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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.

The effects of methionine enkephalin (met-enkephalin) on human immune function are reviewed. This pentapeptide functions to upregulate, or enhance, immune function in the majority of donor samples at low doses and suppresses at high doses. The influence of this molecule is shared by the central nervous, neuroendocrine, and immune systems. Cells from each of these systems possess receptors for met-enkephalin and have the ability to process met-enkephalin from its prohormone, proenkephalin A. Studies have shown that this molecule is capable of enhancing immune function in patients with cancer or AIDS. It is proposed that this molecule be classified as a cytokine.

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.

Cytokine involvement in the immune system's response to stress is now very well documented. Cytokine activity has been implicated in a variety of mental and physical diseases, and has been shown to have a significant role in fueling the vicious circle of depression and illness.The first edition of Cytokines: Stress and Immunity pointed out

Clinical experience shows that cytokines have side effects of a central nervous system origin. Flu-like systems (fever, chills, headache, fatigue) develop rapidly with cytokine therapy. The latter symptoms are followed by neuroasthenia or fatigue syndrome (asthenia, malaise, lethargy, somnolence, headache, low-grade fevers, and Anorexia). Psychomotor, cognitive, and psychiatric changes are also observed (1, 2, 3, 4). Numerous groups have considered the effects of cytokines on the central nervous system (5).

Behavioral Effects of Cytokines: A Psychiatrist's Perspective Ziad Kronfol Worried to Death? Stress, Worry, and Immune Dysregulation in Health and HIV Suzanne C. Segerstrom and Margaret E. Kemeny Psychological Stress and Its Relationship to Cytokines and Inflammatory Diseases Rama Murali, Margaret D. Hanson, and Edith Chen Role of Cytokines in Depression Ghanshyam N. Pandey and Yogesh Dwivedi Loneliness, Dysphoria, Stress, and Immunity: A Role for Cytokines Louise C. Hawkley, Jos A. Bosch, Christopher G. Engeland, Phillip T. Marucha, and John T. Cacioppo Stress, Cytokines, and Peripheral Analgesia Peter John Cabot Alexithymia, Stress, and Immunity Olivier Guilbaud, Maurice Corcos, and Philippe Jeammet Roles of Mu-Opioid Receptor and Endogenous Opiates in Stress-Induced Immunosuppression Jennifer Kelschenbach, Horace H. Loh, and Sabita Roy Stress, Opioid Peptides, and Immune Response Javier Puente, Marion E. Wolf, M. Antonieta Valenzuela, and Aron D. Mosnaim Met-Enkephalin in Oxidative Stress Tihomir Balog, Sandra Soboc? anec, Vis?nja Sc?verko, Helena Haberstock-Debic' and Tatjana Marotti Chronic Stress Induces Death of Lymphocytes Erwei Sun, Lixin Wei, Arthur I. Roberts, Catherine Liu, and Yufang Shi Interleukin- and the Hypothalamic-Pituitary-Adrenal Axis Eric M. Smith, Huolin Tu, and Thomas K. Hughes, Jr. Cytokines, Stress, and Depression Adrian J. Dunn Immunoconversion in Acute Phase Response Istvan Berczi, Andres Quintanar-Stephano, and Kalman Kovacs Interferon in Health and Disease Nachum Dafny, Pamela B. Yang, and Stanley A. Brod Neuropeptide Precursor Processing in Immunocytes: Involvement in Neuroimmune Communication Robert Day and Michel Salzet Clinical Relevance of Opioid-Induced Immunosuppression: Are All Drugs Similar? Paola Sacerdote, Sivia Franchi, and Cataldo Martucci Human Retroviruses and the Cytokine Network Massimo Alfano and Guido Poli Psychiatric Toxicity of Interferon-?: A Model for Understanding the Etiology of Major Depression and Chronic Fatigue Syndrome? Gopinath Ranjith and Carmine Pariante Role of Genetic Predisposition, Cytokines, and Neuroendocrine Response in Development of Thyroid Autoimmunity Antonio Martocchia and Paolo Falaschi Gender Differences, Stress, and Immunity Nicholas P. Plotnikoff and Robert E. Faith

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 neuropeptide, methionine enkephalin (Met-ENK), has been demonstrated to have immunomodulatory activity, most notably increasing CD4+ lymphocyte counts.1 Based on this information-Met-ENK has been administered to virus infected mice and humans in an attempt to treat retroviral diseases.2,3 Bihari and coworkers noted that patients infected with the human immunodeficiency virus (HIV), who either have the acquired immunodeficiency syndrome (AIDS) or AIDS related complex, demonstrated increased CD4+ cell counts.2 However these author’s did not report any long term clinical results for these patients. Specter et al. have reported that BALB/c mice infected with Friend leukemia virus (FLV) or C57BL/6 mice infected with BM5 lymphoproliferative virus complex showed reduced disease progression when treated with a combination of azidothymidine (AZT) and MetENK.3 In this study Met-ENK did not provide any benefit when used alone and AZT was about 50% effective compared to the AZT and Met-ENK combination. The data suggest that AZT and Met-ENK used in combination may be effective for reducing morbidity and mortality of retroviral disease in animal and human infections. It was not clear from the studies cited above whether Met-ENK was providing benefit as an antiviral drug or as an immunostimulatory drug working in combination with an antiviral drug (AZT).

