This study examines the effects of repeated amitriptyline and desipramine administration (10 mg/kg, IP) on the immunoreactivity of saline-injected C57BL/6 mice, as evaluated by the ability of splenocytes to reduce a tetrazolium salt to formazan (MTT test), to proliferate, and to produce cytokines, such as interleukin (IL)–1, IL–2, IL–4, IL–6, IL–10 and interferon gamma (IFN–γ). Desipramine and amitriptyline administered for one or two weeks enhance the biochemical (estimated by MTT test) and proliferative activities of splenocytes. One and two weeks administration of desipramine significantly reduces the secretion of IL–4, an anti-inflammatory cytokine. Amitriptyline administration for four weeks stimulates the proliferative activity of splenocytes and enhances IL–2 bioactivity, whereas four weeks desipramine aministration does not change these parameters in comparison to saline treated control mice. Prolonged desipramine administration (seven and 28 days) significantly increased the bioactivity of IL–1. Four weeks of prolonged administration of amitriptyline and desipramine induces a significant increase in the secretion of IL–10, a cytokine with immunosuppressive and anti-inflammatory activities. The results show that the immunoregulatory effects of tricyclic antidepressants in C57BL/6 mice depend on the drugs used and on the duration of administration.

Objective: In this study, we have evaluated the effects of stress on functional and proteomic changes in submandibular saliva of rats.Design: Male adult rats were divided in three groups: IMO (2 h/day of immobilization for 7 days), LL (constant light during 20 days), C (unstressed controls submitted to 14 h light-10 h dark cycle). Body weight, food intake and the dry weight of submandibular gland were recorded. Saliva samples, collected under anaesthesia following i.p. administration of isoproterenol and pilocarpine (5 mg/kg), were assayed for total proteins (TP), amylase activity and SDS-PAGE electrophoresis.Results: Body weight, food intake and the thy weight of submandibular gland of IMO rats were lower than those of C and LL groups. The salivary volumes secreted in IMO and LL rats, were significantly higher than in controls. The TP output (mu g protein/mu g saliva/mg of dry tissue) and amylase activity output (Au/mu g of saliva/mg of dry tissue) in IMO were significantly higher than in C and LL animals. The electrophoretic pattern of saliva proteins of LL rats, revealed the absence of a protein band of approximately 25 kDa. This band was composed by the common salivary protein-1 and a prolactin-induced protein as identified by peptide mass fingerprinting.Conclusions: Differences in body weight and food intake between IMO and LL might be attributed to the sort and intensity of stressors stimuli. The changes in the volume of secreted saliva could be a compensatory mechanism in response to stressors. The increase of total protein in IMO rats and the absence of 25 kDa proteins in LL, would suggest that the submandibular glands respond to the sympathetic nervous system stimuli induced by the stress with an increase of activity of the sympathetic nerves in IMO and a reduction in LL rats. (C) 2011 Elsevier Ltd. All rights reserved.

The tripeptide FEG (Phe-Glu-Gly), derived from the c-terminus of the salivary gland peptide-T (SGP-T), TDFEGG, and its D-isomer feG are potent anti-inflammatory peptides that reduce type1 immediate hypersensitivity reactions such as vascular leak, intestinal motility disturbances, systemic hypotension, airways constriction and hyper-reactivity, and pulmonary inflammation. They also inhibit LPS-induced shock, cerulein-induced pancreatitis, zymosan-induced arthritis and other acute inflammatory responses. These peptides work by inhibiting neutrophil rolling, chemotaxis and intracellular superoxide production. Using feG and a series of substituted analogues, we have attempted to gain some insight into the structural features of the molecule essential for biological activity.

