Preclinical Assessment of a MUC12-Targeted BiTE (Bispecific T-cell Engager) Molecule.

MUC12 is a transmembrane mucin that is highly expressed in >50% of primary and metastatic colorectal tumors. MUC12 is also expressed by normal epithelial cells of the colon and small intestine. Although MUC12 localization in normal epithelial cells is restricted to the apical membrane, expression in tumors is depolarized and shows broad membrane localization. The differential localization of MUC12 in tumor cells as compared with normal cells makes it a potential therapeutic target. Here, we evaluated targeting of MUC12 with a BiTE (bispecific T-cell engager) molecule. We generated a panel of proof-of-concept half-life extended (HLE) BiTE molecules that bind MUC12 on tumor cells and CD3 on T cells. We prioritized one molecule based on in vitro activity for further characterization in vivo In vitro, the MUC12 HLE BiTE molecule mediated T-cell-redirected lysis of MUC12-expressing cells with half-maximal lysis of 4.4 ± 0.9 to 117 ± 78 pmol/L. In an exploratory cynomolgus monkey toxicology study, the MUC12 HLE BiTE molecule administered at 200 µg/kg with a step dose to 1,000 µg/kg was tolerated with minimal clinical observations. However, higher doses were not tolerated, and there was evidence of damage in the gastrointestinal tract, suggesting dose levels projected to be required for antitumor activity may be associated with on-target toxicity. Together, these data demonstrate that the apically restricted expression of MUC12 in normal tissues is accessible to BiTE molecule target engagement and highlight the difficult challenge of identifying tumor-selective antigens for solid tumor T-cell engagers.

Role of TRPV1 in colonic mucin production and gut microbiota profile.

This study focuses on exploring the role of sensory cation channel Transient Receptor Potential channel subfamily Vanilloid 1 (TRPV1) in gut health, specifically mucus production and microflora profile in gut. We employed resiniferatoxin (ultrapotent TRPV1 agonist) induced chemo-denervation model in rats and studied the effects of TRPV1 ablation on colonic mucus secretion patterns. Histological and transcriptional analysis showed substantial decrease in mucus production as well as in expression of genes involved in goblet cell differentiation, mucin production and glycosylation. 16S metagenome analysis revealed changes in abundance of various gut bacteria, including decrease in beneficial bacteria like Lactobacillus spp and Clostridia spp. Also, TRPV1 ablation significantly decreased the levels of short chain fatty acids, i.e. acetate and butyrate. The present study provides first evidence that systemic TRPV1 ablation leads to impairment in mucus production and causes dysbiosis in gut. Further, it suggests to address mucin production and gut microbiota related adverse effects during the development of TRPV1 antagonism/ablation-based therapeutic and preventive strategies.

EGF-Containing Membrane-Bound Mucins: A Hidden ErbB2 Targeting Pathway?

Membrane-bound mucins belong to a heterogeneous family of large O-glycoproteins involved in numerous cancers and inflammatory diseases of the epithelium. Some of them are also involved in protein-protein interactions, with receptor tyrosine kinase ErbB2, and fundamental and clinical data showed that these complexes have a detrimental impact on cancer outcome, thus raising interest in therapeutic targeting. This paper aims to demonstrate that MUC3, MUC4, MUC12, MUC13, and MUC17 have a common evolutionary origin and share a common structural organization with EGF-like and SEA domains. Theoretical structure-function relationship analysis of the conserved domains indicated that the studied membrane-bound mucins share common biological properties along with potential specific functions. Finally, the potential druggability of these complexes is discussed, revealing ErbB2-related pathways of cell signaling to be targeted.

Differential requirements for FcγR engagement by protective antibodies against Ebola virus.

Ebola virus (EBOV) continues to pose significant threats to global public health, requiring ongoing development of multiple strategies for disease control. To date, numerous monoclonal antibodies (mAbs) that target the EBOV glycoprotein (GP) have demonstrated potent protective activity in animal disease models and are thus promising candidates for the control of EBOV. However, recent work in a variety of virus diseases has highlighted the importance of coupling Fab neutralization with Fc effector activity for effective antibody-mediated protection. To determine the contribution of Fc effector activity to the protective function of mAbs to EBOV GP, we selected anti-GP mAbs targeting representative, protective epitopes and characterized their Fc receptor (FcγR) dependence in vivo in FcγR humanized mouse challenge models of EBOV disease. In contrast to previous studies, we find that anti-GP mAbs exhibited differential requirements for FcγR engagement in mediating their protective activity independent of their distance from the viral membrane. Anti-GP mAbs targeting membrane proximal epitopes or the GP mucin domain do not rely on Fc-FcγR interactions to confer activity, whereas antibodies against the GP chalice bowl and the fusion loop require FcγR engagement for optimal in vivo antiviral activity. This complexity of antibody-mediated protection from EBOV disease highlights the structural constraints of FcγR binding for specific viral epitopes and has important implications for the development of mAb-based immunotherapeutics with optimal potency and efficacy.

