delivered to livers of mice using hydrodynamic injection or high doses of an adenoviral vector; these lead to clearance of HBV. We found that infection of immunocompetent mice with low doses of an adenoviral vector resulted in persistent HBV infection; the mice neither underwent seroconversion to production of antibodies against HBV nor developed a strong HBV-specific effector T-cell response. As in patients with chronic HBV infection, DNA vaccination failed to generate T cells that cleared infection. This model of persistent HBV infection could be used to study the pathogenesis of chronic HBV infection and develop new therapeutic strategies.

Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells

Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca(2+) signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca(2+) mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling 'toolkit' that control the shape of purinergic Ca(2+) responses, and probably several other paracrine Ca(2+)-dependent signals.

Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics

Granule-associated perforin and granzymes (gzms) are key effector molecules of cytotoxic T lymphocytes (Tc cells) and natural killer cells and play a critical role in the control of intracellular pathogens and cancer. Based on the notion that many gzms, including A, B, C, K, H, and M exhibit cytotoxic activity in vitro, all gzms are believed to serve a similar function in vivo. However, more recent evidence supports the concept that gzms are not unidimensional but, rather, possess non-cytotoxic potential, including stimulation of pro-inflammatory cytokines and anti-viral activities. The present study shows that isolated mouse gzmB cleaves the actin-severing mouse protein, cytoplasmic gelsolin (c-gelsolin) in vitro. However, when delivered to intact target cells by ex vivo immune Tc cells, gzmB mediates c-gelsolin proteolysis via activation of caspases 3/7. The NH(2)-terminal c-gelsolin fragment generated by either gzmB or caspase 3 in vitro constitutively severs actin filaments without destroying the target cells. The observation that gzmB secreted by Tc cells initiates a caspase cascade that disintegrates the actin cytoskeleton in target cells suggests that this intracellular process may contribute to anti-viral host defense.

FELASA guidelines for the accreditation of health monitoring programs and testing laboratories involved in health monitoring

Monitoring the health of research animals is an essential part of the research process and helps to ensure that experiments yield reliable and reproducible results. The Federation of European Laboratory Animal Science Associations (FELASA) is one organization that accredits and evaluates health monitoring programs and laboratories involved with health monitoring. In this article, the authors (who are members of the FELASA working group Accreditation Board for Health Monitoring) describe the guidelines of the FELASA health monitoring accreditation process. The ultimate goal of this accreditation program is to make health monitoring reports more thorough and reliable, thereby increasing the standardization of health monitoring of laboratory animals.

Assessing the Search for and Implementation of the Three Rs: A Survey among Scientists

A survey among scientists into the current practice of searching for Replacement, Reduction and Refinement (Three Rs) alternatives, highlights the gap between the statutory required need to apply the Three Rs concept whenever possible and the lack of criteria for searching for Three Rs alternatives. A questionnaire was distributed to 342 scientists (Federation of European Laboratory Animal Science Associations [FELASA] Category C and B individuals), of which 67 responded. These scientists are customers of the Central Animal Laboratory of Radboud University Nijmegen Medical Centre. The results indicate that there is room for improvement in searching effectively for the Three Rs: skills in searching biomedical databases for Three Rs alternatives are limited, knowledge of specialised Three Rs databases is very limited, and satisfaction on the availability and accessibility of Three Rs information is low. None of the respondents allocate budget for a specific Three Rs alternatives search, although 50% do spend, on average, two hours engaged in this search for each application to their animal ethics committees. The majority of the respondents expressed the wish that the search for alternatives could be easier and less time consuming, and prefer to achieve this through the service offered by specialists at the Central Animal Laboratory. On the basis of the results from the questionnaire, the 3R Research Centre was established, with the aim of providing services and support for biomedical scientists, to improve the search for, and subsequent implementation of, the Three Rs.

Proper Perinuclear Localization of the TRIM-like Protein Myospryn Requires Its Binding Partner Desmin

Desmin, the muscle-specific intermediate filament protein, surrounds the Z disks and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles, and the nucleus. In an attempt to explore the molecular mechanisms of these associations, we performed a yeast two-hybrid screening of a cardiac cDNA library. We showed that the desmin amino-terminal domain (N-(1-103)) binds to a 413-kDa TRIM-like protein, myospryn, originally identified as the muscle-specific partner of dysbindin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Binding of desmin with myospryn was confirmed with glutathione S-transferase pulldown assays and coimmunoprecipitation experiments. Western blot analysis revealed that the complex immunoprecipitated by desmin antibodies, in addition to myospryn, contained the BLOC-1 components dysbindin and pallidin. Deletion analysis revealed that only the (N-(1-103)) fragment of desmin binds to myospryn carboxyl terminus and that this association takes place through the 24-amino acid-long carboxyl-terminal end of the SPRY domain of myospryn. Using an antibody against the COOH terminus of myospryn, we demonstrated that myospryn colocalizes with desmin at the periphery of the nucleus, in close proximity to the endoplasmic reticulum, of mouse neonatal cardiomyocytes. In adult heart muscle, the two proteins colocalize, predominantly at intercalated disks and costameres. We also showed that myospryn colocalizes with lysosomes. Using desmin null hearts, we determined that desmin is required for both the proper perinuclear localization of myospryn, as well as the proper positioning of lysosomes, thus suggesting a potential role of desmin intermediate filaments in lysosomes and lysosome-related organelle biogenesis and/or positioning.

