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Nociceptin/orphanin FQ (N/OFQ), added in vitro to murine spleen cells in the picomolar range, suppressed antibody formation to sheep red blood cells in a primary and a secondary plaque-forming cell (PFC) assay. The activity of the peptide was maximal at 10−12 M, with an asymmetric U-shaped dose response curve that extended activity to 10−14 M. Suppression was not blocked by pretreatment with naloxone. Specificity of the suppressive response was shown using affinity purified rabbit antibodies against two N/OFQ peptides, and with a pharmacological antagonist. Antisera against both peptides were active, in a dose related manner, in neutralizing N/OFQ -mediated immunosuppression, when the peptide was used at concentrations from 10−12.3 to 10−11.6 M. In addition, nociceptin given in vivo by osmotic pump for 48 hr suppressed the capacity of spleen cells placed ex vivo to make an anti-sheep red blood cell response. These studies show that nociceptin directly inhibits an adaptive immune response, i.e. antibody formation, both in vitro and in vivo.

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Endomorphin 1 (EM-1) and endomorphin 2 (EM-2) were tested for their capacity to alter immune function. Addition of either of these peptides to murine spleen cells in vitro inhibited antibody formation to sheep red blood cells in a bi-phasic dose dependent manner. Maximal inhibition was achieved at doses in the range of 10(-13) to 10(-15)M. Neither naloxone (general opioid receptor antagonist) nor CTAP (selective mu opioid receptor antagonist) blocked the immunosuppressive effect. To show that there was specificity to the immunosuppressive activity of the peptides, affinity-purified rabbit antibodies were raised against each of the synthetic EM peptides haptenized to KLH and tested for capacity to inhibit immunosuppression. Antibody responses were monitored by a standard solid phase antibody capture ELISA, and antibodies were purified by immunochromatography using the synthetic peptides coupled to a Sepharose 6B resin. Verification of the specificity of affinity-purified antisera was performed by immunodot-blot and solid-phase RIA assays. The antisera specific for both EM-1 and EM-2 neutralized the immunosuppressive effects of their respective peptides in a dose-related manner. Control normal rabbit IgG had no blocking activity on either EM-1 or EM-2. These studies show that the endomorphins are immunomodulatory at ultra-low concentrations, but the data do not support a mechanism involving the mu-opioid receptor.

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