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In the present paper, we show that pineal hormone melatonin interacts with purified cell nuclei from rat spleen and thymus. Binding of 2-[125I]iodomelatonin ([125I]melatonin) by cell nuclei fulfills all criteria for binding to a receptor site. Binding exhibited properties such as dependence on time and temperature as well as reversibility, saturability, high affinity, and specificity. Results suggested binding to single classes of binding sites. The dissociation constants (Kd) for the binding sites in the spleen and thymus nuclei were 68 and 102 pM, respectively. These data are in close agreement with data obtained from kinetic studies, in which the kinetically derived values of the dissociation constant in the spleen and thymus nuclei were 166 and 537 pM, respectively. The affinities for melatonin of these nuclear binding sites suggest that they may recognize the physiological concentrations of melatonin in the tissues. Finally, we have demonstrated that binding of [125I]melatonin by the nuclei is displaced by CGP 52608, a specific ligand of the putative nuclear melatonin receptor RZR/ROR. Results strongly suggest that in addition to membrane receptor-related mechanisms, nuclear receptors may be involved in the regulation of immune system by melatonin.

In the present study, we describe the potential role of melatonin, a pineal hormone, in regulating the activation of the antigen-specific T cell response. Melatonin encouraged the proliferation of Th cells and improved their ability to secrete IL-4, but down-regulated the levels of IL-2 and interferon-gamma (IFN-gamma). Melatonin, however, could not exert any influence on the T cells of unprimed mice. On studying the regulation of subclass of IgG isotype, melatonin specifically enhanced the secretion of antigen-specific IgG1 antibodies and decreased the yield of IgG2a isotype. The results suggest that melatonin possibly acts by selectively activating a Th2-like immune response.

Melatonin is not only produced by the pineal gland, retina and parietal but also by various other tissues and cells from vertebrates, invertebrates, fungi, plants, multicellular algae and by unicells. In plants, many invertebrates and unicells, its concentration often exceeds that found in vertebrate blood by several orders of magnitude. The action of melatonin is highly pleiotropic. It involves firstly, direct effects, via specific binding sites in various peripheral tissues and cells of vertebrates, including immunomodulation; secondly, systemic influences on the cytoskeleton and nitric oxide formation, mediated by calmodulin; and thirdly, antioxidative protection, perhaps also in the context of photoprotection in plants and unicells. In some dinoflagellates, melatonin conveys temperature signals. On the basis of these comparisons, melatonin appears to mediate and modulate influences from several major environmental factors, such as the photoperiod, radiation intensity and temperature.

We analyzed the melatonin signal transduction in human blood lymphocytes. High affinity melatonin receptors were identified by specific binding of 2-[125I]melatonin ([125I]MEL) to human lymphocyte membranes. Scatchard analysis of [125I]MEL binding revealed high affinity receptors, with a dissociation constant (Kd) of 0.45 nM and a binding capacity (Bmax) of 7.8 fmol/mg protein. Specific [125I]MEL binding was reduced markedly by GTP and its nonhydrolyzable analogues guanosine 5'-beta, gamma-imidotriphosphate (Gpp(NH)p) and guanosine 5'-O-(3-triphosphate) (GTP-gamma-S). Gpp(NH)p inhibited in a dose-dependent manner the [125I]MEL specifically bound to human lymphocyte membranes with a half-maximal inhibition (IC50) of 3.5 +/- 0.6 microM Gpp(NH)p. Treatment of human lymphocyte membranes with Gpp(NH)p increased the Kd value (Kd = 1.20 nM). Melatonin inhibited significantly and in a dose-dependent manner forskolin-stimulated cAMP production in intact human lymphocytes. Melatonin was able to stimulate diacylglycerol production in a dose-dependent manner in human lymphocyte membranes. Pertussis toxin treatment inhibited the specific [125I]MEL binding and blocked the ability of melatonin to both inhibit forskolin-stimulated cAMP production and stimulate diacylglycerol production. Pertussis toxin ADP-ribosylation and Western blot experiments demonstrated the protein expression of alpha i1/2, alpha i3/0, and beta gamma complexes of G proteins in human lymphocyte membranes. The results strongly suggest a pertussis toxin-sensitive melatonin signal transduction pathway in human lymphocytes that involves the inhibition of adenylyl cyclase and the stimulation of phospholipase C.

In recent years, melatonin, i.e. the major endocrine product of the pineal gland, was investigated as to its possible regulatory role in the communication between the neuroendocrine and the immune systems. First indications that melatonin may be an endocrine immunomodulator came from early reports about antitumor effects in animals and humans. Since then evidence has accumulated suggesting that melatonin-as a well-preserved molecule during evolution-is indeed involved in the feedback between neuroendocrine and immune functions. At present we begin to discover molecular mechanisms, by which melatonin affects cellular functions in general, and from the variety of possible direct and indirect interactions it appears that melatonin may play a complex physiological role in neuroimmunomodulation. In this article we want to give a critical review of the numerous reports on melatonin influencing immune functions, and to discuss the possible different mechanisms of action, which were suggested recently.

