Kazuhiko Ishihara

Evaluation of bifidobacterial adhesion to acidic sugar chains of porcine colonic mucins

The aim of this study was to assess the adhesion of Bifidobacterium strains to acidic carbohydrate moieties of porcine colonic mucin. Mucins were extracted and purified via gel filtration chromatography followed by density-gradient ultracentrifugation. The presence of sulfated and sialylated carbohydrates in mucins was shown by enzyme-linked immunosorbent assays using PGM34 and HMC31 monoclonal antibodies (mAbs), respectively. Adhesion of Bifidobacterium strains to mucin preparations was markedly affected by the degree of purification. In eight of 22 strains, we observed increased adhesion to mucin preparations purified by ultracentrifugation. Moreover, in some of these eight strains, adhesion to mucin was reduced by pretreatment with sulfatase and/or sialidase, and competitively inhibited by pretreatment with PGM34 and/or HCM31 mAbs. Our results showed that some Bifidobacterium strains adhered to sulfo- and/or sialomucin and were able to recognize carbohydrate structures of the mAbs epitopes. Graphical Abstract The nitrogen source of the culture medium altered the alternative splicing pattern of serine-type carboxypeptidase ocpG mRNA in Aspergillus oryzae.

Induction of Sda-sialomucin and sulfated H-sulfomucin in mouse small intestinal mucosa by infection with parasitic helminth

Mucin is a major component of mucus on gastrointestinal mucosa. Mucin alteration in the host is considered to be the principal event for expulsion of intestinal helminths. However, it is unclear what mucin alterations are induced by various helminth infections. In this study, the alterations of mouse small intestinal mucin after infection with two nematodes, Nippostrongylus brasiliensis and Heligmosomoides polygyrus, which parasitize the jejunal epithelium, and a cestode, Vampirolepis nana, which parasitizes the ileal epithelium, were examined biochemically and histologically using two anti-mucin monoclonal antibodies (mAbs), HCM31 and PGM34, which recognize Sd(a) antigen, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-, and sulphated H type 2 antigen, Fucα1-2Galβ1-4GlcNAc(6SO₃H)β-, respectively. The goblet cell mucins that reacted with HCM31 increased conspicuously on the jejunal mucosa concurrently with expulsion of N.u2009brasiliensis. Increased levels of HCM31-reactive mucins were observed in the jejunal mucosa after H.u2009polygyrus infection, despite the ongoing parasitism. Goblet cell mucins that reacted with PGM34 increased on the ileal mucosa during V.u2009nana parasitism. Small intestinal goblet cells reacting with the two mAbs were not observed in non-infected mice, although sialomucins and sulfomucins were abundantly present. Additionally, the number of ileal goblet cells that reacted with the two mAbs was increased at the time of expulsion of heterophyid trematode. These results indicate that the type of specific acidic mucins expressed after infection varies among species of intestinal helminth, and, furthermore, that the relationship with worm expulsion is also different.

Reactive increase in gastric mucus secretion is an adaptive defense mechanism against low-dose aspirin-induced gastropathy.

BackgroundGastric mucus is considered to play an essential role in gastric mucosal defense mechanisms, especially when irritants are present in the stomach.AimTo investigate the relationship between low-dose aspirin-induced gastropathy and gastric secretory function, especially gastric mucus secretion, in healthy volunteers.MethodsThirty male, asymptomatic, Helicobacter pylori pylori-negative healthy volunteers were asked to take 100xa0mg of enteric-coated aspirin (Bayaspirin) once a day for 10xa0days. Endoscopic examination was performed before and 3 and 10xa0days after drug administration. The extent of endoscopically assessed gastric mucosal injury was semi-quantitatively evaluated according to the modified Lanza score. The pentagastrin-stimulated gastric juice was collected for 10xa0min during the endoscopic examination and subjected to analysis for gastric acid (mEq/10xa0min) or mucus (mg hexose/10xa0min) output.ResultsOverall, the 10-day aspirin treatment significantly increased gastric mucus secretion from 0.8 (interquartile range 1.7) to 1.6 (1.6)xa0mg hexose/10xa0min (Pxa0<xa00.05), with a concomitant and significant decrease in the gastric acid/mucus ratio from 4.3 (5.2) to 2.9 (4.7) (Pxa0<xa00.01). Subsequent analysis of two subgroups of volunteers categorized according to their endoscopic status (“severe gastropathy” vs. “modest gastropathy”) revealed that changes in gastric secretory parameters occurred exclusively in those subjects without severe gastric injury; there was no alteration in these parameters in subjects with severe gastric injury.ConclusionsThe results of this study suggest that the reactive increase in gastric mucus secretion is an adaptive defense mechanism against low-dose aspirin-induced gastropathy. In some individuals, such a response may be insufficient to prevent the development of severe mucosal injury and even ulcers and their complications.