Upregulation of cell-surface mucin MUC15 in human nasal epithelial cells upon influenza A virus infection.

BACKGROUND: Cell-surface mucins are expressed in apical epithelial cells of the respiratory tract, and contribute a crucial part of the innate immune system. Despite anti-inflammatory or antiviral functions being revealed for certain cell-surface mucins such as MUC1, the roles of other mucins are still poorly understood, especially in viral infections. METHODS: To further identify mucins significant in influenza infection, we screened the expression of mucins in human nasal epithelial cells infected by H3N2 influenza A virus. RESULTS: We found that the expression of MUC15 was significantly upregulated upon infection, and specific only to active infection. While MUC15 did not interact with virus particles or reduce viral replication directly, positive correlations were observed between MUC15 and inflammatory factors in response to viral infection. Given that the upregulation of MUC15 was only triggered late into infection when immune factors (including cytokines, chemokines, EGFR and phosphorylated ERK) started to peak and plateau, MUC15 may potentially serve an immunomodulatory function later during influenza viral infection. CONCLUSIONS: Our study revealed that MUC15 was one of the few cell-surface mucins induced during influenza infection. While MUC15 did not interact directly with influenza virus, we showed that its increase coincides with the peak of immune activation and thus MUC15 may serve an immunomodulatory role during influenza infection.

Comparison of Ocular Surface Mucin Expression After Topical Ophthalmic Drug Administration in Dry Eye-Induced Mouse Model.

PURPOSE: To determine the mucinogenic effect of dry eye (DE) treatment drugs currently in use, we compared the levels of mucin production and inflammatory cytokine expression on the ocular surfaces using a DE-induced mice model. METHODS: C57BL/6 mice were separated into 6 groups: a control group, DE-induced mice with the vehicle and treated with cyclosporine A (CsA), rebamipide (Reb), diquafosol tetrasodium (DQS), or prednisolone (Pred). The mRNA expression of MUC 1, 4, 16, 5AC, and proinflammatory cytokines on the corneal epithelia were determined by quantitative real-time polymerase chain reaction. Expression of each MUC was evaluated using flow cytometry and immunohistostaining. Conjunctival goblet cells were analyzed through periodic acid-Schiff (PAS) staining. RESULTS: Desiccating stress significantly decreased both mRNA and protein levels of all MUCs in the cornea. CsA mainly enhanced MUC5AC, with an increase in PAS-positive cells, whereas DQS chiefly increased membrane-associated mucins (MM). However, Reb only minimally increased expression of MUC5AC and Pred only increased MUC4. MUC16 did not show any significant change in any group. On the contrary, the mRNA levels of interleukin (IL)-1ß, -6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were increased in the DE corneas of the control mice and were reduced by all treatments; in particular, IL-6 was significantly suppressed. CONCLUSION: Topical DQS and CsA not only ameliorated ocular surface inflammation under desiccating stress but also upregulated both MM and secretory mucins (SM) and contributed to conjunctival goblet cell recovery, compared to Reb and Pred. Both anti-inflammatory and secretory factors should be considered simultaneously when measuring the treatment effect of DE drugs.

Immunoprevention of Pancreatic Cancer.

