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Unprecedented tandem allylic alkylation/intermolecular Michael addition was used in the preparation of novel bicyclic azalides. NMR spectroscopy was used not only to unambiguously determine and characterize the structures of these unexpected products of chemical reaction but also to investigate the effect the rigid bicyclic modification has on the conformation of the whole molecule. Thus, some of the macrolides prepared showed antibacterial activity in the range of well-known antibiotic drug azithromycin.

This chapter will discuss the recent literature of macrocycles and drug-like property space moving beyond the rule of five (bRo5). Trends in chemical classes that fall within this definition are discussed and the impact of the latest technologies in the field assessed. The physicochemical properties, which have provided both successes and challenges, especially in scale-up, are discussed. A recent patent literature is reviewed and the chapter concludes with a perspective on the future of macrocyclic drug discovery., (© 2018 Elsevier B.V. All rights reserved.)

Three novel spiroketals were prepared by a one-pot transformation of 6-O-methyl-9(E)-hydroxyiminoerythronolide A. We present the formation of a [4.5]spiroketal moiety within the macrolide lactone ring, but also the unexpected formation of a 10-C=11-C double bond and spontaneous change of stereochemistry at position 8-C. As a result, a thermodynamically stable structure was obtained. The structures of two new diastereomeric, unsaturated spiroketals, their configurations and conformations, were determined by means of NMR spectroscopy and molecular modelling. The reaction kinetics and mechanistic aspects of this transformation are discussed. These rearrangements provide a facile synthesis of novel macrolide scaffolds.

The stability in aqueous solution of five classes of coumarin dimers (I-V, compounds 1-29) was studied by HPLC-MS/MS at various pH values. The relationship between chemical structure and stability is discussed. It was found that dimeric compounds with strong electron withdrawing groups (EWGs) on the α-carbon to the bridging C-atom are stable at all pH values, whereas other derivatives undergo retro-Michael addition at rates which are also affected by the substituents on the aromatic rings. In some cases formation of stable isomers or oxidation products was observed. In order to evaluate their developability and potential for progression to in vivo studies, representative compounds were tested in an in vitro microsomal stability assay., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The neuroendocrine system affects the immune system through the neuroendocrine humoral outflow via the pituitary, and through direct neuronal influences via the sympathetic, parasympathetic (cholinergic) and peptidergic/sensory innervation of peripheral tissues. Circulating hormones or locally released neurotransmitters and neuropeptides regulate major immune functions, such as antigen presentation, antibody production, lymphocyte activity, proliferation and traffic, and the secretion of cytokines including the selection of T helper (Th)1 or Th2 cytokine responses. During inflammation, the activation of the stress system, through induction of a Th2 shift protects the organism from systemic "overshooting" with Th1/pro-inflammatory cytokines. Under certain conditions, however, stress hormones, substance P, ATP and the activation of the corticotropin-releasing hormone/substance P-histamine axis may actually facilitate inflammation, through induction of interleukin (IL)-1, IL-6, IL-8, IL-18, tumor necrosis factor (TNF)-alpha and CRP production. Thus, a dysfunctional neuroendocrine-immune interface associated with abnormalities of the 'systemic anti-inflammatory feedback' and/or 'hyperactivity' of the local pro-inflammatory factors may play a role in the pathogenesis of atopic/allergic and autoimmune diseases, obesity, depression and atherosclerosis. Better understanding of the neuroendocrine control of inflammation may provide critical insights into mechanisms underlying a variety of common human immune-related diseases.

Stress is defined as a state of threatened homeostasis. The principal effectors of the stress system include corticotropin-releasing hormone, arginine vasopressin, the glucocorticoids, and the catecholamines norepinephrine and epinephrine. Activation of the stress system leads to adaptive behavioral and physical changes. The principal stress hormones glucocorticoids and catecholamines affect major immune functions such as antigen presentation, leukocyte proliferation and traffic, secretion of cytokines and antibodies, and selection of the T helper (Th) 1 versus Th2 responses. A fully fledged systemic inflammatory reaction results in stimulation of the stress response, which in turn, through induction of a Th2 shift protects the organism from systemic overshooting with Th1/pro-inflammatory cytokines. Stress is often regarded as immunosuppressive, but recent evidence indicates that stress hormones influence the immune response in a less monochromatic way--systemically they inhibit Th1/pro-inflammatory responses and induce a Th2 shift, whereas in certain local responses they promote pro-inflammatory cytokine production and activation of the corticotropin-releasing hormone-mast cell-histamine axis. Through this mechanism a hyper- or hypoactive stress system associated with abnormalities of the systemic anti-inflammatory feedback and/or hyperactivity of the local pro-inflammatory factors may play a role in the pathogenesis of chronic inflammation and immune-related diseases., (Copyright (c) 2006 S. Karger AG, Basel.)

