Recommendations for measuring and standardizing light for laboratory mammals to improve welfare and reproducibility in animal research.

Light enables vision and exerts widespread effects on physiology and behavior, including regulating circadian rhythms, sleep, hormone synthesis, affective state, and cognitive processes. Appropriate lighting in animal facilities may support welfare and ensure that animals enter experiments in an appropriate physiological and behavioral state. Furthermore, proper consideration of light during experimentation is important both when it is explicitly employed as an independent variable and as a general feature of the environment. This Consensus View discusses metrics to use for the quantification of light appropriate for nonhuman mammals and their application to improve animal welfare and the quality of animal research. It provides methods for measuring these metrics, practical guidance for their implementation in husbandry and experimentation, and quantitative guidance on appropriate light exposure for laboratory mammals. The guidance provided has the potential to improve data quality and contribute to reduction and refinement, helping to ensure more ethical animal use.

Beyond Animal Testing Index: Benchmarking tool for a world beyond animal testing.

While the original definition of replacement focuses on the replacement of the use of animals in science, a more contemporary definition focuses on accelerating the development and use of predictive and robust models, based on the latest science and technologies, to address scientific questions without the use of animals. The transition to animal free innovation is on the political agenda in and outside the European Union. The Beyond Animal Testing Index (BATI) is a benchmarking instrument designed to provide insight into the activities and contributions of research institutes to the transition to animal free innovation. The BATI allows participating organizations to learn from each other and stimulates continuous improvement. The BATI was modelled after the Access to Medicine Index, which benchmarks pharmaceutical companies on their efforts to make medicines widely available in developing countries. A prototype of the BATI was field-tested with three Dutch aca­demic medical centers and two universities in 2020-2021. The field test demonstrated the usability and effectiveness of the BATI as a benchmarking tool. Analyses were performed across five different domains. The participating institutes concluded that the BATI served as an internal as well as an external stimulus to share, learn, and improve institutional strategies towards the transition to animal free innovation. The BATI also identified gaps in the development and implementation of 3R technologies. Hence, the BATI might be a suitable instrument for monitoring the effectiveness of policies. BATI version 1.0 is ready to be used for benchmarking at a larger scale.

The use of animals for research is being scrutinized by the public. The transition to animal-free methods is on the political agenda in and outside the European Union. This requires accelerating the development and use of useful and reliable animal-free methods. The Beyond Animal Testing Index (BATI) is designed to provide insight into the activities and contributions of research institutes to the transition to animal-free innovation. The BATI allows participating organizations to learn from each other and stimulates continuous improvement. A prototype of the BATI was field-tested with three Dutch academic medical centers and two universities in 2020-2021. The field test showed that the BATI could be used to monitor how effective policies are and to show where more work is needed towards the full replacement of animals in research.

From Mating to Milk Access: A Review of Reproductive Vocal Communication in Mice.

Vocalisations play a central role in rodent communication, especially in reproduction related behaviours. In adult mice (Mus musculus) the emission of ultrasonic vocalisations (USVs) has been observed in courtship and mating behaviour, especially by males. These have been found to have distinctive individual signatures that influence female choice of mating partner. The most recent findings show that vocal communication also has a role in parental cooperation, in that female mice communicate with male partners in ultrasonic frequencies to induce paternal behaviour. Infant vocalisations form the other important part of reproductive vocal communication. Although born deaf, neonatal mice are capable of producing vocalisations since birth. As an altricial species, successful mother-infant communication is essential for survival, and these vocalisations are important modulators of maternal behaviour. Three main types of infant vocalisations have been identified and characterised. Most research has addressed pure USVs, related to stressful situations (e.g., cold, isolation, handling, presence of unfamiliar males or predators), which usually elicit maternal search and retrieval. In addition, broad-band spectrum signals, emitted post-partum during cleaning of foetal membranes, inhibit biting and injury by adults and "wriggling calls," emitted during suckling, release maternal behaviour (such as licking). Several variables have been identified to modulate vocalisations in mice, including individual characteristics such as strain/genotype, age, sex, and experimental factors such as pharmacological compounds and social context. In recent years, there has been a big increase in the knowledge about the characteristics of vocal communication in rodents due to recent technological advances as well as a growing interest from the neuroscience community. Vocalisation analysis has become an essential tool for phenotyping and evaluating emotional states. In this review, we will (i) provide a comprehensive summary of the current knowledge on mouse reproductive vocal communication and (ii) discuss the most recent findings in order to provide a broad overview on this topic.

Assessable learning outcomes for the EU Education and Training Framework core and Function A specific modules: Report of an ETPLAS WORKING Group.

