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It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrexone, a non-peptidic δ-opioid receptor selective antagonist and opioid receptors on BMDCs have been detected [1]. However, there is little prior data published on naltrexone and DCs. Therefore, we hypothesized that LDN could exert modulating effect on BMDCs. In present study, we studied influence of LDN on both phenotypic and functional maturation of BMDCs. Changes of BMDC post-treatment with LDN were evaluated using conventional light microscope and transmission electron microscopy (TEM); flow cytometry(FCM); cytochemistry; acid phosphatase activity(ACP) test; FITC-dextran bio-assay; mixed lymphocytes and enzyme-linked immunosorbent assay (ELISA). We have found that LDN enhances maturation of BMDCs as evidenced by 1) up-regulating the expression of MHC II, CD40, CD83, CD80 and CD86 molecules on BMDCs; 2) down-regulating the rates of pinocytosis and phagocytosis accompanied by the results of decreased ACP, and FITC-dextran bio-assay; 3) mounting potential of BMDCs to drive T cell; and 4) inducing secretion of higher levels of IL-12 and TNF-α. It is therefore concluded that LDN can efficiently promote the maturation of BMDCs via precise modulation inside and outside BMDCs. Our study has provided meaningful mode of action on the role of LDN in immunoregulation, and rationale on future application of LDN for enhancing host immunity in cancer therapy and potent use in the design of DC-based vaccines for a number of diseases.

AZT (7.5 or 15 mg/kg/dose) and the neuropeptide methionine enkephalin (Met-ENK, 1 or 3 mg/kg/dose) were used in a combined protocol for therapy of established murine retroviral infection. In both models used, Friend virus leukemia (FV) and BM5 complex (lymphadenopathy and immune deficiency), the drug combination was able to reduce mortality and splenomegaly. While increasing mean survival time of those animals that did not survive infection by FV, when compared to infected control mice or mice treated with AZT alone, Met-ENK used alone at 1 and 3 mg/kg/mouse had no effect in reducing morbidity or mortality due to either virus. This suggested that Met-ENK had no direct antiviral effect at the concentrations used. In fact, mice treated with either single drug therapy or the combination still yielded virus in their spleen, even when splenomegaly was absent. The data suggest that Met-ENK, which has been reported to be immunostimulatory, acts in combination to improve the efficacy of AZT in reducing progression of disease in murine retrovirus models for human AIDS.

Previously, this laboratory has demonstrated that azidothymidine used in combination with methionine enkephalin, an opioid pentapeptide, was more effective than AZT alone in inhibiting disease progression due to murine retrovirus infections. In order to study the mechanism(s) by which Met-ENK mediates-antiviral effects, when used in combination with AZT in Friend leukemia virus infected mice, an in vitro focus forming assay was used. AZT at 1 ng/ml inhibited FLV replication by 30-50% in the susceptible Mus dunni cell line. By contrast, the immunostimulatory neuropeptide, Met-ENK, displayed no direct inhibition of viral replication. This suggests that Met-ENK does not have any direct anti-retroviral activity. Subsequent testing of Met-ENK in the presence of AZT showed no ability of this peptide to promote inhibition of viral replication due to AZT. By contrast, in the presence of mouse spleen cells, as a source of lymphocytes, in vitro combination treatments using AZT and Met-ENK reduced FLV replication by 67%, compared to 47% using AZT alone. The inhibition due to Met-ENK was abrogated when spleen cells were pretreated with naloxone, an opioid antagonist. Therefore, we conclude that Met-ENK effects are mediated via opioid receptors on spleen cells and that the observed anti-FLV activity is dependent on the use of Met-ENK stimulated spleen cells in combination with AZT.

The present study in mice demonstrated that TRH when administered over 5 days remained active in the Everett Dopamine Potentiation Test. No evidence of tolerance was observed. In fact, an accumulative effect of TRH appeared to take place. Ablation of the adrenals, ovaries, testes, pineal, spleen, parathyroid, one kidney, or thymus did not disrupt this behavioral potentiation of dopamine by TRH. TRH was found to potentiate the effects of imipramine. T3, T4, and TSH were found to be active in the DOPA potentiation test. No overt toxicity was observed between TRH and pargyline or between TRH and DOPA. Toxicity was seen only when all three agents were used together. TRH was found active in young and old mice. TRH was also found active in potentiating the central effects of serotonin. Biogenic amine brain levels in mice were not altered by TRH when administered for five days. Alpha-methyl-p-tyrosine reduced the activity of TRH in the dopamine potentiation test, suggesting dopaminergic mechanisms are involved by a direct receptor interaction.