Cytokines mediate and control immune and inflammatory responses. Complex interactions exist between cytokines, inflammation and the adaptive responses in maintaining homeostasis, health, and well-being. Like the stress response, the inflammatory reaction is crucial for survival and is meant to be tailored to the stimulus and time. A full-fledged systemic inflammatory reaction results in stimulation of four major programs: the acute-phase reaction, the sickness syndrome, the pain program, and the stress response, mediated by the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Common human diseases such as atopy/allergy, autoimmunity, chronic infections and sepsis are characterized by a dysregulation of the pro- versus anti-inflammatory and T helper (Th)1 versus Th2 cytokine balance. Recent evidence also indicates the involvement of pro-inflammatory cytokines in the pathogenesis of atherosclerosis and major depression, and conditions such as visceral-type obesity, metabolic syndrome and sleep disturbances. During inflammation, the activation of the stress system, through induction of a Th2 shift, protects the organism from systemic 'overshooting' with Th1/pro-inflammatory cytokines. Under certain conditions, however, stress hormones may actually facilitate inflammation through induction of interleukin (IL)-1, IL-6, IL-8, IL-18, tumor necrosis factor-alpha and C-reactive protein production and through activation of the corticotropin-releasing hormone/substance P-histamine axis. Thus, a dysfunctional neuroendocrine-immune interface associated with abnormalities of the 'systemic anti-inflammatory feedback' and/or 'hyperactivity' of the local pro-inflammatory factors may play a role in the pathogenesis of atopic/allergic and autoimmune diseases, obesity, depression, and atherosclerosis. These abnormalities and the failure of the adaptive systems to resolve inflammation affect the well-being of the individual, including behavioral parameters, quality of life and sleep, as well as indices of metabolic and cardiovascular health. These hypotheses require further investigation, but the answers should provide critical insights into mechanisms underlying a variety of common human immune-related diseases., (Copyright (c) 2005 S. Karger AG, Basel)

Evidence accumulated over the last 5-10 years indicates that glucocorticoids (GCs) inhibit the production of interleukin (IL)-12, interferon (IFN)-gamma, IFN-alpha, and tumor necrosis factor (TNF)-alpha by antigen-presenting cells (APCs) and T helper (Th)1 cells, but upregulate the production of IL-4, IL-10, and IL-13 by Th2 cells. Through this mechanism increased levels of GCs may systemically cause a selective suppression of the Th1-cellular immunity axis, and a shift toward Th2-mediated humoral immunity, rather than generalized immunosuppression. During an immune response and inflammation, the activation of the stress system, and thus increased levels of systemic GCs through induction of a Th2 shift, may actually protect the organism from systemic "overshooting" with Th1/pro-inflammatory cytokines and other products of activated macrophages with tissue-damaging potential. However, conditions associated with significant changes of GCs levels, such as acute or chronic stress or cessation of chronic stress, severe exercise, and pregnancy and postpartum, through modulation of the Th1/Th2 balance may affect the susceptibility to or the course of infections as well as autoimmune and atopic/allergic diseases.

Recent evidence indicates that glucocorticoids and catecholamines, the major stress hormones, inhibit the production of proinflammatory cytokines, such as interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma, whereas they stimulate the production of antiinflammatory cytokines, such as IL-10, IL-4, and transforming growth factor (TGF)-beta. Thus, systemically, an excessive immune response, through activation of the stress system, stimulates an important negative feedback mechanism, which protects the organism from an "overshoot" of proinflammatory cytokines and other products of activated macrophages with tissue-damaging potential. Conversely, in certain local responses and under certain conditions, stress hormones actually may boost regional immune responses, through induction of TNF-alpha, IL-1, and IL-8, and by inhibiting TGF-beta production. Therefore, conditions that are associated with significant changes in stress system activity, such as acute or chronic stress, cessation of chronic stress, severe exercise, and pregnancy and the postpartum period, through modulation of the systemic or local pro/antiinflammatory cytokine balance, may suppress or potentiate autoimmune diseases activity and/or progression.

Evidence has recently been obtained that the branches of the autonomic nervous system, mainly, the sympathetic [25], regulate cytokine production. Not only the primary (thymus, bone marrow) and secondary (spleen, tonsils, and lymph nodes) lymphoid organs, but also many other tissues are involved in immune responses and are heavily influenced by noradrenaline (NA) derived from varicose axon terminals of the sympathetic nervous system [25, 100]. Besides NA released from nonsynaptic varicosities of noradrenergic terminals [92], circulating catecholamines (adrenaline, dopamine, NA) are also able to influence immune responses, the production of pro- and anti-inflammatory cytokines by different immune cells. The sympathetic nervous system (catecholamines) and the hypothalamic-pituitary-adrenal (HPA) axis (cortisol) are the major integrative and regulatory components of different immune responses. In our laboratory convincing evidence has been obtained that NA released non-synaptically [90, 92] from sympathetic axon terminals and enhanced in concentration in the close proximity of immune cells is able to inhibit production of proinflammatory (TNF-alpha, IFN-gamma, IL-12, IL-1) and increase antiinflammatory cytokines (IL-10) in response to LPS [25, 91], indicating a fine-tuning control of the production of TNF-alpha and other cytokines by sympathetic innervation under stressful conditions. This effects are mediated via beta2-adrenoceptors expressed on immune cells and coupled to cAMP levels.