Catecholamine receptors of multiple classes have been shown to influence pineal melatonin synthesis in a species-specific manner. In these experiments, the effects of catecholamine receptor agonists on circadian melatonin rhythms of zebrafish (Danio rerio) pineal in vitro were examined. Cyclic application of adrenergic receptor agonists (norepinephrine, phenylephrine, clonidine, and isoproterenol) had no effect on zebrafish pineal melatonin release, nor on the circadian oscillator that regulates melatonin rhythms. Pineal melatonin release was partially suppressed by quinpirole, a D2 dopamine receptor agonist, but cyclic application of quinpirole did not reset the pineal circadian oscillator. Pineal melatonin release was unaffected by either dopamine or SKF38393, a D1 receptor agonist, suggesting that the effects of quinpirole were not mediated by dopamine receptors. The regulatory mechanisms underlying pineal melatonin rhythms appear to differ among teleosts.

Melatonin is a hormone secreted by the pineal gland in response to photoperiods and influences many important biological processes. For one, Melatonin has been shown to produce resistance to cancer and infectious diseases in aged animals. Studies in animals have demonstrated melatonin-related mechanisms of action on immunoregulation. Additionally, melatonin has been successfully used in humans, along with interleukin-2, as a treatment of solid tumors. In vivo and in vitro studies show melatonin enhances both natural and acquired immunity in animals. Despite all of this intriguing evidence, melatonin's mechanism of action on the immune system is only partially defined. It does, however, appear to act through lymphocyte receptors, and perhaps, receptors on other immune tissues, to modulate immune cells. In order to understand immunomodulation and anti-cancer effects, information on melatonin and it's interactions with other endocrine hormones are summarized.

Winter is energetically demanding and stressful; thermoregulatory demands increase when food availability usually decreases. Physiological and behavioral adaptations, including termination of breeding, have evolved among nontropical animals to cope with the energy shortages during winter. Presumably, selection for the mechanisms that permit physiological and behavioral anticipation of seasonal ambient changes have led to current seasonal breeding patterns for many populations. In addition to the well-studied seasonal cycles of mating and birth, there are also significant seasonal cycles of illness and death among field populations of mammals and birds. Energetically challenging winter conditions can directly induce death via hypothermia, starvation, or shock; surviving these demanding conditions likely puts individuals under great physiological stress. The stress of coping with energetically demanding conditions may increase adrenocortical steroid levels that could indirectly cause illness and death by compromising immune function. Individuals would enjoy a survival advantage if seasonally recurring stressors could be anticipated and countered by bolstering immune function. The primary environmental cue that permits physiological anticipation of season is daily photoperiod, a cue that is mediated by melatonin. However, other environmental factors may interact with photoperiod to affect immune function and disease processes. Immune function is compromised during the winter in field studies of birds and mammals. However, laboratory studies of seasonal changes in mammalian immunity consistently report that immune function is enhanced in short day lengths. To resolve this apparent discrepancy, we hypothesize that winter stressors present in field studies counteract short-day enhancement of immune function. Prolonged melatonin treatment mimics short days, and also enhances rodent immune function. Reproductive responsiveness to melatonin appears to affect immune function. In sum, melatonin may be part of an integrative system to coordinate reproductive, immunologic, and other physiological processes to cope successfully with energetic stressors during winter.

The pineal neurohormone melatonin functionally synchronizes the photoperiod in the organism. In the last decade, it has become increasingly clear that the pineal gland and melatonin also play an important immunoregulatory role. T helper (Th) cells bear G-protein-coupled melatonin receptors. Activation of melatonin receptors enhances the release of Th cell cytokines, such as gamma-interferon and interleukin-2, as well as novel opioid cytokines which cross-react immunologically with both interleukin-4 and dynorphin B. These mediators may counteract secondary immunodeficiencies, protect mice against lethal viral and bacterial diseases, synergize with interleukin-2 in cancer patients and affect hematopoiesis. Hematopoiesis is apparently influenced by the action of melatonin-induced opioids on kappa-opioid receptors present on stromal bone marrow cells. Most interestingly, gamma-interferon and colony-stimulating factors may modulate the production of melatonin in the pineal gland. A hypothetical pineal-immune-hematopoietic network is, therefore, taking shape. From the immunopharmacological point of view, there is a need for clinical studies on the effect of melatonin in human immunodeficiency-virus-infected patients and cancer patients. In conclusion, melatonin seems to be an important immunomodulatory hormone which deserves to be further studied to identify its relevance in immune-based diseases, its therapeutic indications and its adverse effects.

