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The development and function of the immune system is regulated by neuroendocrine factors. Immune function may be divided into adaptive and natural immunity. Adaptive immune responses are driven by specific determinants of the antigen (epitopes), require 5-10 d to fully develop, and show an accelerated or memory response after repeated exposure to the same antigen. Natural immunity may be divided into host defense mediated by non-immune factors (e.g., antimicrobial proteins, enzymes, mucus etc.) and polyspecific responses of the immune system. This polyspecific response relies on natural antibodies and on some other serum proteins (e.g., lipopolysaccharide-binding protein-LBP, C-reactive protein-CRP), and on surface receptors of macrophages, natural killer cells and B and T lymphocytes for activation. Highly conserved homologous (crossreactive) epitopes, or homotopes for short, are recognized by the natural immune system. Natural antibodies, LBP, and CRP are capable of activating the entire immune system after combination with the appropriate homotope. During febrile illness natural immune host defense is promptly elevated because of the rapid rise of natural antibodies, LBP, and CRP in the serum. This is known as the acute phase response (APR), which is initiated by a sudden rise of cytokines in the circulation, such as IL-1, IL-6, and TNF-alpha. The cytokines act on the brain, the neuroendocrine system, and on other tissues and organs, which leads to fever and profound hormonal and metabolic changes. The hypothalamus-pituitary adrenal axis is activated and serves as the primary regulator of immune and inflammatory reactions. Insulin, glucagon, and catecholeamine levels are also raised. Bone marrow activity and leukocyte function are high and the liver is converted to the rapid production of acute-phase proteins (APP). APP include LBP, CRP, fibrinogen, some complement components, enzyme inhibitors, and anti-inflammatory proteins, which may rise in the serum from several hundred to a thousand times within 24-48 hr. Therefore, natural immunity is a polyspecific response to homotopes, which functions as an instantaneous defense mechanism in health and which is rapidly boosted by cytokines and hormones during febrile illness. This is a highly successful defense reaction, as in the overwhelming majority of cases, febrile illness leads to recovery and the development of adaptive immunity in man and higher animals.

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A protein of 40 kD molecular weight was isolated from the salivary submandibular glands of male rats. The protein catalyzed the hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester. This esterase activity was inhibitable with the protease inhibitor aprotinin. The sequence of the first 25 amino acids of this protein was identical to that of rat glandular kallikrein (rGK). When added to cultures of murine lymph node cells suboptimally stimulated with the T cell mitogen concanavalin A, rGK markedly stimulated the proliferative activity of these cells. When injected into mice, rGK suppressed the contact sensitivity response to picryl chloride, a form of delayed-type hypersensitivity. Similar in vitro and in vivo effects were induced with GK from porcine pancreas (pGK). Moreover, the aforementioned in vitro and in vivo effects were abolished by aprotinin either added to the tissue culture medium or injected into the animals immediately before rGK or pGK. This demonstrates that the enzymatic activity of rGK and pGK is important for the induction of immunoregulatory effects. These results suggest that rGK is a systemic immunoregulatory enzyme with immunosuppressive potential. GK is the first example for systemic immunoregulation by an enzyme, the secretion of which is under neuroendocrine control.

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In the second part of their article on the emerging field of neuroimmunology, the authors present an overview of the role of neuroimmune mechanisms in defence against infectious diseases and in immune disorders. During acute febrile illness, immune-derived cytokines initiate an acute phase response, which is characterized by fever, inactivity, fatigue, anorexia and catabolism. Profound neuroendocrine and metabolic changes take place: acute phase proteins are produced in the liver, bone marrow function and the metabolic activity of leukocytes are greatly increased, and specific immune reactivity is suppressed. Defects in regulatory processes, which are fundamental to immune disorders and inflammatory diseases, may lie in the immune system, the neuro endocrine system or both. Defects in the hypothalamus-pituitary-adrenal axis have been observed in autoimmune and rheumatic diseases, chronic inflammatory disease, chronic fatigue syndrome and fibromyalgia. Prolactin levels are often elevated in patients with systemic lupus erythematosus and other autoimmune diseases, whereas the bioactivity of prolactin is decreased in patients with rheumatoid arthritis. Levels of sex hormones and thyroid hormone are decreased during severe inflammatory disease. Defective neural regulation of inflammation likely plays a pathogenic role in allergy and asthma, in the symmetrical form of rheumatoid arthritis and in gastrointestinal inflammatory disease. A better understanding of neuroimmunoregulation holds the promise of new approaches to the treatment of immune and inflammatory diseases with the use of hormones, neurotransmitters, neuropeptides and drugs that modulate these newly recognized immune regulators.