Article 23(2) of the European Union Directive 2010/63/EU, which regulates welfare provisions for animals used for scientific purposes, requires that staff involved in the care and use of animals for scientific purposes be adequately educated and trained before they undertake any such work. However, the nature and extent of such training is not stipulated in the Directive. To facilitate Member States in fulfilling their education and training obligations, the European Commission developed a common Education and Training Framework, which was endorsed by the Member States Competent Authorities. An Education & Training Platform for Laboratory Animal Science (ETPLAS) Working Group was recently established to develop further guidance to the Learning Outcomes in the Framework, with the objective to clarify the levels of knowledge and understanding required by trainees, and to provide the criteria by which these Learning Outcomes should be assessed. Using the Framework document as a starting point, assessment criteria for the Learning Outcomes of the modules required for Function A persons (carrying out procedures on animals) for rats, mice and zebrafish were created with sufficient detail to enable trainees, providers and assessors to appreciate the level of knowledge, understanding and skills required to pass each module. Adoption and utilization of this document by training providers and accrediting or approving bodies will harmonize introductory education and training for those involved in the care and use of animals for scientific purposes within the European Union, promote mutual recognition of training within and between Member States and therefore free movement of personnel.

Genetic quality assurance and genetic monitoring of laboratory mice and rats: FELASA Working Group Report.

Genetic quality assurance (QA), including genetic monitoring (GeMo) of inbred strains and background characterization (BC) of genetically altered (GA) animal models, should be an essential component of any QA programme in laboratory animal facilities. Genetic quality control is as important for ensuring the validity of the animal model as health and microbiology monitoring are. It should be required that studies using laboratory rodents, mainly mice and rats, utilize genetically defined animals. This paper, presented by the FELASA Working Group on Genetic Quality Assurance and Genetic Monitoring of Laboratory Murines, describes the objectives of and available methods for genetic QA programmes in rodent facilities. The main goals of any genetic QA programme are: (a) to verify the authenticity and uniformity of inbred stains and substrains, thus ensuring a genetically reliable colony maintenance; (b) to detect possible genetic contamination; and (c) to precisely describe the genetic composition of GA lines. While this publication focuses mainly on mouse and rat genetic QA, the principles will apply to other rodent species some of which are briefly mentioned within the context of inbred and outbred stocks.

A Systematic Review Comparing Experimental Design of Animal and Human Methotrexate Efficacy Studies for Rheumatoid Arthritis: Lessons for the Translational Value of Animal Studies.

Increased awareness and understanding of current practices in translational research is required for informed decision making in drug development. This paper describes a systematic review of methotrexate for rheumatoid arthritis, comparing trial design between 147 animal and 512 human studies. Animal studies generally included fewer subjects than human studies, and less frequently reported randomisation and blinding. In relation to life span, study duration was comparable for animals and humans, but included animals were younger than included humans. Animal studies often comprised males only (61%), human studies always included females (98% included both sexes). Power calculations were poorly reported in both samples. Analyses of human studies more frequently comprised Chi-square tests, those of animal studies more frequently reported analyses of variance. Administration route was more variable, and more frequently reported in animal than human studies. Erythrocyte sedimentation rate and c-reactive protein were analysed more frequently in human than in animal studies. To conclude, experimental designs for animal and human studies are not optimally aligned. However, methotrexate is effective in treating rheumatoid arthritis in animal models and humans. Further evaluation of the available evidence in other research fields is needed to increase the understanding of translational success before we can optimise translational strategies.

The MicroRNA-371 Family as Plasma Biomarkers for Monitoring Undifferentiated and Potentially Malignant Human Pluripotent Stem Cells in Teratoma Assays.

Predicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also identified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.

Formoterol added to low-dose budesonide has no additional antiinflammatory effect in asthmatic patients.

STUDY OBJECTIVE: Adding inhaled long-acting beta2-agonists to a low dose of inhaled corticosteroids (ICSs) results in better asthma control than increasing the dose of ICSs. An important, but as yet unresolved, question is whether this is due to an additional reduction of airway inflammation. DESIGN: Double-blind, parallel-group trial. PATIENTS: Forty asthma patients (FEV1, 50 to 90% predicted; provocative concentration of a substance [methacholine] causing a 20% fall in FEV1 of < 8 mg/mL; no ICSs in the last 4 weeks). INTERVENTIONS: Randomization to 8 weeks of treatment with 100 microg of budesonide bid plus placebo (BUD200) or 100 microg of budesonide bid plus 12 microg of formoterol (BUD200 + F). Then the dose of budesonide (BUD) was increased to 400 microg bid in both groups for another 8 weeks. Bronchial biopsy specimens were collected before, and after 8 and 16 weeks of treatment. Eosinophils (major basic protein [MBP]) and mast cells (tryptase) were analyzed by immunohistochemistry. RESULTS: BUD200 reduced the MBP staining (p = 0.008) and tryptase staining (p = 0.048) in the epithelium compared to baseline levels. There were no significant differences between the BUD200 and BUD200 + F groups. In both groups, increasing the dosage of BUD to 800 microg had no significant additional antiinflammatory effect. CONCLUSIONS: Our results demonstrate that BUD administered at a low dose has significant antiinflammatory effects in patients with mild asthma. No significant additional antiinflammatory effects could be demonstrated either by adding formoterol or by increasing the dose of BUD.