Three pineal methoxyindoles (melatonin (Mel), 5-methoxytryptamine (5-MTA) and 5-methoxytryptophol (5-MTO)) were studied for their ability to influence the proliferative response of human peripheral blood lymphocytes (PBL) and tonsillar lymphocytes (TL) following activation with concanavalin A (ConA) in vitro. The ConA-stimulated DNA synthesis was affected in a different dose-dependent mode by the methoxyindoles tested. Melatonin and 5-MTO inhibited and 5-MTA increased the ConA-induced [3H]thymidine incorporation in PBL and TL. The initial screening for 2-[125I]iodomelatonin binding using a single point assay revealed significantly increased specific binding to PBL and TL after 72-h stimulation with ConA as compared to the non-activated cell cultures. Coincubation of separate lymphocyte cultures with ConA and Mel or 5-MTO resulted in inhibition of the specific 2-[125I]iodomelatonin binding (85% and 74%, respectively). The specific binding determined in the presence of 5-MTA did not differ from control values. Series of saturation and competition experiments were performed to examine the binding characteristics of ConA-stimulated lymphocytes for 2-[125I]iodomelatonin. The radioligand labelled binding sites of high affinity (Kd = 0.14 +/- 0.03 nM) and low capacity (Bmax = 6.8 +/- 1.5 fM/mg protein). Competitive studies with a variety of indoles determined the following order of relative potency for inhibition of 2-[125I]iodomelatonin binding in TL: 2-iodomelatonin > melatonin > > 5-methoxytryptophol. 5-Methoxytryptamine did not show displacement potency for the labelled ligand. Collectively, our data suggest that pineal hormones might be directly involved in the regulation of the T-lymphoproliferative response of human lymphoid cells. We show the availability of melatonin receptors, which seem to be an intrinsic characteristic of activated human lymphocyte populations. While the effects of Mel and 5-MTO can be linked to the binding sites described, it is unlikely that serotonin agonists like 5-MTA may act through the same sites to influence the mitogen-stimulated lymphocyte proliferation.

In addition to the well-documented seasonal cycles of mating and birth, there are also significant seasonal cycles of illness and death among many animal populations. Challenging winter conditions (i.e., low ambient temperature and decreased food availability) can directly induce death via hypothermia, starvation, or shock. Coping with these challenges can also indirectly increase morbidity and mortality by increasing glucocorticoid secretion, which can compromise immune function. Many environmental challenges are recurrent and thus predictable; animals could enhance survival, and presumably increase fitness, if they could anticipate immunologically challenging conditions in order to cope with these seasonal threats to health. The annual cycle of changing photoperiod provides an accurate indicator of time of year and thus allows immunological adjustments prior to the deterioration of conditions. Pineal melatonin codes day length information. Short day lengths enhance several aspects of immune function in laboratory studies, and melatonin appears to mediate many of the enhanced immunological effects of photoperiod. Generally, field studies report compromised immune function during the short days of autumn and winter. The conflict between laboratory and field data is addressed with a multifactor approach. The evidence for seasonal fluctuations in lymphatic tissue size and structure, as well as immune function and disease processes, is reviewed. The role of pineal melatonin and the hormones regulated by melatonin is discussed from an evolutionary and adaptive functional perspective. Finally, the clinically significance of seasonal fluctuations in immune function is presented. Taken together, it appears that seasonal fluctuations in immune parameters, mediated by melatonin, could have profound effects on the etiology and progression of diseases in humans and nonhuman animals. An adaptive functional perspective is critical to gain insights into the interaction among melatonin, immune function, and disease processes.

In the past several years, interest in the immunophysiological role of the pineal gland and melatonin has grown to the extent that now their immunoregulatory role is widely recognized. Melatonin has immunoenhancing properties and it is able to counteract the immunodepression induced by acute stress, drug treatment (i.e., anticancer drugs), and viral infections. Here we review the therapeutic efficacy of melatonin alone or in combination with interleukin-2 (IL-2) in cancer patients who did not respond to standard anticancer chemotherapies and/or refused any aggressive treatment. In this review, we summarize a series of reports from 1986 through 1994 in which patients affected by metastatic solid tumors, metastatic non-small-cell lung cancer, advanced solid neoplasms, myelodysplastic syndrome, hepatocellular carcinoma, and advanced endocrine tumors were studied. The conclusion drawn from these studies is that melatonin protects against IL-2 and synergizes with the IL-2 anticancer action. This combined strategy represents a well tolerated intervention to control tumor growth. In most cases performance status and quality of life seem improved.

