The saliva of the medicinal leech hirudo medicinalis—II. inhibition of platelet aggregation and of leukocyte activity and examination of reputed anaesthetic effects

1. Leech saliva inhibits platelet aggregation induced by collagen, ADP and epinephrine.2. Leech saliva inhibits Superoxide production by neutrophils stimulated by tetradecanoyl phorbol acetate or polyhistidine. The effect is due in part at least to eglin.3. Reputed anaesthetic effects of leech saliva were not detected.     

A potent inhibitor of platelet activating factor from the saliva of the leech Hirudo medicinalis.

Leech saliva is shown to contain protein platelet aggregation inhibitors and a range of selective low molecular weight (LMW) aggregation inhibitors. Gel filtration on Bio-Gel P-2 (cut-off kDa) yields a protein fraction (Fr. I) and three LMW fractions. Fr. I inhibits aggregation induced by collagen, ADP, epinephrine and arachidonic acid. Of all the fractions, only one, Fr. II (LMW) specifically inhibits aggregation induced by platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine). Fr. II also inhibits thrombin-induced platelet aggregation. Fr. III inhibits aggregation induced by ADP, epinephrine and arachidonic acid, and Fr. IV only that induced by arachidonic acid. Fr. II also inhibits PAF- and thrombin-induced thromboxane generation in platelets, but does not inhibit arachidonic acid-induced thromboxane generation. Efforts to separate the anti-PAF from the anti-thrombin activity have been unsuccessful. The inhibition may therefore be due to a single inhibitor, though it may also be due to several inhibitors. Fr. II also inhibits superoxide anion production in formyl Met-Leu-Phe (fMLP)- and ionophore 23187- stimulated neutrophils. This may be due to the inhibition of the effects of PAF generated within the cell. Preliminary results suggest that the Fr. II inhibitor(s) is (are) amphipathic. The interaction of platelets with PAF and their interaction with the inhibitor(s) are mutually exclusive, and the inhibition may be competitive.     

The saliva of the medicinal leech Hirudo medicinalis--I. Biochemical characterization of the high molecular weight fraction

1. A method is described for obtaining dilute Hirudo medicinalis saliva by feeding leeches through a membrane on arginine/saline and squeezing them immediately after from the posterior end forwards. The process can be repeated at intervals. Yields are considerably higher than from salivary gland extracts. 2. Hirudo saliva contains hirudin, eglin, hyaluronidase, collagenase and apyrase. Leech collagenase and apyrase are here reported for the first time. 3. On gel filtration of lyophilized saliva, activity peaks were well defined. Approximate molecular weights were determined. Apyrase appears in two forms with optimum activity around pH 7.5. Collagenase was identified as belonging to the mammalian type.     

Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system

Clem, W. H. (University of Washington, Seattle), and S. J. Klebanoff. Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system. J. Bacteriol. 91:1848-1853. 1966.-Saliva contains an antimicrobial system which inhibits the growth of Lactobacillus acidophilus, as well as a number of other organisms, in complete growth medium. This antimicrobial system consists of the salivary peroxidase (lactoperoxidase) and thiocyanate ions, and requires the presence of H(2)O(2). Saliva inhibits the accumulation of glutamic acid and certain other amino acids by resting cells. This effect of saliva is decreased by dialysis, and thiocyanate ions restore the inhibitory effect of dialyzed saliva. The inhibitory effect of saliva is decreased by heat (100 C, 10 min), and lactoperoxidase restores the inhibitory effect of heated saliva. Thus, the inhibition of glutamic acid accumulation by saliva appears to be due in part to the lactoperoxidase-thiocyanate antimicrobial system. H(2)O(2) increases the inhibitory effect of both saliva and the lactoperoxidase-thiocyanate system on glutamic acid accumulation. The inhibition of glutamic acid accumulation is not preceded by a loss in microbial viability. The glutamic acid accumulated by L. acidophilus under the conditions employed remains largely (over 90%) as free glutamic acid. This suggests that saliva and the lactoperoxidase-thiocyanate-H(2)O(2) system inhibit the net transport of glutamic acid into the cell.     

Oscillations in Superoxide Anion Release by Polymorphonuclear Leukocytes and its Inhibition by Human Saliva

The release of superoxide anion (O 2 - ) by inflammatory polymorphonuclear leukocytes (PMN), obtained from the intraperitoneal cavity of mice, was investigated during two different times of the day. NaF-stimulated PMN at nigth-phase showed greater release of O 2 - than at day. Addition of human saliva in the culture medium dramatically inhibits O 2 - generation by both day- and night-phase cells. Also, the inhibitory effect of diurnal saliva on the cell activation is higher than the nocturnal saliva.     

