A Chondroitin Sulfate Chain of Urinary Trypsin Inhibitor Enhances Protease Inhibitory Activity of the Core Protein.

Human urinary trypsin inhibitor (UTI) is a proteoglycan composed of one core protein covalently linked to one glycosaminoglycan, which is a low sulfated chondroitin 4-sulfate. It is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, functions of the chondroitin sulfate have not been clarified. Recently, we succeeded in remodeling the UTI chondroitin sulfate to hyaluronan to create hyaluronan hybrid UTI, without changing the core protein. Here, we investigated the effect of the remodeled chondroitin sulfate on the activities of serine proteases. Native UTI showed stronger protease inhibitory activity than hyaluronan hybrid UTI or hydrolyzed glycosaminoglycan UTI. Chondroitin 4-sulfate chains with a small peptide derived from the native UTI did not show any protease inhibitory activity. These results suggest that the chondroitin sulfate chain linked covalently to core protein enhances protease inhibitor activity of UTI although the chondroitin sulfate chain itself does not.

A Synbiotic with Tumor Necrosis Factor-α Inhibitory Activity Ameliorates Experimental Jejunoileal Mucosal Injury.

Despite the recent development of biological modifiers for inflammatory bowel diseases (IBD), there continues to be considerable interest in fermented medicines because of its negligible adverse effects. We previously showed that the synbiotic Gut Working Tablet (GWT) alleviates experimental colitis. Here we show that GWT is capable of ameliorating jejunoileal mucosal injury, which is frequently seen with IBD. We created experimental jejunoileal mucositis in rats by injection of methotrexate (MTX) which increases intestinal permeability, a hallmark finding of IBD. Administering GWT to MTX-injected rats restored intestinal integrity by reversing villi shortening, crypt loss, and goblet cell depletion in the mucosa. Also GWT reduced activities of myeloperoxidase and lipid peroxidase and increased superoxide dismutase activity, which is critical for maintaining intestinal function. We further found that GWT suppressed mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) in macrophage and reduced TNF-α mRNA expression in specimens with experimental colitis, which is in contrast to VSL#3 that enhanced TNF-α production. Together, the current and previous animal studies clearly demonstrate the protective role of GWT in chemically induced enterocolitis. Crohn's disease, a well-known IBD, can affect any portion of the intestine, and these results suggest that GWT may be useful as a novel therapeutic or maintenance therapy for IBD.

Enzymatic synthesis of hyaluronan hybrid urinary trypsin inhibitor.

Human urinary trypsin inhibitor is a proteoglycan that has a single low-sulfated chondroitin 4-sulfate chain at the seryl residue in position 10 of the core protein as a glycosaminoglycan moiety, and is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, the functions of the glycosaminoglycan moiety have not yet been elucidated in detail. In the present study, the glycosaminoglycan chains of a native urinary trypsin inhibitor were remodeled to hyaluronan chains, with no changes to the core protein, using transglycosylation as a reverse reaction of the hydrolysis of bovine testicular hyaluronidase, and the properties of the hybrid urinary trypsin inhibitor were then analyzed. The trypsin inhibitory activitiy of the hyaluronan hybrid urinary trypsin inhibitor was similar to that of the native type; however, its inhibitory effect on the hydrolysis of hyaluronidase were not as strong as that of the native type. This result demonstrated that the native urinary trypsin inhibitor possessed hyaluronidase inhibitory activity on its chondroitin sulfate chain. The hyaluronan hybrid urinary trypsin inhibitors obtained affinity to a hyaluronan-binding protein not exhibited by the native type. The interactions between the hyaluronan hybrid urinary trypsin inhibitors and phosphatidylcholine (abundant in the outer layer of plasma membrane) were stronger than that of the native type. Hyaluronan hybrid urinary trypsin inhibitors may be useful for investigating the functions of the glycosaminoglycan chains of urinary trypsin inhibitors and hyaluronan, and our hybrid synthesizing method may be used widely in research for future medical applications.

Study of the mechanisms of a Japanese traditional fermented medicine in the improvement of constipation.

BACKGROUND AND AIM: Gut Working Tablet (GWT) is a Japanese traditional fermented medicine based on Aspergillus oryzae NK-fermented grain germ. Although GWT has been used by patients with constipation, the mechanism has not been investigated. The aim of this study was to examine the possible mechanisms of the effect of GWT on constipation. METHODS: The effect of GWT water extracts on gut contractility using ileum strips from guinea pig and on the growth of Bifidobacterium longum were examined in vitro. The 14 Sprague Dawley rats were administered loperamide at 10 mg/day per kg for 3 days. They were fed with and without 5% of GWT before and during administration of loperamide. Number of stools and weight of feces were measured before and during administration of loperamide. The concentrations of short-chain fatty acids (SCFAs) in the feces and cecal contents were measured by gas chromatography. RESULTS: GWT water extracts dose-dependently induced ileal contractile responses, which were inhibited by atropine. The growth of B. longum was increased in the presence of GWT water extracts in a dose-dependent manner (P < 0.01 vs control). The decrease in both the number and weight of feces caused by loperamide was improved by GWT administration (P < 0.05 vs loperamide). The decrease in the butyric acid concentration in feces and cecal contents induced by the administration of loperamide was inhibited by GWT (P = 0.035 and 0.018). CONCLUSION: GWT water extracts may induce cholinergic-like stimulation and promote the growth of probiotics. Furthermore, GWT water extract contributed to normalization of colonic SCFAs. These results may explain, at least in part, the beneficial effects of GWT on constipation.