Clinical observations indicate that some autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis, frequently remit during pregnancy but exacerbate, or have their onset, in the postpartum period. The immune basis for these phenomena is poorly understood. Recently, excessive production of IL-12 and TNF-alpha was causally linked to rheumatoid arthritis and multiple sclerosis. We studied 18 women with normal pregnancies in their third trimester and during the early postpartum period. We report that during the third trimester pregnancy, ex vivo monocytic IL-12 production was about 3-fold and TNF-alpha production was approximately 40% lower than postpartum values. At the same time, urinary cortisol and norepinephrine excretion and serum levels of 1,25-dihydroxyvitamin were 2- to 3-fold higher than postpartum values. As shown previously, these hormones can directly suppress IL-12 and TNF-alpha production by monocytes/macrophages in vitro. We suggest that a cortisol-, norepinephrine-, and 1,25-dihydroxyvitamin-induced inhibition and subsequent rebound of IL-12 and TNF-alpha production may represent a major mechanism by which pregnancy and postpartum alter the course of or susceptibility to various autoimmune disorders.

Peripherally produced CRH acts as a local auto/paracrine proinflammatory agent. Urocortin is a new member of the CRH family that acts through the family of CRH receptors. In this study, we demonstrated that the expression of urocortin mRNA in synovia of patients with rheumatoid arthritis was greater than that of patients with osteoarthritis. Also, we detected urocortin and CRH receptor immunoreactivity in the synovial lining cell layer, subsynovial stromal cells, blood vessel endothelial cells, and mononuclear inflammatory cells from the joints of rheumatoid arthritis and osteoarthritis patients. The expression of immunoreactive urocortin was significantly greater in rheumatoid arthritis than osteoarthritis (P < 0.0001) and correlated with the extent of inflammatory infiltrate. CRH receptor immunoreactivity was strong in mononuclear inflammatory cells of rheumatoid arthritis synovia. Urocortin stimulated IL-1beta and IL-6 secretion by human peripheral blood mononuclear cells in vitro. These findings suggest that, like CRH, urocortin is present in peripheral inflammatory sites, such as rheumatoid synovium, and acts as an immune-inflammatory mediator.

The brain and the immune system are the two major adaptive systems of the body. During an immune response the brain and the immune system "talk to each other" and this process is essential for maintaining homeostasis. Two major pathway systems are involved in this cross-talk: the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). This overview focuses on the role of SNS in neuroimmune interactions, an area that has received much less attention than the role of HPA axis. Evidence accumulated over the last 20 years suggests that norepinephrine (NE) fulfills the criteria for neurotransmitter/neuromodulator in lymphoid organs. Thus, primary and secondary lymphoid organs receive extensive sympathetic/noradrenergic innervation. Under stimulation, NE is released from the sympathetic nerve terminals in these organs, and the target immune cells express adrenoreceptors. Through stimulation of these receptors, locally released NE, or circulating catecholamines such as epinephrine, affect lymphocyte traffic, circulation, and proliferation, and modulate cytokine production and the functional activity of different lymphoid cells. Although there exists substantial sympathetic innervation in the bone marrow, and particularly in the thymus and mucosal tissues, our knowledge about the effect of the sympathetic neural input on hematopoiesis, thymocyte development, and mucosal immunity is extremely modest. In addition, recent evidence is discussed that NE and epinephrine, through stimulation of the beta(2)-adrenoreceptor-cAMP-protein kinase A pathway, inhibit the production of type 1/proinflammatory cytokines, such as interleukin (IL-12), tumor necrosis factor-alpha, and interferon-gamma by antigen-presenting cells and T helper (Th) 1 cells, whereas they stimulate the production of type 2/anti-inflammatory cytokines such as IL-10 and transforming growth factor-beta. Through this mechanism, systemically, endogenous catecholamines may cause a selective suppression of Th1 responses and cellular immunity, and a Th2 shift toward dominance of humoral immunity. On the other hand, in certain local responses, and under certain conditions, catecholamines may actually boost regional immune responses, through induction of IL-1, tumor necrosis factor-alpha, and primarily IL-8 production. Thus, the activation of SNS during an immune response might be aimed to localize the inflammatory response, through induction of neutrophil accumulation and stimulation of more specific humoral immune responses, although systemically it may suppress Th1 responses, and, thus protect the organism from the detrimental effects of proinflammatory cytokines and other products of activated macrophages. The above-mentioned immunomodulatory effects of catecholamines and the role of SNS are also discussed in the context of their clinical implication in certain infections, major injury and sepsis, autoimmunity, chronic pain and fatigue syndromes, and tumor growth. Finally, the pharmacological manipulation of the sympathetic-immune interface is reviewed with focus on new therapeutic strategies using selective alpha(2)- and beta(2)-adrenoreceptor agonists and antagonists and inhibitors of phosphodiesterase type IV in the treatment of experimental models of autoimmune diseases, fibromyalgia, and chronic fatigue syndrome.