Oscillations in Superoxide Anion Release by Polymorphonuclear Leukocytes and its Inhibition by Human Saliva

The release of superoxide anion (O 2 -) by inflammatory polymorphonuclear leukocytes (PMN), obtained from the intraperitoneal cavity of mice, was investigated during two different times of the day. NaF-stimulated PMN at nigth-phase showed greater release of O 2 - than at day. Addition of human saliva in the culture medium dramatically inhibits O 2 - generation by both day- and night-phase cells. Also, the inhibitory effect of diurnal saliva on the cell activation is higher than the nocturnal saliva.     

Activation of Neutrophils is Daily Inhibited by Saliva

The circadian release of superoxide anion (O 2 ?) by inflammatory neutrophils was investigated with mice kept under a constant light regime. Neutrophils were elicited to the peritoneal cavity by injection of 12% sodium caseinate-PBS solution, collected and stimulated to release O 2 ? to the culture milieu upon the addition of phorbol 12-myristate 13-acetate (PMA). The night phase neutrophils were able to release three fold more O 2 ? than day phase cells. Furthermore, the addition of human saliva in neutrophils cultures dramatically inhibits O 2 ? generation. Diurnal human saliva inhibits more effectively neutrophils activation than the nocturnal saliva. The PMA stimulation induces the synthesis of 13 kDa and 14 kDa molecular mass proteins detected in autoradiograms, and saliva additions decrease their synthesis. These two proteins are well known to be essential to the NAD(P)H oxidase assembly complex and, therefore, making neutrophils able to exert their main function as phagocytic cells.     

Activation of Neutrophils is Daily Inhibited by Saliva

The circadian release of superoxide anion (O 2 ? ) by inflammatory neutrophils was investigated with mice kept under a constant light regime. Neutrophils were elicited to the peritoneal cavity by injection of 12% sodium caseinate-PBS solution, collected and stimulated to release O 2 fito the culture milieu upon the addition of phorbol 12-myristate 13-acetate (PMA). The night phase neutrophils were able to release three fold more O 2 fithan day phase cells. Furthermore, the addition of human saliva in neutrophils cultures dramatically inhibits O 2 figeneration. Diurnal human saliva inhibits more effectively neutrophils activation than the nocturnal saliva. The PMA stimulation induces the synthesis of 13 kDa and 14 kDa molecular mass proteins detected in autoradiograms, and saliva additions decrease their synthesis. These two proteins are well known to be essential to the NAD(P)H oxidase assembly complex and, therefore, making neutrophils able to exert their main function as phagocytic cells.     

Saliva of the tick Ixodes dammini inhibits neutrophil function

Pilocarpine-induced saliva of adult female Ixodes dammini ticks inhibits the function of peritoneal-derived rat neutrophils, as measured by anaphylatoxin-induced aggregation, FMLP-induced granule enzyme secretion, zymosan-induced superoxide secretion, and phagocytosis of Borrelia burgdorferi spirochetes. Inhibition ranged from 40 to 80% when saliva was diluted 20 times into the assay medium. This neutrophil-inhibiting activity of I. dammini saliva may aid in tick feeding and facilitate pathogen transmission.     

Mechanisms of inhibition of vascular-platelet hemostasis by salivary gland secretion of the medicinal leech Hirudo medicinalis

The mechanism of inhibition of the vascular-platelet stage of hemostasis by medicinal leech salivary gland secretion was studied. It was shown that the secretion blocks platelet adhesion on the surface of collagens belonging to different genetic classes, inhibits the primary attachment of platelets and completely suppresses their spreading on collagen surface. Whatever its antithrombin activity, the leech secretion inhibits platelet aggregation stimulated by various inductors, e. g., ADP, prostaglandin endoperoxide analog U-46619, Ca2+ ionophore A23187, arachidonic acid. The secretion possessing the antithrombin activity causes a greater inhibition of the thrombin-stimulated aggregation than that devoid of this activity. Leech secretion stimulates adenylate cyclase of platelet membranes in a receptor-mediated fashion and increases the level of cAMP. The active substance is a low molecular weight, thermostable trypsin-resistant fraction of the secretion. Stimulation of adenylate cyclase is not mediated by adenosine receptors. It is supposed that the mechanism of this activating effect involves platelet prostaglandin receptors.     