An experiment was conducted to define the seasonal changes in LH secretion in ovariectomized does with oestrogen implants and the effect of immunization against melatonin. Fifteen mature Australian cashmere goats were ovariectomized and given either no further treatment or one or two implants containing oestradiol; another similar group of 15 does were immunized against melatonin before ovariectomy and oestrogen treatment. LH concentrations and livemasses were recorded every week for 2 years. Livemasses of both groups showed a distinct seasonal pattern with a summer maximum and a winter minimum irrespective of treatment. LH concentrations also showed distinct seasonal patterns with a significant interaction between the number of implants and the time of the year. In the nonimmunized does, the presence of a constant low dose of oestrogen (one implant) resulted in low concentrations of LH except from May to August, the normal period of spontaneous ovulatory activity in intact does. In contrast, nonimmunized does receiving a high dose of oestrogen (two implants) showed a rise in LH concentrations in February, and concentrations remained high until August. Immunization against melatonin abolished this differential LH secretory pattern, and both doses of oestrogen were associated with a short period of high LH concentration between May and September. These results indicate that a negative feedback effect of oestrogen results in low LH secretion for most of the year and that hypothalamic sensitivity to LH decreases for only a short period between May and August.(ABSTRACT TRUNCATED AT 250 WORDS)

An experiment was conducted to determine whether active immunization against melatonin could modify the perception of abrupt photoperiodic changes in ewes. Two groups each containing six intact Ile-de-France ewes were submitted to alternate periods of short days for 2.5 months and long days for 2.5 days for about 70 weeks. Three series of active immunizations against a melatonin conjugate were carried out during the first of the three long-day periods. Control ewes were actively immunized at the same time against human serum albumin. Blood samples were taken once a week throughout the experiment to measure antibody titre and affinity, and prolactin and progesterone concentrations. Sera of all treated ewes demonstrated higher antibody titres than those of control ewes. Antisera were highly specific, as evidenced by the absence of displacement of iodinated melatonin in the presence of ten melatonin metabolites. Significant effects of photoperiod and of the interaction between treatment and photoperiod on prolactin concentration were detected. Prolactin concentrations in plasma of the control ewes were high during long days and low during short days. However, in the treated ewes, with the exception of the first period of long days, prolactin concentrations were not influenced by photoperiodic changes. Ovulatory activity of control ewes, as demonstrated by progesterone measurements, was stimulated by short days and inhibited by long days. In contrast, ovulatory activity of treated ewes, after a response identical to that of control ewes after the first photoperiodic shift from long to short days, showed a complete desynchronization of ovulatory activity relative to photoperiodic changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripubertal male mink (Mustela vison) were treated with prolactin, melatonin or antibodies against melatonin to determine the effects of altered circulating concentrations of prolactin and melatonin throughout one season of testicular development. Treatment began on 1 October and continued until 4 March. Administration of 0.5 mg ovine prolactin day-1 by minipump increased the circulating concentration of prolactin for the duration of the study and increased serum concentrations of LH. This treatment had no effect on the testosterone concentration or on testis size. Neither chronic treatment with melatonin throughout the period of crudescence nor passive immunization against melatonin for 79 days affected the circulating concentrations of prolactin, LH, testosterone or testis size. These results show clearly that, unlike in other seasonally breeding species, prolactin does not play a significant role in testis growth in the mink. Administration of melatonin to male mink in October did not affect testis growth, presumably because the melatonin signal that cues photoperiodic events had already been received. Administration of antibodies against melatonin did not affect any of the features measured, suggesting that melatonin may have neural but not peripheral effects. Further support for this view can be found in the absence of an influence of melatonin on testis growth or on the plasma concentration of testosterone.

The function of the pars tuberalis as a mediator of the action of melatonin remains elusive. As a direct method of assessing the potential role of secretory proteins, ovine pars tuberalis cells have been cultured and radiolabelled with 35S-methionine, and the accumulation of specific radioactive products in the medium, measured after separation by SDS-PAGE and fluorography. The synthesis and secretion of a number of labelled proteins are increased by forskolin (1 microM) and inhibited dose dependently by melatonin (IC50, 300 pM), although consistently a 72-kD protein (p72), is the most intensely labelled of these. Thus, 72 acts as a useful marker of cellular activity for melatonin, whereas prolactin (p23) provides a melatonin non-responsive marker in ovine pars tuberalis cell cultures. The synthesis and secretion of p72 and other melatonin-sensitive proteins is regulated through the cyclic AMP/protein kinase A second-messenger pathway, as analogues of cyclic AMP mimic the action of forskolin, yet 1,9-dideoxyforskolin, a forskolin analogue that is not active on adenylate cyclase, has no effect. However, the phorbol ester, phorbol-12,13-myristate acetate, also regulates the synthesis and secretion of the same profile of proteins as forskolin indicating a potential role for protein kinase C, which occurs through an independent rather than a synergistic pathway. The differential effects of nocadazole (1 microM) and extracellular calcium depletion upon p72 and prolactin secretion indicates that p72 is secreted by a calcium and microtubule independent pathway, in contrast to prolactin. These observations in conjunction with the absence of dense-core storage vesicles in melatonin-responsive cells of the ovine PT are consistent with constitutive secretion of p72 from the latter and regulated secretion of prolactin from melatonin non-responsive cells. Using immunoprecipitation de novo synthesis and secretion of either LH or LH-like proteins from ovine pars tuberalis cells could not be detected under the conditions used. The absence of 125I-(Des-Gly10[D-Ala6]-LHRH-ethylamide) binding over most, but not all, of the ovine pars tuberalis supports the contention that the majority of the cells of the ovine pars tuberalis are not gonadotrophs. These results provide further support for the unique function for the pars tuberalis.

