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Objective: The aim of this study was to determine if there is an association between the salivary protein profile and disease control in asthma., Methods: Thirty asthmatic patients (17 adults and 13 children) participated in this study. Saliva samples were collected from healthy subjects, controlled and uncontrolled asthmatics. Individual samples from each group were combined to form a pooled sample, from which proteomic analysis was performed using gel-based quantitative proteomics., Results: Fourteen out of thirty asthmatics were classified to be controlled asthma. Most of asthmatics received inhaled corticosteroids as the controller medications. SDS-PAGE showed predominant bands at high molecular weight in asthmatic saliva compared to that of the controls. Shotgun proteomic analyses indicated that 193 salivary proteins were expressed in both controlled and uncontrolled asthmatics. They were predicted to associate with proteins involved in pathogenesis of asthma including IL-5, IL-6, MCP-1, VEGF, and periostin and asthma medicines (Cromolyn, Nedocromil, and Theophylline). Nucleoside diphosphate kinase (NME1-NME2) only expressed in controlled asthmatics whereas polycystic kidney and hepatic disease 1 (PKHD1)/fibrocystin, zinc finger protein 263 (ZNF263), uncharacterized LOC101060047 (ENSG00000268865), desmoglein 2 (DSG2) and S100 calcium binding protein A2 (S100A2) were only found in uncontrolled asthma. Therefore, the six proteins were associated with disease control in children and adults with asthma., Conclusion: Our findings suggest that NME1-NME2, PKHD1, ZNF 263, uncharacterized LOC101060047, DSG 2 and S100 A2 in saliva are associated with disease control in asthma.

Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide-selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one-pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against α-thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1 mg kg -1 . The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein-protein interactions, in addition to an active site., (© 2020 Wiley-VCH GmbH.)

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

Saliva contains a large number of proteins that play various crucial roles to maintain the oral health and tooth integrity. This oral fluid is proposed to be one of the most important host factors, serving as a special medium for monitoring aspects of microorganisms, diet and host susceptibility involved in the caries process. Extensive salivary proteomic and peptidomic studies have resulted in considerable advances in the field of biomarkers discovery for dental caries. These salivary biomarkers may be exploited for the prediction, diagnosis, prognosis and treatment of dental caries, many of which could also provide the potential templates for bioactive peptides used for the biomimetic management of dental caries, rather than repairing caries lesions with artificial materials. A comprehensive understanding of the biological function of salivary proteins as well as their derived biomimetic peptides with promising potential against dental caries has been long awaited. This review overviewed a collection of current literature and addressed the majority of different functions of salivary proteins and peptides with their potential as functional biomarkers for caries risk assessment and clinical prospects for the anti-caries application., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The objective of this study is to assess the occurrence of and risk factors for serious infections in rheumatoid arthritis (RA)-associated interstitial lung disease (ILD). All patients with RA-ILD (ACR 1987 criteria for RA) seen at a single center from 1998 to 2014 were identified and manually screened for study inclusion. Follow-up data were abstracted until death or December 31, 2015. Serious infection was defined as requiring antimicrobial therapy and hospitalization. Risk of infection was analyzed by person-year (py) methods using time-dependent covariates started when the medication was first used and stopped 30 days after the medication was discontinued. Of the 181 included patients, 87 (48 %) were female. The mean age at ILD diagnosis was 67.4 (±9.9) years, and median follow-up time was 3.1 (range: 0.01 to 14.8) years. Higher infection rates were observed during the first year after ILD diagnosis (14.1 per 100 py) than subsequently (5.7 per 100 py; p = 0.001). Pneumonia was the most common (3.9 per 100 py). Overall infection risk was higher in organizing pneumonia (OP) (27.1 per 100 py) than usual interstitial pneumonia (7.7 per 100 py) or non-specific interstitial pneumonia (5.5 per 100 py) (p < 0.001). The highest infection rate observed was with a daily prednisone use >10 mg per day (15.4 per 100 py). Patients with RA-ILD are at risk of serious infection. Prednisone use >10 mg per day was associated with higher rates of infection. Immunosuppressive drug use governed by concern for risk of infection in patients with ILD resulting in channeling bias cannot be excluded.

Renal cell carcinoma (RCC), also called kidney cancer or renal adenocarcinoma, is highly resistant to current treatments. It has been previously reported that a Kunitz-type inhibitor domain-containing protein, isolated from the salivary glands of the Amblyomma cajennense tick, triggers apoptosis in murine renal adenocarcinoma cells (Renca) by inhibiting the proteasome and endoplasmic reticulum stress. Of note, Amblyomin-X is the corresponding recombinant protein identified in the cDNA library from A. cajennense salivary glands. Herein, using orthotopic kidney tumors in mice, we demonstrate that Amblyomin-X is able to drastically reduce the incidence of lung metastases by inducing cell cycle arrest and apoptosis. The in vitro assays show that Amblyomin-X is capable of reducing the proliferation rate of Renca cells, promoting cell cycle arrest, and down-regulating the expression of crucial proteins (cyclin D1, Ki67 and Pgp) involved in the aggressiveness and resistance of RCC. Regarding non-tumor cells (NIH3T3), Amblyomin-X produced minor effects in the cyclin D1 levels. Interestingly, observing the image assays, the fluorescence-labelled Amblyomin-X was indeed detected in the tumor stroma whereas in healthy animals it was rapidly metabolized and excreted. Taken the findings together, Amblyomin-X can be considered as a potential anti-RCC drug candidate., Competing Interests: The authors declare no conflicts of interest.