The inhibitory effect of whole and deproteinized saliva on mutagenicity and clastogenicity resulting from a model nitrosation reaction

The objective of this study was to simulate in vitro at least some of the conditions that prevail in man during ingestion of nitrate and nitrosable compounds. Human saliva has been chosen because most chemicals ingested through food will interact with saliva. The nitrosation of methylurea was used as a model because the nitrosation products can be readily detected by their mutagenic (his+ revertants of S. typhimurium) and clastogenic (chromosome aberrations in CHO cells) properties. The results show that human saliva inhibits the formation of mutagenic and clastogenic nitrosation products when present during nitrosation. A 50% inhibition of mutagenicity results from the addition of a saliva sample diluted at 5% of the original concentration. In the test system used a similar inhibitory effect was obtained by 2.5 mM ascorbic acid or 2.0 mM chlorogenic acid. The main inhibitory agents seem to reside in a deproteinized fraction which was filtered through an ultrafilter UM2 (greater than 1000 MW). At strong acid levels (below pH 2) the saliva loses its inhibitory effect on the nitrosation of methylurea. The contribution of saliva to the inhibition of endogenous nitrosation within the oral cavity or stomach is discussed.     

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity,a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host''s immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.     

Saliva of the soft tick, Ornithodoros moubata, contains anti-platelet and apyrase activities.

1. Pilocarpine-induced saliva of the soft tick Ornithodoros moubata inhibits platelet aggregation induced by ADP or collagen, even when diluted 2000 times into platelet rich plasma. 2. Saliva contains apyrase (ATP-diphosphohydrolase) activity, which has an optimal pH of 7.0 for ADP and of 8.0 for ATP hydrolysis, respectively. Both Ca2+ and Mg2+ activate the reactions. 3. The mean specific activities for ATP and ADP hydrolysis at pH 7.5 were 0.97 and 0.74 mumoles orthophosphate/min/mg protein. 4. These results, which demonstrate for the first time such activities in the saliva of soft ticks, support the hypothesis that the saliva of blood sucking arthropods serves an anti-hemostatic role during feeding and that large amounts of salivary apyrase activity have evolved independently in hematophagous arthropods.     

Tick saliva inhibits the chemotactic function of MIP-1alpha and selectively impairs chemotaxis of immature dendritic cells by down-regulating cell-surface CCR5

Ticks are blood-feeding arthropods that secrete immunomodulatory molecules through their saliva to antagonize host inflammatory and immune responses. As dendritic cells (DCs) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC migration and function. Bone marrow-derived immature DCs pre-exposed to tick saliva showed reduced migration towards macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and regulated upon activation, normal T cell expressed and secreted (RANTES) chemokines in a Boyden microchamber assay. This inhibition was mediated by saliva which significantly reduced the percentage and the average cell-surface expression of CC chemokine receptor CCR5. In contrast, saliva did not alter migration of DCs towards MIP-3beta, not even if the cells were induced for maturation. Next, we evaluated the effect of tick saliva on the activity of chemokines related to DC migration and showed that tick saliva per se inhibits the chemotactic function of MIP-1alpha, while it did not affect RANTES, MIP-1beta and MIP-3beta. These data suggest that saliva possibly reduces immature DC migration, while mature DC chemotaxis remains unaffected. In support of this, we have analyzed the percentage of DCs on mice 48h after intradermal inoculation with saliva and found that the DC turnover in the skin was reduced compared with controls. Finally, to test the biological activity of the saliva-exposed DCs, we transferred DCs pre-cultured with saliva and loaded with the keyhole limpet haemocyanin (KLH) antigen to mice and measured their capacity to induce specific T cell cytokines. Data showed that saliva reduced the synthesis of both T helper (Th)1 and Th2 cytokines, suggesting the induction of a non-polarised T cell response. These findings propose that the inhibition of DCs migratory ability and function may be a relevant mechanism used by ticks to subvert the immune response of the host.     

Identification of an IL-2 binding protein in the saliva of the Lyme disease vector tick, Ixodes scapularis

A potent inhibitor of mitogen-stimulated T cell proliferation exists in the saliva of several species of hard ticks, including the Lyme disease vector tick, Ixodes scapularis. Our characterization of this phenomenon has led to the identification of a possible mechanism for the T cell inhibitory activity of I. scapularis saliva. The T cell inhibitor can overcome stimulation of mouse spleen cells with anti-CD3 mAb; however, a direct and avid interaction with T cells does not appear to be necessary. Tick saliva inhibits a mouse IL-2 capture ELISA, suggesting that a soluble IL-2 binding factor is present in the saliva. This hypothesis was verified by using a direct binding assay in which plate-immobilized tick saliva was shown to bind both mouse and human IL-2. Elimination of the IL-2 binding capacity of saliva in the in vitro assays by trypsin digestion demonstrated that the IL-2 binding factor is a protein. These experiments comprise the first demonstration of the existence of such a secreted IL-2 binding protein from any parasite or pathogen. This arthropod salivary IL-2 binding capacity provides a simple mechanism for the suppression of T cell proliferation as well as for the activity of other immune effector cells that are responsive to IL-2 stimulation. Relevance of the tick T cell inhibitory activity to the human immune system is demonstrated by the ability of tick saliva to inhibit proliferation of human T cells and CTLL-2 cells grown in the presence of human IL-2.     