We describe a solid-phase competitive enzyme immunoassay for determination of melatonin in serum. The detection limit of the assay is 1.0 fmol/well. Low cross-reactivity of the antiserum with other indoles, parallel serum extract dilution and melatonin standard curves, good analytical recovery, and within- and between-assay CVs of 6.4-14.4% provide validation of the assay. Linear regression analysis of melatonin concentrations measured with this assay (y) and with a commercial 3H RIA (x) in 88 sera yielded the relation y = 0.62 x - 0.76, Sy/x = 0.03. Values for melatonin in serum samples from healthy subjects are lower during the day than during the night. Melatonin response in rat serum and pineal gland to isoproterenol injection is similar to published RIA data. The analytical procedure is also simple. Thus, the assay should have practical applications in investigation of pineal function in both clinical and basic studies.

The administration of melatonin to thymocyte-infused nude mice during the evening (dark cycle) of the circadian period restores transplantation immunity permanently, whereas inoculation during the morning cycle is quite ineffective. Transplantation of an intact pineal gland from normal donors into the mediastinal (athymic) tissue of nude mice leads to restoration of the capacity to reject skin grafts. Findings such as these attest to the genesis of transplantation immunity as linked to a thymus-directed establishment of a pineal circadian cyclicity.

Four kinds of monoclonal antibodies (moAbs) specific to paraquat, diquat, methamphetamine (MA) and melatonin (MT) were produced. 1-Methyl,1'-hexanoic acid-4,4'-bi-pyridinium, 4-amino-D,L-phenylalanine-diazo-coupled diquat, p-amino-methamphetamine as haptens were synthesized and coupled to bovine serum albumin (BSA) directly or to BSA via glutaraldehyde, and an antigen of MT was prepared by binding MT to BSA with the Mannich reaction. These immunogens of BSA conjugates were immunized i.p. to BALB/C mice. Usable moAbs were established 4 to 6 months after the first immunization. Amounts of paraquat, diquat, MA and MT causing a 50% of inhibition by enzyme-linked immunosorbent assay or radioimmunoassay with one of the most specific moAbs from each hapten were 1.1 ng, 0.67 nmol, 10.0 ng and 1.4 ng, respectively.

[125I]-labelled iodomelatonin binding sites in membrane preparations of duck thymus were studied. The specific binding of [125I]-labelled iodomelatonin in the duck thymus was stable, saturable, reversible and of high affinity. Scatchard analysis of the binding of [125I]-labelled iodomelatonin in the duck thymus collected at midlight had an equilibrium dissociation constant (Kd) of 34.8 +/- 9.4 pmol/l and a maximum number of binding sites (Bmax) of 0.98 +/- 0.07 fmol/mg protein. Two-point diurnal study demonstrated that the Bmax in samples collected at midlight was 42.0% higher (p < 0.05) than that at middark, but there was no significant difference (p > 0.05) between the midlight and middark Kd values. Competition inhibition studies showed that only melatonin, 2-iodomelatonin, 6-chloromelatonin, 6-hydroxymelatonin, N-acetylserotonin, 5-methoxytryptophol, and 5-hydroxytryptamine showed significant inhibition of the [125I]iodomelatonin binding in duck thymus membrane preparations, while the other compounds had no significant inhibition. Our results suggested a direct action of melatonin on the thymus and, thus, the cellular immune system.

Four vaccination protocols were utilized to investigate the effects of immunoneutralizing circulating melatonin on the annual cashmere growth cycle and cashmere production in Australian feral goats. A fluctuating anti-melatonin antibody response, achieved by repeated booster vaccinations, resulted in an acceleration of the growth cycle in goats which exhibited a significant immune response, compared to sham-immunized controls. Responding goats showed two cycles of cashmere length growth in the first 16 months and increased annual cashmere production in the first year. However, in the second year, these effects were no longer apparent, suggesting either some form of desensitization to melatonin, or a diminished response due to declining antibody titre. The effects of immunization were observed in both sexes; the effect on cashmere length was greater in wethers than in does. Cashmere fibre growth in response to a continuously declining plane of specific antibody showed increased cycle frequency, albeit with a decreased amplitude; guard hair growth cycles were affected to a much lesser extent. Small transient peaks of specific immunity at the summer or winter solstice were without significant effect on cashmere growth. Immunization to provoke a persistent anti-melatonin antibody response at the winter solstice resulted in significantly increased greasy fleece weight, % cashmere yield, and mass of cashmere produced, but no change in fibre diameter in both sexes. Thus the timing of cashmere growth cycles in goats may be, at least transiently, altered by appropriately timed immunization against melatonin. The mechanism of pineal-mediated regulation of cashmere growth cycles may involve (i) entrainment of an endogenous rhythm by melatonin, or (ii) seasonal alteration of cashmere follicle sensitivity to the effect of melatonin.