Opioid peptides are potent analgesics with therapeutic potential in the treatment of acute and chronic pain. Their efficacy is limited by peptidases (enkephalinases). Opiorphin pentapeptide (QRFSR) is the first characterized human endogenous inhibitor of enkephalinases. The peptide is able to increase the binding and affinity of endogenous opiates to mu opioid receptors; thus, the mechanism of opiorphin may provide a new therapeutic approach in pain management. The analgesic effect of opiorphin was proven in several earlier published in vitro and in vivo studies. Our aim was to test the transfer of opiorphin through a blood-brain barrier model for the first time. The flux of opiorphin was tested on a blood-brain barrier culture model consisting of rat brain endothelial, glial and pericyte cells. Brain endothelial cells in this triple co-culture model form tight monolayers characterized by transendothelial electrical resistance measurement. Relative quantity of the peptide was estimated by mass spectrometry. The transfer of opiorphin through the blood-brain barrier model was estimated to be ∼3%, whereas the permeability coefficient was 0.53 ± 1.36 × 10(-6) cm/s (n = 4). We also observed rapid conversion of N-terminal glutamine into pyroglutamic acid during the transfer experiments. Our results indicate that opiorphin crosses cultured brain endothelial cells in the absence of serum factors in a significant amount. This is in agreement with previous in vivo data showing potentiation of enkephalin-mediated antinociception. We suggest that opiorphin may have a potential as a centrally acting novel drug to treat pain., (Copyright © 2015 IMSS. Published by Elsevier Inc. All rights reserved.)

Objective: To kill urothelial cancer cells while preserving healthy cells, this study used photothermal therapy (PTT). PTT techniques target urothelial cancer cells using gold nanoparticles (GNPs) and a green light laser., Materials and Methods: The GNPs were conjugated with anti-Mucin 7 antibodies, which acted as a probe for targeting tumor cells. Conjugated GNPs were exposed to a green light laser (532 nm) with sufficient thermal energy to kill the transitional cell carcinomas (TCCs)., Results: According to our results, nanoparticles conjugated with Mucin 7 antibodies damaged all types of cancer cells (MBT2, T24, 9202, and 8301) at relatively low energy levels (i.e., 500 laser shots at 10 W/cm(2) in power, 1.6 Hz in frequency, and 300 ms in duration). Nonconjugated nanoparticles required 30 W/cm(2) or more to achieve the same effect. Cell damage was directly related to irradiation time and applied laser energy., Conclusions: The minimally invasive PTT procedure combined with Mucin 7 targeted GNPs is able to kill cancer cells and preserve healthy cells. The success of this treatment technique can likely be attributed to the lower amount of energy required to kill targeted cancer cells compared with that required to kill nontargeted cancer cells. Our in vitro pilot study yielded promising results; however, additional animal studies are required to confirm these findings.

Human opiorphin is a recently identified endogenous pentapeptide, encoded by ProL1 multigenes family that contributes to cardiovascular modulation. The aim of this study was to evaluate the effect of opiorphin through intravenous injection (i.v.) on mean arterial pressure (MAP) regulation. To investigate the bioactivity of opiorphin, a rat cannulation model was developed for MAP measurement and blood sampling. In our present study, opiorphin (200-700 nmol/kg) increased MAP in dose-related and time-dependent manner in conscious rats, which associated highly with the elevation of angiotensin II (AngII) levels in serum. Furthermore, the MAP elevation induced by opiorphin was completely blocked by AngII receptor antagonist valsartan and partially attenuated by angiotensin-converting enzyme inhibitor captopril. Finally, we tested the effect of opiorphin in hypoxia condition, which exhibited that opiorphin reversed hypoxia induced hypotension in conscious rats. Taken together, these results indicated that opiorphin may play an important role in the modulation of blood pressure through AngII dependent pathway, which may help future development of potent clinical therapeutics for emergency treatment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cancer is linked to hypercoagulability, and many studies have shown that anticoagulant drugs affect tumor progression. In this study was demonstrated that the Amblyomin-X (which is a recombinant protein that exerts similarity to the Kunitz-type inhibitors and shows pro-apoptotic effects in different tumor cell lines) and heparin (a classic anticoagulant) have similar effects on cancer progression and on normalization of the hypercoagulable state. However, Amblyomin-X showed a distinct mechanism in triggering its effects in vitro, because it exerted a cytotoxic effect in cancer cells by inducing apoptosis and promoting cell cycle arrest., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57BL/6 mice. Ixolaris (250 μg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 μg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Clinical Relevance: Late complications can occur with use of current antimetabolites to prevent scarring following glaucoma filtration surgery (GFS). Safer, more targeted, anti-fibrosis agents are sought., Objectives: The protein saratin has been shown to exhibit anti-fibrotic and anti-thrombotic properties in response to injury, but had not been used for glaucoma surgery. The goal of this study was to compare the efficacy of saratin with that of the widely accepted mitomycin-C (MMC) in prolonging bleb survival following GFS in the rabbit model. Two saratin delivery routes were compared; a single intraoperative topical application versus a combination of intraoperative topical application with two additional postoperative injections., Methods: Twenty-four New Zealand White rabbits underwent GFS and received either intraoperative topical saratin, intraoperative topical saratin plus two injections on post-operative days 4 and 8, balanced saline solution (BSS), or MMC. The bleb tissues and their elevation durations were compared based on clinical and histological findings., Results: Rabbits receiving topical+injections of saratin had a mean bleb survival of 33.6±8.5 days, significantly higher than the negative BSS controls, which averaged 17.4±6.0 days (p = 0.018). No improvement over BSS was seen for rabbits receiving topical saratin only (15.5±4.8 days, p = 0.749). Rabbits receiving saratin did not develop bleb avascularity and thinning associated with MMC treatment and there were no apparent clinical signs of toxicity., Conclusions: Treatment with a single intraoperative topical application plus two additional postoperative injections significantly prolonged bleb elevation comparable to MMC, but without toxicity; however, topical application alone was ineffective.