Adenosine (ADO) exerts potent anti-inflammatory and immunosuppressive effects. In this paper we address the possibility that these effects are partly mediated by inhibition of the secretion of IL-12, a proinflammatory cytokine and a major inducer of Th1 responses. We demonstrate that 5'-N-ethylcarboxamidoadenosine (NECA), a nonspecific ADO analogue, and 2-p-(2-carbonyl-ethyl)phenylethylamino-5'-N-ethylcarboxamidoadenos ine (CGS-21680), a specific A2a receptor agonist, dose-dependently inhibited, in whole blood ex vivo and monocyte cultures, the production of human IL-12 induced by LPS and Stapholococcus aureus Cowan strain 1. However, the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine and the A3 receptor agonists N6-Benzyl-NECA and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-d -ribofuranuronamide expressed only weak inhibitory effects. On the other hand, NECA and CGS-21680 dose-dependently potentiated the production of IL-10. The differential effect of these drugs on monocyte IL-12 and IL-10 production implies that these effects are mediated by A2a receptor signaling rather than by intracellular toxicity of ADO analogue's metabolites. Moreover, CGS-21680 inhibited IL-12 production independently of endogenous IL-10 induction, because anti-IL-10 Abs failed to prevent its effect. The selective A2a antagonist 8-(3-Chlorostyryl) caffeine prevented the inhibitory effect of CGS-21680 on IL-12 production. The phosphodiesterase inhibitor Ro 20-1724 dose-dependently potentiated the inhibitory effect of CGS-21680 and, furthermore, Rp-cAMPS, a protein kinase A inhibitor, reversed the inhibitory effect of CGS-21680, implicating a cAMP/protein kinase A pathway in its action. Thus, ligand activation of A2a receptors simultaneously inhibits IL-12 and stimulates IL-10 production by human monocytes. Through this mechanism, ADO released in excess during inflammatory and ischemic conditions, or tissue injury, may contribute to selective suppression of Th1 responses and cellular immunity.

Interleukin-12 and tumor necrosis factor (TNF)-alpha promote T-helper (Th) 1 responses and cellular immunity, whereas IL-10 suppresses Th1 activities and stimulates Th2 and humoral immune responses. Recent evidence indicates that glucocorticoids, norepinephrine, epinephrine, histamine, and adenosine inhibit the production of human IL-12 and TNF-alpha, whereas they do not affect or even stimulate the production of IL-10. Through this mechanism these neuroendocrine mediators may cause a selective suppression of Th1 responses and a Th2 shift rather than generalized Th suppression. The substantial Th2-driving force of endogenous stress mediators, as well as histamine and adenosine, can be amplified to a great extent during certain conditions and may play a role in increased susceptibility of the organism to various infections that are normally cleared by Th1 responses. In addition, conditions that contribute to a substantial increase or decrease of local or systemic concentrations of these mediators via modulation of IL-12, TNF alpha/IL-10 balance may also play a role in induction, expression, and progression of certain autoimmune diseases, allergic/atopic reactions, and tumor growth. These conditions include: acute or chronic stress; cessation of chronic stress or chronic hypoactivity of the stress system; severe exercise; serious surgical procedures or traumatic injuries; major burns; severe ischemia or hypoxia; pregnancy and the postpartum period. Thus, better understanding of the neuroendocrine regulation of IL-12, TNF-alpha/IL-10 balance might help the development of new therapeutic strategies for the treatment of Th1- and Th2-mediated human diseases.

Recent evidence indicates that glucocorticoids and catecholamines, the end-products of the stress system, and histamine, a product of activated mast cells, might selectively suppress cellular immunity, and favour humoral immune responses. This is mediated by a differential effect of stress hormones and histamine, on T helper 1 (Th1)/Th2 patterns and type 1/type 2-cytokine production. Thus, systemically, stress might induce a Th2 shift, while, locally, under certain conditions, it might induce pro-inflammatory activities through neural activation of the peripheral corticotropin-releasing factor-mast cell-histamine axis. Through the above mechanisms, stress may influence the onset and/or course of infectious, autoimmune/inflammatory, allergic and neoplastic diseases.

In general, stress has been regarded as immunosuppressive. Recent evidence, however, indicates that acute, subacute or chronic stress might suppress cellular immunity but boost humoral immunity. This is mediated by a differential effect of stress hormones, the glucocorticoids and catecholamines, on T helper 1 (Th1)/Th2 cells and type 1/type 2 cytokine production. Furthermore, acute stress might induce pro-inflammatory activities in certain tissues through neural activation of the peripheral corticotropin-releasing hormone-mast cell-histamine axis. Through the above mechanisms, stress might influence the onset and/or course of infectious, autoimmune/inflammatory, allergic and neoplastic diseases.

Rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) frequently develop and progress in settings in which sympathoadrenomedullary and gonadal hormone levels are changing, e.g., during pregnancy, postpartum period, menopause, estrogen administration. This paper addresses the view that adrenal and gonadal hormonal deficiency facilitates excessive macrophage production of TNF-alpha and IL-12 that characterizes RA, whereas excessive estrogen action is suggested to play an essential role in the production of IL-10 in patients with SLE. Disease activity in SLE, in contrast to RA, appears to be associated with high-level production of IL-10, relative to the proinflammatory cytokines, TNF-alpha and IL-12. Accumulating data suggest that novel therapeutic approaches may ultimately be developed from continued investigation of the role of the neuroendocrine factors in RA and SLE.

Corticotropin-releasing hormone (CRH) influences the immune system indirectly, through activation of the hypothalamic-pituitary-adrenal axis and sympathetic system, and directly, through local modulatory actions of peripheral (immune) CRH. We recently demonstrated that catecholamines and histamine potently inhibited interleukin (IL)-12 and stimulated IL-10, whereas glucocorticoids suppressed IL-12, but did not affect IL-10 production ex vivo. Thus, both glucocorticoids and catecholamines, the end products of the stress system, and histamine, a product of activated mast cells, may selectively suppress cellular immunity and favor humoral immune responses. We localized immunoreactive CRH in experimental carrageenin-induced aseptic inflammation and, in humans, in inflamed tissues from patients with several autoimmune disease. In addition, we demonstrated that CRH activated mast cells via a CRH receptor type 1-dependent mechanism, leading to release of histamine and hence vasodilatation and increased vascular permeability. Thus, activation of the stress system, through direct and indirect effects of CRH, may influence the susceptibility of an individual to certain autoimmune, allergic, infectious or neoplastic diseases. Antalarmin, a novel nonpeptide CRH antagonist, prevented several proinflammatory effects of CRH, thus revealing its therapeutic potential in some forms of inflammation.