Protective Effects of Gastric Mucus

The gastric mucosa is continuously exposed to many noxious factors and substances. How the gastric mucosa maintains structural integrity and resists auto-digestion by substances such as acid and pepsin puzzled clinicians and investigators for more than 200 years. The gastric epithelium must also resist damage from extrinsic agents, including Helicobacter pylori (H. pylori) and noxious ingestions such as ethanol and nonsteroidal anti-inflammatory drugs (NSAIDs). The luminal surface of the stomach is covered by a viscoelastic mucus gel layer that acts as a protective barrier against the harsh luminal environment. The structural characteristics of this barrier are primary indicators of its physiological function and changes of its composition have been identified in gastrointestinal pathologies. This chapter presents recent insights into the implication of the gastric mucus barrier as “no mucus, no protection”. While acid, pepsin, and H. pylori are thought to be major factors in the pathophysiology of gastritis, the importance of the mucosal defense system has also been emphasized. Gastric ‘cytoprotection’ refers to a reduction or prevention of chemically induced acute hemorrhagic erosions by compounds such as prostaglandin (PG) and SH derivatives without inhibiting acid secretion in rodents (Robert, 1979; Szabo et al., 1981). Since the concept of ‘cytoprotection’ was introduced, increasing attention has been paid to the effect of medications on the gastric mucosal defensive mechanisms. Although the exact mechanisms of the mucosal defense system are unknown, it involves one or more of the naturally occurring gastric mucosal defensive factors such as mucus metabolism. For estimation of the gastroprotective function, many drugs have been investigated for their activity to protect the gastric mucosa from a variety of necrotizing agents such as ethanol and HCl. Considerable information has accumulated about the gastroprotective function of the mucus that covers the mucosal surface of the stomach.

Interleukin-13/interleukin-4 receptor pathway is crucial for production of Sda-sialomucin in mouse small intestinal mucosa by Nippostrongylus brasiliensis infection

Mucin is a major component of mucus in gastrointestinal mucosa. Increase of specific sialomucins having Sda blood group antigen, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-, is considered to be associated with expulsion of the parasitic intestinal nematode Nippostrongylus brasiliensis. In this study, we examined the relationship between interleukin (IL)-13 pathway and expression of Sda-sialomucins in small intestinal mucosa with N. brasiliensis infection. Nematode infection induced marked increases in small intestinal mucins that reacted with anti-Sda antibody in wild type (wt) mice. However, this increase due to infection was supressed in IL-4 receptor α deficient (IL-4Rα-/-) mice, which lack both IL-4 and IL-13 signaling via IL-4R, and severe combined immunodeficient (SCID) mice, which have defects in B- and T-lymphocytes. Analysis using tandem mass spectroscopy showed that Sda-glycans were not expressed in small intestinal mucins in IL-4Rα-/- and SCID mice after infection despite the appearance of Sda-glycans in the infected wt mice. Inoculation of recombinant IL-13 into the infected SCID mice restored expression of Sda-glycan. Our results suggest that the IL-13/IL-4R axis is important for the production of Sda-sialomucins in the host intestinal mucosa with parasitic nematode infection.

Heat-shock protein 72 promotes platelet aggregation induced by various platelet activators in rats

Increase of thrombus in the coronary arteries is positively correlated with the level of heat-shock protein 72 (HSP72) in the blood of patients with acute myocardial infarction (AMI). Platelet aggregation participates in thrombus formation on ruptured plaque in AMI. In this study, we aimed to clarify the role of HSP72 in thrombus formation by evaluating the effects of HSP72 on platelet aggregation. Platelet aggregation activities were measured in platelet-rich plasma obtained from male Sprague-Dawley rats with or without the platelet activators, such as adenosine diphosphate (ADP), collagen, thrombin receptor-activating peptide-6 (TRAP-6), ristocetin, and arachidonic acid. Changes in aggregation were estimated by the co-addition of recombinant HSP72 and anti-HSP72 antibodies. Our results showed that addition of HSP72 increased platelet aggregation in the presence of low concentrations of ADP, collagen, TRAP-6, ristocetin, and arachidonic acid. Increased platelet aggregation stimulated by ADP and HSP72 was reduced by the co-addition of anti-HSP72 antibodies. Thus, these findings suggested that HSP72 was released extracellularly in response to stress, promoting thrombus formation and AMI. Additionally, treatment with anti-HSP72 antibodies may control platelet aggregation induced by extracellular HSP72.

Discrimination of rat Brunner's gland carbohydrate antigens by site-specific monoclonal antibodies.

Mucus produced and secreted by gastrointestinal mucosa contains various types of mucins equipped with unique sugar chains considered to play critical roles in protecting mucous membranes; therefore, the identification and verification of mucin sugar chains is important for understanding the underlying mechanisms. In our previous work, we generated three monoclonal antibodies (mAbs), RGM22, RGM26, and RGM42, which recognize sugar chains in rat gastric mucin. Here, we immunohistochemically analyzed the rat gastrointestinal mucosa and found that the antigens recognized by RGM22 and RGM42 were expressed in the rat antrum and Brunners glands, whereas that recognized by RGM26 was detected in the antrum, but rarely in Brunners glands. We found that these antibodies reacted with porcine gastric mucin-derived oligosaccharides bearing a common structure: GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-6GalNAc-ol. Moreover, epitope analysis revealed that RGM42 and RGM22 recognized α-linked GalNAc and GalNAcα1-3Gal, respectively, on the GalNAcα1-3(Fucα1-2)Gal structure, whereas RGM26 was specific for GalNAcα1-3(Fucα1-2)Gal. These results indicate that rat Brunners glands express specific antigens bearing GalNAcα1-3Gal that are recognized by RGM22 and RGM42. Thus, RGM22, RGM26, and RGM42 with their unique antigen specificities could be useful tools for investigation of oligosaccharide diversity among mucins.