Salivary glands from haematophagous animals express a notable diversity of negative modulators of platelet function. Triplatin is an inhibitor of collagen-induced platelet aggregation which has been described as an antagonist of glycoprotein VI (GPVI). Because triplatin displays sequence homology to members of the lipocalin family of proteins, we investigated whether triplatin mechanism of action could be explained by interaction with pro-haemostatic prostaglandins. Our results demonstrate that triplatin inhibits platelet aggregation induced by low doses of collagen, thromboxane A2 (TXA(2)) mimetic (U46619), and arachidonic acid (AA). On the other hand, it does not inhibit platelet aggregation by convulxin, PMA, or low-dose ADP. Isothermal titration calorimetry (ITC) revealed that triplatin binds AA, cTXA(2), TXB(2), U46619 or prostaglandin (PG)H(2) mimetic (U51605). Consistent with its ligand specificity, triplatin induces relaxation of rat aorta contracted with U46619. Triplatin also interacts with PGF(2α) and PGJ(2), but not with leukotrienes, AA or biogenic amines. Surface plasmon resonance experiments failed to demonstrate interaction of triplatin with GPVI; it also did to inhibit platelet adhesion to fibrillar or soluble collagen. Because triplatin displays sequence similarity to apolipoprotein D (ApoD) - a lipocalin associated with high-density lipoprotein, ApoD was tested as a putative TXA(2)-binding molecule. ITC failed to demonstrate binding of ApoD to all prostanoids described above, or to AA. Furthermore, ApoD was devoid of inhibitory properties towards platelets activation by AA, collagen, or U46619. In conclusion, triplatin mechanism of action has been elucidated without ambiguity as a novel TXA(2)- and PGF(2α)- binding protein. It conceivably blocks platelet aggregation and vasoconstriction, thus contributing to successful blood feeding at the vector-host interface.

Experimental evidence suggests that C inhibition and more particularly combined inhibition of C and the TLR coreceptor CD14 may be of therapeutic benefit in sepsis and other inflammatory conditions. A barrier to the testing and further development of many inhibitors is that their activity is species specific. Pig is a relevant species for experimental models of human disease, and this study undertakes a comprehensive comparison of the inhibitory efficacy of the C5 inhibitor Ornithodoros moubata C inhibitor (OmCI) in human and porcine whole blood ex vivo models of Escherichia coli-induced sepsis. The effect of OmCI on complement activity in pigs undergoing E. coli sepsis was also examined. Porcine and human serum, and whole blood anticoagulated with lepirudin, was incubated with E. coli and the effect of OmCI investigated. The ex vivo results were virtually identical in pig and human. OmCI completely ablated the activity of all three C pathways at 0.64 μM. E. coli-induced C activation and expression of CD11b (wCD11R3 in the pig), was abolished ex vivo at 0.32 μM OmCI. Combining anti-CD14 and OmCI reduced the formation of IL-8 and TNF-α more potently than the single inhibitors. OmCI also efficiently bound E. coli-induced leukotriene B(4) in pig and human plasma. In support of our ex vivo findings, in vivo the activity of all C pathways was inhibited at 0.6 mg OmCI/kg pig. In conclusion, OmCI efficiently inhibited pig and human C activation, has accompanying anti-inflammatory effects and is a promising candidate inhibitor for further in vivo studies of sepsis.

Aims: The goal of this review is to identify the antimicrobial proteins in the oral fluids, saliva and gingival crevicular fluid and identify functional families and candidates for antibacterial treatment., Results: Periodontal biofilms initiate a cascade of inflammatory and immune processes that lead to the destruction of gingival tissues and ultimately alveolar bone loss and tooth loss. Treatment of periodontal disease with conventional antibiotics does not appear to be effective in the absence of mechanical debridement. An alternative treatment may be found in antimicrobial peptides and proteins, which can be bactericidal and anti-inflammatory and block the inflammatory effects of bacterial toxins. The peptides have co-evolved with oral bacteria, which have not developed significant peptide resistance. Over 45 antibacterial proteins are found in human saliva and gingival crevicular fluid. The proteins and peptides belong to several different functional families and offer broad protection from invading microbes. Several antimicrobial peptides and proteins (AMPs) serve as templates for the development of therapeutic peptides and peptide mimetics, although to date none have demonstrated efficacy in human trials., Conclusions: Existing and newly identified AMPs may be developed for therapeutic use in periodontal disease or can serve as templates for peptide and peptide mimetics with improved therapeutic indices., (© 2011 John Wiley & Sons A/S.)

