The Effect of Mucin in Artificial Saliva on Erosive Rehardening and Demineralization.

The effect of mucin in artificial saliva on rehardening (RE-experiment) and inhibition (DE-experiment) of erosion was evaluated. The treatment groups were: artificial saliva with mucin, artificial saliva without mucin, human saliva, and water. For the RE-experiment, after immersion of enamel blocks in citric acid (4 min), hardness was measured and blocks were subjected to treatment for 2 h. For the DE-experiment, sound blocks were subjected to treatment for 2 h and immersed in citric acid (4 min). Percentages of hardness recovery (RE) and loss (DE) were analyzed (ANOVA/Tukey's test). The salivas promoted similar rehardening, but only the saliva with mucin was similar to human saliva with regard to enamel protection against erosion.

α-Selective glycosylation affords mucin-related GalNAc amino acids and diketopiperazines active on Trypanosoma cruzi.

This work addresses the synthesis and biological evaluation of glycosyl diketopiperazines (DKPs) cyclo[Asp-(αGalNAc)Ser] 3 and cyclo[Asp-(αGalNAc)Thr] 4 for the development of novel anti-trypanosomal agents and Trypanosoma cruzi trans-sialidase (TcTS) inhibitors. The target compounds were synthetized by coupling reactions between glycosyl amino acids αGalNAc-Ser 7 or αGalNAc-Thr 8 and the amino acid (O-tBu)-Asp 17, followed by one-pot deprotection-cyclisation reaction in the presence of 20% piperidine in DMF. The protected glycosyl amino acid intermediates 7 and 8 were, in turn, obtained by α-selective, HgBr2-catalysed glycosylation reactions of Fmoc-Ser/Thr benzyl esters 12/14 with αGalN3Cl 11, being, subsequently, fully deprotected for comparative biological assays. The DKPs 3 and 4 showed relevant anti-trypanosomal effects (IC50 282-124 µM), whereas glycosyl amino acids 1 and 2 showed better TcTS inhibition (57-79%) than the corresponding DKPs (13-25%).

Differential expression of cruzipain- and gp63-like molecules in the phytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins.

Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain, respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cellular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry and confocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, presenting co-localization in membrane domains and intracellular regions. Subsequently, we showed that exogenous proteins modulated the production of both protease classes, but in different ways. Regarding the metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surface exposition (≈30%). Conversely, the cruzipain-like molecule was differentially modulated according to the proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively; mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surface exposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation in the cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secreted cruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the parasites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expression and the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cruzipain-like molecules.

Antinociceptive and anti-inflammatory activities of lectin from the marine green alga Caulerpa cupressoides.

The search for new compounds for controlling pain and inflammation, with minimal side effects has focused on marine algae. The aim of this work was to investigate the effect of the purified lectin from the green marine alga Caulerpa cupressoides (CcL) in classical models of nociception and inflammation. Male Swiss mice received i.v. CcL 30 min prior to receiving 0.8% acetic acid (10 ml/kg; i.p); 1% formalin (20 microl; s.c.) or were subjected to thermal stimuli. We observed that CcL (3, 9 or 27 mg/kg) significantly reduced the number of writhes induced by acetic acid by 37.2%; 53.5% and 86.0%, respectively. CcL (27 mg/kg) also reduced the second phase of the formalin test. However, CcL (27 mg/kg) did not present significant antinociceptive effects in the hot plate test, when compared to morphine, suggesting that its antinociceptive action occurs predominantly through a peripheral mechanism. The antinociceptive effects were abolished when CcL was pre-incubated with mucin (20mg/kg; i.v.). When CcL (9 mg/kg) was administered i.v. in Wistar rats 30 min before carrageenan administration, neutrophil counts were reduced by 65.9%. CcL also inhibited paw edema in all time intervals, especially at the third hour. Finally, CcL (9 mg/kg) administered i.v. in mice did not cause hepatic or renal alterations and did not affect body mass or macroscopy of the organs examined. In conclusion, CcL appears to have important antinociceptive and anti-inflammatory activities and could represent an important agent for future studies.

Mucins in the host defence against Naegleria fowleri and mucinolytic activity as a possible means of evasion.

Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM). This parasite invades its host by penetrating the olfactory mucosa. During the initial stages of infection, the host response is initiated by the secretion of mucus that traps the trophozoites. Despite this response, some trophozoites are able to reach, adhere to and penetrate the epithelium. In the present work, we evaluated the effect of mucins on amoebic adherence and cytotoxicity to Madin-Darby canine kidney (MDCK) cells and the MUC5AC-inducing cell line NCI-H292. We showed that mucins inhibited the adhesion of amoebae to both cell lines; however, this inhibition was overcome in a time-dependent manner. N. fowleri re-established the capacity to adhere faster than N. gruberi. Moreover, mucins reduced the cytotoxicity to target cells and the progression of the illness in mice. In addition, we demonstrated mucinolytic activity in both Naegleria strains and identified a 37 kDa protein with mucinolytic activity. The activity of this protein was inhibited by cysteine protease inhibitors. Based on these results, we suggest that mucus, including its major mucin component, may act as an effective protective barrier that prevents most cases of PAM; however, when the number of amoebae is sufficient to overwhelm the innate immune response, the parasites may evade the mucus by degrading mucins via a proteolytic mechanism.

Interaction with host factors exacerbates Trypanosoma cruzi cell invasion capacity upon oral infection.

Outbreaks of severe acute Chagas' disease acquired by oral infection, leading to death in some cases, have occurred in recent years. Using the mouse model, we investigated the basis of such virulence by analyzing a Trypanosoma cruzi isolate, SC, from a patient with severe acute clinical symptoms, who was infected by oral route. It has previously been shown that, upon oral inoculation into mice, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium by engaging the stage-specific surface glycoprotein gp82, whereas the surface molecule gp90 functions as a down-modulator of cell invasion. We found that, when orally inoculated into mice, metacyclic forms of the SC isolate, which express high levels of gp90, produced high parasitemias and high mortality, in sharp contrast with the reduced infectivity in vitro. Upon recovery from the mouse stomach 1h after oral inoculation, the gp90 molecule of the parasites was completely degraded, and their entry into HeLa cells, as well as into Caco-2 cells, was increased. The gp82 molecule was more resistant to digestive action of the gastric juice. Host cell invasion of SC isolate metacyclic trypomastigotes was augmented in the presence of gastric mucin. No alteration in infectivity was observed in T. cruzi strains CL and G which were used as references and which express gp90 molecules resistant to degradation by gastric juice. Taken together, our findings suggest that the exacerbation of T. cruzi infectivity, such as observed upon interaction of the SC isolate with the mouse stomach components, may be responsible for the severity of acute Chagas' disease that has been reported in outbreaks of oral T. cruzi infection.

Inhibition of a p38/stress-activated protein kinase-2-dependent phosphatase restores function of IL-1 receptor-associate kinase-1 and reverses Toll-like receptor 2- and 4-dependent tolerance of macrophages.

Pretreatment of macrophages with Toll-like receptor (TLR)2 or TLR4 agonists leads to a stage of cell hyporesponsiveness to a second stimulation with TLR agonists. This tolerance state is accompanied by the repression of IL-1 receptor-associated kinase-1, mitogen-activated protein kinases, and IkappaB phosphorylation and expression of genes encoding proinflammatory cytokines, like IL-1beta and TNF-alpha. In this report, we demonstrated that mucin-like glycoprotein (tGPI-mucin) of Trypanosoma cruzi trypomastigotes (TLR2 agonist) and LPS (TLR4 agonist) induce cross-tolerance in macrophages and we addressed the role of phosphatase activity in this process. Analysis of the kinetic of phosphatase activity induced by tGPI-mucin or LPS revealed maximum levels between 12 and 24 h, which correlate with the macrophage hyporesponsiveness stage. The addition of okadaic acid, an inhibitor of phosphatase activity, reversed macrophage hyporesponsiveness after exposure to either LPS or tGPI-mucin, allowing phosphorylation of IL-1R-associated kinase-1, mitogen-activated protein kinases, and IkappaB and leading to TNF-alpha gene transcription and cytokine production. Furthermore, pretreatment with either the specific p38/stress-activated protein kinase-2 inhibitor (SB203580) or the NF-kappaB translocation inhibitor (SN50) prevented the induction of phosphatase activity and hyporesponsiveness in macrophage, permitting cytokine production after restimulation with LPS. These results indicate a critical role of p38/stress-activated protein kinase-2 and NF-kappaB-dependent phosphatase in macrophage hyporesponsiveness induced by microbial products that activate TLR2 and TLR4.

Identification and characterization of a novel mouse gene encoding a Ras-associated guanine nucleotide exchange factor: expression in macrophages and myocarditis elicited by Trypanosoma cruzi parasites.

