Biological Activity of Porcine Gastric Mucin on Stress Resistance and Immunomodulation.

Purified porcine gastric mucin (PGM) is an alternative biomaterial to native mucin which displays multifunctional properties for exploring a wide range of biomedical applications. The present study evaluated the in vitro (RAW 264.7 macrophage cells) and in vivo (zebrafish embryos and larvae) bioactivities of PGM. The median lethal concentration (LC50) of PGM was 197.9 µg/mL for embryos, while it was non-toxic to RAW 264.7 cells, even at 500 µg/mL. Following PGM exposure (100 µg/mL), a higher embryo hatching rate (59.9%) was observed at 48 h post fertilization, compared to the control (30.6%). Protective effects of PGM from pathogenic Aeromonas hydrophila were demonstrated by high larvae survival rates of 85.0% and 94.0% at 50 and 100 µg/mL of PGM exposure, respectively. Heat tolerance effect of PGM (50 and 100 µg/mL) on larvae (40 °C for 48 h) was confirmed by 75% and 100% of survival rates, respectively. Additionally, PGM reduced the A. hydrophila-induced reactive oxygen species (ROS) generation in larvae. The qRT-PCR results in PGM exposed larvae exhibited induction of immune-related genes (tlr5a and tlr5b, myd88, c-rel, il1ß, tnf-α, il6, il10, cxcl18b, ccl34a.4, defbl1, hamp, ctsd, muc2.1, muc5.1, muc5.2, and muc5.3), stress response (hsp70, hsp90aa1.1, and hsp90ab1), and antioxidant genes (cat and sod1). Moreover, our results revealed that PGM involved in the regulation of transcriptional gene induction increases Hsp90 protein in the zebrafish larvae. Furthermore, upregulation of Il6, Il10, Tnfα, Ccl3, Defa-rs2, Defa21 and Camp and antioxidant genes (Sod2 and Cat) were observed in PGM-exposed RAW 264.7 cells. Overall findings confirmed the activation of immune responses, disease resistance against pathogenic bacteria, heat tolerance, and ROS-scavenging properties by PGM, which may provide insights into new applications for PGM as a multifunctional immunomodulator.

Assessment of the Applicability of Capsid-Integrity Assays for Detecting Infectious Norovirus Inactivated by Heat or UV Irradiation.

Human noroviruses are the leading cause of viral gastroenteritis. In the absence of a practical culture technique for routine analysis of infectious noroviruses, several methods have been developed to discriminate between infectious and non-infectious viruses by removing non-viable viruses prior to analysis by RT-qPCR. In this study, two such methods (RNase and porcine gastric mucin) which were designed to remove viruses with compromised capsids (and therefore assumed to be non-viable), were assessed for their ability to quantify viable F-specific RNA bacteriophage (FRNAP) and human norovirus following inactivation by UV-C or heat. It was found that while both methods could remove a proportion of non-viable viruses, a large proportion of non-viable virus remained to be detected by RT-qPCR, leading to overestimations of the viable population. A model was then developed to determine the proportion of RT-qPCR detectable RNA from non-viable viruses that must be removed by such methods to reduce overestimation to acceptable levels. In most cases, nearly all non-viable virus must be removed to reduce the log overestimation of viability to within levels that might be considered acceptable (e.g. below 0.5 log10). This model could be applied when developing alternative pre-treatment methods to determine how well they should perform to be comparable to established infectivity assays.

Role of sialic acids in feline enteric coronavirus infections.

To initiate infections, many coronaviruses use sialic acids, either as receptor determinants or as attachment factors helping the virus find its receptor underneath the heavily glycosylated mucus layer. In the present study, the role of sialic acids in serotype I feline enteric coronavirus (FECV) infections was studied in feline intestinal epithelial cell cultures. Treatment of cells with neuraminidase (NA) enhanced infection efficiency, showing that terminal sialic acid residues on the cell surface were not receptor determinants and even hampered efficient virus-receptor engagement. Knowing that NA treatment of coronaviruses can unmask viral sialic acid binding activity, replication of untreated and NA-treated viruses was compared, showing that NA treatment of the virus enhanced infectivity in untreated cells, but was detrimental in NA-treated cells. By using sialylated compounds as competitive inhibitors, it was demonstrated that sialyllactose (2,6-α-linked over 2,3-α-linked) notably reduced infectivity of NA-treated viruses, whereas bovine submaxillary mucin inhibited both treated and untreated viruses. In desialylated cells, however, viruses were less prone to competitive inhibition with sialylated compounds. In conclusion, this study demonstrated that FECV had a sialic acid binding capacity, which was partially masked by virus-associated sialic acids, and that attachment to sialylated compounds could facilitate enterocyte infections. However, sialic acid binding was not a prerequisite for the initiation of infection and virus-receptor engagement was even more efficient after desialylation of cells, indicating that FECV requires sialidases for efficient enterocyte infections.

Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.

Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA) appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.

Mucin biopolymers as broad-spectrum antiviral agents.

Mucus is a porous biopolymer matrix that coats all wet epithelia in the human body and serves as the first line of defense against many pathogenic bacteria and viruses. However, under certain conditions viruses are able to penetrate this infection barrier, which compromises the protective function of native mucus. Here, we find that isolated porcine gastric mucin polymers, key structural components of native mucus, can protect an underlying cell layer from infection by small viruses such as human papillomavirus (HPV), Merkel cell polyomavirus (MCV), or a strain of influenza A virus. Single particle analysis of virus mobility inside the mucin barrier reveals that this shielding effect is in part based on a retardation of virus diffusion inside the biopolymer matrix. Our findings suggest that purified mucins may be used as a broad-range antiviral supplement to personal hygiene products, baby formula or lubricants to support our immune system.

Natural antibiotic function of a human gastric mucin against Helicobacter pylori infection.

Helicobacter pylori infects the stomachs of nearly a half the human population, yet most infected individuals remain asymptomatic, which suggests that there is a host defense against this bacterium. Because H. pylori is rarely found in deeper portions of the gastric mucosa, where O-glycans are expressed that have terminal alpha1,4-linked N-acetylglucosamine, we tested whether these O-glycans might affect H. pylori growth. Here, we report that these O-glycans have antimicrobial activity against H. pylori, inhibiting its biosynthesis of cholesteryl-alpha-D-glucopyranoside, a major cell wall component. Thus, the unique O-glycans in gastric mucin appeared to function as a natural antibiotic, protecting the host from H. pylori infection.

Oral administration of swine gastric mucin alters levels of serum and urine nitric oxide, and lipid metabolism in mice.

Mice were fed with swine gastric mucin in a basal diet for 5 weeks. In 5 week-old mice, a 2% mucin diet significantly decreased nitric oxide levels of serum and liver. Reduction of serum total cholesterol and triglyceride and increase of HDL-cholesterol level were also significant with the mucin diet. In 14 month-old mice, the mucin diet was less effective.

Acid-dependent adherence of Helicobacter pylori urease to diverse polysaccharides.

BACKGROUND & AIMS: The significance of acid-primed recognition of ligands by Helicobacter pylori urease is unknown. This study aimed to further characterize the specificity of urease adherence in vitro and verify whether specific inhibition will translate into in vivo suppression of colonization. METHODS: A highly sensitive competitive enzyme-linked ligand capture assay was used to quantify the capacity of each test inhibitor to compete with labeled mucin for binding sites on immobilized native urease. A model polymer that strongly bound urease was used in an in vivo trial using euthymic hairless mice as an infection model. RESULTS: The blockage of urease-gastric mucin interaction by certain inhibitors revealed an acid-functional lectin-like activity by urease, specifically recognizing bacterial lipopolysaccharides and certain species of polysaccharides, nonbacterial glycolipids, and glycoproteins. Dextran sulfate significantly (P < 0.01) suppressed colonization of mice by H. pylori when given before and/or after challenge. CONCLUSIONS: The acid-driven high-affinity adherence of H. pylori urease to mucin and lipopolysaccharides contributes to gastric mucosal colonization by the bacterium based on in vivo targeting experiments using specific polysaccharides in a mouse model with acute infection. Acid-functional urease-homing polysaccharides that can interfere with urease-mucin or H. pylori whole cell-mucin interaction in vitro can significantly interfere with colonization by the bacterium in vivo.

Inhibition of Helicobacter pylori sialic acid-specific haemagglutination by human gastrointestinal mucins and milk glycoproteins.