IL-12 and IL-10, respectively, stimulate Th1 and Th2 immune responses. The development of some allergic reactions, infections, and tumors are associated with excessive histamine production and a shift toward Th2 responses. Here we address the possibility that this association is causally linked, at least in part, to modulation of IL-12 and IL-10 production by histamine. We report that histamine dose-dependently inhibited the secretion of human IL-12 (p70) and increased the production of IL-10 in LPS-stimulated whole blood cultures. These effects of histamine were antagonized by cimetidine, an H2 receptor antagonist, but not by selective H1 and H3 receptor blockers, and were mimicked by an H2 receptor agonist. The effects of histamine on IL-12 and IL-10 secretion were independent of endogenous secretion of IL-10 or exogenous addition of IL-12, while Ro 20-1724, a phosphodiesterase inhibitor, potentiated the effects of histamine on IL-12 and IL-10 production, implicating cAMP in its actions. Similar modulatory effects of histamine on IL-12 and IL-10 production, which were reversed by the H2 antagonist cimetidine, were observed in PBMC and isolated monocytes stimulated by Staphylococcus aureus Cowan strain 1 and LPS, respectively. Thus, histamine, via stimulation of H2 receptors on peripheral monocytes and subsequent elevation of cAMP, suppresses IL-12 and stimulates IL-10 secretion, changes that may result in a shift of Th1/Th2 balance toward Th2-dominance. This may represent a novel mechanism by which excessive secretion of histamine potentiates Th2-mediated allergic reactions and contributes to the development of certain infections and tumors normally eliminated by Th1-dependent immune mechanisms.

Corticotropin-releasing hormone (CRH) is a major regulator of the hypothalamic-pituitary-adrenal axis (HPA) and principal coordinator of the stress response. As in stress, intracerebroventricular administration of CRH suppresses the immune system indirectly, via glucocorticoid and/or sympathetic system-mediated mechanisms. Also, during inflammatory stress, the cytokines TNF alpha, IL-1, and IL-6 stimulate hypothalamic CRH and/or vasopressin secretion as a way of preventing the inflammatory reaction from overreacting. Recently, CRH receptors were described in peripheral sites of the immune system, and CRH was found to promote several immune functions in vitro. We demonstrated a direct role of CRH in the inflammatory immune process in vivo, by first studying the effect of systemic CRH immunoneutralization in an experimental model of carrageenin-induced aseptic inflammation in Spague-Dawley rats. We extended these observations to other forms of experimental inflammation, including streptococcal cell wall polysaccharide- and adjuvant-induced arthritides and peptide R16 (epitope of the interphotoreceptor retinoid-binding protein)-induced uveitis in Lewis rats. We also studied human disease states, including rheumatoid arthritis, Hashimoto thyroiditis, and ulcerative colitis. Inflamed tissues contained large amounts of IR CRH, reaching levels similar to those observed in the hypophyseal portal system. We also demonstrated the presence of CRH mRNA and CRH receptors in inflammatory cells and identified the mast cells as a major immune target for CRH. In addition to production by immune cells, the peripheral nervous system, including the postganglionic sympathetic neurons and the sensory fibers type C, appears to contribute to IR CRH production in inflammatory sites. The production of CRH from the postganglionic sympathetic neurons may be responsible for the stress-induced activation of allergic/autoimmune phenomena, such as asthma and eczema, via mast cell degranulation. Antalarmin, a novel nonpeptide CRH receptor antagonist, displaced 125I-labeled ovine CRH binding in rat pituitary, frontal cortex, and cerebellum, but not heart, consistent with antagonism at the CRHR1 receptor. In vivo antalarmin significantly inhibited CRH-stimulated ACTH release and carrageenin-induced subcutaneous inflammation in rats. Thus, antalarmin and other related compounds that antagonize CRH at the level of its own receptor have therapeutic potential in some forms of inflammation directly mediated by type 1 CRH receptors and promise to enhance our understanding of the many roles of CRH in immune/inflammatory reactions.

Pregnancy and the postpartum period are associated with significant changes in levels of several hormones, such as estrogen, progesterone, cortisol and possibly catecholamines. Moreover, several autoimmune diseases such as rheumatoid arthritis tend to remit, develop or exacerbate during pregnancy or the postpartum period. Thus, the question arises: are the changes in the hormones and the expression of autoimmune diseases during these periods causally linked, or are these associations an epiphenomenon? Here we suggest that a causal link might be provided through differential neuroendocrine regulation of Th1-type and Th2-type cytokine production.