The biological effects of cannabinoids (CB) are mediated by CB 1 and CB 2 receptors. The role of CB 2 receptors in the gastrointestinal tract is uncertain. In this study, we examined whether CB 2 receptor activation is involved in the regulation of gastrointestinal transit in rats. Basal and lipopolysaccharide (LPS)‐stimulated gastrointestinal transit was measured after instillation of an Evans blue‐gum Arabic suspension into the stomach, in the presence of specific CB 1 and CB 2 agonists and antagonists, or after treatment with inhibitors of mediators implicated in the transit process. In control rats a CB 1 (ACEA; 1 mg kg −1 ), but not a CB 2 (JWH‐133; 1 mg kg −1 ), receptor agonist inhibited basal gastrointestinal transit. The effects of the CB 1 agonist were reversed by the CB 1 antagonist AM‐251, which alone increased basal transit. LPS treatment increased gastrointestinal transit. This increased transit was reduced to control values by the CB 2 , but not the CB 1 , agonist. This inhibition by the CB 2 agonist was dose dependent and prevented by a selective CB 2 antagonist (AM‐630; 1 mg kg −1 ). By evaluating the inhibition of LPS‐enhanced gastrointestinal transit by different antagonists, the effects of the CB 2 agonist (JWH‐133; 1 mg kg −1 ) were found to act via cyclooxygenase, and to act independently of inducible nitric oxide synthase (NOS) and platelet‐activating factor. Interleukin‐1 β and constitutive NOS isoforms may be involved in the accelerated LPS transit. The activation of CB 2 receptors in response to LPS is a mechanism for the re‐establishment of normal gastrointestinal transit after an inflammatory stimulus. British Journal of Pharmacology (2004) 142 , 1247–1254. doi: 10.1038/sj.bjp.0705889

The anti-inflammatory tripeptide feG was tested in the conscious allergic sheep asthma model for its effects on antigen-induced early (0-3h) and late (5-8h) increases in specific lung resistance (SRL; bronchoconstriction) and airway hyperresponsiveness (AHR; 24h). Intravenous feG (1 mg/kg) had no effect on the early response but reduced late-phase increase in SRL by 73 % and restored AHR to 90 % of the control response. Orally administered feG (2 mg/kg), given twice, 24h and 30 min before antigen challenge, was also ineffective on the early response but reduced late increases in SRL by 78 % and re-established AHR to 95 % of the control response. Inhaled feG (30 mg/sheep) reduced both early- and late- increases in SRL by 83 % and 88 %, respectively, and AHR was restored to normal (90 % of control). Airway administration of feG offers the advantage of inhibiting the early response, possibly by acting on a target that is not available with intravenous and oral administration of the peptide. The ability of feG to block the late response and AHR, when given by three different routes of administration, to sheep suggests that this peptide may be an effective anti-asthmatic agent.

Interactions between the neuro-endocrine system and immune system help maintain health. One interaction involves the superior cervical ganglia (SCG), which regulate the prohormone submandibular rat 1 (SMR1) produced by the submandibular gland (SMG). A peptide derived from SMR1, feG, has anti-inflammatory activity, and modification to D-isomer feG enhances bioactivity. We tested feG as a therapeutic agent for airways inflammation, using rats sensitized by OVA or Nippostrongylus brasiliensis (Nb). Treatment with feG but not fdG down-regulated OVA-challenge-induced increases in bronchoalveolar lavage (BAL)-derived macrophages, eosinophils and PMN (neutrophils) by 44%, 69% and 67%, respectively, at 24 h. We found that feG also reduced ICAM-1 on BAL-derived macrophages and eosinophils by 27% and 65%, and L-selectin on PMN by 55% following OVA challenge. Furthermore, feG but not fdG reduced the OVA-induced TNF increase in BAL fluid. We showed that feG also down-regulated both hyper-responsiveness to methacholine (by 27%) and microgranulomata formation in the lung parenchyma. In Nb-challenged rats, feG treatment inhibited ex vivo allergen-induced contraction of tracheal smooth muscle by up to 73%. In conclusion, feG, which is a mimetic of a peptide derived from a rat salivary gland prohormone, has anti-inflammatory properties in allergic airways inflammation in Brown-Norway rats. The role of the SCG-SMG neuro-endocrine pathway in allergic asthma and other inflammatory diseases requires additional study.

A D-enantiomeric analog of the submandibular gland rat-1 tripeptide FEG (Seq: NH3+-Phe-Glu-Gly-COO-) called feG (Seq: NH3+- D-Phe-D-Glu-Gly-COO-) was examined by molecular dynamics simulations in water. Previous in vacuo simulations suggested a conformation consisting predominantly of interactions between the Phe side chain and glutamylcarboxyl group and a carboxyl/amino termini interaction. The solvated peptide was simulated using two approaches which were compared - a single 400-ns simulation and a "simulation tree.'' The "tree'' approach utilized 45 10-ns simulations with different conformations used as initial structures for given trajectories. We demonstrate that multiple short duration simulations are able to describe the same conformational space as that described by longer simulations. Furthermore, previously described in vacuo interactions were confirmed with amendments: the previously described head-to-tail arrangement of the amino and carboxyl termini, was not observed; the interaction between the glutamyl carboxyl and Phe side chain describes only one of a continuum of conformations present wherein the aromatic residue remains in close proximity to the glutamyl carbonyl group, and also interacts with either of the two available carboxyl groups. Finally, utilizing only two separate 10-ns trajectories, we were able to better describe the conformational space than a single 60-ns trajectory, realizing a threefold decrease in the computational complexity of the problem.