Benefit from anti-inflammatory treatment during clinical remission of atopic asthma.

STUDY OBJECTIVES: Subjects with atopic asthma often experience a disappearance of symptoms around puberty. However, airway inflammation and remodeling may persist. It is unknown whether those findings warrant prolonged anti-inflammatory treatment despite the absence of symptoms. In this study, we investigated whether a short course of combined anti-inflammatory treatment would, also in this specific patient population, diminish airway inflammation and/or remodeling. DESIGN: A double-blind, randomized placebo-controlled trial was conducted in 28 asymptomatic subjects with a history of atopic asthma, with established bronchial hyperresponsiveness to methacholine (MCh) as non-invasive indicator of ongoing airway pathology. INTERVENTIONS: Intervention consisted of the salmeterol/fluticasone propionate combination (SFC) product (50/250 microg bid via the Diskus inhaler) or placebo for 3 months. MEASUREMENTS: The change in lung function (FEV1), bronchial response to MCh and adenosine monophosphate (AMP), the fraction of nitric oxide in exhaled air (FENO) and quality of life (QOL) scores were measured. Also, bronchial biopsies were taken and cryo sections immunostained for eosinophils (major basic protein, MBP) and mast cells (tryptase and chymase) before and after treatment. The change in reticular basement membrane (RBM) thickness, one of the parameters of airway remodeling, was also determined. RESULTS: SFC treatment improved hyperresponsiveness to MCh (P = 0.014) as well as AMP (P = 0.011), and reduced FENO (P < 0.001) significantly as compared with placebo. Lung function tended to improve (NS). Furthermore, SFC treatment reduced tryptase in the subepithelium of bronchial biopsy specimens (P = 0.01), and slightly reduced RBM thickness (P = 0.05). However, eosinophils in (sub)epithelium were not significantly affected; neither were chymase levels, blood eosinophils or QOL scores. CONCLUSIONS: We found that 3 months of treatment with fluticasone propionate and salmeterol reduced airway hyperresponsiveness, FENO and tryptase density in the airway mucosa as markers of airway inflammation. MBP density in the airway mucosa and QOL were, however, unchanged. The clinical relevance of these findings, especially with respect to the long-term outcome, has not been determined yet.

Alternative methods for the use of non-human primates in biomedical research.

The experimental use of non-human primates (NHP) in Europe is tightly regulated and is only permitted when there are no alternatives available. As a result, NHP are most often used in late, pre-clinical phases of biomedical research. Although the impetus for scientists, politicians and the general public to replace, reduce and refine NHP in biomedical research is strong, the development of 3Rs technology for NHP poses specific challenges. In February 2014 a workshop on "Alternative methods for the use of NHP in biomedical research" was organized within the international exchange program of EUPRIM-Net II, a European infrastructure initiative that links biomedical primate research centers. The workshop included lectures by key scientists in the field of alternatives as well as by experts from governmental and non-governmental organizations. Furthermore, parallel sessions were organized to stimulate discussion on the challenges of advancing the use of alternative methods for NHP. Subgroups voted on four statements and together composed a list with opportunities and priorities. This report summarizes the presentations that were held, the content of the discussion sessions and concludes with recommendations on 3Rs development for NHP specifically. These include technical, conceptual as well as political topics.

Asthma remission: does it exist?

Subjects believed to have grown out of asthma often develop symptoms again later in life. Ongoing airway inflammation may determine the risk of relapse, although the mechanisms involved are still misunderstood. Additionally, patients with asthma during childhood may develop irreversible airflow obstruction ( airway remodeling) as a result of chronic airway inflammation. Recently, airway inflammation and remodeling could be demonstrated in bronchial biopsy specimens from young adults who considered themselves grown out of asthma. It is also shown that evidence of airway inflammation and remodeling can be obtained noninvasively, thereby providing the opportunity to monitor disease activity. If chronic airway inflammation and/or remodeling are consistent findings in asymptomatic subjects with a history of atopic asthma, the question arises whether natural history can be positively altered with prolonged antiinflammatory therapy. Benefits of long-term prognosis are, however, not yet shown. Since epidemiologic work has demonstrated that a certain percentage of subjects with apparently outgrown atopic asthma remains asymptomatic without needing therapy for the rest of their lives, it can be argued that "asthma remission does exist." The question is whether this percentage can be increased with prolonged antiinflammatory therapy and regular control.