Neuroendocrine-immune interactions are profoundly regulated by corticotropin-releasing hormone (CRH) indirectly, through activation of a global stress response, and directly, through pro-inflammatory actions on peripheral immune functions. The indirect effects of stress on immune/inflammatory responses occur via the stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic/adrenomedullary system. We have demonstrated that glucocorticoids and catecholamines favor T helper 2 (TH2) over T helper 1 (TH1) immune cells and mediators, by controlling the production of specific key regulatory cytokines. This could explain the influences of chronic stress on the development, course, and pathology of certain allergic, autoimmune/inflammatory, infectious, and neoplastic diseases. We have also shown that 'immune CRH' is secreted peripherally and plays a direct immunomodulatory role as an autocrine or paracrine mediator of inflammation. Upon release from immune cells and peripheral sensory afferent and/or postganglionic sympathetic nerves, CRH acts locally to elicit pro-inflammatory responses. This would explain the triggering or exacerbation of certain allergic or vasokinetic states by acute stress.

Morphine treatment modulates a variety of immunological parameters, including tumor necrosis factor-alpha (TNF-alpha) production by activated macrophages in vitro. The aim of our study was to clarify the effect of morphine on lipopolysaccharide (LPS)-induced TNF-alpha production in vivo. Plasma TNF-alpha levels of mice were determined by ELISA. Subcutaneous injection of morphine decreased LPS-induced TNF-alpha production throughout the response, an effect that was dose-dependent and reversible by naloxone. Blockade of the sympathetic transmission by chlorisondamine prevented the inhibitory effect of morphine on TNF-alpha production. It is concluded that (i) systemic administration of morphine inhibits LPS-induced TNF-alpha production in vivo via 'classic' opioid receptors; (ii) this effect requires intact sympathetic outflow. Since the increased incidence of bacterial and viral infections in opioid addicts is well documented, it is suggested that the inhibitory effect of morphine on TNF-alpha production might play a substantial role in the increased vulnerability of these individuals to certain infections.

Unusual fatty acids, with up to 34 carbon atoms and containing one or two bis-methylene-interrupted double-bond systems, have been identified in the sponge Haliclona cinerea from the Black Sea. These include the dienes-5,9-16:2, 7,11-18:2, 9,13-20:2, 13,17-24:2, 15,19-26:2, 17,21-28:2, 19,23-30:2 and 21,25-32:2; trienes-5,9,23-30:3, 5,9,24-31:3, 5,9,25-32:3, and 5,9,27-34:3; and the tetraenes-5,9,19,23-30:4, 5,9,21,25-32:4, and 5,9,23,27-34:4. In addition, 5,9,13-eicosatrienoic acid was present. Many of these do not appear to have been described before, and only 5,9-16:2 and 5,9,23-30:3 are found often in sponges. They were identified by using silver-ion high-performance liquid chromatography to simplify the complex mixture of fatty acids for subsequent analysis by gas chromatography-mass spectrometry as picolinyl ester derivatives. Deuteration with Wilkinson's catalyst in homogeneous solution confirmed the structures. We speculate that the di- and tetraenoic fatty acids arise by chain elongation of 5,9-hexadecadienoic acid, also a major component of the lipids, followed by further insertion of double bonds in the 5 and 9 positions. The trienes may be formed from 9-hexadecenoic acid by similar mechanisms.

Interleukin-12 (IL-12) is a key inducer of differentiation of uncommitted T helper (TH) cells toward the TH1 phenotype, which regulates cellular immunity, whereas IL-10 inhibits TH1 functions and potentiates TH2-regulated responses (i.e., humoral immunity). To examine the potential effects of stress on TH1/TH2 balance, we studied the ability of three prototype stress hormones-dexamethasone (a synthetic glucocorticoid) and the catecholamines norepinephrine and epinephrine-to alter the production of IL-12 (p70) and IL-10 induced by bacterial lipopolysaccharide (LPS) in human whole blood. Dexamethasone inhibited LPS-induced bioactive IL-12 production in a dose-dependent fashion and at physiologically relevant concentrations; it had no effect on IL-10 secretion. The glucocorticoid-induced reduction of IL-12 production was antagonized by RU 486, a glucocorticoid-receptor antagonist, suggesting that it was mediated by the glucocorticoid receptor. Norepinephrine and epinephrine also suppressed IL-12 production in a dose-dependent fashion and at physiological concentrations; both catecholamines, however, dose-dependently increased the production of IL-10. The effects of either catecholamine on IL-12 or IL-10 secretion were blocked completely by propranolol, a beta-adrenoreceptor antagonist, indicating that they were mediated by the beta-adrenergic receptor. These findings suggest that the central nervous system may regulate IL-12 and IL-10 secretion and, hence, TH1/TH2 balance via the peripheral end-effectors of the stress system. Thus, stress may cause a selective suppression of TH1 functions and a shift toward a TH2 cytokine pattern rather than generalized TH suppression. The TH1-to-TH2 shift may be responsible for the stress-induced susceptibility of the organism to certain infections. Through the same or a reciprocal mechanism, states associated with chronic hyperactivity or hypoactivity of the stress system might influence the susceptibility of an individual to certain autoimmune, allergic, infectious, or neoplastic diseases.