Guidelines for the Human Transportation of Research Animals

Transportation of research animals is a major component of the research enterprise but one that can have a substantial negative impact on animal well-being. Both environmental conditions and the novelty of the transportation experience can cause stress, which can result in short-term changes in the physiological and psychological condition of the animals and thus affect subsequent research outcomes, especially if the animals are utilized immediately after transportation. Therefore, the primary goal for all individuals involved in animal transport should be to move them in a manner that does not jeopardize their well-being and ensures their safe arrival at their destination in good health and with minimal distress. While shipments from breeders to research institutions are generally well executed through the use of company-owned fleets of environmentally controlled vehicles, arranging humane transport from vendors without established transport systems, or between research institutions, can be challenging for research facility staff without extensive experience. International transport of nonhuman primates (hereafter primates) is particularly challenging because their shipment and quarantine require adherence to the legislation of each country involved and because few airlines are willing to carry them. What is needed to meet these challenges is comprehensive guidance detailing the potential negative effects of the transportation process on research animal well-being and the steps necessary to minimize adverse effects for the main species transported, as well as the relevant local, national and international regulations and the various organizations involved. Although a working group of the United Kingdom’s Laboratory Animal Science Association (LASA) has recently considered these issues and developed guidance for Europe (Swallow et al. 2005), no single source of such information existed for the United States of America (USA) until publication of this book from the National Research Council’s Institute for Laboratory Animal Research (ILAR).

Principles and practice in ethical review of animal experiments across Europe: summary of the report of a FELASA working group on ethical evaluation of animal experiments

This paper summarizes a more detailed report produced by the Federation of European Laboratory Animal Science Associations (FELASA 2005), which describes and explores a set of principles for the conduct of ethical review of laboratory animal use. It presents a synopsis of results from a questionnaire that elicited information on how each of 20 countries represented in FELASA currently approaches such ethical review. This information suggests that, although local practices differ, there is an emerging consensus on the key elements that any ethical review process should involve. Drawing on the questionnaire findings, this summary also includes a brief discussion to support and amplify a series of recommendations, covering the objectives of ethical review; legal requirements; the scope of work reviewed and the 'level' at which review is approached; general principles for the organization of ethical review processes; the factors considered in the review; needs for ongoing review after initial authorization; participants in the review process; wider impacts of the review process; and strategies that can help to ensure quality and consistency of review outcomes. For further information and examples of current practice, as well as more detailed discussion to support the recommendations, readers are urged to refer to the complete report, available at http://www.lal.org.uk/pdffiles/FELASA_ethics_FULL_Report. pdf or via: http://www.felasa.eu/recommendations.htm.

Federation of European laboratory animal science associations (FELASA)

Federation of European laboratory animal science associations (FELASA)

Suffering and severity: report of a session at the Laboratory Animal Science Association's winter meeting

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Training for Reduction in Laboratory Animal Use

Reduction, refinement and replacement of laboratory animals wherever possible, are the guiding principles of the Federation of European Laboratory Animal Science Associations (FELASA) education guidelines. Of these, reduction is probably the least understood. Reduction and refinement are dependent upon each other. In the reduction "axis", there is a window of appropriate numbers of animals; too few and the experiment will lack power--too many and animals will be wasted. Reduction must be understood as the appropriate number of animals required for each experiment. The refinement "axis" is more straightforward. Better welfare is always desirable. Any factor can interfere with a study in two ways. If it changes the mean, this may not be serious, because it should do so in all groups. If it causes a change in variation, then this is far more troublesome, because this is bound to alter the appropriate number of animals for an experiment. Scientists are definitely concerned about the variation of the characters that they are working with. It is obvious that changes in variation may be study-specific, which makes the formulation of overall guidelines difficult. Indeed, instead of trying to assess the impact of housing and procedures on every possible character, it could be more productive to look at the effects of welfare indicators on variation, with the understanding that low variation here is likely to be reflected by low variation in most other characters, while aiming to achieve the most uniform welfare of the animals in the study.

