Studies supported by this grant have unequivocally demonstrated that Adriamycin (ADM) has immunomodulating effects, which are multiple, but can be essentially reduced to stimulation of macrophage and T cell activation and functions. These effects result in maintenance of immune responses otherwise depressed as tumor grows. They also have therapeutic relevance when the drug is used in combination with IL2 or TNF. Based on the information acquired it is now important to explore the mechanisms of ADM action on the immune system and to develop therapeutic regimens exploiting the ADM actions. In view of recent advances in methodologies allowing single cell assays, the former group of studies is now possible and should contribute to basic understanding of the molecular pharmacology of the immune system and of drug interactions with cytokine function. In the current proposal we seek to determine which of the effects of ADM on the function of lymphocyte populations and of macrophages, represents a primary effect. Using cytofluorometry and cell sorting techniques, cell separation, identification and recombination experiments will be carried out to identify cells which are the primary determinants of responses to ADM. Using PCR, levels of mRNA for either critical cytokines or their receptors will be assessed to see whether ADM-induced effects are related to transcription phenomena. Using confocal microscopy and HPLC, ADM will be localized and quantitated intracellularly to identify possible cellular sites of action. The redistribution of spectrin, a structural protein, will be studied to see whether it correlates with and contributes to the stabilization of activated cells and the retention of antitumor response. The potential applications of ADM in combination with selective agents and cytokines known to affect T cells or macrophages in the EL4/C57B1/6 model will be explored. Correlations will be further verified between in vivo antitumor effects and the selective elevation of effectors of anti-EL4 host responses and/or the presence of selected markers in the effector cell populations. The outstanding therapeutic effects of ADM in combination with IL2 will be studied further and additional leads pursued through combinations of ADM with other cytokines (e.g., INFgamma, TNF or IL1) or with modifiers of immunomodulation such as indomethacin; in addition, the effects of replacing ADM with cyclophosphamide in some of the systems studied will be evaluated towards a further clarification of the differences observed between these two agents.
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