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A novel scientific discipline that examines the complex interdependence of the neural, endocrine and immune systems in health and disease has emerged in recent years. In health, the neuroimmunoregulatory network is fundamental to host defence and to the transfer of immunity to offspring; the network also plays important roles in intestinal physiology and in tissue regeneration, healing and reproduction. The proliferation of lymphocytes in primary lymphoid organs (bone marrow, bursa of Fabricius [in birds] and thymus) and in secondary lymphoid organs (spleen, lymph nodes and mucosal lymphoid tissue) depends on prolactin and growth hormone. These hormones allow immune cells to respond to antigen and to soluble mediators, called cytokines. Immune-derived cytokines are capable of inducing fever and of altering neuro-transmitter activity in the brain and hormone secretion by the pituitary gland. The activation of the hypothalamus-pituitary-adrenal axis by cytokines leads to immunosuppression. Lymphoid organs are innervated, and tissue mast cells respond to neurologic stimuli. In general, acetylcholine and substance P exert immunostimulatory and proinflammatory effects, whereas epinephrine and somatostatin are immunosuppressive and anti-inflammatory. In this article, the authors predict that novel approaches to immunomodulation will be possible by altering the level or efficacy of immunoregulatory hormones and neurotransmitters.

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The evidence for the integration of the submandibular gland (SMG) into the neuroimmunoregulatory network has been reviewed. In laboratory rodents, factors extracted from the SMG were shown to stimulate lymphocyte proliferation, to affect the weight of the thymus, spleen and lymph nodes and to induce immunosuppression in several in vivo animal models. The SMG produces significant quantities of nerve growth factor (NGF), epidermal growth factor (EGF), transforming growth factor-beta and kallikreins, which are secreted into the saliva and affect immune and mucosal tissues and nerve endings in the gastrointestinal tract. These factors play a role in regulating mucosal immuno/inflammatory response and in regeneration and healing. The major salivary glands also produce antimicrobial proteins and secretory IgA antibodies which are essential factors in mucosal host defense. SMG-derived NGF, EGF and glandular kallikrein are delivered into the bloodstream where they may act as important systemic immunoregulators and also have major regulatory influences on the central neuroendocrine system. There is evidence to indicate that EGF is involved in the regulation of gonadal function. Growth hormone, prolactin, androgens, thyroid hormone and corticosteroids regulate protein synthesis in the SMG, whereas secretory activity is regulated by sympathetic (alpha- and beta-adrenergic) parasympathetic (muscarinic) and peptidergic (substance P and vasoactive intestinal peptide) nerve fibers. Fluid and electrolyte secretion is promoted by parasympathetic, whereas protein secretion is stimulated by sympathetic nerve impulses. Steroid hormones and cytokines (interleukin-1 alpha, -beta, tumor necrosis factor, interferon-gamma) have a major regulatory influence on protein secretion, including the secretion of immunoglobulin into the saliva. The SMG interacts with the mucosal and systemic compartments of the immune system, with the central and peripheral nervous systems, with the pituitary gland, and with peripheral endocrine organs. These interactions enable the SMG to exert regulatory influences on immune/inflammatory reactions in the gastrointestinal tract, in the lungs, and possibly elsewhere. It is suggested that these functions make this gland a key regulatory organ in the neuroimmunoregulatory network. Evidence is increasing that the major salivary glands fulfill similar functions in other species, including humans.

