Advocating for Generalizability: Accepting Inherent Variability in Translation of Animal Research Outcomes.

Advancing scientific discovery requires investigators to embrace research practices that increase transparency and disclosure about materials, methods, and outcomes. Several research advocacy and funding organizations have produced guidelines and recommended practices to enhance reproducibility through detailed and rigorous research approaches; however, confusion around vocabulary terms and a lack of adoption of suggested practices have stymied successful implementation. Although reproducibility of research findings cannot be guaranteed due to extensive inherent variables in attempts at experimental repetition, the scientific community can advocate for generalizability in the application of data outcomes to ensure a broad and effective impact on the comparison of animals to translation within human research. This report reviews suggestions, based upon work with National Institutes of Health advisory groups, for improving rigor and transparency in animal research through aspects of experimental design, statistical assessment, and reporting factors to advocate for generalizability in the application of comparative outcomes between animals and humans.

Low-dose intraperitoneal Freund's adjuvant: toxicity and immunogenicity in mice using an immunogen targeting amyloid-beta peptide.

Complete Freund's adjuvant (CFA) is effective for potentiating immune responses in mice when administered subcutaneously, and is often more potent when given intraperitoneally (i.p.). However, the the potential toxicity of i.p. administration in mice has led investigators and Institutional Animal Care and Use committees to increasingly view the use of CFA i.p. with reservation. We evaluated whether an 80% reduction in the dose of CFA administered i.p. to mice, compared to the i.p. doses used in a previous analysis, could abrogate the untoward effects associated with its use, while still maintaining adjuvanticity. Using a novel immunogen targeting the N-terminus of the 42-amino acid amyloid-beta peptide, we compared low dose CFA administered i.p., with three other commonly used adjuvants given i.p.: alum, incomplete Freunds adjuvant (IFA) and monophoshoryl lipid A + trehalose dicorynomycolate (MPL + TDM). The results of the study showed that, though the reduction in intraperitoneal dose of CFA mitigated transient weight loss and leukocytosis observed previously with higher doses of i.p. CFA, all mice administered CFA or IFA i.p. developed abdominal adhesions and granulomatous peritonitis. Mice from all adjuvant groups, however, appeared to tolerate the respective adjuvants well and excellent comparative immunogenicity was observed in mice immunized with the Freunds and MPL + TDM adjuvants. Consequently, we conclude that though a high-titered, humoral response may be generated using low dose CFA administered i.p., the accompanying toxicity remains significant, and thus alternative adjuvants and/or routes should be considered.

Feline leukemia virus envelope sequences that affect T-cell tropism and syncytium formation are not part of known receptor-binding domains.

The envelope protein is a primary pathogenic determinant for T-cell-tropic feline leukemia virus (FeLV) variants, the best studied of which is the immunodeficiency-inducing virus, 61C. We have previously demonstrated that T-cell-tropic, cytopathic, and syncytium-inducing viruses evolve in cats infected with a relatively avirulent, transmissible form of FeLV, 61E. The envelope gene of an 81T variant, which encoded scattered single-amino-acid changes throughout the envelope as well as a 4-amino-acid insertion in the C-terminal half of the surface unit (SU) of envelope, was sufficient to confer the T-cell-tropic, cytopathic phenotype (J. L. Rohn, M. S. Moser, S. R. Gwynn, D. N. Baldwin, and J. Overbaugh, J. Virol. 72:2686-2696, 1998). In the present study, we examined the role of the 4-amino-acid insertion in determining viral replication and tropism of FeLV-81T. The 4-amino-acid insertion was found to be functionally equivalent to a 6-amino-acid insertion at an identical location in the 61C variant. However, viruses expressing a chimeric 61E/81T SU, containing the insertion together with the N terminus of 61E SU, were found to be replication defective and were impaired in the processing of the envelope precursor into the functional SU and transmembrane (TM) proteins. In approximately 10% of cultured feline T cells (3201) transfected with the 61E/81T envelope chimeras and maintained over time, replication-competent tissue culture-adapted variants were isolated. Compensatory mutations in the SU of the tissue culture-adapted viruses were identified at positions 7 and 375, and each was shown to restore envelope protein processing when combined with the C-terminal 81T insertion. Unexpectedly, these viruses displayed different phenotypes in feline T cells: the virus with a change from glutamine to proline at position 7 acquired a T-cell-tropic, cytopathic phenotype, whereas the virus with a change from valine to leucine at position 375 had slower replication kinetics and caused no cytopathic effects. Given the differences in the replication properties of these viruses, it is noteworthy that the insertion as well as the two single-amino-acid changes all occur outside of predicted FeLV receptor-binding domains.