Characterization and usefulness of stool antigen tests using a monoclonal antibody to Helicobacter pylori catalase.

BACKGROUND AND AIM: Two types of stool antigen tests have been used in the management of Helicobacter pylori infection. Testmate Pylori Antigen enzyme immunoassay (TPAg EIA) is a direct sandwich enzyme immunoassay (EIA) while Testmate Rapid Pylori Antigen (Rapid TPAg) is performed using immunochromatography. The aim of this study was to study the characterization and usefulness of these tests. METHODS: Accuracy of both tests was studied using 111 fecal samples obtained from H. pylori-positive or -negative patients. Cross-reactivity was examined with four other Helicobacter spp. and five fecal bacteria in humans. To estimate the sensitivity of both kits, we tested H. pylori clinical strains. We also examined the diagnostic performances of both tests after the storage for 12 months. RESULTS: The accuracy of both Testmate kits was 100% in fecal samples from 111 patients. No cross-reactivity was observed in both Testmate kits in five fecal bacteria and four other Helicobacter spp. TPAg EIA and Rapid TPAg showed positive results in 1342 of 1344, and 483 of 485 clinical strains, respectively. Diagnostic performances was maintained for 12 months when TPAg EIA was stored at 4°C and Rapid TPAg at 30°C. CONCLUSIONS: We examined the details of high accuracy of TPAg EIA and Rapid TPAg. The diagnostic performance of both kits was maintained after storage for up to 1 year. The two types of tests would be useful in the management of H. pylori infection.

Serodiagnosis of Helicobacter hepaticus infection in patients with liver and gastrointestinal diseases: western blot analysis and ELISA using a highly specific monoclonal antibody for H. hepaticus antigen.

BACKGROUND: Helicobacter hepaticus infection might be associated with liver and biliary tract diseases. To investigate its pathogenic role, the properties of anti-H. hepaticus serum antibody in patients with liver and diseases were elucidated. METHODS: Serum samples were collected from 166 patients-69 with liver diseases, 38 with upper gastrointestinal diseases, 17 with lower gastrointestinal diseases, 26 with biliary tract diseases, and 16 with pancreas diseases; 30 control sera were obtained from 30 healthy blood donors. Serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA) and western blot using the new monoclonal antibody HR II-51. RESULTS: Anti-H. hepaticus serum antibody concentrations in patients with liver disease (n = 69) were significantly increased compared with those in other disease groups (p = 0.014 to <0.001). Particularly, liver cirrhosis (n = 19) showed a significantly higher antibody level compared with other liver diseases (n = 50, p = 0.005) and healthy donors (n = 30, p = 0.0005), as well as a higher seroprevalence (68.4%) compared with other liver diseases (p = 0.05) and healthy donors (p = 0.004). Furthermore, the ELISA value in liver cirrhosis (n = 19) was significantly higher than that in patients with hepatitis B virus (HBV)-and/or hepatitis C virus (HCV)-infected chronic hepatitis (n = 15) (0.389 ± 0.084 vs. 0.350 ± 0.084, p = 0.029). However, there was no relationship between the total immunoglobulin concentration and the anti-H. hepaticus antibody level in each liver disease (Spearman's rank correlation coefficient [rs] < 0.225). CONCLUSIONS: H. hepaticus infection might play a role in the development of liver diseases; in particular, it might increase the risk of the development of HBV- and/or HCV-infected liver diseases.

Serological analysis of Helicobacter hepaticus infection in patients with biliary and pancreatic diseases.

BACKGROUND AND AIMS: Infection with Helicobacter hepaticus has been associated with development of hepatocellular carcinoma and gallstones in animal models. In humans, however, the association of H. hepaticus infection with biliary and pancreatic diseases has not been elucidated. The aim of this study was to serologically examine the prevalence of H. hepaticus infection in patients with biliary and pancreatic diseases. METHODS: Serum samples obtained from 55 patients with cholelithiasis, 18 with bile duct or gallbladder cancer and 19 with pancreatic cancer were studied. Sera were obtained from 34 control subjects who underwent endoscopy and were diagnosed as not having peptic ulcers or cancers. Seropositivity of H. hepaticus was examined by western blot analysis using a H. hepaticus-specific antigen. To validate the specificity, positive sera were also tested after absorption with H. hepaticus whole-cell sonicate. Serum samples were also tested for the presence of anti-Helicobacter pylori antibody. RESULTS: Prevalence of antibody to H. hepaticus-specific antigen in patients with bile tract cancer was 38.8% and was significantly higher than in control subjects (13.1%, P < 0.05). Prevalence of antibody to H. hepaticus-specific antigen was 18.2% and 10.5% in patients with cholelithiasis and pancreatic cancer, respectively. Seropositivity for H. pylori was similar in all groups. Detection of the H. hepaticus-specific band was significantly decreased after the sera were absorbed with H. hepaticus whole-cell sonicate. CONCLUSION: Infection with H. hepaticus might be associated with bile duct cancer. Results obtained from absorbed sera suggested high specificity of the western blot analysis.

