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The purpose of these studies was to evaluate bone marrow from male CD rats following exposure to known hematotoxins using flow cytometry (FC) and a monoclonal antibody to the cell surface antigen CD71. Rats were treated with either CHO (300 mg/kg for 10 days) or DAUN (10 mg/kg for 1 day). Control groups received the appropriate vehicle. Half of the animals from each group were euthanized at the end of the dosing schedule and the remaining animals were euthanized after a recovery period. Hematology analyses were completed prior to the onset of each study and on the day of necropsy. Marrow was isolated from the tibia, stained with R-phycoerythrin-conjugated mouse anti-rat CD71 (transferrin receptor on proliferating cells) monoclonal antibody, and then analyzed by FC for myeloid:erythroid (M:E) ratios. FC determinations of myeloid and erythroid population percentages and M:E ratios from untreated rats were confirmed by microscopic examination of marrow cytospins and selected flow cell sorts. M:E ratios for control animals determined by FC were not significantly different between the two studies (1.83 vs 1.89). CHO treatment caused a significant (p < 0.01) decrease in M:E ratios (0.96 for CHO vs 1.48 for control) at day 11 due to increased erythroid cells. M:E ratios were significantly increased (p < 0.05) with DAUN treatment at day 3 (5.07 for DAUN vs 1.70 for control) and corresponded to generalized depletion of all marrow cell lines, especially erythroid cells. After recovery, M:E ratios of CHO and DAUN rats were similar to controls. Hematological values corroborated changes in marrow myeloid and erythroid populations evaluated by this FC technique. Using FC and a monoclonal antibody to the cell surface antigen CD71, this study confirmed the reversible selective toxicity on myeloid and erythroid marrow populations following in vivo exposure to CHO or DAUN. This FC procedure provides a rapid, sensitive method for bone marrow analysis compared to conventional cytological examination. [Copyright &y& Elsevier]

The direct popliteal lymph node assay (PLNA) is a predictive test used to detect the immune-stimulating potential of pharmaceuticals and other low molecular weight compounds (LMWCs) with known autoimmunogenic or sensitizing properties. Two limitations in the PLNA are the existence of false negatives and the inability of the assay to provide mechanistic information. Recently the direct PLNA was modified by incorporating reporter antigens (RA), either TNP-Ficoll or TNP-OVA. In the RA-PLNA, immune stimulation is detected by measuring IgM or IgG TNP-specific antibody-forming cells (AFC) using an enzyme-linked immunospot (ELISPOT) assay. The RA-PLNA, when using potent, known autoimmunogenic compounds, may provide greater sensitivity compared to the direct PLNA and might distinguish LMWCs that have intrinsic adjuvant activity from those that create neo-antigens, using TNP-OVA and TNP-Ficoll, respectively. The purpose of this study was to rigorously compare the two assays. Our first objective was to investigate the interlaboratory reproducibility of the RA-PLNA using four autoimmunogenic LMWC models, plus one negative control LMWC. Subsequently, we tested seven LMWCs with known sensitizing properties and compared the results from the direct and modified assay. The test group included LMWCs thought to be mechanistically distinct and similar to compounds typically encountered in preclinical safety assessment. All control and treatment AFC plaques were collected (76 total), pooled, coded to conceal their source, and counted. The interlaboratory reproducibility of the RA-PLNA was demonstrated with the model autoimmunogenic compounds HgCl2, diphenylhydantoin, D-penicillamine, and the negative control compound phenobarbital, by detecting TNP-specific IgM and polyclonal IgG production to both reporter antigens. Additionally, the sensitizing effects of streptozotocin were identified using an IgG2a ELISPOT with both TNP-OVA and TNP-Ficoll. With the extended test group, the sensitizing effects of aniline, a false negative LMWC in the direct PLNA, was not detected in this study when using the direct PLNA. However, there was an increase of IgG1 AFCs using TNP-OVA, when compared to control (508 ± 113 vs. 12 ± 4 respectively). Glafenine, diclofenac, and ibuprofen, all associated with drug-induced anaphylaxis in humans, produced significant increases in IgG1 production to TNP-OVA. Of these three LMWCs, only diclofenac, which has been documented to induce neo-antigen formation, was detected with TNP-Ficoll, Hydralazine immunomodulation could be detected only with the direct PLNA although significant increases in IgM were identified with the co-injection of either reporter antigen. Isoniazid and methyldopa consistently produced negative responses in both assays. In summary, this study has demonstrated acceptable interlaboratory reproducibility of the RA-PLNA, using model autoimmunogenic LMWCs. Additionally, it demonstrated that an advantage of the RA-PLNA was that it identified all anaphylactic-associated LMWCs tested, detected the false negative compound aniline, and revealed what is thought to be the mechanism(s) associated with diclofenac-induced immunostimulation. [ABSTRACT FROM PUBLISHER]