The cellular parts of the immune system (macrophages, T cells, and B cells) are regulated in their functions, to a large extent, by factors produced by these cells. These factors produced by the cells of the immune system are referred to as cytokines in the generic sense. More specifically, factors produced by lymphocytes are known as lymphokines, whereas those produced by macrophages are known as monokines. Recent progress in biotechnology is based on the production of large amounts of these factors, allowing for clinical evaluation in cancer and AIDS patients as well as other conditions involving the immune system (Figure 3.1).

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.

Publisher Summary This chapter reviews the hypothalamic releasing hormones and catecholamines. Ten women, ages 25-45, with primary, unipolar depression but in good physical health were treated in a double-blind, placebo-controlled, crossover design. Thyrotropin releasing hormone (TRH) acted alone rather than in combination with Imipramine, and that a single injection acted very rapidly though transiently. In this experiment as well as in the experiments with normal and schizophrenic women, psychological and behavioral changes were demonstrated in an environment characterized by pleasant stability and the absence of environmental noise such as may be introduced by intensive psychotherapeutic intervention. That behavioral effects occur in humans, at least with TRH seems certain. The effects have been shown to occur in normal and depressed women and are suggested by a single-blind study of schizophrenic women. The effects of a single injection are rapid, occurring within a few hours, and lasting not more than a few days. They are best demonstrated in a pleasant, stable, and relatively noise free environment. The effects are subtle, and it seems likely that intrinsic hormonal effects may be amplified or dampened by the environment.

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.

Clinical studies of the immunological effects of methionine enkephalin in normal volunteers, cancer, and AIDS patients are summarized. The major immunology changes seen were increases in T cell subsets, natural killer activity, as well as mitogen blastogenesis. Clinically, the cancer and ARC patients did not develop infections.

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.

Early studies suggested that peptides from the pituitary gland might have affects on the central nervous system (CBS). Murphy and Milicr [I] and Miller and Ogawa [r’] showed that administration of corticotropin (ACTH) resulted in resistance to extinction of a conditioned avoidance response. Dc Wied [3] used this test involving electrical shock to demonstrate that mel~~nocyte-stimulating hormone (MSH) can have the same effuct as AC‘TH and that the active sequence is ACTH/MSH -l II) [AJ. Approaching the problem for the first time from a direct interest in MSH, several different behavioral tests in the rat and man as well as studies of electrical activity (EEG) in the brain of these species were initiated in I966 by Kastin and colleagues. The results from these investigations arc consistent with the hyp~~thesis that MSH results in improved visual memory and sustained levels of attention [S. h]. Thcsc studlcs differed in approach from the previous ones of Krivoy 01 al. [7,8]. who examined the spinal cord and electrical discharge. and from those of Fcrrari OT rri. 19. 10]. who tested stretching activity and yawning. In an unrelated series of iilvestig~~tiol~s, Wilson rt trl. [ 1 I] were investigating the interactions of tricyclic antidepressants and thyroid hormones. Their early findings suggested that the time of onset of antidepressant activity was shortened by the use of thyroid hormone [I 11 (T,, tri-iodothyronine) as well as thyrotropin (TSH) [I?]. a pitlti~lry hormone whose release is stimulated by TRH (thyrotropin-rel~sing hormone). Thus, the stage was sot for the next logical series of studies to dctcrmine whether the relatively newly identified and isolated hypothalamic releasing factors [ l3]. MIF--1 (melanocyte-stimulating hormone release i?lhibiting factor. Pro-L~Ll-Gly-~H~) and TRH. had direct effects in the central nervous system. Later. other hypothalamic hormones like growth hormone release inhibiting hormone (GH-RIH) and gonadotropin-releasing hormone (GnRH) were also tested.

Publisher Summary This chapter examines the effects of prolyl-leucyl-glycine amide (PLG) on 3, 4-dihydroxy-phenylalanine (DOPA) potentiation and brain biogenic amine levels in rats and mice following acute and repeated doses. The central nervous system activity of TRH (thyrotropin-releasing hormone) and MIF (melanocyte-stimulating hormone-release inhibiting factor, PLG was discovered by means of the Everett brain dopamine potentiation test for antidepressant activity and the Everett oxotremorine antagonism test for antiparkinson activity. The finding that thyrotropin-releasing hormone (TRH) is active in the pargyline-DOPA mouse activation test formed in part the motive for trials of the hormone as a remedy for depression. When either TRH or MIF was administered over 5 days the original level of activity was maintained in the dopamine potentiation test. The central effects of TRH or MSH release from the pituitary. Furthermore, ablation of the adrenals, ovaries, testes, pineal, spleen, parathyroid, one kidney, or thymus, did not disrupt the behavioral potentiation of dopamine by TRH or MIF. Biogenic amine brain levels in mice and rats were not altered by TRH or MIF.

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.