BACKGROUND: Pancreatic cancer (PC) is considered an incurable disease due to late diagnosis, rapid spread and negligible response treatment methods, with a 5-year survival rate of only 7%. Hence, there is an urgency in developing novel strategies for PC prevention. This review is focused on discussing the challenges in understanding complex immune functions in tumor microenvironment and host-induced immune responses against tumors, selection of antigens for development of preventive vaccines, lessons from immunoprevention clinical trials and challenges in developing future vaccines. METHODS: 65 original articles were referenced from various sources, based on immunoprevention or criteria pertaining to tumor antigens and immune responses in PC. All these articles were analyzed for the method details and results obtained, and the existing challenges were derived for successful development of clinical immunoprevention strategies. RESULTS: The analysis of these articles and our experience with preclinical efficacy evaluations of various preventive approaches against PC helped in identifying specific tumor antigens as targets which can overcome tumor cell immune suppression. This review discussed the status of primary, secondary and tertiary preventive vaccines and reasons for failure of therapeutic vaccines. The key parameters for effective vaccination were identified, including stage of the disease for vaccination efficacy, use of appropriate animal models for development of preventive vaccines. Potential of chemopreventive agents as adjuvants in immunoprevention was discussed. This review identified new challenges for development of immunopreventive vaccines. CONCLUSION: This review analyzed various aspects of vaccine development for immunoprevention of PC and emphasized the challenges for development of immunoprevention strategies.

A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone.

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1ß upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1ß in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex.

Gastrointestinal Endogenous Protein-Derived Bioactive Peptides: An in Vitro Study of Their Gut Modulatory Potential.

A recently proposed paradigm suggests that, like their dietary counterparts, digestion of gastrointestinal endogenous proteins (GEP) may also produce bioactive peptides. With an aim to test this hypothesis, in vitro digests of four GEP namely; trypsin (TRYP), lysozyme (LYS), mucin (MUC), serum albumin (SA) and a dietary protein chicken albumin (CA) were screened for their angiotensin-I converting (ACE-I), renin, platelet-activating factor-acetylhydrolase (PAF-AH) and dipeptidyl peptidase-IV inhibitory (DPP-IV) and antioxidant potential following simulated in vitro gastrointestinal digestion. Further, the resultant small intestinal digests were enriched to obtain peptides between 3-10 kDa in size. All in vitro digests of the four GEP were found to inhibit ACE-I compared to the positive control captopril when assayed at a concentration of 1 mg/mL, while the LYS < 3-kDa permeate fraction inhibited renin by 40% (±1.79%). The LYS < 10-kDa fraction inhibited PAF-AH by 39% (±4.34%), and the SA < 3-kDa fraction inhibited DPP-IV by 45% (±1.24%). The MUC < 3-kDa fraction had an ABTS-inhibition antioxidant activity of 150 (±24.79) µM trolox equivalent and the LYS < 10-kDa fraction inhibited 2,2-Diphenyl-1-picrylhydrazyl (DPPH) by 54% (±1.62%). Moreover, over 190 peptide-sequences were identified from the bioactive GEP fractions. The findings of the present study indicate that GEP are a significant source of bioactive peptides which may influence gut function.

Novel Therapies to Inhibit Mucus Synthesis and Secretion in Airway Hypersecretory Diseases.

BACKGROUND: In asthma and chronic obstructive pulmonary disease (COPD), airway mucus hypersecretion contributes to impaired mucociliary clearance, mucostasis and, potentially, the development of mucus plugging of the airways. SUMMARY: Excess mucus production can be targeted via therapies that focus on inhibition mucin synthesis, via reducing expression of mucin (MUC) genes, and/or inhibition of mucin secretion into the airways. KEY MESSAGES: This review discusses a number of therapeutic approaches to reduce airway mucus in asthma and COPD, including the use of synthetic and natural products. In particular, it highlights areas where clinical trials of inhibitors of particular target molecules are lacking. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are an example of a targeted therapy that has been researched to reduce mucus synthesis, as have inhibitors of EGFR's downstream signalling pathways, for example, mitogen-activated protein kinase-13 and hypoxia inducible factor-1. However, their efficacy and safety profiles are currently not up to the mark. There is clinical potential in Bio-11006, which reduces mucus secretion via the inhibition of myristoylated alanine-rich C-kinase substrate and is currently in Phase IIb trial.

Dual effects of a lectin from the green seaweed Caulerpa cupressoides var. lycopodium on inflammatory mediators in classical models of inflammation.