Submandibular gland peptide-T (SGP-T) is a potent anti-chemotactic agent for human neutrophils possessing anti-inflammatory properties. Biologically active analogues of SGP-T have been synthesized and a biotinylated form (KG6-SGP-T; Bio-KG6-SGP-T) was utilized to identify binding sites on isolated human neutrophils. Neutrophils incubated with Bio-KG6-SGP-T followed by phycoerythrin (PE)–avidin secondary reagent were fixed and visualized using histochemistry and flow cytometry. At doses of 10−8 and 10−9 M, Bio-KG6-SGP-T was shown to bind to neutrophils. The binding of Bio-KG6-SGP-T, at doses of 10−8 and 10−9 M, to neutrophils was abolished by a 100-fold excess of non-biotinylated peptide (KG6-SGP-T), but not by 100-fold excess of SGP-T. However, all peptides, dose-dependently reduced the binding of a CD16b antibody (LNK16 clone) to isolated human neutrophils. This discrepancy probably results from different preferred conformations for Bio-KG6-SGP-T, KG6-SGP-T and SGP-T, since exhaustive conformational searches revealed a high degree of overlap between α-Bio-KG6-SGP-T and KG6-SGP-T that was not seen with SGP-T.

This study examined the structure activity relationship of NH(3)-Phe-Glu-Gly-COO(-) (FEG), a potent inhibitor of intestinal anaphylaxis. The inhibition by FEG analogues of antigen-provoked contractions of isolated ileal segments obtained from ovalbumin-sensitized rats was determined and molecular modeling performed. A combination of aromaticity of the first residue, minimal extension of the carboxyl group on residue 2, and underivatized N and C termini were essential for biological activity. FEG, WEG, WDG and the d-enantiomeric forms of FEG (feG) and YEG (yeG) retained biological activity. By considering dipole moments, the structural and conformational features critical to biological activity were established as the glutamyl-carboxyl group/Phe side chain and carboxyl/amino termini interactions. Analysis of Ramachandran plots for position 1 sidechains indicate that mobility of the aromatic sidechain must be restricted to retain biological activity. The anti-anaphylactic effects of FEG, characterized by specific structural and conformational restrictions, indicate a selective interaction with a receptor for this peptide in the intestine.

About a decade ago, we published the first of a series of studies, which eventually led us to develop the concept of a sympathetic-superior cervical ganglion-submandibular gland axis, which appeared to regulate the release of immunosuppressive substances from the submandibular glands of rats. These putative mediators could suppress anaphylaxis and LPS-induced shock. We eventually discovered that the agents released from the salivary glands capable of producing, the suppression of these inflammatory responses were novel, small molecular weight peptides and our current work has focused on the actions of one of these (submandibular gland peptide-T: SGP-T) and its analogs on animal models of anaphylaxis and LPS-induced inflammation. SGP-T, as well as the C terminal tri-peptide FEG, are both potent inhibitors of intestinal and cardiovascular anaphylaxis in egg albumen-sensitized Hooded-Lister or Sprague Dawley rats. They also inhibit endotoxin-induced hypotension in Sprague Dawley rats. These results are a striking demonstration of the ability of these salivary gland peptides to inhibit early phase immune responses. We have shown that the D-isomer of FEG (feG) prevents the infiltration of leukocytes following injection of LPS into the peritoneum. Similarly, in another presentation, at this meeting, we show that feG can also block late phase responses in anaphylaxis by preventing infiltration of pulmonary tissue by leukocytes. In other models, we have been able to show that these peptides inhibit carrageenan-induced neutrophilia within the skin and inhibit leukocyte rolling and adhesion in mesenteric venules. Current work is focusing on the molecular mechanisms which lead to recruitment and activation of leukocytes into intestinal tissue following anaphylaxis and LPS activation. We will present data showing inhibition of expression of identified cell markers involved in chemotaxis and activation in both these models, which reveals some interesting differences between the two, suggesting that these peptides may act on different receptor subtypes. In summary, our collective work to date implies that there is an important sympathetic pathway, involving the superior cervical ganglion, that regulates the release of novel peptides from the submandibular glands which play an important part in early and late phase immune responses in anaphylaxis and LPS-induced inflammation. In future work we hope, not only to reveal the molecular mode of action of these peptides, but also to return to the point where we began and use our increasing molecular knowledge to study the neuroregulation of their production and release.

