The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

KIT D816V-mutated bone marrow mesenchymal stem cells in indolent systemic mastocytosis are associated with disease progression.

Multilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V-mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P = .03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P ≤ .003). Overall, these results support the notion that ISM patients with mutated MSCs may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSCs and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome.

Diagnosis and classification of mastocytosis in non-specialized versus reference centres: a Spanish Network on Mastocytosis (REMA) study on 122 patients.

The diagnosis of 'rare diseases', such as mastocytosis, remains a challenge. Despite this, the precise benefits of referral of mastocytosis patients to highly specialized reference centres are poorly defined and whether patients should be managed at non-specialized versus reference centres remains a matter of debate. To evaluate the quality and efficiency of diagnostic procedures performed at the reference centres for mastocytosis in Spain (REMA) versus other non-reference centres, we retrospectively analysed a series of 122 patients, for the overall degree of agreement obtained for the World Health Organization (WHO) diagnostic and classification criteria betwen the referring and REMA centres. Our results showed that not all WHO diagnostic criteria were frequently investigated at the referring centres. Among the five WHO diagnostic criteria, the highest degree of agreement was obtained for serum tryptase levels [median 90% (95% confidence interval 84-96%)]; in turn, the overall agreement was significantly lower for the major histopathological criterion [80% (72-89%)], and the other three minor criteria: cytomorphology [68% (56-80%)] immunophenotyping of BM mast cells [75% (62-87%)] and detection of the KIT mutation [34% (8-60%)]. Referral of patients with diagnostic suspicion of mastocytosis to a multidisciplinary reference centre improves diagnostic efficiency and quality.

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications.

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)--with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)--as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

Gene expression profile of highly purified bone marrow mast cells in systemic mastocytosis.

BACKGROUND: Despite the fact that a great majority (>90%) of patients with systemic mastocytosis (SM) carry a common genetic lesion, the D816V KIT mutation, little is known regarding the molecular and biological pathways underlying the clinical heterogeneity of the disease. OBJECTIVE: We sought to analyze the gene expression profile (GEP) of bone marrow mast cells (BMMCs) in patients with SM and its association with distinct clinical variants of the disease. METHODS: GEP analyses were performed by using DNA-oligonucleotide microarrays in highly purified BMMCs from patients with SM carrying the D816V KIT mutation (n=26) classified according to the diagnostic subtype of SM versus normal/reactive BMMCs (n=7). Validation of GEP results was performed with flow cytometry in the same set of samples and in an independent cohort of 176 subjects. RESULTS: Overall, 758 transcripts were significantly deregulated in patients with SM, with a common GEP (n=398 genes) for all subvariants of SM analyzed. These were characterized by upregulation of genes involved in the innate and inflammatory immune response, including interferon-induced genes and genes involved in cellular responses to viral antigens, together with complement inhibitory molecules and genes involved in lipid metabolism and protein processing. Interestingly, aggressive SM additionally showed deregulation of apoptosis and cell cycle-related genes, whereas patients with indolent SM displayed increased expression of adhesion-related molecules. CONCLUSION: BMMCs from patients with different clinical subtypes of SM display distinct GEPs, which might reflect new targetable pathways involved in the pathogenesis of the disease.

CD30 expression by bone marrow mast cells from different diagnostic variants of systemic mastocytosis.

AIMS: CD30 expression by bone marrow (BM) mast cells (MC) has been reported recently in systemic mastocytosis (SM) patients. The aim of this study was to investigate the potential diagnostic and prognostic value of CD30 expression in SM as assessed by multiparameter flow cytometry. METHODS AND RESULTS: A total of 163 consecutive BM samples corresponding to 142 SM patients and 21 non-mastocytosis cases were studied. CD30 was positive in most SM patients (80%), but in only one non-mastocytosis case (4.8%). When combined with CD25, CD30 contributed to an improved accuracy over that of CD25 alone (98% versus 93%) mainly because most (eight of nine) of the well-differentiated SM (WDSM), who lacked CD25, were CD30(+). Similar levels of expression of CD30 were observed among all different subgroups of SM except mast cell leukaemia; among indolent SM (ISM) patients, no significant association was observed between the levels of CD30 expression and other clinical and biological features of the disease. CONCLUSIONS: The increased expression of CD30 associated with absence of CD25 contributes to the diagnosis of WDSM and its distinction from other subtypes of SM. By contrast, CD30 expression did not contribute either to prognostic stratification of ISM or to the differential diagnosis between ISM and aggressive SM cases.