OBJECTIVE: Wide biotechnological investigations of only a limited number of seaweed lectins have been performed. We previously demonstrated the anti-nociceptive and anti-inflammatory effects of a lectin isolated from the green seaweed Caulerpa cupressoides var. lycopodium (CcL). Herein, we further studied the mechanisms of action of CcL. METHODS: Classical acute inflammation models induced by different flogistic agents were used to evaluate the anti-inflammatory action of CcL. CcL was injected locally into the rat paw to verify a possible pro-inflammatory outcome. RESULTS: CcL (0.1, 1 or 10 mg/kg; i.v.) reduced the carrageenan-induced rat paw edema and neutrophilic infiltration, which was not altered by either mucin (inhibitor of CcL carbohydrate-binding site) or ZnPP-IX (specific HO-1 inhibitor). Immunohistochemical analyses showed that CcL (1 mg/kg) reduced the expression of the cytokines IL-1ß, TNF-α, IL-6 and COX-2. CcL (0.1, 1 or 10 mg/kg) inhibited dextran, and CcL (1 mg/kg) inhibited histamine-induced rat paw edema. Both effects were reversed by mucin inhibition. CcL (1 mg/kg) was ineffective for the treatment of serotonin- and bradykinin-induced rat paw edema. When injected via the i.pl. route, CcL (10 mg/kg) elicited rat paw edema involving a wide range of mediators. CONCLUSIONS: The anti-inflammatory action of CcL involves the inhibition of IL-1ß, TNF-α, IL-6 and COX-2 expression and histamine H1 receptors. When locally administered, CcL exerts pro-inflammatory actions.

Depletion of mucin in mucin-producing human gastrointestinal carcinoma: Results from in vitro and in vivo studies with bromelain and N-acetylcysteine.

Aberrant expression of membrane-associated and secreted mucins, as evident in epithelial tumors, is known to facilitate tumor growth, progression and metastasis, and to provide protection against adverse growth conditions, chemotherapy and immune surveillance. Emerging evidence provides support for the oncogenic role of MUC1 in gastrointestinal carcinomas and relates its expression to an invasive phenotype. Similarly, mucinous differentiation of gastrointestinal tumors, in particular increased or de novo expression of MUC2 and/or MUC5AC, is widely believed to imply an adverse clinicopathological feature. Through formation of viscous gels, too, MUC2 and MUC5AC significantly contribute to the biology and pathogenesis of mucin-secreting gastrointestinal tumors. Here, we investigated the mucin-depleting effects of bromelain (BR) and N-acetylcysteine (NAC), in nine different regimens as single or combination therapy, in in vitro (MKN45, KATOIII and LS174T cell lines) and in vivo (female nude mice bearing intraperitoneal MKN45 and LS174T) settings. The inhibitory effects of the treatment on cancer cell growth and proliferation were also evaluated in vivo. Our results suggest that a combination of BR and NAC with dual effects on growth and mucin products of mucin-expressing tumor cells is a promising candidate towards the development of novel approaches to gastrointestinal malignancies with the involvement of mucin pathology. This capability supports the use of this combination formulation in locoregional approaches for reducing the adverse effects of the aberrantly secreted gel-forming mucins, as in pseudomyxoma peritonei and similar pathologies with ectopic production of mucin.

A newly developed anti-Mucin 13 monoclonal antibody targets pancreatic ductal adenocarcinoma cells.

Pancreatic cancer is one of the most severe forms of malignancy. Patients with unresectable or metastatic pancreatic cancer usually receive chemotherapy that causes various adverse effects. Antibody-drug conjugates (ADCs), drugs developed by conjugating an anticancer agent to a monoclonal antibody (mAb), can alleviate the side effects of chemotherapy because ADCs selectively bind to cancer cells expressing a particular antigen. We recently developed the recombinant protein DT3C comprising diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). The mAb-DT3C conjugates can be used to select mAbs that are internalized by cells, because the conjugates decrease cell viability only when they are internalized by cells through Ab-antigen reactions. We developed a new mAb to be internalized by TCC-PAN2 cells, a pancreatic carcinoma cell line. The mAb, designated TCC56, recognized Mucin 13 (MUC13), while TCC56­DT3C conjugates induced cell death in TCC-PAN2 cells expressing MUC13. We found that MUC13 was expressed, at least partially, in all 40 pancreatic ductal carcinoma tissues and adjacent non-cancerous tissues analyzed. The expression levels of MUC13 in pancreatic cancer tissues were greater than those in normal tissues. Our findings suggest that MUC13 can be a target molecule for pancreatic cancer treatment. ADCs, including mAb TCC56, could be promising anticancer agents to alleviate the adverse effects of chemotherapy.

Suppressive effects of coixol, glyceryl trilinoleate and natural products derived from Coix Lachryma-Jobi var. ma-yuen on gene expression, production and secretion of airway MUC5AC mucin.