Laboratory Animal Science Association (LASA) Alternatives Section Meeting — Designing to Reduce and Refine: Strategies and Successes

Laboratory Animal Science Association (LASA) Alternatives Section Meeting — Designing to Reduce and Refine: Strategies and Successes

The European Science Foundation: Three Rs Policy and Education

The European Science Foundation (ESF) has adopted a position paper on the use of animals in research. In this document, the ESF sets out its view on conditions that must be met to make the use of animals morally acceptable. The ESF explicitly states that laboratory animals have an intrinsic value that must be respected. The consequences of recognising the intrinsic value have been elaborated in the position paper and include, among others, that ESF Member Organisations are encouraged to endorse the reduction, refinement and replacement principles. Prior to the performance of an animal experiment, the protocol should be subjected to independent expert review, including the weighing of the likely benefit versus the likely animal suffering. Also, the development and organisation of accredited courses on laboratory animal science, including information on animal alternatives, welfare and ethics, are encouraged. The Council of Europe and the Federation of Laboratory Animal Science Associations have issued recommendations as to the education and training of persons involved in animal-based research. The ESF guidelines are in line with these recommendations. The recommendations are explained, and some details on the implementation in The Netherlands are presented.

179IMPORTANCE OF EMBRYO TRANSFERS IN TRANSGENIC MOUSE FACILITIES

With the increasing demand for and production of transgenic and mutant mice for biomedical research, embryo transfer plays a paramount role. The purpose of performing embryo transfer in this species is to generate transgenic mice via blastocyst injection of embryonic stem cells or pronuclear injection of DNA constructs, to revitalize cryopreserved sperm and embryos, and to generate mouse lines that meet specific pathogen-free health standards for breeding in barrier areas (rederivation). We present results from two years of carrying out embryo transfers for rederivation purposes in the large mouse breeding facility of the GSF—National Research Center for Environment and Health, Neuherberg, Germany. Pathogens to be eradicated from inbred transgenic (C57BL/6 background) and mutant (C3H background) mouse lines included mouse hepatitis virus, mouse minute virus, and mouse parvovirus. In vitro- and in vivo-produced two-cell embryos were washed 3 times in M2 medium. A total of 20 embryos each were transferred to the oviduct of 8- to 12-week-old specific pathogen-free pseudopregnant (Day 0.5) Swiss recipients under aseptic conditions. Mice were then kept singly in individually ventilated cages and manipulated in a Class II laminar flow hood. From each transfer to one to five recipients with embryos originating from the same mouse line, one recipient was tested for the presence of microorganisms 6 to 12 weeks after embryo transfer, i.e. at 0 to 6 weeks after weaning, according to the FELASA (Federation of European Laboratory Animal Science Associations) Guidelines. A total of 290 embryo transfers were performed for revitalization of cryopreserved sperm from 52 mouse lines, cryopreserved two-cell embryos from 18 mouse lines and rederivation of 12 mouse lines using freshly collected two-cell embryos. From these 290 embryo transfers, 59 mouse lines were re-established (40 from cryopreserved sperm, 7 from cryopreserved embryos and 12 from in vivo-produced embryos). Health monitoring of 54 recipients showed that all mouse lines generated were free of all pathogens stated in the FELASA list. The results presented here show that all 12 (100%) mouse lines were re-established after transfer of freshly collected two-cell embryos whereas 77% and 39% success rates were observed for revitalization of cryopreserved sperm and embryos, respectively. The success of embryo transfer in eradicating pathogens depends on the inability of these pathogens to transverse the zona pellucida and enter and/or infect embryonic cells. In our mouse facility, embryo transfer provided an efficient method to successfully revitalize cells of the mouse germ line as well as to eradicate prevalent murine pathogens. Furthermore, the results demonstrate the efficiency of transferring embryos of different origins and thereby obtaining and maintaining specific pathogen-free health standards in our mouse colonies.

FELASA recommendations for the accreditation of laboratory animal science education and training.

Article 26 of the Council of Europe Convention, requires that `Persons who carry out procedures, or take part in procedures, or take care of animals used in procedures, including supervision, shall have had appropriate education and training’ (CE 1986 ). Similarly, Article 14 of EU Directive 86=609=EEC states that `Persons who carry out experiments or take part in them and persons who take care of animals used for experiments, including duties of a supervisory nature, shall have appropriate education and training’ (Of®cial J EC 1986 ). Both statutes refer to this as `competence’. The term competence was initially rather poorly de®ned and this caused some confusion. The Federation of European Laboratory Animal Science Associat ions (FELASA) has developed the notion of competencies, and made them clearer and comprehensive. FELASA identi®es four categories of competence (FELASA 1995 ), which are also adopted by the Council of Europe (1993):

Bases metodológicas e neurofuncionais da avaliação de ocorrência de dor / sofrimento em animais