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Extracts of the submandibular gland (SMG) of rats contain fractions that stimulate the in vitro proliferation of Con A-treated lymphocytes. One of the stimulatory fractions was also shown to induce in vivo immunosuppression in rats and mice in several experimental models. Since many other biologically active factors of the SMG had been found to be hormone dependent, we investigated the effects on the immunosuppressive factor of hypophysectomy (Hx) and of hormonal reconstitution in male Fischer rats. Hx induced a marked atrophy of the SMG together with an almost complete disappearance of both the in vitro lymphocyte-stimulating activity and the in vivo immunosuppressive activity, the latter assayed with the contact sensitivity reaction in mice. The treatment of the Hx rats with pituitary hormones demonstrated that prolactin (PRL), thyroid stimulating hormone (TSH), and luteinizing hormone (LH) induced a significant reconstitution of these biological activities, growth hormone led to the recovery of the lymphocyte-stimulating activity but not of the immunosuppressive activity, while follicle-stimulating hormone, and adrenocorticotropic hormone did not induce any recovery of these biological activities. In view of the positive results obtained with TSH and LH further experiments were done to compare the effects of thyroid and sex hormones with those of PRL. The results demonstrated that testosterone and thyroid hormones induced significant recovery of the lymphocyte-stimulating and the immunosuppressive activity. The combination of these two hormones with PRL produced the most effective results. On the other hand, estrogens and progesterone had no significant effects. These results confirm the effectiveness of androgens and thyroid hormones in stimulating the production of biologically active factors by the SMG. Moreover, they demonstrate that PRL, a hormone not previously considered to increase the activity of the SMG, stimulates the production of immunoregulatory factors in Hx animals.

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It had previously been suggested that the submandibular gland (SMG) of mice and rats may contain in vivo immunosuppressive factor(s). To identify such factor(s), we used a multi-step purification procedure of rat SMG extracts. Gel filtration chromatography of the SMG crude extract resulted in two pools of fractions with significant effects on lymphocyte reactivity in the in vitro concanavalin A (Con A) bioassay. Of these two pools, only the one with lower molecular weight resulted in the prolongation of murine skin allograft survival, the suppression of the delayed-type hypersensitivity (DTH) response to picryl chloride and the decrease in number of direct (IgM) plaque-forming cells against sheep red blood cells. Fractionation of this low molecular weight (LMW) pool through hydrophobic interaction chromatography resulted in three protein fractions designated A, B and C. Of these fractions only fraction A produced significant suppression of the DTH response. Further purification of fraction A with anion exchange chromatography produced two fractions with immunosuppressive activity in the DTH response. One fraction demonstrated on SDS-PAGE a single component of 40,000 MW, while the other had two components of 30,000 and 40,000 MW respectively.

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This study was undertaken to determine whether early pregnancy factor secreted by preimplantation embryos has immunosuppressive properties. Human early pregnancy factor was purified from embryo growth media of in vitro fertilized ova with ion-exchange and gel filtration chromatography. During each step of purification the fractions were tested for (1) early pregnancy factor activity with the rosette inhibition assay, (2) immunosuppressive properties with a concanavalin A-stimulated lymphocyte proliferation assay, and (3) purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicate that (1) human early pregnancy factor has a basic molecular weight of 14 kd, (2) early pregnancy factor has immunosuppressive activity, (3) polymers of early pregnancy factor also appear to be present in the embryo growth media, and (4) immunosuppressive factors other than early pregnancy factor are also secreted by preimplantation human embryos. Early pregnancy factor and other factor(s) produced by the preimplantation embryo may play a role in suppressing maternal cellular immune responses, thereby preventing maternal rejection of the embryo.