In this study, we investigated whether natural products including coixol derived from Coix Lachryma-Jobi var. ma-yuen affect MUC5AC mucin gene expression, production and secretion from airway epithelial cells. Confluent NCI-H292 cells were pretreated with oleic acid, linoleic acid, glyceryl trilinoleate, beta-stigmasterol or coixol for 30 min and then stimulated with PMA (phorbol 12-myristate 13-acetate), EGF (epidermal growth factor) or TNF-α (tumor necrosis factor-α) for 24 h. The MUC5AC mucin gene expression, mucin protein production and secretion were measured by RT-PCR and ELISA. The results were as follows: (1) Oleic acid, linoleic acid, glyceryl trilinoleate, beta-stigmasterol and coixol inhibited the expression of MUC5AC mucin gene induced by PMA from NCI-H292 cells; (2) Oleic acid, linoleic acid, glyceryl trilinoleate, beta-stigmasterol and coixol also inhibited the production of MUC5AC mucin protein induced by the same inducers from NCI-H292 cells; (3) Coixol inhibited the expression of MUC5AC mucin gene and production of MUC5AC mucin protein, induced by EGF or TNF-α from NCI-H292 cells; (4) Coixol decreased PMA-induced MUC5AC mucin secretion from NCI-H292 cells. This result suggests that coixol, the characteristic component among the examined five natural products derived from C. Lachryma-Jobi var. ma-yuen, can regulate gene expression, production and secretion of mucin, by directly acting on airway epithelial cells.

The eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1): a novel therapeutic target for eosinophilic disorders.

BACKGROUND: Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in all subjects. Additional approaches are clearly needed. OBJECTIVE: We sought to explore the potential of the human eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1) as a therapeutic target for eosinophilic disorders. METHODS: EMR1 expression was assessed in blood and bone marrow specimens from eosinophilic and healthy subjects, cell lines, CD34(+) cells differentiated in vitro, and tissue biopsy specimens by using flow cytometry, quantitative PCR, and immunostaining. Eosinophil targeting by a novel, humanized, afucosylated anti-EMR1 IgG1 was evaluated in vitro by using a natural killer cell-mediated killing assay and in vivo in cynomolgus monkeys. RESULTS: Analysis of blood and bone marrow cells from healthy and eosinophilic donors and in vitro-differentiated CD34(+) cells confirmed restriction of human EMR1 surface and mRNA expression to mature eosinophils. Tissue eosinophils also expressed EMR1. Although EMR1 was highly expressed on eosinophils from all subjects, surface expression was negatively correlated with absolute eosinophil counts (r = -0.46, P < .001), and soluble plasma levels correlated positively with absolute eosinophil counts (r = 0.69, P < .001), suggesting modulation of EMR1 in vivo. Nevertheless, afucosylated anti-EMR1 mAb dramatically enhanced natural killer cell-mediated killing of eosinophils from healthy and eosinophilic donors and induced a rapid and sustained depletion of eosinophils in monkeys. CONCLUSION: EMR1 expression is restricted to mature blood and tissue eosinophils. Targeting of eosinophils with afucosylated anti-EMR1 antibody shows promise as a treatment for eosinophilic disorders.

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species.

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

Mucin-based targeted pancreatic cancer therapy.

The prognosis of pancreatic cancer (PC) patients is very poor with a five-year survival of less than 5%. One of the major challenges in developing new therapies for PC is the lack of expression of specific markers by pancreatic tumor cells. Mucins are heavily Oglycosylated proteins characterized by the presence of short stretches of amino acid sequences repeated several times in tandem. The expression of several mucins including MUC1, MUC4, MUC5AC, and MUC16 is strongly upregulated in PC. Recent studies have also demonstrated a link between the aberrant expression and differential overexpression of mucin glycoproteins to the initiation, progression, and poor prognosis of the disease. These studies have led to increasing recognition of mucins as potential diagnostic markers and therapeutic targets in PC. In this focused review we present an overview of the therapies targeting mucins in PC, including immunotherapy (i.e. vaccines, antibodies, and radioimmunoconjugates), gene therapy, and other novel therapeutic strategies.

Carcinoma mucins trigger reciprocal activation of platelets and neutrophils in a murine model of Trousseau syndrome.

