Expanding the 3R principles

Aske, Kim Charles; Waugh, Courtney

doi:10.15252/embr.201744428

Cited by 65 publications

(24 citation statements)

References 9 publications

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“…ARRIVE guidelines) and regulations (e.g. 3R regulations) to minimize animal suffering and distress in the laboratory ( 29 , 30 ). Seventy female BALB/c nude mice (6 weeks old, 15–18 g) were purchased from Charles River (Kanagawa, Japan).…”

Section: Methodsmentioning

confidence: 99%

A defucosylated anti‑CD44 monoclonal antibody 5‑mG2a‑f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma

et al. 2020

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CD44 is widely expressed on the surface of most tissues and all hematopoietic cells, and regulates many genes associated with cell adhesion, migration, proliferation, differentiation, and survival. CD44 has also been studied as a therapeutic target in several cancers. Previously, an anti-CD44 monoclonal antibody (mAb), C 44 Mab-5 (IgG 1 , kappa) was established by immunizing mice with CD44-overexpressing Chinese hamster ovary (CHO)-K1 cells. C 44 Mab-5 recognized all CD44 isoforms, and showed high sensitivity for flow cytometry and immunohistochemical analysis in oral cancers. However, as the IgG 1 subclass of C 44 Mab-5 lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), the antitumor activity of C 44 Mab-5 could not be determined. In the present study, we converted the mouse IgG 1 subclass antibody C 44 Mab-5 into an IgG 2a subclass antibody, 5-mG 2a , and further produced a defucosylated version, 5-mG 2a-f, using FUT8-deficient ExpiCHO-S (BINDS-09) cells. Defucosylation of 5-mG 2a-f was confirmed using fucose-binding lectins, such as AAL and PhoSL. The dissociation constants (K D) for 5-mG 2a-f against SAS and HSC-2 oral cancer cells were determined through flow cytometry to be 2.8x10-10 M and 2.6x10-9 M, respectively, indicating that 5-mG 2a-f possesses extremely high binding affinity. Furthermore, immunohistochemical staining using 5-mG 2a-f specifically stained the membranes of oral cancer cells. In vitro analysis demonstrated that 5-mG 2a-f showed moderate ADCC and CDC activities against SAS and HSC-2 oral cancer cells. In vivo analysis revealed that 5-mG 2a-f significantly reduced tumor development in SAS and HSC-2 xenografts in comparison to control mouse IgG, even after injection seven days post-tumor inoculation. Collectively, these results suggest that treatment with 5-mG 2a-f may represent a useful therapy for patients with CD44-expressing oral cancers.

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Section: Methodsmentioning

confidence: 99%

A defucosylated anti‑CD44 monoclonal antibody 5‑mG2a‑f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma

et al. 2020

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“…The use of animals in scientific experiments has many key advantages taking into consideration as it does, the complexity of the complete living organism however, there are also intrinsic challenges including a moral imperative to follow a “Replace, Reduce and Refine” philosophy [1,2], and to maximize data acquisition and usefulness in all experimental systems involving them. An absolute requirement for any experimental system is reproducibility [3].…”

Section: Introductionmentioning

confidence: 99%

Towards large scale automated cage monitoring – Diurnal rhythm and impact of interventions on in-cage activity of C57BL/6J mice recorded 24/7 with a non-disrupting capacitive-based technique