The role of environmental enrichment in the aetiopathogenesis of Staphylococcus aureus facial, periorbital and retroorbital abscesses in mice.

In this observational retrospective study, an outbreak of Staphylococcus aureus abscesses was correlated with the presence of sharp edges in damaged plastic environmental enrichment within the cages. In 2010, Lawson reported cases of S. aureus mandibulofacial and maxillofacial abscess in mice and proposed excessive barbering or grooming, leading to the mastication and fragmentation of hair, as an aetiopathogenesis of S. aureus abscesses. In contrast, in this study, the presence of hair was not found in any of the histopathology, and abscesses were present in the periorbital area. S. aureus colonises the skin, nasopharynx and intestines, and may cause pyogenic infections if a breach in local defences promotes staphylococcal invasion. Whole genome sequencing and analysis supported the hypothesis that this outbreak resulted from clonal expansion of S. aureus infected C57BL6/J mice imported into the area and infection transmission from humans to mice was ruled out. An additional aetiopathogenesis is proposed for S. aureus abscesses with the sharp edges of damaged plastic environmental enrichment items leading to oral mucosal injury allowing S. aureus entrance into tissues, its carriage into the submucosa, followed by abscess formation.

Quantification of Hair Corticosterone, DHEA and Testosterone as a Potential Tool for Welfare Assessment in Male Laboratory Mice.

Steroids, providing information regarding several biological patterns including stress and sexual behavior, have been investigated in different matrices in laboratory mice. Data regarding hair quantification, indicative of longer timespans when compared to blood and saliva, are lacking. The aim of the work was to analyze the hormonal hair profile of laboratory male mice and to investigate potential relationships with age and housing, as a potential tool for welfare assessment. Fifty-six adult male C57BL/6J and C57BL/6OlaHsd substrain mice were included in the study, housed in pairs or groups. Testosterone (T) and dehydroepiandrosterone (DHEA) were quantified by radioimmunoassay, corticosterone (CORT) by ELISA. Mean hormone levels were 6.42 pg/mg for T, 23.16 pg/mg for DHEA and 502.1 pg/mg for CORT. Age influenced all hormones by significantly increasing T and DHEA levels and decreasing CORT; only DHEA, significantly higher in grouped mice, was influenced by housing conditions. The influence of age indicates the need for accurate age-related reference intervals, while the higher levels of DHEA in grouped animals suggests that such housing practice may be beneficial for social interactions. In conclusion, it seems that hair hormones quantification may be a good tool for welfare assessment in laboratory mice and may help in refining husbandry.

Difficulties in the molecular diagnosis of Helicobacter rodent infections.

Molecular diagnostic methods using the polymerase chain reaction (PCR) are the gold standard in Helicobacter diagnostics. Most rely on the amplification of parts of the 16S rRNA gene sequence. Therefore, the validity and accuracy of results depends heavily on the PCR design and the time of its publication because new sequences are continually being submitted to databases. Here we report the presence of helicobacter in commercially bred mice supposedly free of this infection. Furthermore, three out of six different commercial laboratories performing helicobacter testing on the same spiked faecal samples failed to detect and identify H. hepaticus. We designed a simple generic PCR assay that amplifies a 261bp amplicon spanning two of the seven variable regions in the 16S rRNA of helicobacter. Using this assay together with an established generic assay designed by Bohr [Bohr, U.R., Primus, A., Zagoura, A., Glasbrenner, B., Wex, T., Malfertheiner, P., 2002. A group-specific PCR assay for the detection of Helicobacteraceae in human gut. Helicobacter 7, 378-383] and then cloning and sequencing their products, we detected the H. hepaticus used in the study that three commercial laboratories failed to detect. We think these assays together could detect all the currently known species of helicobacter and hopefully the new ones as well. In addition, we have been able to identify different species of helicobacter and their relative proportions infecting a single animal. This information has also shown that some helicobacters may have a much broader host range than originally reported.

Mycobacterium gordonae infection in a colony of African clawed frogs (Xenopus tropicalis).

Mycobacterium gordonae is an occasional human pathogen associated with cutaneous infections and nodular granulomatous skin lesions. A case of cutaneous nodular infection caused by M. gordonae in a colony of African clawed frogs (Xenopus tropicalis) is described and confirms this organism to be an opportunistic frog pathogen.