Lipopolysaccharide-induced suppression of airway Th2 responses does not require IL-12 production by dendritic cells.

The prevalence of atopic asthma, a Th2-dependent disease, is reaching epidemic proportions partly due to improved hygiene in industrialized countries. There is an inverse correlation between the level of environmental endotoxin exposure and the prevalence of atopic sensitization. As dendritic cells (DC) have been implicated in causing sensitization to inhaled Ag, we studied the effect of endotoxin on Th2 development induced by bone marrow DC in vitro and by intratracheal injection in vivo, with particular emphasis on the role played by the polarizing cytokine IL-12. Bone marrow-derived DC stimulated with Escherichia coli O26:B6 LPS produced IL-12p70 for a limited period of time, after which production became refractory to further stimulation with CD40 ligand, a phenomenon previously called "exhaustion." The level of IL-12 production of DC did not correlate with Th1 development, as exhausted OVA-pulsed DC were still capable of shifting the cytokine pattern of responding OVA-specific Th cells toward Th1 in vitro and in vivo. When mice were first immunized by intratracheal injection of OVA-DC and subsequently challenged with OVA aerosol, prior in vitro stimulation of DC with LPS reduced the development of airway eosinophilia and Th2 cytokine production. Most surprisingly, the capacity of LPS to reduce Th2-dependent eosinophilic airway inflammation was IL-12-independent altogether, as IL-12p40 knockout DC had a similar reduced capacity to prime for Th2 responses. These results suggest that LPS reduces sensitization to inhaled Ag by reducing DC-driven Th2 development, but that IL-12 is not necessary for this effect.

Allergen-induced accumulation of airway dendritic cells is supported by an increase in CD31(hi)Ly-6C(neg) bone marrow precursors in a mouse model of asthma.

Airway dendritic cells (DCs) are held responsible for inducing sensitization to inhaled antigen, leading to eosinophilic airway inflammation, typical of asthma. However, less information is available about the role of these cells in ongoing inflammation. In a mouse model of asthma, sensitization to ovalbumin (OVA) was induced by intratracheal injection of myeloid OVA-pulsed DCs. Upon OVA aerosol challenge and induction of eosinophilic airway inflammation in sensitized mice, there was a time-dependent and almost 100-fold increase in the number of MHCII(+) CD11b(+) CD11c(+) endogenous airway DCs as well as CD11b(+) blood DCs. The mechanism of this increase was studied. Adoptive transfer experiments demonstrated that accumulation of airway DCs was not due to reduced migration to the mediastinal lymph nodes. Rather, the massive increase in airway and lymph node DCs was supported by an almost 3-fold expansion of myeloid CD31(hi)Ly-6C(neg) hematopoietic precursor cells in the bone marrow (BM). There was no change in any of the other 5 populations revealed by CD31/Ly-6C staining. When these CD31(hi)Ly-6C(neg) BM precursors were sorted and grown in granulocyte macrophage-colony-stimulating factor, they differentiated into MHCII(+) CD11c(+) DCs. The same CD31(hi)Ly-6C(neg) precursors also expressed the eotaxin receptor CCR3 and differentiated into eosinophils when grown in interleukin 5. Serum levels of eotaxin were doubled in mice with inflammation. These findings in an animal model of asthma suggest that the BM increases its output of myeloid precursors to meet the enhanced demand for DCs and eosinophils in inflamed airways.

Proteomic analysis of exosomes secreted by human mesothelioma cells.

Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins present in exosomes secreted by tumor cells have been poorly defined. This study describes the protein composition of mesothelioma cell-derived exosomes in more detail. After electrophoresis of exosome preparations, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) was used to characterize the protein spots. MHC class I was found to be present together with the heat shock proteins HSC70 and HSP90. In addition, we found annexins and PV-1, proteins involved in membrane transport and function. Cytoskeleton proteins and their associated proteins ezrin, moesin, actinin-4, desmoplakin, and fascin were also detected. Besides the molecular motor kinesin-like protein, many enzymes were detected revealing the cytoplasmic orientation of exosomes. Most interesting was the detection of developmental endothelial locus-1 (DEL-1), which can act as a strong angiogenic factor and can increase the vascular development in the neighborhood of the tumor. In conclusion, mesothelioma cells release exosomes that express a discrete set of proteins involved in antigen presentation, signal transduction, migration, and adhesion. Exosomes may play an important role in the interaction between tumor cells and their environment.