The present paper demonstrates the effect of melatonin on cyclic AMP production in human lymphocytes from peripheral blood. Melatonin by itself did not influence cyclic AMP accumulation in these cells at any dose studied; however, the drug potentiated the effect of vasoactive intestinal peptide (VIP) on the cyclic nucleotide production. In the presence of physiological concentrations of VIP (either 1, 10 or 100 pM), melatonin potentiated cyclic AMP production. However, at high doses of VIP (either 1, 10 or 100 nM), melatonin exhibited no such effect. The results suggest that human lymphocytes are a target for melatonin and that it may participate, jointly with VIP, in the regulation of immune function.

Melatonin, the main hormone of the pineal gland, when chronically injected into young mice or mice immunodepressed by aging or by cyclophosphamide treatment, is able to enhance the antibody response to a T-dependent antigen. The enhancement of the antibody response is associated with increased induction of T helper cell activity and IL-2 production, as evidenced in mice immunodepressed by aging or by cyclophosphamide treatment. These observations suggest that melatonin treatment may be successfully used in the therapy of immunodepressive conditions.

A stereotactic electrolytic lesion of the anterior hypothalamic area in mice produces a rapid involution of the thymus and a reduction of lymphocytes in the peripheral blood. This effect on the thymus and blood lymphoid compartment can be prevented by postoperational administration of thyrotropin-releasing hormone (TRH) or melatonin. These activities of TRH or melatonin are antagonized by the opioid receptor blocker naltrexone. They do not seem to depend on stimulation of the thyroid gland or of the endogenous opioid system but rather on a direct activity of TRH on thymic targets or binding sites on lymphocytes.

Clinical, epidemiological, biochemical, immunological, and radiological studies suggest that the pineal gland may be implicated in the pathophysiology of multiple sclerosis (MS). The following communication is concerned with the association among MS, pregnancy, the postpartum period, and melatonin secretion and illustrates, based on a clinical case report, the influence of the pineal gland on the clinical course of MS. This association is noteworthy since MS may worsen during the postpartum period and melatonin secretion is reported to be altered most dramatically by pregnancy and delivery. Since melatonin secretion is cyclical, undergoing diurnal, weekly, seasonal, and annual variations, it is proposed that the pineal gland may be the "prime mover" underlying the spontaneous exacerbations and remissions in MS.

Cultured human peripheral blood mononuclear leucocytes (PBML) were able to synthesize indoleamines, including melatonin, and were also able to convert melatonin taken up from the incubation medium into N-acetyl-5-hydroxytryptamine (NAHT) and 5-hydroxytryptamine (5-HT). These compounds were analysed by h.p.l.c., and melatonin was additionally characterized by two-dimensional t.l.c., mass spectrometry and radioimmunoassay. Only hydroxyindoles were detected by h.p.l.c. in unstimulated PBML culture. Sustained stimulation by melatonin or interferon-gamma (IFN-gamma) increased markedly the basal production of 5-HT. IFN-gamma- or 5-HT-stimulated (but not resting) cells produced NAHT and melatonin. Furthermore, the addition of melatonin to the culture medium strongly enhanced NAHT and 5-HT production without affecting tryptophan hydroxylation, suggesting the possibility of direct or indirect transformation of melatonin into NAHT and 5-HT.

Our previous work showed that the pineal neurohormone melatonin induces activated T lymphocytes to release opioid peptides with immunoenhancing and anti-stress properties. Here we present evidence that these peptides crossreact with anti-beta-endorphin and anti-met-enkephalin antisera, and bind specifically to thymic opioid receptors. Furthermore, the same antisera injected in prednisolone treated mice prevented the normal recovery of thymus cellularity and of the capacity to mount a primary antibody response against T-dependent antigens. Surgical pinealectomy, i.e. inhibition of endogenous melatonin and absence of antigen activation negated the effect of such antisera demonstrating the physiological relevance of this melatonin-immuno-opioids network. It is proposed that function of this network may be that of driving a correct immune recovery after the depression caused by the elevated corticosteroids level associated with immune responses and/or stressful situations.

