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Suppression of in vitro antibody production by dimethylnitrosamine in mixed cultures of mouse primary hepatocytes and mouse splenocytes
Authors:D H Kim  K H Yang  K W Johnson  M P Holsapple
Affiliation:1. Department of Oncology and Molecular Medicine, Tumor Immunology Unit, Istituto Superiore di Sanità, Rome, Italy;2. Institute for Photonics and Nanotechnology, Italian National Research Council, Rome, Italy;3. Department of Civil Engineering and Computer Science, University of Rome Tor Vergata, Rome, Italy;4. Department of Electronic Engineering, University of Rome Tor Vergata, Rome, Italy;1. Department of Physiology, University of Tennessee Health Science Center, Memphis, TN;2. Cleveland Clinic Foundation, Cleveland, OH
Abstract:
The suppression of in vitro antibody responses by dimethylnitrosamine (DMN) was produced in a mouse hepatocyte and splenocyte co-culture system. Mouse hepatocytes were isolated from female B6C3F1 mice and cultured for 20-24 hr to allow the formation of a monolayer on collagen-coated plastic petri dishes. Spleen cells were isolated from the same hybrid and were co-cultured with the hepatocytes along with DMN. Cyclophosphamide (CP), an immunosuppressive agent requiring metabolic activation that was included as an initial positive control, produced a marked suppression of the in vitro antibody responses to LPS, DNP-Ficoll, and SRBCs in 4 hr in the co-culture system. Under comparable conditions DMN markedly suppressed the response to SRBCs, marginally suppressed the response to DNP-Ficoll, and did not suppress the polyclonal response to LPS. The suppression by DMN was related to the rocking speed during the 4-hr co-culture period and was optimally produced when the cultures were not rocked. Addition of serum into the medium (10% fetal calf serum) during the co-culture period did not change the effects of DMN on the antibody response. However, the addition of extracellular DNA (1 mg calf thymus DNA/ml) prevented the suppression of the antibody response by DMN. These results suggest that DNA represents the primary macromolecular target for the reactive intermediate of DMN, and indicate that a syngeneic co-culture system can be used to characterize the in vitro immunosuppression
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