Exogenous pyrogens, e.g., bacterial lipopolysaccharides (LPS), are thought to stimulate macrophages to release endogenous pyrogens, e.g., TNF alpha, IL-1 beta, and IL-6, which act in the hypothalamus to produce fever. We studied the effect of different alpha 1- and alpha 2-adrenoceptor subtype antagonists, applied intraperitoneally, on the febrile response induced by LPS in rabbits. Evidence was obtained that prazosin, an alpha 1- and alpha 2B/2C-adrenoceptor antagonist; WB-4101, an alpha 1- and alpha 2A-adrenoceptor antagonist; CH-38083, a highly selective alpha 2-adrenoceptor antagonist (alpha 2:alpha 1 > 2000); BRL-44408, an alpha 2A-adrenoceptor antagonist; and ARC-239, an alpha 2B/2C- and also alpha 1-adrenoceptor antagonist, blocked the increase of colonic temperature of the rabbit produced by 2 micrograms/kg LPS administered intravenously without being able in themselves to affect colonic temperature. In addition, prazosin, WB-4101 and CH-38083 antagonized the fall in skin temperature that occurred at the time when the colonic temperature was rising in control animals injected with LPS. All these results suggest that norepinephrine, through stimulation of both alpha 1- and alpha 2- (alpha 2A- and alpha 2B/2C-) adrenoceptor subtypes, is involved in producing fever in response to bacterial LPS.

The object of these experiments was to investigate whether noradrenaline is the signal neurotransmitter between the sympathetic nervous system and rat thymocytes. Using immunocytochemistry, evidence was obtained that the rat thymus (thymic capsule, subcapsular region and connective tissue septa) is innervated by noradrenergic varicose axons terminals (tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunostained nerve fibres). This innervation is mainly associated with the vasculature and separately from vessels along the thymic tissue septa it branches into the thymic parenchyma. Using electron microscopy, classical synapses between thymocytes and neuronal elements were not observed. The neurochemical study revealed that these nerve terminals are able to take up, store and release noradrenaline upon axonal stimulation in a [Ca2+]o-dependent manner. The release was tetrodotoxin (1 microM)-sensitive, and reserpine pretreatment prevented axonal stimulation to release noradrenaline, indicating vesicular origin of noradrenaline. In addition, it was found that the release of noradrenaline was subjected to negative feedback modulation via presynaptic alpha 2-adrenoreceptors. Using a patch-clamp technique, electrophysiological evidence was obtained showing that noradrenaline inhibits in a concentration-dependent manner outward voltage-dependent potassium (k+) current recorded from isolated thymocytes. Since noradrenergic varicose axon terminals enter the parenchyma thymocytes and the boutons are not in close apposition to their target cells, noradrenaline released from these terminals diffuses away from release site to reach its targets, thymocytes, and to exert its inhibitory effect on voltage-dependent K+ -current. Since K+ channels are believed to be involved in T cell proliferation and differentiation, the modulation of K+ channel gating by noradrenaline released in response to axonal activity suggests that signals from blood-born or locally released hormones and cytokines. In this respect, noradrenaline released from non-synaptic neuronal varicosities and exerting its effect within the radius of diffusion may serve as a chemical link between the sympathetic nervous system and thymocytes and may have physiological and pathological importance in the thymus during stress and inflammatory/immune responses.

In a previous study we demonstrated that mice pretreated with the highly selective alpha 2-adrenoceptor antagonist CH-38083 showed blunting of the tumor necrosis factor-alpha (TNF-alpha) response induced by bacterial lipopolysaccharide (LPS). In the present study, the effect of a selective block of alpha 2-adrenoreceptors and the role of the sympathetic nervous system (SNS) in the regulation of LPS-induced TNF-alpha production was explored further using different selective adrenoceptor antagonists and agonists. While adrenalectomy did not prevent the effect of CH-38083, the block of the sympathetic transmission by chlorisondamine fully abolished the inhibitory effect of CH-38083 on LPS-induced TNF-alpha production, suggesting that the effect of the alpha 2-adrenoceptor blocking agent is corticosteroid-independent, but it requires intact sympathetic activity. Since the selective block of alpha 2-adrenoceptors results in an increased sympathetic activity and an increase of the release of noradrenaline (NA) in both the central and the peripheral nervous systems, and in our experiments propranolol, a non-selective beta-adrenoceptor antagonist, and atenolol, a selective antagonist of beta 1-adrenoceptors, prevented the effect of alpha 2-adrenoceptor blockade by CH-38083 of the TNF-alpha response induced by LPS, it seems likely that the excessive stimulation by NA of beta 1-adrenoceptors is responsible for this action. The role of beta-adrenoceptors and endogenous catecholamines is further substantiated by the finding that pretreatment of animals with propranolol alone resulted in a dose-dependent increase of the TNF-alpha response induced by LPS, and that isoproterenol, a non-selective beta-adrenoceptor agonist, decreased it.(ABSTRACT TRUNCATED AT 250 WORDS)

In a previous study we have shown that, in response to electrical stimulation, there is a substantial release of noradrenaline (NA) from the sympathetic nerve terminals of the rat thymus which is of axonal, vesicular origin. In the present study neurochemical evidence was obtained that the release of NA is subject to presynaptic modulation. This modulation operates through stimulation of alpha 2B-adrenoreceptors, N-nicotinic, P1-purinergic and prostaglandin E2 presynaptic receptors. Through these receptors the release of NA, i.e., the message from the central nervous system to the thymus, can be affected by endogenous ligands or drugs. A novel, potent and highly selective competitive antagonist of the alpha 2-adrenoreceptor, CH-38083, significantly enhanced the release of NA, suggesting that its release in the thymus is under tonic inhibitory control exerted by endogenously released NA. Since adrenoreceptors on thymocytes involved in the modulation of certain thymocyte functions have recently been described, it is suggested that the presynaptic modulation of the release of NA in the thymus is involved in neuro-immune communication.