The aim of this study was to evaluate the anti-tumor activity of Amblyomin-X, a serine protease Kunitz-type inhibitor. Amblyomin-X induced tumor mass regression and decreased number of metastatic events in a B16F10 murine melanoma model. Alterations on expression of several genes related to cell cycle were observed when two tumor cell lines were treated with Amblyomin-X. PSMB2, which encodes a proteasome subunit, was differentially expressed, in agreement to inhibition of proteasomal activity in both cell lines. In conclusion, our results indicate that Amblyomin-X selectively acts on tumor cells by inducing apoptotic cell death, possibly by targeting the ubiquitin-proteasome system., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment., Objectives: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression., Methods and Results: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization., Conclusion: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Objective: Complement mediated injury of the neuromuscular junction is considered a primary disease mechanism in human myasthenia gravis and animal models of experimentally acquired myasthenia gravis (EAMG). We utilized active and passive models of EAMG to investigate the efficacy of a novel C5 complement inhibitor rEV576, recombinantly produced protein derived from tick saliva, in moderating disease severity., Methods: Standardized disease severity assessment, serum complement hemolytic activity, serum cytotoxicity, acetylcholine receptor (AChR) antibody concentration, IgG subclassification, and C9 deposition at the neuromuscular junction were used to assess the effect of complement inhibition on EAMG induced by administration of AChR antibody or immunization with purified AChR., Results: Administration of rEV576 in passive transfer EAMG limited disease severity as evidenced by 100% survival rate and a low disease severity score. In active EAMG, rats with severe and mild EAMG were protected from worsening of disease and had limited weight loss. Serum complement activity (CH(50)) in severe and mild EAMG was reduced to undetectable levels during treatment, and C9 deposition at the neuromuscular junction was reduced. Treatment with rEV576 resulted in reduction of toxicity of serum from severe and mild EAMG rats. Levels of total AChR IgG, and IgG(2a) antibodies were similar, but unexpectedly, the concentration of complement fixing IgG(1) antibodies was lower in a group of rEV576-treated animals, suggesting an effect of rEV576 on cellular immunity., Interpretation: Inhibition of complement significantly reduced weakness in two models of EAMG. C5 inhibition could prove to be of significant therapeutic value in human myasthenia gravis.

Peptides with broad-spectrum antimicrobial activity are found in the mucosal surfaces at many sites in the body, including the airway, the oral cavity, and the digestive tract. Based on their in vitro antimicrobial and other immunomodulatory activities, these host defense peptides have been proposed to play an important role in the innate defense against pathogenic microbial colonization. The genes that encode these peptides are up-regulated by pathogens, further supporting their role in innate immune defense. However, the differences in the local microbial environments between the generally sterile airway and the highly colonized oral cavity suggest a more complex role for these peptides in innate immunity. For example, beta-defensin genes are induced in the airway by all bacteria and Toll-like receptor (TLR) agonists primarily through an NF-kappaB-mediated pathway. In contrast, the same genes are induced in the gingival epithelium by only a subset of bacteria and TLR ligands, via different pathways. Furthermore, the environments into which the peptides are secreted--specifically saliva, gingival crevicular fluid, and airway surface fluid--differ greatly and can effect their respective activities in host defense. In this review, we examine the differences and similarities between host defense peptides in the oral cavity and the airway, to gain a better understanding of their contributions to immunity.

The recently emerged Vcsa1 gene is one member of the variable coding sequence (VCS) multigene family of Rattus norvegicus. This gene encodes the precursor prohormone SMR1 (submandibular rat-1), which on enzymatic processing gives rise to several 5 to 11 amino acid peptides that modulate a variety of physiological functions. The analgesic pentapeptide sialorphin and anti-inflammatory heptapeptide submandibular gland peptide-T (TDIFEGG) are the most intensively studied. Although the Vcsa1 gene and its protein product are unique to rats, TDIFEGG or a derivative acts on all species examined to date, including human cells, in functions related to allergic reactions and inflammation. In this review, the patent and academic literature on SMR1 and its natural peptides and their derivatives are reviewed for consideration of biological targets and relevance to the development of novel therapeutic agents. The VCS gene family is discussed and we speculate on possible human homologs of these potent anti-inflammatory rat-derived peptides. The biologically active peptide products of SMR1 are considered and the mechanism of action and structure-activity relationships of the anti-inflammatory submandibular gland peptide-T and its derivatives are discussed.

