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

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

Neutrophil responses to Aspergillosis : new roles for old players

Neutrophils are professional phagocytic cells that play a crucial role in innate immunity. Through an assortment of antifungal effector mechanisms, neutrophils are essential in controlling the early stages of fungal infection. These mechanisms range from the production of reactive oxygen intermediates and release of antimicrobial enzymes to the formation of complex extracellular traps that aid in the elimination of the fungus. Their importance in antifungal immunity is supported by the extreme susceptibility to infection of patients with primary (e.g., chronic granulomatous disease) or acquired (e.g., undergoing immunosuppressive therapy) neutrophil deficiency. More recently, common genetic variants affecting neutrophil antifungal capacity have also been disclosed as major risk factors for aspergillosis in conditions of generalized immune deficiency. The present review revisits the role of neutrophils in the host response against Aspergillus and highlights the consequences of their deficiency in susceptibility to aspergillosis.This work was supported by a Research Grant from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Cristina Cunha was supported by the Fundacao para a Ciencia e Tecnologia, Portugal (contract SFRH/BPD/96176/2013)

Lack of the Delta Subunit of RNA Polymerase Increases Virulence Related Traits of Streptococcus mutans

The delta subunit of the RNA polymerase, RpoE, maintains the transcriptional specificity in Gram-positive bacteria. Lack of RpoE results in massive changes in the transcriptome of the human dental caries pathogen Streptococcus mutans. In this study, we analyzed traits of the ΔrpoE mutant which are important for biofilm formation and interaction with oral microorganisms and human cells and performed a global phenotypic analysis of its physiological functions. The ΔrpoE mutant showed higher self-aggregation compared to the wild type and coaggregated with other oral bacteria and Candida albicans. It formed a biofilm with a different matrix structure and an altered surface attachment. The amount of the cell surface antigens I/II SpaP and the glucosyltransferase GtfB was reduced. The ΔrpoE mutant displayed significantly stronger adhesion to human extracellular matrix components, especially to fibronectin, than the wild type. Its adhesion to human epithelial cells HEp-2 was reduced, probably due to the highly aggregated cell mass. The analysis of 1248 physiological traits using phenotype microarrays showed that the ΔrpoE mutant metabolized a wider spectrum of carbon sources than the wild type and had acquired resistance to antibiotics and inhibitory compounds with various modes of action. The reduced antigenicity, increased aggregation, adherence to fibronection, broader substrate spectrum and increased resistance to antibiotics of the ΔrpoE mutant reveal the physiological potential of S. mutans and show that some of its virulence related traits are increased

Two Genes on A/J Chromosome 18 Are Associated with Susceptibility to Staphylococcus aureus Infection by Combined Microarray and QTL Analyses

Although it has recently been shown that A/J mice are highly susceptible to Staphylococcus aureus sepsis as compared to C57BL/6J, the specific genes responsible for this differential phenotype are unknown. Using chromosome substitution strains (CSS), we found that loci on chromosomes 8, 11, and 18 influence susceptibility to S. aureus sepsis in A/J mice. We then used two candidate gene selection strategies to identify genes on these three chromosomes associated with S. aureus susceptibility, and targeted genes identified by both gene selection strategies. First, we used whole genome transcription profiling to identify 191 (56 on chr. 8, 100 on chr. 11, and 35 on chr. 18) genes on our three chromosomes of interest that are differentially expressed between S. aureus-infected A/J and C57BL/6J. Second, we identified two significant quantitative trait loci (QTL) for survival post-infection on chr. 18 using N2 backcross mice (F1 [C18A]×C57BL/6J). Ten genes on chr. 18 (March3, Cep120, Chmp1b, Dcp2, Dtwd2, Isoc1, Lman1, Spire1, Tnfaip8, and Seh1l) mapped to the two significant QTL regions and were also identified by the expression array selection strategy. Using real-time PCR, 6 of these 10 genes (Chmp1b, Dtwd2, Isoc1, Lman1, Tnfaip8, and Seh1l) showed significantly different expression levels between S. aureus-infected A/J and C57BL/6J. For two (Tnfaip8 and Seh1l) of these 6 genes, siRNA-mediated knockdown of gene expression in S. aureus–challenged RAW264.7 macrophages induced significant changes in the cytokine response (IL-1 β and GM-CSF) compared to negative controls. These cytokine response changes were consistent with those seen in S. aureus-challenged peritoneal macrophages from CSS 18 mice (which contain A/J chromosome 18 but are otherwise C57BL/6J), but not C57BL/6J mice. These findings suggest that two genes, Tnfaip8 and Seh1l, may contribute to susceptibility to S. aureus in A/J mice, and represent promising candidates for human genetic susceptibility studies

Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus

The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus–triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with potentially conserved roles also in mammals

Neutrophils can Promote Clotting via FXI and Impact Clot Structure via Neutrophil Extracellular Traps in a Distinctive Manner in vitro

Neutrophils and neutrophil extracellular traps (NETs) have been shown to be involved in coagulation. However, the interactions between neutrophils or NETs and fibrin(ogen) in clots, and the mechanisms behind these interactions are not yet fully understood. In this in vitro study, the role of neutrophils or NETs on clot structure, formation and dissolution was studied with a combination of confocal microscopy, turbidity and permeation experiments. Factor (F)XII, FXI and FVII-deficient plasmas were used to investigate which factors may be involved in the procoagulant effects. We found both neutrophils and NETs promote clotting in plasma without the addition of other coagulation triggers, but not in purified fibrinogen, indicating that other factors mediate the interaction. The procoagulant effects of neutrophils and NETs were also observed in FXII- and FVII-deficient plasma. In FXI-deficient plasma, only the procoagulant effects of NETs were observed, but not of neutrophils. NETs increased the density of clots, particularly in the vicinity of the NETs, while neutrophils-induced clots were less stable and more porous. In conclusion, NETs accelerate clotting and contribute to the formation of a denser, more lysis resistant clot architecture. Neutrophils, or their released mediators, may induce clotting in a different manner to NETs, mediated by FXI