The immunophenotype of mast cells and its utility in the diagnostic work-up of systemic mastocytosis.

SM comprises a heterogeneous group of disorders, characterized by an abnormal accumulation of clonal MCs in 1 or more tissues, frequently involving the skin and BM. Despite the fact that most adult patients (>90%) carry the same genetic lesion (D816V KIT mutation), the disease presents with multiple variants with very distinct clinical and biologic features, a diverse prognosis, and different therapeutic requirements. Recent advances in the standardization of the study of BM MC by MFC allowed reproducible identification and characterization of normal/reactive MCs and their precursors, as well as the establishment of the normal MC maturational profiles. Analysis of large groups of patients versus normal/reactive samples has highlighted the existence of aberrant MC phenotypes in SM, which are essential for the diagnosis of the disease. In turn, 3 clearly distinct and altered maturation-associated immunophenotypic profiles have been reported recently in SM, which provide criteria for the distinction between ISM patients with MC-restricted and multilineage KIT mutation; thus, immunphenotyping also contributes to prognostic stratification of ISM, particularly when analysis of the KIT mutation on highly purified BM cells is not routinely available in the diagnostic work-up of the disease.

Molecular characterization of a virulent canine coronavirus BGF strain.

Molecular characterisation of a canine coronavirus (CCoV) isolate (BGF), associated with an outbreak of diarrhoea in puppies, showed 92.7% identity with attenuated Insavc-1 strain. Canine coronavirus BGF revealed a full length non-structural protein 3b (nsp 3b), associated with virulence in other coronaviruses, and a highly divergent region at the amino terminal domain of the membrane protein that may be implicated in avoiding the host immune reaction. This new canine coronavirus strain could help to identify virulence factors in coronavirus.

XY female marmoset (Callithrix jacchus).

A marmoset (Callithrix jacchus) with atypical external genitalia was phenotypically and genetically characterized. Testosterone concentration correlated with that of female marmosets. Externally, there was only one opening for the urethra. Internal genitalia were characteristic of those of female marmosets, and consisted of ovaries, with follicles in various developmental stages, and uterus. Microscopically, a normal vaginal structure was found. An XX/XY chimerism and high steroid hormone values are normally found in common marmosets. Genetic analysis was used for in vivo determination of sex. The Y-linked zinc finger protein gene (ZFY) last intron, and sex-determining region Y gene (SRY) exon were found by use of polymerase chain reaction and posterior sequencing analyses, indicating that this marmoset had Y-linked chromosome sequences. Normal SRY exons can, therefore, be associated with female internal sexual organs in marmosets; this may be the first XY female described in non-human primates.

Immunophenotypic characterization of bone marrow mast cells in mastocytosis and other mast cell disorders.

Mastocytosis is a term used to designate a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow (BM), liver, spleen, and lymph nodes, among others. Recent advances in our understanding of mast cell biology and disease resulted in the identification of important differences in the expression of mast cell surface antigens between normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts lead to the inclusion of this immunophenotypic abnormality in the World Health Organization diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients lacking histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this chapter, we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, for their phenotypic characterization, and the criteria currently used for correct interpretation of the immunophenotypic results obtained.

Genotoxic endpoints in the earthworms sub-lethal assay to evaluate natural soils contaminated by metals and radionuclides.

Eisenia andrei was exposed, for 56 days, to a contaminated soil from an abandoned uranium mine and to the natural reference soil LUFA 2.2. The organisms were sampled after 0, 1, 2, 7, 14 and 56 days of exposure, to assess metals bioaccumulation, coelomocytes DNA integrity and cytotoxicity. Radionuclides bioaccumulation and growth were also determined at 0 h, 14 and 56 days of exposure. Results have shown the bioaccumulation of metals and radionuclides, as well as, growth reduction, DNA damages and cytotoxicity in earthworms exposed to contaminated soil. The usefulness of the comet assay and flow cytometry, to evaluate the toxicity of contaminants such as metals and radionuclides in earthworms are herein reported. We also demonstrated that DNA strand breakage and immune cells frequency are important endpoints to be employed in the earthworm reproduction assay, for the evaluation of soil geno and cytotoxicity, as part of the risk assessment of contaminated areas. This is the first study that integrates DNA damage and cytotoxicity evaluation, growth and bioaccumulation of metals and radionuclides in a sub lethal assay, for earthworms exposed to soil contaminated with metals and radionuclides.