The ability of Trypanosoma cruzi to activate macrophages is, at least in part, attributed to the glycosylphosphatidylinositol-anchored mucin-like glycoproteins (GPI-mucins) expressed in the surface of the trypomastigote stage of the parasite. The differential display reverse transcriptase-polymerase chain reaction and the reverse Northern blot were used to study modulation of gene expression in murine macrophages exposed to GPI-mucins and in cardiac tissues from mice infected with T. cruzi. Among several cDNAs that were more abundant in lanes corresponding to macrophages stimulated with GPI-mucins as compared with resting cells, we confirmed the differential expression of A1, interleukin-18, and GPIgamma4. Some of these genes were also shown to have enhanced expression in the cardiac tissue (DAP-12, A1, and GPIgamma4) from infected animals. The expression of GPIgamma4 was also enhanced in human monocytes stimulated with GPI-mucins or bacterial lipopolysaccharides. The complete sequence of the GPIgamma4 transcript and its gene including the 5' upstream region was defined. GPIgamma4 was encoded by a novel, single copy gene present in mouse as well as human genomes and showed conserved homology to different members of the guanine nucleotide exchange factor family.

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes induce in vivo leukocyte recruitment dependent on MCP-1 production by IFN-gamma-primed-macrophages.

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins from Trypanosoma cruzi trypomastigotes (tGPI-mucins) activate macrophages in vitro to produce proinflammatory cytokines, chemokines, and nitric oxide. These effects of tGPI-mucins may be important in the ensuing immune response to T. cruzi. Here, we have sought evidence for a role of tGPI-mucins in mediating leukocyte recruitment in vivo. tGPI-mucins are highly effective in promoting cell recruitment in the pleural cavity of mice primed with IFN-gamma-inducing agents but not in naïve mice. Maximal recruitment was observed at a dose between 250 and 1250 ng tGPI-mucins. There was a significant elevation in the levels of MCP-1 in the pleural cavity of primed animals injected with tGPI-mucins, and in vivo neutralization of MCP-1 abolished leukocyte recruitment. Pretreatment with anti-MIP-1alpha or anti-RANTES had no effect on the recruitment induced by tGPI-mucins. MCP-1 immunoreactivity was detected in pleural macrophages, and macrophages produced MCP-1 in vitro, especially after priming with IFN-gamma. Finally, tGPI-mucins induced significant leukocyte recruitment in primed C3H/HeJ but not in TLR2-deficient mice. Together, our results suggest that T. cruzi-derived GPI-mucins in conjunction with IFN-gamma may drive tissue chemokine production and inflammation and bear a significant role in the pathogenesis of Chagas disease.

Differential inhibitory mechanism of cyclic AMP on TNF-alpha and IL-12 synthesis by macrophages exposed to microbial stimuli.

Microbial stimuli such as bacterial lipopolysaccharide (LPS) or glycosylphosphatidylinositol-mucins derived from Trypanosoma cruzi trypomastigotes (tGPI-mucins) are effective stimulators of the synthesis of cytokines by macrophages. Here, we evaluated the ability of cyclic AMP mimetic or elevating agents to modulate TNF-alpha and IL-12 synthesis by murine inflammatory macrophages. Cholera Toxin (ChTx) inhibited tGPI-mucins (2.5 nM) or LPS (100 ng ml(-1)) induced TNF-alpha and IL-12(p40) synthesis in a concentration-dependent manner. Similarly, the cyclic AMP mimetics, 8-bromo cyclic AMP or dibutyryl cyclic AMP, or prostaglandin (PG) E2 inhibited the synthesis of both cytokines by macrophages exposed to microbial stimuli. The protein kinase A inhibitor H-89 partially reversed the inhibitory effects of dibutyryl cyclic AMP and PGE2 on both IL-12(p40) and TNF-alpha synthesis. Pretreatment of macrophages with dibutyryl cyclic AMP or ChTx augmented the synthesis of IL-10 triggered by microbial products. Elevation of cyclic AMP inhibited the synthesis of TNF-alpha, but not IL-12(p40), by inflammatory macrophages from IL-10 knockout mice. Kinetic studies showed that synthesis of both TNF-alpha and IL-10 peaked at 8 h and IL-12 at 24 h after stimulation with microbial stimuli. Together, our findings favour the hypothesis that the cyclic AMP inhibitory activity on IL-12(p40) but not on TNF-alpha synthesis is dependent on de novo protein synthesis, most likely involving IL-10, by macrophages stimulated with microbial products. Accordingly, dibutyryl cyclic AMP inhibited IL-12(p40) synthesis only when added before or at the same time of the stimuli. In contrast, the effect of this cyclic AMP analogue on TNF-alpha synthesis was protracted and observed even 2 h after the addition of the stimuli.