Background: The C-terminal of the prohormone submandibular rat 1 protein (SMR-1) contains several small peptides that reduce the severity of allergic inflammation and septic shock, and are part of the cervical sympathetic trunk-submandibular gland (SMG) axis of neuroendocrine immunology. These peptides include the heptapeptide, submandibular gland peptide-T and the tripeptide FEG. The D-isomeric form of this tripeptide, feG, which is active when administered orally, reduces LPS-provoked leukocyte rolling on mesenteric venules and influx of inflammatory cells into the peritoneum and intestinal muscle. Methods: To investigate the mechanism of action of these peptides, the influx of inflammatory molecules into the airways, and several properties of human neutrophils were examined. Results: Oral feG (1 mg/kg) inhibited the influx of inflammatory cells into the airways lumen of allergen challenged, sensitized Brown Norway rats. This inhibition occurred whether feG was given 30 min prior to 6 h post allergen challenge. Moreover, feG in picomolar to nanomolar concentrations inhibited PAF elicited chemotaxis by 30–40%, but the peptides did not affect superoxide production or phagocytosis by neutrophils. feG reduced PAF-stimulated expression of CD11b. Conclusions: feG may exert its anti-inflammatory effects by modulating the expression and functions of β2 integrins. The CST-SMG axis may be a major neuroendocrine pathway that modulates allergic asthma and other inflammatory responses.

The role of the D-isomeric form of the salivary gland tripeptide FEG (feG) and its carboxyl-amidated derivative, feG(NH2), in regulating leukocyte adherence to nonfixed atrial slices from Sprague-Dawley rats was examined under static conditions. Optimal binding of the leukocytes was seen if the leukocytes were treated with platelet activating factor (PAF; 10(-9)M). The increased adherence of PAF-treated peripheral blood leukocytes was totally inhibited by both feG and feG(NH2) (10-9M), as well as by antibodies against CD18 and CD49d. In contrast, the binding of peritoneal leukocytes was blocked only by CD49d antibody. Circulating leukocytes obtained from lipopolysaccharide (LPS) treated (2 mg/kg ip) rats did not bind to atrial slices obtained from normal hearts, but readily bound to atrial slices obtained from LPS-treated rats. This leukocyte binding was inhibited by in vivo feG treatment (100 microg/kg ip, 24 h before harvest) or by treating the isolated cells with feG (10(-9)M). The amidated peptide feG(NH2) reduced neutrophil accumulation in the atrium elicited by ip injection of LPS, whereas feG was ineffective. The reduction in neutrophil infiltration into the myocardium by feG(NH2) and the prevention of leukocyte interaction with myocytes seen with both feG and feG(NH2) probably results in hindered leukocyte migration in the inflamed heart, resulting in less tissue damage. The inhibition by these tripeptides on neutrophil adhesion to myocytes suggests that salivary glands hormones regulate the severity of cardiac inflammation.

The cervical sympathetic trunk-submandibular gland neuroendocrine axis plays an integral role in physiological adaptations and contributes to the maintenance of systemic homeostasis, particularly under the 'stress conditions' seen with tissue damage, inflammation, and aggressive behavior. The variety of polypeptides, whose release from acinar and ductal cells is under sympathetic nervous system control, offers coordinated and progressive levels of endocrine communication. Proteolytic enzymes (e.g. the kallikreins and furin maturases) are involved in the conversion of inactive precursors (e.g. Pro-EGF and SMR1) into biologically active molecules (e.g. EGF, SMR1-pentapeptide), which act on local or distant targets and thereby modulate the homeostatic process.