The effect of selective block of alpha 2-adrenoreceptors on plasma levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and corticosterone induced by bacterial lipopolysaccharide (LPS) was investigated in mice using ELISA and RIA. It was found that the LPS-induced TNF-alpha response was significantly blunted in mice pretreated with CH-38083, a novel and highly selective alpha 2-adrenoreceptor antagonist (the alpha 2/alpha 1 ratio is > 2000). In contrast, LPS-induced increases in both corticosterone and IL-6 plasma levels were further increased by CH-38083. Since it has recently been shown that the selective block of alpha 2-adrenoreceptors located on noradrenergic axon terminals resulted in an increase in the release of noradrenaline (NA), both in the central and peripheral nervous systems, and, in our experiments, that propranolol prevented the effect of alpha 2-adrenoreceptor blockade on TNF-alpha plasma levels induced by LPS, it seems likely that the excessive stimulation by NA of beta-adrenoreceptors located on cytokine-secreting immune cells is responsible for this action. Since it is generally accepted that increased production of TNF-alpha is involved in the pathogenesis of inflammation and endotoxin shock on the one hand, and corticosterone and even IL-6 are known to possess anti-inflammatory properties on the other hand, it is suggested that the selective block of alpha 2-adrenoreceptors might be beneficial in the treatment of inflammation and/or endotoxin shock.

The effect of tumor necrosis factor-alpha (TNF-alpha) on the stimulation-evoked release of noradrenaline (NA) from isolated rat median eminence (ME) was investigated, using a low-volume perfusion system. Median eminence, loaded with [3H]noradrenaline, was superfused with Krebs solution and stimulated electrically (2 Hz, 120 shocks). The effect of TNF-alpha was studied on the S2/S1 ratio. It was found that stimulation-evoked release of NA from noradrenergic axon terminals in the isolated rat ME was inhibited by TNF-alpha and this effect was concentration-dependent. In contrast, TNF-alpha had no effect on the release of [3H]NA from the spleen. Since NA released in the ME might be involved in the modulation of corticotropin-releasing factor (CRF) production, it is suggested that TNF-alpha, through presynaptic modulation of NA release from noradrenergic nerve terminals in the ME, might regulate CRF and other neurohormone release in this hypothalamic structure.

Stimulation of the immune system or experimental conditions (bacterial lipopolysaccharide (LPS) treatment) provoke a broad spectrum of physiological responses. It was recently shown that one of them is the activation of the hypothalamic-pituitary-adrenal (HPA) axis. The mechanism and the site or sites through which LPS stimulates the HPA axis are not well understood. To establish whether the effect of bacterial LPS is related in vivo to the presence of hypothalamic hypophysiotrophic peptides (corticotrophin-releasing factor-41, arginine vasopressin, etc.), plasma ACTH and corticosterone levels were monitored in intact and sham-operated rats, and in rats with paraventricular nucleus lesions in order to remove the main source of these neuropeptides. Evidence was obtained that 4 h after treatment, LPS was able to activate the hypophysial-adrenal system in the absence of hypophysiotrophic neuropeptides of paraventricular origin. It is suggested that, in vivo, LPS could have a direct effect on the pituitary gland or that it acts through an extrapituitary, non-paraventricular pathway to activate the HPA axis.

The immune system and the hypothalamic-pituitary-adrenal (HPA) axis play important role in the overall inflammatory response. The mechanism through which lipopolysaccharide (LPS, endotoxin) stimulates the HPA axis is not well understood. In order to clarify the role of hypophysiotropic peptides of paraventricular origin in the effect of LPS on ACTH and corticosterone secretion, the effect of LPS was studied on rats with lesions of hypothalamic paraventricular nucleus (PVN). It was shown that 90 min after 2 mg/kg LPS i.p. the ACTH, but not the corticosterone response was effectively blunted in PVN-lesioned rats, as compared to sham operated animals. However, in PVN-lesioned rats 240 min after treatment with LPS a significantly higher plasma ACTH and corticosterone level was monitored. It is, therefore, suggested that in response to LPS activation of HPA both CRF(s)-dependent and CRF(s)-independent mechanisms are involved, even a direct effect of the adrenal cortex should be taken into account.

Strips of rat spleen, loaded with [3H]NA, were superfused with Krebs' stimulated electrically at different frequencies (2, 8 and 32 Hz) with the same number of pulses and the effect of different receptor agonists and antagonists was studied on the S2/S1 ratio. Evidence has been obtained that the sympathetic nerve terminals in the spleen of the rat are equipped with presynaptic alpha 2B-adrenoreceptors, M-muscarinic, N-nicotinic and P1-purinoreceptors. Clonidine, an alpha 2-adrenoreceptor agonist, did not change the release of NA at low frequency but increased it at a high frequency of stimulation. It is suggested that clonidine is a partial agonist and reveals its antagonistic property when the biophase concentration of the full agonist, NA, released endogenously large. A novel, potent and highly selective competitive antagonist of the alpha 2-adrenoreceptor, CH-38083, enhanced significantly the release of NA, removing presynaptic negative feedback control of NA. It is suggested that the release of NA is subject to presynaptic modulation, through different presynaptic receptors and that it is involved in the communication between the central nervous system and a secondary lymphoid organ, the spleen.