A sialic acid-binding lectin from ovine placenta: purification, specificity and interaction with actin.

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since it binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of actin to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.

Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity.

The complement of sialyloligosaccharides present on the surface of human tracheal epithelium has been implicated as an important factor in the selection of hemagglutinin receptor specificity of human influenza A virus. Human strains of influenza A virus preferentially recognize host cell receptors bearing SA alpha 2,6Gal sequences, a sequence which is found on the surface of ciliated tracheal epithelium. A fluorescently-labelled H3 human virus strain bound avidly to the apical surface of human tracheal epithelium, while a fluorescently-labelled receptor variant strain, which preferentially binds SA alpha 2,3Gal sequences, showed little binding to the epithelial surface and localized primarily to intracellular mucin droplets. Extracts of human bronchial mucin, which is known to contain sialic acid primarily in the SA alpha 2,3Gal linkage, was a potent inhibitor of the binding of the receptor variant strain to trachea sections, while the binding of the parent strain was unaffected by the presence of mucin. Human bronchial mucin also inhibited the binding of the receptor variant strains, but not the parent virus strains, to human erythrocytes derivatized to contain SA alpha 2,6Gal sequences. These results suggest that a combination of selection pressures present in the respiratory tract environment have resulted in the evolution of a hemagglutinin receptor specificity in human influenza A virus strains which optimizes recognition of, binding to and infection of host cells.

Mucin allows survival of Salmonella typhi within mouse peritoneal macrophages.

Salmonella typhi is a facultative intracellular human specific pathogen. Both immunocompetent and immunodeficient mice are resistant to S. typhi. However, when they are infected with S. typhi suspended in mucin, the bacteria become pathogenic and infect peritoneal phagocytic cells. The LD50 for mice was 10(5) bacteria suspended in 5% mucin; mouse survival was approximately 48 hours after injection. A high number of bacteria was recovered from peritoneal cells; transmission electron microscopy disclosed a large number of vesicles filled with S. typhi cells in peritoneal cells from infected animals. The addition of mucin to cultures of the reticuloendothelial cell line J774.3 also allowed invasion of the mammalian cells with S. typhi. These data indicate that mucin allows intracellular survival of S. typhi.

Mucin allows survival of Salmonella typhi within mouse peritoneal macrophages

Salmonella typhi is a facultative intracellular human specific pathogen. Both immunocompetent and immunodeficient mice are resistant to S. typhi. However, when they are infected with S. typhi suspended in mucin, the bacteria become pathogenic and infect peritoneal phagocytic cells. The LD50 for mice was 10(5) bacteria suspended in 5 percent mucin; mouse survival was approximately 48 hours after injection. A high number of bacteria was recovered from peritoneal cells; transmission electron microscopy disclosed a large number of vesicles filled with S. typhi cells in peritoneal cells from infected animals. The addition of mucin to cultures of the reticuloendothelial cell line J774.3 also allowed invasion of the mammalian cells with S. typhi. These data indicate that mucin allows intracellular survival of S. typhi

Inhibition of cholera toxin binding to membrane receptors by pig gastric mucin-derived glycopeptides: differential effect depending on the ABO blood group antigenic determinants.

The capacity of pig gastric mucin-derived glycopeptides to interfere with the binding of cholera toxin (CT) to membrane receptors was studied. Two types of glycopeptide preparations with or without human blood group A antigenic activity were assayed for comparison in a system in which the target for the toxin was rat erythrocyte ghosts. Blood group A-active glycopeptides (A+ glycopeptides) were more potent inhibitors for the toxin binding than those lacking group A activity (A- glycopeptides). The mean values of the 50% inhibitory dose revealed that the A+ glycopeptide preparations were 6.6-fold-more potent inhibitors than the A- ones (P less than 0.001). The inhibitory capacity of the different A+ glycopeptide preparations was not directly proportional to the group A antigenic titer. The A+ glycopeptides showed a higher capacity than the A- glycopeptides to interact with the toxin as revealed by CT-glycopeptide complex formation, which could be detected by Sephacryl S-400 chromatography. This result suggests that glycopeptide inhibition of CT binding to the erythrocyte ghosts is mediated by a competition between the GM1 receptors and the glycopeptides for the toxin. The differential effect between both types of glycoconjugates was independent of the way of measuring the amount of glycopeptides used (dry weight, carbohydrate or protein content). The existence in the gastrointestinal tract of mucins not carrying or carrying different ABO blood group determinants, which could behave as more or less potent inhibitors of CT binding to membrane receptors, may help to explain the relationship between ABO blood groups and severity of cholera.