Activation of Th2 CD4(+) T cells is necessary and sufficient to elicit allergic airway disease, a mouse model with many features of human allergic asthma. Effectively controlling the activities of these cells could be a panacea for asthma therapy. Blood-feeding parasites have devised remarkable strategies to effectively evade the immune response. For example, ticks such as Ixodes scapularis, which must remain on the host for up to 7 days to feed to repletion, secrete immunosuppressive proteins. Included among these proteins is the 15-kDa salivary protein Salp15, which inhibits T cell activation and IL-2 production. Our objective for these studies was to evaluate the T cell inhibitory properties of Salp15 in a mouse model of allergic asthma. BALB/cJ mice were Ag sensitized by i.p. injection of OVA in aluminum hydroxide, with or without 50 mug of Salp15, on days 0 and 7. All mice were challenged with aerosolized OVA on days 14-16 and were studied on day 18. Compared with control mice sensitized with Ag, mice sensitized with Ag and Salp15 displayed significantly reduced airway hyperresponsiveness, eosinophilia, Ag-specific IgG1 and IgE, mucus cell metaplasia, and Th2 cytokine secretion in vivo and by CD4(+) T cells restimulated with Ag in vitro. Our results demonstrate that Salp15 can effectively prevent the generation of a Th2 immune response and the development of experimental asthma. These studies, and those of others, support the notion that a lack of ectoparasitism may contribute to the increasing prevalence of allergic asthma.

Objectives: Few epidemiological studies have confirmed the antifungal activity of histatin 5 in saliva against Candida species. The purpose of this study was to examine the relationship between concentrations of histatin 5 and the number of cultivable Candida in saliva samples from elderly., Methods: Whole saliva samples were obtained from 124 elderly people, 65 years or older, living in a rural community. The concentrations of histatin 5 in saliva samples were determined by the enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody. Total colony-forming units (CFUs) were counted on a selective medium for Candida. Multiple linear regression analysis was performed to determine the independent contribution of explanatory variables to Candida CFUs using age, sex, histatin 5 concentration and type of denture prosthesis as independent variables., Results: Saliva samples from 104 subjects (84%) were candidal colony-positive. The youngest group (65-69 years old) showed significantly smaller Candida CFU counts than those in the older group. The mean Candida CFU count of denture wearers was significantly higher than that of non-denture wearers. Significantly negative associations were found between Candida CFU counts and histatin 5 level in the oldest group (p < 0.05) and in the full-denture wearers (p < 0.01). Multiple linear regression analysis revealed that Candida CFU counts were mostly associated with type of dentures, followed by histatin 5 concentration., Conclusion: Possible activity of histatin 5 against Candida in whole saliva of elderly people was epidemiologically confirmed. The area covered by the prostheses was a strong factor associated with the Candida CFU count.

Excessive platelet activation and accumulation can lead to vessel occlusion and thus present major therapeutic challenges in cardiovascular medicine. Apyrase, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ADP and ATP released from platelets and endothelial cells, thereby reducing platelet activation and recruitment. In the present study, we expressed a 68-kDa recombinant mosquito (Aedes aegypti) salivary apyrase using a baculovirus/insect cell expression system and purified it to homogeneity using anion-exchange chromatography on a large scale. A yield of 18 mg of purified recombinant apyrase was obtained from 1 litre of the medium. Kinetic analysis indicated that the recombinant apyrase had a K(m) of 12.5 microM for ADP and a K(m) of 15.0 microM for ATP. The recombinant apyrase inhibited ADP-, collagen- and thrombin-induced human platelet aggregation in a dose-dependent manner, indicating that the recombinant protein retained nucleotidase activity in a whole cell system, which suggests that it may serve as a therapeutic agent for inhibition of platelet-mediated thrombosis.

Tick histamine-binding proteins bind histamine with high affinity and specificity. This is attained by a novel binding mechanism, whereby histamine is sequestered within a binding cavity of the lipocalin fold. The histamine binding proteins and related protein family members are currently under investigation as potential therapeutic agents for the treatment of various diseases, including conjunctivitis, allergic rhinitis, carcinoid syndrome and rheumatoid arthritis. While these proteins show great therapeutic potential, they are part of a diverse family of tick lipocalin proteins, some of which have been implicated in tick-host rejection and host pathogenesis. As such, the therapeutic mining of tick lipocalins should be considered within the framework of the rest of the family.

Background: Morbidity and mortality from Pseudomonas aeruginosa sepsis remain high despite the availability of antibiotics to which the microorganism is sensitive., Methods: The in vitro activity of histatin derivative P-113d was investigated against Pseudomonas aeruginosa. In addition, its in vivo efficacy was studied in 3 rat models of infection: intraperitoneal injection of 1 mg of P. aeruginosa 10 lipopolysachharide, intraperitoneal injection of 2 x 10(10) cfu of P. aeruginosa ATCC 27853, and intra-abdominal sepsis induced by cecal ligation and puncture. Rats received isotonic sodium chloride solution parenterally (control groups), 1 mg of P-113d/kg of body weight, 1 mg of polymyxin B/kg of body weight, or 20 mg of imipenem/kg of body weight. Main outcomes measured were abdominal exudate and plasma bacterial growth, plasma concentrations of endotoxin and tumor necrosis factor (TNF)- alpha, and lethality., Results: The in vivo studies showed that all compounds reduced lethality, when compared with results for the control group. Overall, P-113d exhibited a slightly lower antimicrobial activity than did imipenem, even though P-113d achieved a substantial decrease in plasma concentrations of endotoxin and TNF- alpha, compared with the imipenem. No statistically significant differences for antimicrobial and antiendotoxin activities were noted between P-113d and polymyxin B., Discussion: These results provide evidence for double antiendotoxin and antimicrobial activity for P-113d and point to its potential use for the treatment of severe infections.

Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4-1 microM and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100-200 mcicrog/kg doses of sialorphin required the activation of mu- and delta-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.

Two new classes of anticoagulants in development at the present time [anti-factor Xa and anti-factor IIa (direct antithrombin) agents] should change our future strategies for prevention and treatment of venous thromboembolic events. Among the anti-factor Xa, the pentasaccharides are initiating their clinical use. Fondaparinux, the synthetic form of the natural pentasaccharide is active in prevention and treatment of venous thromboembolic and coronary thrombotic events. A modified form (idraparinux) whose pharmacokinetics allows one administration only once a week should have the same type of efficacy. Among direct antithrombin agents, hirudin and derivatives have been developed in the past decade burt are not routinely used. Synthetic direct antithrombins allowing oral route are currently developed: Exanta with the most advanced development, is active in prevention and treatment of venous thromboembolic and coronary thrombotic events. It could allow (if confirmed by clinical trials) a complete oral treatment of deep vein thrombosis without any biological monitoring. Exanta is also active in the prevention of arterial thromboembolic events on atrial fibrillation. Other molecular forms of synthetic per os direct antithrombin are also in development. But molecules aimed at other targets are also tested: the most advanced are those antagonizing the initial phase of tissue factor activation of factor VII but other strategies are being tested such as stimulation of fibrinolysis.

Innate human salivary defence proteins, lysozyme, lactoferrin and peroxidase, are known to exert a wide antimicrobial activity against a number of bacterial, viral and fungal pathogens in vitro. Therefore, these proteins, alone or in combinations, have been incorporated as preservatives in foods and pharmaceuticals as well as in oral health care products to restore salivas' own antimicrobial capacity in patients with dry mouth. These antimicrobials used in oral health care products, such as dentifrices, mouth-rinses, moisturizing gels and chewing gums, have been purified from bovine colostrum. In this review I critically evaluate the clinical efficacy and safety of this kind of preventive approach against various oral diseases and symptoms.

Purpose: Post-carotid endarterectomy, thrombosis, and intimal hyperplasia may be decreased by the inhibition of platelet adhesion and activation. In this study, a novel agent, saratin, was used to inhibit platelet-to-collagen adhesion in a rat carotid endarterectomy model. Saratin is a recombinant protein isolated from the saliva of the medicinal leech Hirudo medicinalis, which is thought to act by binding to collagen, and inhibits von Willebrand factor-collagen interaction under conditions of increased shear and therefore, the adherence and activation of platelets at the vessel wall. Saratin has the advantage of being a nonsystemic, site-specific topical application., Methods: A rat carotid endarterectomy model was used in which an open technique with arteriotomy and intimectomy was used. Saratin was applied to the endarterectomized surface of the carotid artery before arterial closure. End point measurements included platelet adhesion, thrombosis rate, intimal hyperplasia development, bleeding times, and platelet counts. Electron micrographs of carotid arteries were used for quantitative analysis of platelet aggregation and platelet counts. Intimal hyperplasia and thrombosis were assessed with computer-assisted morphometric analysis of elastin-stained carotid artery sections with direct measurement of the intimal hyperplasia area., Results: The topical application of saratin significantly decreased platelet adhesion compared with controls at 3 hours after carotid endarterectomy (64 +/- 17 vs 155 +/- 33 platelets per grid, P = .05), and 24 hours after carotid endarterectomy (35 +/- 11 vs 149 +/- 37 platelets per grid, P = .0110), respectively. A percent luminal stenosis, as a measure of intimal hyperplasia, was significantly decreased with saratin application compared with controls (10.9% +/- 1.8% vs 29.8% +/- 6.8%, P = .0042). This decrease in intimal hyperplasia formation correlated with the inhibition of platelet adhesion. Thirty-three percent of control arteries were found to be thrombosed 2 weeks after carotid endarterectomy compared with a 0% thrombosis rate in the saratin-treated group (P = .0156). No increased bleeding was encountered along the arterial suture line in the saratin group. Bleeding times and systemic platelet counts were not found to change significantly in the saratin-treated rats compared with control rats at 3 and 24 hours after endarterectomy., Conclusion: Saratin significantly decreased platelet adhesion, intimal hyperplasia, luminal stenosis, and thrombosis after carotid endarterectomy in rats. Saratin did not increase suture line bleeding or bleeding times, and did not decrease platelet counts. Saratin may serve as a topical agent to be used for the site-specific inhibition of thrombosis and intimal hyperplasia after vascular manipulation.

