Capture ELISA and flow cytometry methods for toxicologic assessment following immunization and cyclophosphamide challenges in beagles

Abstract

The purpose of this subacute 22-day study was to evaluate methods for canine circulating immunoglobulins (IgM, IgG, and IgE) and select B- and T-lymphocyte populations (CD4-helpers, CD8-suppressors, pan-T and pan-B) for immunotoxicity testing using an organ system (concordance) approach. The challenge substance for immunoglobulin testing was repeated immunization with six-way distemper vaccination (DHLAPP), while the challenge substance for leukocyte subpopulations was treatment with cyclophosphamide. Immunoglobulin measurements were made by capture enzyme-linked immunosorbent assay (ELISA), and leukocyte immunophenotyping by fluorescein isothiocyanate/phycoerythrin conjugation (flow cytometry). A battery of parameters that would be used in a typical regulatory study were taken to aid interpretation of the data generated by these methods. Body weights, food consumption, clinical observations, complete clinical chemistry and urinalysis measurements were taken. Gross pathology and micropathology of sternal bone marrow, spleen, mesenteric and retropharyngeal lymph nodes, thymus, liver and kidney were completed. The ELISA method demonstrated acceptable intra-assay reproducibility for IgM, IgG and IgE, with values in good agreement as reported for radial immunodiffusion. The immunologic challenge demonstrated a biological trend of an increase in IgM that preceded an increase in IgG with no discernible trend in IgE response, and no abnormalities in lymphocyte subpopulations. Principle flow cytometry findings related to cyclophosphamide were that the relative percent of B cells decreased dramatically and progressively after compound administration; being statistically decreased in males on day 22 compared with day -5. The relative percent CD4 and CD8 contribution increased, but the CD4/CD8 ratio remained relatively unchanged as total white blood cells decreased progressively. The increase in relative percent CD4 (males only) was statistically significant according to a two-sample t-test on days 17, 20 and 22 when compared with the pre-treatment day -5. There was a relative percent increase in CD5-panT, but absolute numbers were dramatically decreased. We conclude that an organ system approach to assessment of the immune system which incorporates humoral antibody, enumeration of lymphocyte populations and pathologic evaluation of the lymphoreticular organs assists in the interpretation of an adverse toxicological response. The ELISA method for measurement of Igs detected the expected levels of IgG, IgM and IgE due to repeated vaccinations and to cyclophosphamide treatment. The flow cytometry method was acceptable for measuring select canine lymphocyte populations and detecting the expected decrease in B cells due to cyclophosphamide treatment. Both methods may be added to a testing battery for assessing immunotoxicityl in canine regulatory studies.

Evaluation of canine T-cell dependent antibody response to the primary and secondary immunization with keyhole limpet hemocyanin

“…Despite the importance of utilization of dogs as a non-rodent species in the immunotoxicity assessment of drug candidates, only primary response data have been available on techniques and methods applicable to the canine models so far (Jones et al, 2000;Finco-Kent and Kawabata, 2005;Thiem et al, 1988;Lebrec et al, 2012;Legrand et al, 2013). To develop a practical study design incorporating both the primary and secondary responses in a TDAR assay in dogs, we evaluated kinetics of anti-KLH IgM and IgG antibody responses following primary and secondary immunizations with KLH (10 mg/dog) by intravenous or intramuscular route.…”

Immunotoxic effects of cyclophosphamide and cyclosporine in the dog

“…Despite the key role of dog studies in the non-clinical safety evaluation of drug candidates, few data are available on techniques and methods applicable to immunotoxicity dog studies (Thiem et al, 1988;Jones et al, 2000;Finco-Kent and Kawabata, 2005;Lebrec et al, 2012). The aim of the present study was to test whether widely accepted immunotoxicity endpoints including lymphocyte subset immunophenotyping, the anti-KLH TDAR assay, and histological examination of the main lymphoid organs were reliable to detect immunosuppression induced by cyclosporine and cyclophosphamide in dogs and could therefore be used for non-clinical immunotoxicity evaluation in this species.…”