A program for successful muskellunge management—A Minnesota success story

Minnesota has established a successful muskellunge, Esox masquinongy, stocking program as a result of a series of research studies that enabled area fisheries managers to make informed management decisions. The previous propagation and stocking program (pre-1982) reared muskellunge progeny from Shoepack Lake near the Minnesota–Ontario border, but these fish were not attaining trophy size. Protein electrophoresis showed there were two different muskellunge strains in the state. A controlled study was conducted on progeny from natural muskellunge populations from Leech and Shoepack lakes. Fish from these populations, together, were used to stock two other Minnesota lakes, and their growth rates, age of maturity, and maximum size attained were compared. Leech Lake muskellunge grew faster and attained a larger maximum size than the Shoepack Lake strain. As a result, a new stocking program based on the Leech Lake strain was developed. Because it was difficult to collect Leech Lake gametes, the Minnesota Department of Natural Resources created seven brood stock lakes using Leech Lake progeny. As a result of combining genetic considerations, new research on spawning sites, more restrictive harvest regulations, and catch-and-release fishing, anglers’ catch of trophy-sized muskellunge in Minnesota has increased.     

Saliva plays a dual role in oxidation process in stomach medium

The aim of this study was to evaluate the role of saliva in the oxidation process under the acidic condition of the stomach. Saliva specimens played varied roles in the lipid peroxidation process of heated muscle tissue in simulated gastric fluid: pro-oxidant effects, no effects, and antioxidant effects. To elucidate these differences, selected saliva components were examined. The pseudoperoxidase activity of lactoperoxidase increased lipid peroxidation, while thiocyanate and nitrite-reduced lipid peroxidation. The effect of a saliva specimen on lipid peroxidation was correlated with the concentration of nitrite in the specimen, but not with that of other saliva components. The inhibitory effect of nitrite may be due to its conversion to NO. Elucidation of the antioxidant effect of saliva on co-oxidation of d-alpha-tocopherol in gastric fluid, demonstrated that saliva alone cannot protect d-alpha-tocopherol from co-oxidation, although it partially protected against lipid peroxidation. The presence of red wine polyphenols in stomach medium totally inhibits food lipid peroxidation and d-alpha-tocopherol co-oxidation.     

Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from the tick Ixodes scapularis

Here we report the ability of the tick Ixodes scapularis, the main vector of Lyme disease in the United States, to actively and specifically affect the host proteolytic activity in the sites of infestation through the release of a cystatin constituent of its saliva. The cystatin presence in the saliva was verified both biochemically and immunologically. We named the protein sialostatin L because of its inhibitory action against cathepsin L. We also show that the proteases it targets, although limited in number, have a prominent role in the proteolytic cascades that take place in the extracellular and intracellular environment. As a result, sialostatin L displays an antiinflammatory role and inhibits proliferation of cytotoxic T lymphocytes. Beyond unraveling another component accounting for the properties of tick saliva, contributing to feeding success and pathogen transmission, we describe a novel tool for studying the role of papain-like proteases in diverse biologic phenomena and a protein with numerous potential pharmaceutical applications.     

Lung and salivary scavenger receptor glycoprotein-340 contribute to the host defense against influenza A viruses

The lung scavenger receptor-rich protein glycoprotein-340 (gp-340) is present in bronchoalveolar lavage (BAL) fluids and saliva and mediates specific adhesion to and aggregation of bacteria. It also binds to surfactant proteins A and D (SP-A and -D). Prior studies demonstrated that SP-A and SP-D contribute to innate defense against influenza A virus (IAV). We now show that lung and salivary gp-340 inhibit the hemagglutination activity and infectivity of IAV and agglutinate the virions through a mechanism distinct from that of SP-D. As in the case of SP-A, the antiviral effects of gp-340 are mediated by noncalcium-dependent interactions between the virus and sialic acid-bearing carbohydrates on gp-340. Gp-340 inhibits IAV strains that are resistant to SP-D. Concentrations of gp-340 present in saliva and BAL fluid of healthy donors are sufficient to bind to IAV and inhibit viral infectivity. On the basis of competition experiments using competing saccharide ligands, it appears that SP-D does not entirely mediate that anti-IAV activity of BAL fluid and contributes little to that of saliva. Furthermore, removal of gp-340 from BAL fluid and saliva significantly reduced anti-IAV activity. Hence, gp-340 contributes to defense against IAV and may be particularly relevant to defense against SP-D-resistant viral strains.