This study examines the effects of repeated administration of the selective serotonin reuptake inhibitors (SSRIs), fluoxetine and citalopram (10 mg/kg, i.p.), on immunoreactivity in C57BL/6 mice. Immune functions were evaluated by the ability of splenocytes to reduce a tetrazolium salt to formazan (MTT test), to proliferate, and to produce cytokines, including interleukin (IL)-1, IL-2, IL-4, IL-6, IL-10 and interferon gamma (IFNγ). Citalopram administered for 1, 2 and 4 weeks stimulates the proliferative activity of splenocytes and suppresses their ability to secrete the anti-inflammatory cytokine IL-4. Fluoxetine administration for 1 and 2 weeks, but not 4 weeks, stimulates the proliferative activity of splenocytes, whereas a 4-week administration of fluoxetine suppresses the secretion of IL-4. Four weeks of prolonged administration of citalopram and fluoxetine induces a significant increase in the production of IL-6 and IL-10, a cytokine with immunosuppressive and anti-inflammatory activities. The results show that, in C57BL/6 mice, the immunomodulatory effects of SSRIs depend on the SSRI used and the duration of administration.

A novel peptide hormone isolated from salivary glands, submandibular gland peptide-T (SGP-T), protects against the enhanced hypotensive response to intravenously administered lipopolysaccharide (LPS; 3.5 mg/kg; Salmonella typhosa) in rats with their submandibular glands removed (sialadenectomy). In this study, we examined the effects of SGP-T on LPS-provoked perturbations of heart function. Sialadenectomy did not alter basal heart function, although the sialadenectomized rats exhibited more pronounced reductions in ventricular peak systolic pressure (VPSP), ventricular pressure at max dP/dt (Pmax dP/dt), and maximum ventricular negative dP/dt (-dP/dt) relative to unoperated controls following LPS challenge. These changes were primarily due to changes in afterload (blood pressure). However, in sialadenectomized rats LPS-induced changes in Pmax -dP/dt (ventricular pressure at maximum -dP/dt) did not correlate with changes in VPSP, which suggests that sialadenectomy may alter the systolic component of the heart beat. The peptide SGP-T, at doses of 1 and 3.5 microg/kg, corrected Pmax -dP/dt and all other endotoxin-induced changes in heart function. Since Pmax -dP/dt is a measure of relaxation during diastole, the submandibular glands appear to play a role in protecting the heart against the adverse effects of acute endotoxin administration on ventricular relaxation. These effects of the submandibular glands may be mediated by the release of the peptide SGP-T.

The heptapeptide submandibular gland peptide-T (SGP-T; sequence = Thr-Asp-Ile-Phe-Glu-Gly-Gly) was isolated from rat submandibular glands based on its ability to reduce endotoxic hypotension. Since these glands also modulate neutrophil function, the effects of SGP-T on neutrophil function were investigated. I.v. SGP-T (35 and 100 micrograms/kg), when administered prior to and after the s.c. implantation of a carrageenan soaked sponge into rats, significantly reduced the accumulation of neutrophils in the sponge. Neutrophils retrieved from saline soaked sponges generated substantial amounts of superoxide anion in response to both phorbol myristate acetate (PMA) and N-fornyl-methionyl-leucyl-phenylalanine (fMLP), whereas those obtained from carrageenan impregnated sponges were refractory to these oxidative stimuli. Treatment with SGP-T promoted a dose-dependent recovery in the ability of neutrophils obtained from carrageenan soaked sponges to generate superoxide anion. This study, by identifying SGP-T as a regulator of neutrophil function, supports the concept that salivary glands are involved in the regulation of inflammatory responses.

Neuro-endocrine immunology, a field arising from curiosity about the mind-body connection, is evolving rapidly. From intriguing, but seemingly unexplainable observations with human infections and disease, experimental systems have been developed that provide a solid scientific basis for new understanding. There have been major efforts to understand influences of the nervous system on immune and inflammatory responses, e.g., innervation of the immune system, molecular communication pathways, and complex phenomena such as conditioning of immune responses and mechanisms of host defenses. In turn, the immune system communicates with the neuro-endocrine systems. Imbalances in the neuro-endocrine-immunologic circuitry are relevant in host defenses and in injury and repair. Examples of these themes in neuro-endocrine-immunology arise in several host-parasite models of neurogenic inflammation, immediate hypersensitivity responses, and granuloma formation. The hypothalamic-pituitary-adrenal axis and the cervical sympathetic trunk-submandibular gland axis provide important models to enhance understanding of this poorly known component of the host-parasite relationship.