Monoclonal antibodies(moAbs) reactive with melatonin(MT) were produced using MT, coupled to bovine serum albumin(BSA) with the Mannich reaction, as immunogen and conventional hybridoma techniques. Hybridoma clones secreting the moAbs were selected by an enzyme-linked immunosorbent assay system using MT-carboxymethylchitin and BSA as screening antigens. The moAbs from 6 clones were characterized by a cross-reactivity test using radioimmunoassay with 125I-labelled MT. The moAbs recognized MT but hardly recognized other analogues except for N-acetylserotonin with a crossreactivity of 0.81%. An inhibition curve for MT was obtained in the range of 50 pg to 100 ng and 1.4 ng of MT inhibited the value of the assay by half. There is interference from some unknown source in human serum.

In rats, intraventricularly injected antimelatonin antibody caused the appearance of transitory epileptiform abnormalities. Epileptic activity arose from and was limited to the cortical mantle of the hemisphere ipsilateral to the injection side. The occasional occurrence of lateralized seizures has also been observed. Control injection of saline, of the vehicle (rabbit serum), or of antibody saturated with melatonin induced flattening and desynchronization of the electroencephalogram but not epileptiform activity. Repeated antimelatonin injections caused reappearance of the same type of epileptic abnormalities that lasted slightly longer than the first time. Melatonin may play an inhibitory role in neuronal excitability.

The effects of active immunization against a range of gonadal steroids on pineal beta-adrenoceptors were studied in cycling Merino ewes. In selected cases, the effects of exogenous steroids were also investigated. Immunization against progesterone had no detectable effect on pineal beta-adrenoceptor density or ligand binding affinity. Immunization against estrone or 17 beta-estradiol significantly increased binding affinity and decreased beta-receptor density. Testosterone immunization in ewes caused similar effects but to a lesser degree. In contrast, immunization against androstenedione resulted in a decreased affinity and an increase in receptor density in ewes. In subsequent experiments, some parameters of pineal function in intact cycling and ovariectomized ewes were compared; a significant decrease in beta-receptor density and an increase in binding affinity were noted in the ovariectomized animals. Androstenedione-releasing implants decreased beta-receptor density and increased beta-receptor affinity in pineals from intact ewes, but these implants had no effect on pineal parameters in ovariectomized ewes. Immunization of ewes against 17 beta-estradiol had no significant effects on basal or isoprenaline-stimulated N-acetyltransferase or adenyl cyclase activities. Immunization of ewes against melatonin, or in vitro incubation of pineal glands with the hormone had no effects on the pineal parameters studied. It is concluded that estrogens affect the pinealocyte beta-receptors in ewes, while specific androgens may act indirectly on these receptors to modify their interaction with estrogens.

Melatonin binding in the pineal gland of albino rats is estimated using an immunohistochemical procedure. Binding is saturable, has relatively high affinity (Apparent KD = 2.7 nM), and competition studies indicate binding of indoleamines possessing an N-acetyl group on the terminus of the side chain (N-acetylserotonin and melatonin). These data are consistent with the interpretation that immunohistochemically determined melatonin in unfixed pineal tissue is assessing binding of N-acetylated indolealkylamines to pineal cell components. In albino rats maintained on 12-hour light: 12-hour dark cycles, melatonin binding exhibits a diurnal rhythm with low levels of saturation (30%) early in the light and saturation by endogenous melatonin near the onset of darkness. An annual rhythm of melatonin binding was observed in albino rats with low levels during the summer and high levels during the winter. Other rats were maintained on 12-hour light:dark cycles and fed for 2 hours either early in the light period or early in the dark period. For both morning- and evening-fed animals, melatonin binding was high prior to feeding and dropped immediately after feeding. Changes in melatonin binding that occur in response to alterations of feeding and time of year suggest the possibility that this binding reflects a functional site for melatonin.

Plasma melatonin was determined in samples of patients with colorectal carcinoma and in controls using an iodinated radioimmunoassay. Both groups showed large individual variability in absolute melatonin levels. However, during the night, melatonin concentration in cancer patients was significantly lower than in controls.

A radioimmunoassay of melatonin using a new iodinated derivative has been developed. Simple chemical treatments have then been designed to convert serotonin, N-acetylserotonin, and 5-methoxytryptamine to melatonin. Thus these four molecules, belonging to the same metabolic pathway, were separately assayed in the same radioimmunological system at the same sensitivity level (0.01 pmol). Some biological results on the circadian variations of melatonin and serotonin in blood, pineal, and miscellaneous brain structures are presented.

Rabbits immunized with human albumin conjugates of N-(p-chlorobenzoyl)-2-methyl-5-methoxyindole-3-acetic acid (indomethacin), 5-methoxytryptamine, and 2-methyl-5-methoxyindole-3-acetic acid produced antibodies that bound [3-H] melatonin. The serologic specificities for the binding of melatonin with antibodies from four antisera to these three immunogens were determined. Melatonin was measurable even at the 0.1 picomole level by a radioimmunoassay. Serotonin, N-acetylserotonin, 5-methoxytryptophol, 5-methoxytryptamine and 6-hydroxymelatonin can be ruled out as immunoreactive material with the use of the multiple antisera of different specificities.