Three new analogs of dimethylheptylpyran (DMHP) (SP-141, SP-143, and SP-175) were found to exhibit significant anticonvulsant activity against audiogenic, supramaximal electroshock, and maximal pentylentetrazol induced seizures in mice. In rats, all three compounds were found to be more active than diphenylhydantoin in the supramaximal electroshock test. In particular, a different profile of anticonvulsant activity was demonstrated for SP-175 compared to DMHP or delta-9-THC. It was discovered in 5-day studies that dilantin, phenobarbital, DMHP, and SP-175 do not exhibit tolerance development against audiogenic seizures in mice.

This study demonstrates that TRH potentiated the behavioral effects of DOPA-pargyline in thyroidectomized rats as well as in normal rats. This behavioral effect of TRH therefore can be considered to be independent of the thyroid gland in the DOPA potentiation test. Possible mechanisms and clinical implications are discussed.

A pentapeptide, methionine-enkephalin, which interacts with opiate receptors in the brain, was found to markedly potentiate the behavioral effects of DOPA when administered intraperitoneally into mice. These effects, which were even more striking with D-alanine-2-methionine-5-enkephalin but less with morphine, persisted at least two hours after systemic injection of the peptide. Only a weak effect of enkephalin was seen in a serotonin potentiation test. Systemic injections of enkephalin resulted in a significant reduction of footshock-induced fighting and slight reduction in audiogenic seizures in mice. The results suggest that the CNS effects observed after systemic administration of enkephalin may involve the dopaminergic receptor mechanism.

Thyrotropin releasing hormone (TRH) antagonizes the behavioral and temperature reducing effects of pentobarbital in rodents. The hormone is effective whether given before or after the barbiturate. This antagonism by TRH of the effects of pentobarbital probably does not depend upon thyroid hormone release as L-triiodothyronine administration is ineffective.

The pituitary peptide hormone α-MSH was found to potentiate the behavioral effects of DOPA but not serotonin. It slightly reduced footshock-induced fighting and decreased audiogenic seizures in susceptible mice, but was inactive in reducing the tremors induced by oxotremorine. The pattern of activity of α-MSH in these neuropharmacological tests differed from that of several other peptides. The results support our concept that naturally occuring peptides exert direct actions on the central nervous system independent of endocrine effects.

Both thyrotropin-releasing hormone (TRH) and amphetamine antagonize pentobarbital. They are more effective in the day than at night. This is true for TRH even when the dose of pentobarbital is increased at night to prolong sedation. Under this condition the day-night difference is lost for amphetamine. Both substances are more effective in cold ambient temperatures (18 degrees C) and less effective in warm temperatures, but their activity at warmer temperatures (37 degrees C) is still substantial. In contrast, somatotropin release-inhibiting factor (SRIF) augments the effects of pentobarbital at room temperature. This action is unaffected by time of day. However, the increase in sleeping time is lost in both a warm environment and in a cold environment.

The hypothalamic hormone GnRH was found to potentiate behavioral effects of DOPA and serotonin. In addition, GnRH was observed to reduce slightly audiogenic seizures as well as spontaneous motor activity. No significant effects were observed against footshock induced fighting or oxotremorine effects. These studies support our concept of the actions of hypothalamic peptides on the central nervous system.

The present study in mice has demonstrated that MIF greatly potentiates the behavioral effects of DOPA. The potentiation by MIF was found to be independent of MSH since MIF was found to potentiate DOPA effects in hypophysectomized mice.

The present study demonstrates that pemoline enhances performance of acquisition in a maze test in young immature rats. Toxicity studies in such rats indicate a wide safety margin. The mechanism of this performance enhancement by pemoline was proposed to be via dopaminergic mechanisms since the psychostimulant effects were prevented by a-methyl-p-tyrosine, a known depletor of brain catechol amines.

Synthetic thyrotropin releasing hormone (TBH) was administered to albino rats in order to determine the effects of this drug on norepinephrine-H3 metabolism in the brain. With the possible exception of a slight enhancement of release, acute or chronic administration of TRH had little effect on the disposition and metabolism of norepinephrine-H3 in rat brain. In addition, no significant changes were found in brain levels of endogenous norepinephrine, serotonin or dopamine following the injection of TRH. Thus, little evidence was found to support a possible relationship between the reported clinical antidepressant activity of TRH and its effects on norepinephrine metabolism in brain.

Significant potentiation of the behavioral effects of DOPA were observed in mice pretreated with GH-RIH. In addition, a slight reduction of oxotremorine induced symptoms was seen. No significant effects of GH-RIH were observed in several other tests involving the central nervous system (CNS). The results support our concept of the CNS actions of peptides.

SummaryMIF was demonstrated to antagonize the central and peripheral effects of oxotremorine in normal as well as in hypophysectomized mice. Its effectiveness in hypophysectomized mice confirms our earlier report that MIF exerts actions upon the CNS which are independent of its MSH release-inhibiting activity.The authors thank Dr. E. Kimura and Mr. P. Young for the anticholinergic studies in the isolated guinea pig ileum.