Candida albicans is the predominant species of yeast isolated from patients with oral candidiasis, which is frequently a symptom of human immunodeficiency virus infection and is a criterion for staging and progression of AIDS. Salivary histatins (Hsts) are potent in vitro antifungal agents and have great promise as therapeutic agents in humans with oral candidiasis. The molecular mechanisms by which Hsts kill yeast cells are not known. We report here, that unlike other antimicrobial proteins, Hsts do not display lytic activities to lipid membranes, measured by release and dequenching of the fluorescent dye calcein. Analysis of the magnitude and time course of Hst-induced calcein release from C. albicans cells further showed that loss of cell integrity was a secondary effect following cell death, rather than the result of primary disruption of the yeast cell membrane. 125I-Hst 5 binding studies indicated that C. albicans expressed a class of saturable binding sites (KD = 1 microM), numbering 8.6 x 10(5) sites/cell. Both Hst 3 and Hst 4 competed for these binding sites with similar affinities, which is consistent with the micromolar concentration of Hsts required for candidacidal activity. Specific 125I-Hst 5 binding was not detected to C. albicans spheroplasts, which were 14-fold less susceptible to Hst 5 killing, compared with intact cells in candidacidal assays. In overlay experiments, 125I-Hst 5 bound to a 67-kDa protein detected in C. albicans whole cell lysates and crude membrane fractions, but not in the yeast cell wall fraction. Consistent with the overlay data, cross-linking of 125I-Hst 5 to C. albicans resulted in the appearance of a specific 73-kDa 125I-Hst 5-containing complex that was not detected in the cell wall. 125I-Hst 5-binding protein of similar size was also observed in susceptible S. cerevisiae strain TI#20. This is the first description of Hst 5 binding sites on C. albicans which mediate cell killing and identification of a 67-kDa yeast Hst 5-binding protein. The binding characteristics of Hst 5 are in agreement with the observed potency of its biological effect and provide crucial information to the use of Hst 5 as a therapeutic agent. The presence of a specific C. albicans Hst 5-binding protein provides further insight into the potential mechanism of yeast killing and suggests a basis for differential activity between yeast killing and the nontoxic nature of Hsts to humans.

Histatins, histidine-rich proteins found within parotid and submandibular secretions, are a novel class of endogenous peptides with antimicrobial properties. This masked, randomized, placebo-controlled preclinical investigation examined the effect of 3 topical histatins on the development of plaque and gingivitis in beagle dogs. 16, female, 1-year-old beagles were brought to optimal gingival health by mechanical scaling and polishing followed by rigorous daily tooth brushing. At the conclusion of this pretreatment period, dogs were randomly divided into 4 groups for the application of test formulations, and were placed on a plaque-promoting diet. Test agents included 3 synthetic salivary histatins (histatin 5, P-113 and P-113D) which were incorporated in hydroxypropyl methylcellulose gel at a concentration of 0.125%, and a placebo, or negative control, which was the gel vehicle alone. Throughout the 10-week treatment period, test formulations (2.0 ml) were applied 2 x daily to all premolar teeth using a Monojet syringe. Plaque formation and gingival inflammation were assessed using the plaque (PI) and gingival (GI) indices on days 0, 7, 14, 21, 28, 42, 56 and 70. Furthermore, bleeding to probing was recorded as a percent of sites (%BOP) and according to the modified sulcus bleeding index (mSBI). Comparisons among groups and between group pairs (active versus placebo) were made with Kruskal-Wallis tests with the average of data over the interval, days 14-42, being the primary focus of the analysis. From baseline to day 7, all groups expressed similar indices. Thereafter, overall significant differences among the groups were noted at day 42 for PI, at days 21, 28, 42 and 70 for GI, and at days 14 and 28 for %BOP (p < 0.05). In particular, beagles treated with P-113 demonstrated significantly lower PI scores at day 42 (p < 0.05), significantly lower GI scores from days 21 through 42 (p < 0.05), and significantly lower %BOP scores at days 14 and 28 (p < 0.05) compared to beagles treated with placebo. Beagles treated with P-113D exhibited significantly lower GI at day 42 compared to the placebo (p < 0.05). For the primary analysis conducted over the midtreatment interval (days 14-42), significant differences were detected for all parameters except mSBI (p < 0.05). Accordingly, significantly lower PI scores were found for P-113, lower GI scores for P-113 and P-113D, and lower %BOP for P-113 and P-113D compared to placebo (p < 0.05). These data indicate that in the beagle model, salivary histatins, P-113 and P-113D, topically applied, can significantly reduce clinical signs of plaque formation and gingival inflammation.

Patients who have undergone irradiation for head and neck tumors commonly have xerostomia. Loss of the protective constituents normally found in saliva leaves patients at greater risk for development of significant dental pathologic disorders, including gingival and periodontal disease. Periodontal disease and tooth extractions are currently accepted as etiologic factors for the development of osteoradionecrosis. This double-blind crossover trial was conducted to assess the efficacy of a dentifrice containing salivary peroxidase elements in the reduction of gingivitis in a population of patients with irradiated cancer. Subjects were instructed to brush with the dentifrice provided. Plaque and gingival index values were obtained and statistically compared with baseline values. A weak positive effect was found between use of the dentifrice and a reduction in gingival inflammation. Patient compliance was a limiting factor in this treatment effect. The results suggest possible efficacy for the dentifrice in augmenting traditional measures of postradiation oral health maintenance.