Helicobacter pylori, a human gastric pathogen causing chronic gastritis and duodenal ulcer disease, has been found in large amounts in gastric mucous gel layer. Mucin preparations, separated from human gastric juices and isolated from different colon regions, were examined for their ability to inhibit haemagglutination of H. pylori with the emphasis on evaluating the role of sialic acid-dependent haemagglutinins of the bacteria in colonisation of the stomach. The mucins showed high inhibitory activity for H. pylori, which was significantly decreased after the removal of sialic acids from the mucins. The inhibitory potencies using high molecular mass mucin-like components from bovine milk were comparable with those obtained for gastric mucins, suggesting their possible role in the prevention of H. pylori infection.

The effects of reductive dissociation of gastric mucin isolated in the presence of proteinase inhibitors.

Mucin was purified by the gel filtration method on columns with high porous molecular sives in buffers with SDS and proteinase inhibitors. The addition of proteinase inhibitors distinctly inhibited proteolytic activity. It was found that the obtained mucin, after disulphide-bound reduction, is dissociated to mucin subunits and N-glycosylated glycoprotein of molecular weight about 75 kDa. This protein has carbohydrate and amino acid composition different from high molecular fraction. The 75 kDa protein is strongly associated with high molecular mass mucin subunits and can be separated either during electrophoresis or fractionation in buffers with 2-mercaptoethanol.

Macromolecular mechanisms of sputum inhibition of tobramycin activity.

Tobramycin, an aminoglycoside antibiotic, is used in the treatment of Pseudomonas aeruginosa infections in cystic fibrosis patients. Tobramycin bioactivity, however, is antagonized by sputum. Glycoproteins (mucins) and high-molecular-weight DNA make up 2 to 3% (P. L. Masson and J. F. Heremans, p. 412-475, In M. J. Dulfano, ed., Sputum: Fundamentals and Clinical Pathology, 1973) and 3 to 10% (W. S. Chernick and G. J. Barbero, Pediatrics 24:739-745, 1959, and R. Picot, I. Das, and L. Reid, Thorax 33:235-242, 1978) of the dry weight of sputum, respectively. tobramycin binds to both mucins and DNA obtained from sputum (R. Ramphal, M. Lhermitte, M. Filliat, and P. Roussel, J. Antimicrob. Chemother. 22:483-490, 1988). In vitro, recombinant human DNase (rhDNase) hydrolyzes high-molecular-weight DNA of > 50 kb within sputum to fragments of 2 to 4 kb. Studying dialyzable tobramycin, we examined drug binding to whole sputum and to "mock sputum," which consisted of porcine gastric mucin and calf thymus DNA. We also studied the effects of rhDNase treatments of sputum, mock sputum, and calf thymus DNA on tobramycin binding. We found that treatments of sputum, mock sputum, and calf thymus DNA with rhDNase did not significantly increase the tobramycin bioactivity within the dialysates; surprisingly, sputum binding of tobramycin was increased by rhDNase. We conclude that rhDNase does not increase the bioactivity of tobramycin in sputum.

In vitro adsorption of a hydrophobic mutagen to gastrointestinal mucus glycoprotein (mucin) and dietary fibre.

The adsorption of mutagens by some dietary fibres has been suggested as one mechanism by which dietary fibres protect against colorectal cancer. It is thought that these dietary fibres carry the mutagen out of the digestive tract, decreasing the effective mutagen concentration to which epithelial cells are exposed. The ability of gastrointestinal mucin to alter the extent to which the hydrophobic mutagen 1,8-dinitropyrene (DNP) adsorbs in vitro onto the insoluble dietary fibre alpha-cellulose, was investigated. It was found that crude and purified human ileal mucins themselves adsorbed DNP and decreased the adsorption of DNP onto alpha-cellulose. Purified mucin which had been treated with trypsin also adsorbed DNP. These studies suggest that in the digestive tract there would be competition for the adsorption of DNP between mucin and insoluble dietary fibres, such as alpha-cellulose. This factor must be considered in predictions about the distribution of hydrophobic, mutagenic carcinogens in the digestive tract and their role in the etiology of colorectal cancer.

An antibiotic-free medium for the xenic cultivation of Entamoeba gingivalis.