Identificar e consequentemente evitar situações que sujeitem animais a sofrimentos têm sido um objetivo bem definido, em Bioética. Entretanto, o assunto é complexo, basicamente por duas razões (lASP - Intemational Association for the Study of Pain- SHORT; POZNAN, 1992, p. 64): 1. dor/sofrimento são fenômenos de vivência subjetiva (McGRATH; FINLEY, 2000. p. 15-19); 2. os animais, assim como os bebês humanos, não nos informam verbalmente a respeito do que sentem. Propõe-se, assim, uma visão abrangente da ciência, sustentada pelo método racional, que possa embasar a avaliação das mais diversas situações em que são utilizados animais: experimentação científica, uso para fins didáticos, produção, exibição em circos, parques e zoológicos, entre outros, particularizando-se a polêmica questão da possibilidade de o uso do sedém e de esporas causar dor / sofrimento nos animais durante treinamentos e provas de rodeio. Para tanto, analisa-se a similitude de configuração morfofuncional das vias neurais da dor, comparativamente ao ser humano, recorrendo-se, assim, ao Princípio da Homologia, preconizado pela LASA - Laboratory Animal Science Association. Para se identificar a vivência de dor / sofrimento em animais, são também analisadas as possibilidades (Anais do Simpósio "The Detection and Relief of Pain in Animais" - B. V.A. - British Veterinarian Association) de ocorrência de Sinais Fisiológicos, bem como de Comportamento Sugestivo dessa vivência. Formas substitutivas de procedimentos que envolvem a ocorrência de dor / sofrimento de animais, nas mais variadas situações em que são utilizados, devem ser buscadas, pretendendo-se sempre preservar suas condições de bem-estar. Essa postura não apenas é compatível como direito natural dos animais, de não serem sujeitados a sofrimentos, como ainda é coerente com a condição de dignidade que pretendemos merecer, como seres humanos.

Communication of the Three Rs within Establishments: Some Problems and Solutions

A meeting was organised by the Laboratory Animal Science Association (LASA) Alternatives Section to provide a forum in which ideas for strategies to raise awareness of the Three Rs could be discussed. One of the main objectives of the meeting was to identify effective strategies which could feasibly be implemented at establishments. In general, there are two main ways in which Three Rs information can be obtained and communicated. Individuals can be assigned to a role with specific responsibilities (for example, a named alternatives expert), or committees can be established, with a remit to find ways to reduce, refine and/or replace animal use (for example, an alternatives committee). The meeting involved invited speakers who are engaged in obtaining and communicating Three Rs information, and this report presents a summary of the advantages and limitations of various approaches that are being undertaken.

Ethical review process in academia

The UK Government issued a requirement on 1 April 1998 that all establishments designate under the 1986 Act should have an ethical review process (ERP) satisfactorily installed by 1 April 1999. This document, which has been circulated by the Laboratory Animal Science Association (LASA) to its members, is a report of a meeting held to formulate guidelines to assist establishments in the process of setting up an ERP. The document builds on the experience of participants who had already had experience of either establishing or running an ERP, and addresses the requirements of an ERP, taking into account that different establishments will have different needs and that therefore the structure of ERPs may differ. The booklet provides a number of excellent bullet points addressing the roles of those involved in an ERP, how to review an existing ERP, and concerns that people may have about such issues as confidentiality or possible delays to experiments. LASA is to be commended for producing such a useful document in a comparatively short time. Moreover, as the ERP is seen as an evolving concept, the booklet should continue to be useful for a number of years to come.

The Production of Polyclonal Antibodies in Laboratory Animals. The Report and Recommendations of ECVAM Workshop 35.

This is the report of the thirty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). This joint ECVAM/FELASA (Federation of European Laboratory Animal Science Associations) workshop on The Immunisation of Laboratory Animals for the Production of Polyclonal Antibodies was held in Utrecht (The Netherlands), on 20-22 March 1998, under the co-chairmanship of Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) and Wim de Leeuw (Inspectorate for Health Protection, The Netherlands). The participants, all experts in the fields of immunology, laboratory animal science, or regulation, came from universities, industry and regulatory bodies. The aims of the workshop were: a) to discuss and evaluate current immunisation procedures for the production of polyclonal antibodies (including route of injection, animal species and adjuvant ); and b) to draft recommendations and guidelines to improve the immunisation procedures, with regard both to animal welfare and to the optimisation of immunisation protocols. This report summarises the outcome of the discussions and includes a number of recommendations and a set of draft guidelines (included in Appendix 1).

Health monitoring of non-human primate colonies

Recommendations of the Federation of European Laboratory Animal Science Associations (FELASA) Working Group on Non-Human Primate Health accepted by the FELASA Board of Management, 21 November 1998 FELASA Working Group on Non-Human Primate Health: H. Weber (Convenor), E. Berge, J. Finch, P. Heidt, F.-J. Kaup, G. Perretta, B. Verschuere & S. Wolfensohn