Variability of Spleen and Mesenteric Lymph Node in Control Cynomolgus Monkeys ( Macaca fascicularis) from Nonclinical Safety Studies: A Retrospective Assessment.

We assessed the variability of spleen and mesenteric lymph node (MLN) microscopic observations and the correlations of these observations with other study data from 478 control cynomolgus monkeys from 53 routine nonclinical safety studies. Spleen weight parameters (absolute and relative to body or brain weights) were highly variable both within a control group on an individual study (up to 5.11-fold) and among animals with the same light microscopic observation. Grades for microscopic observations were also highly variable. The most frequent microscopic observations for spleen were changes in the size and number of germinal centers (58%), acidophilic (hyaline) material in lymphoid follicles (52%), and compound lymphoid follicles (20%). The most frequent microscopic observations in the MLN were eosinophil infiltrates (90%), changes in size and number of germinal centers (42%), and brown pigment (21%). The only meaningful relationships ( r2 > 0.3) were positive correlations between reticuloendothelial hyperplasia and malarial pigment in the spleen and between each of these observations and spleen weight parameters. We conclude that determination of test article-related effects on the immune system in routine monkey toxicology studies requires careful consideration and a weight-of-evidence approach due to the low numbers of animals/group, the inherent variability in spleen and MLN parameters, and the infrequent correlation among immune system-related end points.

Scientific and Regulatory Policy Committee Review: Review of the Organisation for Economic Co-operation and Development (OECD) Guidance on the GLP Requirements for Peer Review of Histopathology.

In 2014, the Organisation for Economic Co-operation and Development (OECD) issued guidance no. 16, Guidance on the GLP Requirements for Peer Review of Histopathology. The stated purpose of the guidance document is "to provide guidance to pathologists, test facility management, study directors and quality assurance personnel on how the peer review of histopathology should be planned, managed, documented, and reported in order to meet Good Laboratory Practice (GLP) expectations and requirements." On behalf of and in collaboration with the global societies of toxicologic pathology, the Society of Toxicologic Pathology initiated a review of OECD guidance no. 16. The objectives of this review are to provide a unified interpretation of the guidance, to recommend compliant processes for organizations to implement, and to avoid inconsistent process adaptations across the industry. This review of the guidance document is the product of a global collaboration with other societies of toxicologic pathology and provides a section-by-section international consensus view and interpretation of the OECD guidance on peer review.

Off-target platelet activation in macaques unique to a therapeutic monoclonal antibody.

AMG X, a human neutralizing monoclonal antibody (mAb) against a soluble human protein, caused thrombocytopenia, platelet activation, reduced mean arterial pressure, and transient loss of consciousness in cynomolgus monkeys after first intravenous administration. In vitro, AMG X induced activation in platelets from macaque species but not from humans or baboons. Other similar mAbs against the same pharmacological target failed to induce these in vivo and in vitro effects. In addition, the target protein was known to not be expressed on platelets, suggesting that platelet activation occurred through an off-target mechanism. AMG X bound directly to cynomolgus platelets and required both the Fab and Fc portion of the mAb for platelet activation. Binding to platelets was inhibited by preincubation of AMG X with its pharmacological target or with anti-human Fc antibodies or by preincubation of platelets with AMG X F(ab')(2) or human immunoglobulin (IVIG). AMG X F(ab')(2) did not activate platelets. Thus, platelet activation required both recognition/binding of a platelet ligand with the Fab domain and interaction of platelet Fc receptors (i.e., FcγRIIa) with the Fc domain. These findings reflect the complexity of the mechanism of action of mAbs and the increasing awareness of potential for unintended effects in preclinical species.

Molecular characterization of Helicobacter pylori strains isolated from cynomolgus monkeys (M. fascicularis).

We recently reported the occurrence of natural infection with H. pylori in a group of cynomolgus monkeys with chronic active gastritis and gastric erosions. The goal of the present study was to characterize and to compare strains isolated from animals originating from two different geographical areas. Gross and microscopic pathology determined at the time of necropsy was similar in all animals. H. pylori were isolated from specimens harvested in five monkeys (four from Vietnam and one from the Philippines) with gastritis. Isolates from monkeys bred in Vietnam had a similar DNA fingerprint pattern, which was distinct from that of isolates from a monkey bred in the Philippines. All strains were of the s1a vacA subtype, but all the 'Vietnamese' strains were cagA+ and all but one were iceA1 whereas the 'Philippino' strains were cagA- and iceA2. The sequences of the 16S rRNA of the Vietnamese and Philippino strains shared 98% homology and both clustered with H. pylori sequences present in the NCBI database. In conclusion, cynomolgus monkeys can be naturally colonized by H. pylori, and the strains isolated from these animals appear to vary according to the geographical origin, thus indicating probable infection prior to importation. Since some of the cynomolgus monkeys developed antral erosions during natural infection, we propose that this animal model may be used to investigate the role of H. pylori in ulcerogenesis.

T-cell-dependent antibody response: assay development in cynomolgus monkeys.

The current study was designed to develop and test a T-cell dependent antibody response to keyhole limpet hemocyanin (KLH) in cynomolgus monkeys. In an optimization experiment, monkeys (3/sex) were given a single intramuscular injection of KLH at 10 mg/animal to evaluate the kinetics of the antibody response. Serum samples were collected pretest, and on Days 4, 6, 8, 11, 15 and 22 for measurement of anti-KLH IgM and IgG endpoint titers. In a subsequent experiment, female monkeys (3/group) were treated once daily by gavage with the immunosuppressive agent cyclosporine (Neoral) at 0, 10 and 50 mg/kg for 21 days, and the effects of drug treatment on anti-KLH IgM and IgG responses were determined. The effects of cyclosporine on hematology, biochemistry, bone marrow, organ weights, gross and histopathology, and peripheral lymphocyte subsets also were evaluated. Robust anti-KLH IgM and IgG responses were seen in monkeys given a single intramuscular injection of KLH at 10 mg/animal, with peak antibody responses at approximately 10-14 days post-immunization for anti-KLH IgM, and 14-21 days for anti-KLH IgG. Decreases in anti-KLH IgG endpoint titers were seen in 1 monkey given cyclosporine at 10 mg/kg, and 1 monkey dosed at 50 mg/kg. Relative to vehicle control animals, mild lymphoid depletion was evident in lymph nodes and tonsil of monkeys with suppressed anti-KLH IgG titers. Collectively, these findings in individual animals provided evidence of cyclosporine-induced immunosuppression. Cyclosporine at 10 and 50 mg/kg did not alter anti-KLH IgM production, hematology, biochemistry, bone marrow, organ weights, or peripheral lymphocyte subsets. Lastly, the results of this study demonstrated that KLH immunization at 10 mg/animal did not alter the standard toxicity endpoints evaluated in control animals.