Diamond's TYI-S-33 (Trypticase-Yeast Extract-Iron-Serum) medium was used as the basis for a new antibiotic-free medium for xenic growth of Entamoeba gingivalis. Nutritional requirements of the oral protozoan were determined in an effort to optimize growth. TYI-S-33 medium did not support E. gingivalis growth prior to modification. The changes included: (a) deletion of L-cysteine.HCl and thioctic acid, (b) substitution of glucose for dextran I (mol. wt 185,000) or rice starch, (c) reduction of concentrations of tryptone (2.5 g l-1), yeast extract (1.25 g l-1) and dextran I (1 g l-1), (d) increased concentration of ferric ammonium citrate (0.2 g l-1), and (e) addition of gastric mucin (2.4 g l-1). Dextran I was chosen as the major carbon source; its use in the medium limited growth of accompanying bacteria. This new antibiotic-free medium significantly increased E. gingivalis growth (16-20 E. gingivalis trophozoites observed per field) as compared to growth in Diamond's TYSGM-9 (Trypticase-Yeast Extract-Serum-Gastric Mucin) medium (six to 10 E. gingivalis trophozoites observed per field).

Promotion of Pasteurella haemolytica infection in mice by iron.

Mice given 60 micrograms iron, as aqueous ferric ammonium citrate, intravenously were more susceptible than untreated controls to intraperitoneal infection with T serotypes of Pasteurella haemolytica as shown by significant reductions in LD50 values. Iron injection has advantages over administration of bacteria suspended in mucin for studies of P haemolytica infection in mice.

Hemoglobin enhancement of experimental infection of mice with Pasteurella haemolytica.

A 2% hemoglobin preparation injected into mice along with Pasteurella haemolytica culture was found to be as good an enhancer of virulence as a 7% swine gastric mucin preparation. The hemoglobin preparation was easier to prepare and was less toxic than mucin for mice when injected intraperitoneally.

[Effect of the sialic acids of mucin on its ability to increase the virulence of serotype A meningococci]. / Vliianie sialovykh kislot mutsina na ego sposobnost' povyshat' virulentnost' meningokokka serogruppy A.

The influence of the content of bound sialic acids in mucin on its capacity to decrease the infective dose of meningococcal culture has been studied; as a result, the direct relationship with a high degree of correlation between these two characteristics has been revealed. The direct relationship between the content of bound sialic acids and the viscosity of aqueous mucin solutions has also been revealed in this study. The above-mentioned biological activity of mucin is not observed in the presence of free sialic acids.

The dynamics of experimental bacterial infections in muscles of mice and the influence of hog gastric mucin.

NMRI-mice were infected in the posterior of the thigh muscles of the right hind leg by injecting one of two isogenic strains of E. coli, one of which was passaged from mouse to mouse for 20 years [mouse-pathogenic (MP) strain], whereas the other was transferred on agar slants only for the same period of time (lab strain). Both strains cause persistent infections only if given as undiluted overnight culture, the MP-strain seeming slightly more virulent. Injection of lower numbers of bacteria results in a corresponding decrease of bacterial cell counts in the animals' thighs. The elimination of the bacteria seems to be all the more rapid the smaller the inoculum. If the infection was administered in mucin suspension, the MP-strain multiplies in the host tissue to a greater degree than the lab strain, irrespectively of the dose injected. The number of cells per muscle reaches a maximum level of about 10(9) even after injecting 10(2) cells of the MP-strain in mucin. To reach the same level, 10(5) cells of the lab strain are needed. Four days after infection, the bacteria start to die off in those animals originally receiving the lower infection doses. Possible reasons are discussed.

Mucin model for group B type III streptococcal infection in mice.

An experimental murine infection was established by the intraperitoneal injection of a log-phase culture of a laboratory reference strain of Streptococcus agalactiae, Lancefield group B, type III (strain SS620), suspended in sterile hog gastric mucin. The enhancement of streptococcal virulence was measured by a significantly increased mortality in outbred ICR Swiss mice. An inbred C57BL6 strain of mice was resistant to the mucin-bacterial combination. Mucin, treated with Desferal to chelate the iron, did not lose the capacity to enhance the virulence of group B, type III streptococci in ICR Swiss mice. Iron-dextran was not a suitable substitute for mucin and failed to enhance the virulence of group B, type III streptococci. The results of these studies indicate that iron is not the resistance-lowering factor in this group B, type III streptococci-mucin model.