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With the sequential use of ammonium sulfate precipitation, gel filtration and chromatofocusing, we have partially purified from extracts of the submandibular glands of rats a factor (referred to as submandibular gland's immunosuppressive factor or SMG-ISF) capable of inhibiting the in vitro proliferation of mitogen- and antigen-stimulated murine lymphocytes. The semi-purified suppressor fractions had an isoelectric point of 4.4 to 4.5 and consisted of at least three molecular species. These active fractions suppressed the mitogenic effects of Concanavalin A phytohemagglutinin, and lipopolysaccharide. In vitro immune reactions such as the mixed lymphocyte culture MLC reaction and the production of cytotoxic T lymphocytes (CTL) across major histocompatibility barriers in mice were also suppressed. These in vitro immunosuppressive effects required the addition of the suppressor fractions early after the initiation of the cultures and were reversed if the factor was removed from the cultures at least 48 to 72 hr before the completion of the assays. The active fractions did not affect the proliferation of CTLL 2 cells induced by interleukin 2 (IL 2), but inhibited the mitogenic and co-stimulatory effects of IL 1 on mouse thymocytes, and in this effect showed a dose-response relation suggestive of a competitive mechanism. These characteristics of SMG-ISF indicate a specific inhibition of the activity of IL 1.

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The injection of BCG vaccine in C57BL/6J mice results in the suppression of the generation of cytotoxic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC) and of mitogenic reactions to concanavalin A (Con A). Suppression is mediated by macrophage-like suppressor cells. Since previous work had indicated that suppression involved the inhibition of the production of interleukin-2 (IL-2), the effects of BCG on interleukin-1 (IL-1), a monokine required for IL-2 production, were investigated. It was found that the release of IL-1-like activity in spleen cell cultures stimulated with LPS or Con A was increased by previous BCG treatment of the cell donors. In MLC, the release of IL-1-like activity was also increased by BCG. However, the detection of IL-1-like activity in MLC supernatants was prevented by the presence of a suppressor factor. In this case, the IL-1-like activity could be separated with gel filtration from the suppressor factor which had higher molecular weight. The production of IL-1-like activity by CBA/J spleen cells, which are not suppressed by BCG, was not significantly different from that of C57BL/6J cells, which are markedly suppressed. Moreover, the addition of IL-1 to the BCG-suppressed cultures not only did not restore normal reactivity, but actually further suppressed CTL formation. It was concluded that BCG-induced suppression cannot be attributed to decreased IL-1 activity. The suppressor factor discovered during these investigations may have a role in this type of suppression.

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The addition of crude extracts from rat submandibular (SM) glands to murine spleen and lymph node cell cultures stimulated with concanavalin A (Con A) induced either suppression (at high concentrations) or further stimulation (at lower concentrations) or further stimulation (at lower concentrations) of proliferative activity. Gel filtration of the extracts revealed that suppressive activity was due to factors of molecular weight in the 50,000-96,000 range, while stimulation was due to factors in the 13,000-35,000 molecular weight range. The suppressor activity of the higher molecular weight fractions was not due to a reduction of cell viability or of the uptake of tritiated thymidine. This was demonstrated by the fact that the addition of IL-2 to the cultures completely reversed the suppressive effect. Further fractionation of the suppressive and of the stimulatory gel filtration fractions with the chromatofocusing technique led to the identification of a single fraction with suppressor activity and of multiple discrete fractions with stimulatory activity.

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Donor mice were treated IV with BCG and after various time intervals the spleens from these animals were injected into syngeneic recipients which were simultaneously challenged with an allogeneic tumour. The spleen cells from the BCG-treated donors, but not untreated donors, conferred on the recipients an ability to induce a potentiated CMC reaction against the tumour. The transference of BCG-induced potentiating activity could not be explained by the transference of viable BCG organisms, but was mediated by a cell that was anti-Thy.1-sensitive, silica-resistant, plastic-nonadherent, and nylon wool-adherent, and was sensitive in vivo to anti-thymocyte serum but resistant to hydrocortisone. By the use of congenic strains of mice that differed at the Thy.1 allele, it was shown that the cells responsible were not precursors of the cytotoxic lymphocytes but were cells that produced an amplification of the response of the recipient host's precursor cytotoxic T cells.