Trousseau syndrome is classically defined as migratory, heparin-sensitive but warfarin-resistant microthrombi in patients with occult, mucinous adenocarcinomas. Injecting carcinoma mucins into mice generates platelet-rich microthrombi dependent on P- and L-selectin but not thrombin. Heparin prevents mucin binding to P- and L-selectin and mucin-induced microthrombi. This model of Trousseau syndrome explains resistance to warfarin, which inhibits fluid-phase coagulation but not selectins. Here we found that carcinoma mucins do not generate microthrombi in mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte ligand for P- and L-selectin. Furthermore, mucins did not activate platelets in blood from PSGL-1-deficient mice. Mucins induced microthrombi in radiation chimeras lacking endothelial P-selectin but not in chimeras lacking platelet P-selectin. Mucins caused leukocytes to release cathepsin G, but only if platelets were present. Mucins failed to generate microthrombi in cathepsin G-deficient mice. Mucins did not activate platelets in blood from mice lacking cathepsin G or protease-activated receptor-4 (PAR4), indicating that cathepsin G activates platelets through PAR4. Using knockout mice and blocking antibodies, we found that mucin-triggered cathepsin G release requires L-selectin and PSGL-1 on neutrophils, P-selectin on platelets, and Src family kinases in both cell types. Thus, carcinoma mucins promote thrombosis through adhesion-dependent, bidirectional signaling in neutrophils and platelets.

Inhibition of secretion, production and gene expression of mucin from cultured airway epithelial cells by prunetin.

This study investigated whether prunetin significantly affects the secretion, production and gene expression of mucin from cultured airway epithelial cells. Confluent primary rat tracheal surface epithelial (RTSE) cells were pretreated with adenosine triphosphate (ATP) for 5 min and then chased for 30 min in the presence of prunetin to assess the effect on mucin secretion using enzyme-linked immunosorbent assay (ELISA). At the same time, confluent NCI-H292 cells were pretreated with prunetin for 30 min and then stimulated with epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) for 24 h, respectively. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription-polymerase chain reaction (RT-PCR) and ELISA. The results were as follows: (1) prunetin significantly suppressed ATP-induced mucin secretion from cultured RTSE cells; (2) prunetin inhibited the production of MUC5AC mucin protein induced by EGF or PMA from NCI-H292 cells; (3) prunetin also inhibited the expression of MUC5AC mucin gene induced by EGF or PMA from NCI-H292 cells. This result suggests that prunetin can regulate the secretion, production and gene expression of mucin, by directly acting on airway epithelial cells.

Macrolide antibiotics inhibit mucus secretion and calcium entry in Swine airway submucosal mucous gland cells.

Macrolide antibiotics such as erythromycin (EM) and azithromycin (AZM) are beneficial in the treatment of mucus hypersecretion in inflammatory pulmonary diseases. Several indirect and direct mechanisms of action have been proposed. This study investigates the direct effect of macrolides on secretory function of isolated submucosal mucous gland cells (SMGCs). We hypothesize that macrolides inhibit the calcium influx necessary for evoked mucus secretion. To test this, we quantified mucin protein release using enzyme-linked immunosorbent assay, calcium-activated K(+) (K(Ca)), and calcium-activated Cl(-) (Cl(Ca)) currents. We measured nonselective cation current (NSCC) using whole-cell patch-clamp techniques; intracellular calcium concentration ([Ca(2+)](i)) was measured using fura-2 Ca(2+) imaging. We found that both EM and AZM are agonists at muscarinic receptors. EM (10 µM) evoked a small but significant increase in mucin release but inhibited the mucin release induced by subsequent acetylcholine (ACh) treatment. Both EM and AZM (10 µM) evoked K(Ca) and Cl(Ca) whole-cell currents, which were blocked by atropine. EM and AZM also accelerated the decay of inositol trisphosphate-induced K(Ca) and Cl(Ca) currents without changing the peak current amplitudes. Likewise, internal application of AZM (10 µM) enhanced the decay rate of ACh-induced K(Ca) and Cl(Ca) currents. EM (1-10 µM) and AZM (0.1-10 µM) slowly (over 25-30 min) inhibited thapsigargin (TG)-induced Ca(2+) entry when applied during the plateau phase of Ca(2+) entry but blunted TG-induced Ca(2+) entry by 70% after a 5-min pretreatment before initiating calcium entry. EM blocked TG-induced NSCC. We conclude that macrolide antibiotics are partial agonists at muscarinic receptors but inhibit stimulated mucus release by inhibiting calcium entry in SMGCs.