Having prepared antisera to serotonin and to melatonin, the authors were able to show that, in the pineal gland, the behaviour of these two antisera in immunohistochemical studies differs. The antiserum raised against 5HT, actually bound to the molecule formed by condensation of formaldehyde on 5HT and therefore could be used to reveal 5HT in tissue fixed with formaldehyde. On the other hand, the anti-melatonin antiserum bound to melatonin, and could therefore be used to reveal its presence in fresh tissue.

A new radioimmunoassay (RIA) using [125I]-melatonin as tracer for determination of melatonin in biological fluids has been developed. Melatonin antisera were raised in rabbits by immunization with bovine thyroglobulin conjugate of N-acetyl-5- methoxytryptophan . Two high-affinity and specific antisera were obtained. Unlike in previous studies melatonin was radioiodinated directly. Iodo-Gen was used as the oxidant. Radioiodinated melatonin was purified by TLC for use in RIA. Melatonin was extracted from plasma, serum and urine samples (1 ml) with chloroform. Using the extraction the sensitivity of the RIA method was 18 fmol/ml of original sample. Plasma, serum and urine extracts diluted parallelly with synthetic melatonin in RIA. HPLC analysis of plasma and serum extracts showed only one immunoreactive peak co-eluting with synthetic melatonin. Majority of urine immunoreactivity co-eluted with synthetic melatonin, but 7-23% contaminating immunoreactivity was also observed. Daytime values for rat plasma, human serum and urine melatonin were 30-60, 20-40 and 50-130 fmol/ml and the respective night values were 160-300, 180-370 and 230-470 fmol/ml. Thus a characteristic diurnal rhythm of melatonin was observed in all cases. The urinary excretion of immunoreactive melatonin during the day was 3-9 and during the night 11-28 pmol/h. Thus we have developed a specific and valid RIA method for the determination of plasma and serum melatonin. Despite the incomplete specificity of the RIA for urine determinations, a clear diurnal rhythm for urine melatonin was observed. The distinct advantage of the utilization of [125I]melatonin as tracer is that the costly and cumbersome scintillation counting can be avoided.

Melatonin immunofluorescence in the rat colon was measured with a cadmium sulfide photometer in conjunction with an immunohistological double antibody procedure. The method proved to be very reliable with 5-10% intra- and interassay reliabilities. In addition, manipulation of the primary antibody concentration, the incubation time, and the nature of the first antibody produced predicted changes in fluorescence intensity. Treatment with p-chlorophenylalanine led to a marked reduction of melatonin-specific fluorescence.

Major environmental variables such as daily and seasonal changes of light and temperature regulate the daily circadian variations of synthesis and release of the pineal neurohormone N-acetyl-5-methoxytryptamine (melatonin). Melatonin has now been shown to be a potent immunoregulatory agent, and to be able to antagonize the immunosuppressive effects of acute anxiety stress in mice, as measured by antibody production, by thymus weight, and by the capacity of stressed- and evening-melatonin-treated mice to react against a lethal virus. Both psychogenic factors and infectious agents such as viruses can act as "stressors" and induce an immunosuppression. Their combination is a determinant for the course of infectious diseases and, perhaps, cancer. Circadian (evening) melatonin possesses thus the singular ability to up-regulate the immunosuppression of stressed mice. This effect of melatonin is not exerted directly on immunocompetent cells, but mediated via the endogenous opioid system upon antigen-activation of T cells. Melatonin being a short-lived hormone with negligible side-effects which is rapidly degraded and eliminated by the body, the use of melatonin offers a new approach to the physiological control of stress and stress-related infectious diseases. In addition, melatonin could be used for the potentiation of primary immunization (vaccination) against antigens of the most varied nature which do not evoke a robust or longlasting secondary (memory) response. The regulatory function of pineal melatonin is discussed also in relation to hematopoiesis, to its oncostatic effects, and to its possible dual role in reproduction physiology and generation of immunocompetence and tolerance during ontogeny.

A structural immunochemical study has shown that fixation of melatonin on bovine serum albumin by formaldehyde occurs via reaction of both an indole NH group and the side chain, allowing extremely specific antibody production. Isotopic in vitro investigations and radioimmunological estimation of this hormone in man and in the rat shows that it is transported by plasmatic albumin in a 1/10(6) ratio and is eliminated in the urine as native material, 6-hydroxy melatonin and as an as yet unidentified metabolite.

The sites of fixation to bovine serum albumin of melatonin and of mono- and dimethylated melatonin identified by mass spectrometry and nuclear magnetic resonance, have been determined. The binding sites appear to be important in the conservation of the structural features necessary for the antigenicity of the molecule.