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The interexperimental variation often observed following the repeated monitoring of immune phenomenon was analysed using the alloimmune cellular response that developed in the spleens of B6AF mice following a challenge with Mastocytoma P815-X2 and intervention with BCG. The response, mediated solely by T cells, could be defined in terms of the involvement of nylon-wool adherent and nonadherent T cells. The relative contribution of each cell type to the response was found to vary considerably and could be use to define the interexperimental variation. Hence when a given treatment resulted in a poor response, the major contributors were nonadherent to nylon-wool, while when the same treatment yielded a good response, adherent cells became the predominant contributors. It was concluded that the inter-experiment variations observed here were a biological event, rather than an experimental artefact, being a function of the relative contribution of different T-cell subpopulations.

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The study was undertaken of the ability of Bacillus Calmette-Guérin (BCG), used in combination with a live tumor cell vaccine, to potentiate systemic antitumor immunity in rats. BCG was administered in two injections, with the first one given i.v. and the second one given intradermally mixed with the tumor cell vaccine 12 days later at the time of implant of an s.c. tumor, which served to monitor antitumor immunity. Both these BCG injections were necessary to obtain maximal protection measured in terms of increased survival rates of the rats and of decreased tumor growth rates. The dose of BCG in both the first and second injections was critical for optimal protection, since low doses (125 to 250 micrograms) afforded protection, while higher doses (over 1500 micrograms) decreased or abolished the protective effect. These results strongly suggest that BCG, only if administered under the appropriate conditions, is able to potentiate systemic tumor immunity and to provide a significant level of protection for a tumor-bearing animal.

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The effect of BCG on the development of cell-mediated cytotoxicity (CMC) in the spleens of allogeneic tumor-bearing female (C57BL/6Jfemale x A/Jmale)F1 mice was studied. BCG-treated tumor-bearing animals elicited a stronger CMC response than did the untreated tumor-bearing animals. Furthermore, the degree of activation was dependent on the time, route of injection, and dose of BCG. Therefore, if the BCG was administered at suboptimal conditions, a less marked stimulation of the CMC resulted. A combination of a high BCG dose given iv 12 days before implant of P815 tumor cells and a low, second dose given at the time of implant produced the most marked activation of the CMC response. By the use of in vitro technique, the specific CMC was mediated mostly by T-cells, with macrophages making a less significant contribution.

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The effectiveness of selected BCG regimens to produce an activation of cell-mediated cytotoxicity (CMC) in an allogeneic tumor system was compared with its ability to cause tumor regression in syngeneic tumor systems. In the tumor system selected in both inbred LEW rats and inbred C57BL/6J and (C57BL/6Jfemale x A/Jmale)F1 mice, a correlation was observed in that BCG treatments that caused marked CMC activation in the allogeneic tumor systems also effectively caused tumor regression and increased animal survival in syngeneic systems. It was concluded that the allogeneic CMC reaction can be used to predict the capacity of BCG to cause tumor rejection.

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Lethally irradiated F1 hybrid mice were given an i.v. injection of parental strain spleen cells. Six days later, their spleen cells were used as the effector cells to measure the in vitro cell-mediated cytotoxicity (CMC) of the parental cells. The treatment of the donors with hydrocortisone resulted in a marked decrease of the capacity of their spleen cells to produce a CMC reaction, whereas the treatment with antithymocyte serum (ATS) resulted in an almost complete loss of such activity. The mixing of spleen cells from hydrocortisone-treated parental donors with the spleen cells from ATS-treated parental donors before injection resulted in a synergistic amplification of the cytotoxic response. The anti-Thy-1 serum treatment of either spleen cell population abolished the synergism completely. These results indicate that cortico-resistant T cells act as precursors of cytotoxic lymphocytes and that ATS-resistant T cells produce an amplification of their reaction.

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The effect of BCG on the development of cell-mediated cytotoxicity (CMC) in the spleens of allogeneic tumor-bearing (C57BL/6 X A)F1 mice was studied. BCG-treated tumor-bearing animals elicited a stronger CMC response than did the untreated tumor-bearing animals. Furthermore, the degree of activation was dependent on different doses of BCG, so that if the BCG was administered at doses above or below the optimum dose, a less marked stimulation of the CMC resulted. The combination of BCG given before the tumor implant plus an intratumor inoculation resulted in a significantly higher CMC than did either treatment alone.

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