Iannello²,

et al. 2019

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Background and aimsAutomated recording of laboratory animal’s home cage behavior is receiving increasing attention since such non-intruding surveillance will aid in the unbiased understanding of animal cage behavior potentially improving animal experimental reproducibility.Material and methodsHere we investigate activity of group held female C57BL/6J mice (mus musculus) housed in standard Individually Ventilated Cages across three test-sites: Consiglio Nazionale delle Ricerche (CNR, Rome, Italy), The Jackson Laboratory (JAX, Bar Harbor, USA) and Karolinska Insititutet (KI, Stockholm, Sweden). Additionally, comparison of female and male C57BL/6J mice was done at KI. Activity was recorded using a capacitive-based sensor placed non-intrusively on the cage rack under the home cage collecting activity data every 250 msec, 24/7. The data collection was analyzed using non-parametric analysis of variance for longitudinal data comparing sites, weekdays and sex.ResultsThe system detected an increase in activity preceding and peaking around lights-on followed by a decrease to a rest pattern. At lights off, activity increased substantially displaying a distinct temporal variation across this period. We also documented impact on mouse activity that standard animal handling procedures have, e.g. cage-changes, and show that such procedures are stressors impacting in-cage activity.These key observations replicated across the three test-sites, however, it is also clear that, apparently minor local environmental differences generate significant behavioral variances between the sites and within sites across weeks. Comparison of gender revealed differences in activity in the response to cage-change lasting for days in male but not female mice; and apparently also impacting the response to other events such as lights-on in males. Females but not males showed a larger tendency for week-to-week variance in activity possibly reflecting estrous cycling.ConclusionsThese data demonstrate that home cage monitoring is scalable and run in real time, providing complementary information for animal welfare measures, experimental design and phenotype characterization.

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“…FCM has been recognized as a powerful tool for MNT (Harada, Matsuzaki, Takeiri, Tanaka, & Mishima, 2013), which solves the problems of time-consuming and subjective difference in traditional microscopy, improves the sensitivity and reproducibility, and achieves high throughput (Avlasevich et al, 2011;Laingam, Froscio, & Humpage, 2008;Witt et al, 2008). In addition, MNT in rat peripheral blood using FCM could be more easily integrated into conventional animal toxicological tests, which is particularly important for the evaluation of chemical substances under the 3R principle (reduction, replacement, and refinement) (Aske & Waugh, 2017).…”

mentioning

confidence: 99%

Development of a novel flow cytometry‐based approach for reticulocytes micronucleus test in rat peripheral blood

Chen

Huo

Liu

et al. 2020

J of Applied Toxicology

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The micronucleus test (MNT) is the most widely applied short-term assay to detect clastogens or spindle disruptors. The use of flow cytometry (FCM) has been reported for micronucleated erythrocytes scoring in peripheral blood. The aim of this study was to develop a novel and practical protocol for MNT in rat peripheral blood by FCM, with the method validation. CD71-fluorescein isothiocyanate and DRAQ5 were adopted for the fluorescent staining of proteins and DNA, respectively, to detect micronuclei. To validate the method, groups of male Sprague-Dawley rats (five per group) received two oral gavage doses at 0 and 24 h of six chemicals (four positive mutagens: ethyl methanesulphonate [EMS], cyclophosphamide [CP], colchicine [COL], and ethyl nitrosourea [ENU]; two nongenotoxic chemicals: sodium saccharin and eugenol). Blood samples were collected from the tail vein before and on the five continuous days after treatments; all of which were analyzed for micronuclei presence by both the manual (Giemsa staining) and FCM methods. The FCMbased method consistently demonstrated highly sensitive responses for micronucleus detection at all concentrations and all time points for EMS, CP, COL, and ENU. Sodium saccharin and eugenol could be identified as negative in this protocol. Results obtained with the FCM-based method correlated well with the micronucleus frequencies (r = 0.659-0.952), and the proportion of immature erythrocytes (r = 0.915-0.981) tested by Giemsa staining. The method reported here, with easy operation, low background, and requirement for a regular FCM, could be an efficient system for micronucleus scoring.

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Expanding the 3R principles

Abstract: The implementation of the 3R in research using animals has already improved animal welfare and benefited science. Further progress could be achieved by expanding the 3R to include “Robustness” and “Reproducibility”.

Cited by 65 publications

References 9 publications

A defucosylated anti‑CD44 monoclonal antibody 5‑mG2a‑f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma

A defucosylated anti‑CD44 monoclonal antibody 5‑mG2a‑f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma

Towards large scale automated cage monitoring – Diurnal rhythm and impact of interventions on in-cage activity of C57BL/6J mice recorded 24/7 with a non-disrupting capacitive-based technique

Development of a novel flow cytometry‐based approach for reticulocytes micronucleus test in rat peripheral blood

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