Interaction between exposed collagen and platelets and/or von Willebrand factor is believed to be one of the initiating events for thrombus formation at sites of damaged endothelium. Interference with this mechanism may provide an anti-thrombotic potential. Calin, a product from the saliva of the leech Hirudo medicinalis, was tested in vitro and for its in vivo activity in a thrombosis model in hamsters. Calin specifically and dose dependently (IC50:6.5 to 13 micrograms/mL) inhibited human platelet aggregation induced by collagen. In addition, specific platelet adhesion onto microtiter wells coated with collagen and detected with a monoclonal antiglycoprotein IIb/IIIa antibody-conjugated with horseradish peroxidase, could be completely prevented with Calin (IC50:22 micrograms/mL). A dose-response curve was constructed in groups of six hamsters in whom a standardized trauma was induced on the femoral vein. Thrombus formation was followed continuously using video recording and processing of the image obtained upon transillumination of the vessel. Intravenous Calin dose-dependently inhibited platelet-rich thrombus formation in this model with an ED50 of 0.07 mg/kg and complete inhibition with 0.2 mg/kg. No effects were seen on coagulation tests or bleeding times, whereas ex vivo aggregation induced by collagen was inhibited dose dependently. Local application of leech saliva, Calin, hirudin, or the combination of the latter two into the bleeding time wound of hamsters resulted in a mild prolongation of the bleeding time (twofold to threefold). A similar experiment in baboons did not cause any prolongation of the bleeding time. This is in sharp contrast with the long-lasting bleeding after a leech bite itself in both species. Calin from the leech Hirudo medicinalis is able, by binding to collagen, to effectively interfere with platelet-collagen interaction, which results in an antithrombotic effect observed in a platelet-rich thrombosis model in hamsters.

Leech antiplatelet protein (LAPP) is a specific inhibitor of collagen-induced human platelet aggregation and adhesion to collagen under static conditions. Recombinant LAPP (rLAPP) and L-366,763 (acetylated-Cys-Asn-Pro-Arg-Gly-Asp-Cys-NH2), a peptidyl fibrinogen receptor antagonist, were evaluated in an anesthetized baboon thrombosis model using a collagen-coated graft segment of an arteriovenous shunt to elicit thrombus formation. Animals were randomized to receive systemic intravenous administration of rLAPP (100 micrograms.kg-1 x min-1; n = 5), L-366,763 (8.5 micrograms.kg-1 x min-1; n = 3), or saline (n = 3). Despite complete and selective inhibition of type I collagen-induced ex vivo aggregation of platelets, rLAPP had no significant effect on the rate or the extent of 111-In-labeled platelet deposition onto the collagen graft and no effect on template bleeding time. In contrast, L-366,763 completely prevented platelet deposition, maintained blood flow, and significantly prolonged bleeding time at the dosage that inhibited ex vivo aggregation in response to all agonists studied. In this study, the absence of an antithrombotic benefit of rLAPP contrasted sharply with the efficacy of the fibrinogen receptor antagonist. These results demonstrate that specific inhibition of collagen-mediated platelet aggregation alone is not sufficient to prevent platelet-dependent thrombosis in this baboon model.

Votive tablets found during the excavation of shrines of the Graeco-Roman god of medicine (Asklepios or Aesculapius) associate the healing of superficial lesions with contact with the oral cavity of non-poisonous serpents. We suggest that this may have been the empirical exploitation of the healing properties of salivary growth factors. By immunohistochemistry and immunoblotting we demonstrate the expression of the epidermal growth factor and its receptor in the oral, upper digestive, and salivary epithelia of Elaphe quatuorlineata, a species probably used in healing rituals.

Six denture stomatitis patients, all found to have Candida albicans on their maxillary denture and palatal tissue surfaces, volunteered in this preliminary study to test the in vivo efficacy of human salivary antifungal histidine-rich polypeptides (HRPs) in treating their oral disease. The patients were equally divided among the Newton types classification and, as expected, the severity of the inflammation was greatest in the Newton type III patients and least in the Newton type I patients. Patients received sterile solutions of either HRP-3 or HRP-4, which they used both as a mouthrinse and as a denture soak for a period of 1 week. Agar replicas of the tissue-fitting surface of the maxillary dentures revealed HRP reduction and/or elimination of C. albicans from the denture; in one Newton type II individual, this finding directly correlated with a site-specific reduction in palatal inflammation. In the Newton type II and type III individuals alike, there was a significant generalized decrease in inflammation suggesting the therapeutic efficacy of the HRPs. Killing of this yeast species by the HRPs, as determined by scanning electron microscopy (SEM), was probably responsible for the observed clinical benefits noted in this investigation. In the SEM, HRP-treated blastospores appeared severely deflated, as if they had been emptied of significant quantities of intracellular material.

The purpose of this study was to prepare a protein solution to simulate natural saliva using mucin extracted from bovine submaxillary gland. The extraction process was done in keeping with Drans & Leung's (1980) modified technique. Once the bovine submaxillary gland was extracted (approximately 500 g), it is cut and stored in distilled water for 12 hrs at 4 degrees C in order to eliminate any tissue or blood remaining in the section. The tissue is then homogenized and suspended in a phosphate buffer solution with a pH of 7 for 48 hours. The solution is then centrifuged and the supernatant is treated to extract the protein present in the solution. The remaining solution is dialyzed with distilled water, changed three times and then lyophilized. The lyophilized product is diluted in distilled water (1 g/1) taking aliquots to determine the pureness of the proteins. The solution obtained has a viscosity and protein composition which is very similar and offers great advantages for use in patients who suffer from severe xerostomy.