Characterization and application of a new monoclonal antibody with high specificity for Helicobacter hepaticus.

BACKGROUND AND AIMS: Infection with Helicobacter hepaticus is suggested to play a role in the pathogenesis of chronic liver disease in humans. However, reactive antigens among Helicobacter species make the development of an H. hepaticus ELISA test with high specificity difficult. A new monoclonal antibody from a hybridoma clone (HRII-51) showed high specificity to H. hepaticus without cross-reaction to other gastrointestinal bacteria. METHODS: The molecular weight of HRII-51 immunoreactive antigen was examined by Western blot of H. hepaticus probed with the monoclonal antibody HRII-51. A HRII-51-immunoreactive antigen capture ELISA was prepared in which the specific antigen was anchored by HRII-51-immobilized ELISA plate. Accuracy of HRII-51 antigen capture ELISA was examined using sera obtained from mice inoculated with Helicobacter species. Specificity of HRII-51 antigen capture ELISA was compared to that of H. hepaticus antigen-based ELISA using human sera with absorption by H. pylori cell lysate. RESULTS: HRII-51 immunoreactive antigen had a molecular weight of 15 kDa. Sensitivity and specificity of HRII-51 antigen capture ELISA were 87.0% and 97.6% in mice inoculated with Helicobacter species. In human sera, modification of the results by absorption with H. pylori lysate was smaller in HRII-51 antigen capture ELISA comparing with H. hepaticus-antigen-based ELISA. CONCLUSION: Use of the HRII-51 antigen capture ELISA would be a useful approach for the serodiagnosis of H. hepaticus infection in both experimental animals and humans.

Free radicals in phacoemulsification and aspiration procedures.

OBJECTIVES: To detect free radicals in phacoemulsification and aspiration procedures using electron-spin resonance and to observe the effect of ophthalmic viscosurgical devices (viscoelastic agents) on free radical intensity. METHODS: (1) A test tube containing BSS Plus (Alcon Laboratories, Inc, Fort Worth, Tex) with 1% of the spin-trapping agent, 5,5'-dimethyl-1-pyrroline N-oxide, without irrigation and aspiration, was exposed to ultrasound (100% for 20 seconds). A preparation of hyaluronate sodium (Healon [a cohesive agent that contains 1% hyaluronate sodium] (Pharmacia, Uppsala, Sweden) or Viscoat [a dispersive agent that contains 3% hyaluronate sodium and 4% chondroitin sulfate] (Alcon Laboratories, Inc)) was added to the solution to observe inhibitory effects. (2) To simulate a clinical procedure, an eye model with irrigation and aspiration of a combination of 1% 5,5'-dimethyl-1-pyrroline N-oxide and BSS Plus, 25 mL/min, as the irrigating solution was exposed to ultrasound (for 10, 20, or 30 seconds). Healon or Viscoat was injected into the anterior chamber. Free radicals were measured by an electron-spin resonance spectrometer. RESULTS: (1) A characteristic signal corresponding to hydroxyl radicals was detected. Similar inhibition by Healon and Viscoat was observed. (2) Two ophthalmic viscosurgical devices similarly suppressed the signal at 10 seconds. The inhibition by Healon ceased at 20 seconds, whereas Viscoat suppressed the signal throughout the time course. CONCLUSIONS: Phacoemulsification produces hydroxyl radicals in the anterior chamber even with irrigation and aspiration. The effect of ophthalmic viscosurgical devices on free radicals depends on the retention of the materials within the anterior chamber. CLINICAL RELEVANCE: There are complications associated with phacoemulsification.

Catalase, a specific antigen in the feces of human subjects infected with Helicobacter pylori.

Recently, we reported the production of three new monoclonal antibodies with high specificity for a Helicobacter pylori antigen suitable for diagnosis of H. pylori infection. The aim of the present study was to identify the antigen recognized by these monoclonal antibodies concerning both H. pylori and the feces of human subjects infected with H. pylori. The cellular antigen was purified from an H. pylori cell extract by immunoaffinity column chromatography with the monoclonal antibody as a ligand. The amino-terminal amino acid sequences (eight residues) of the purified antigen and H. pylori catalase were the same. The molecular weights of native and subunit, specific catalase activity, and UV and visible spectra of the purified antigen were in good agreement with those of H. pylori catalase. The human fecal antigens were purified from two fecal samples of two H. pylori-positive subjects by ammonium sulfate precipitation, CM-Sephadex C(50) chromatography, and the same immunoaffinity chromatography used for the H. pylori cellular antigen. The fecal antigens had catalase activity. The amino-terminal amino acid sequences (five residues) of the human fecal antigen and H. pylori catalase were the same. The monoclonal antibodies reacted with the native cellular antigen, but did not react with the denatured antigen, human catalase, and bovine catalase. The results show that the target antigen of the monoclonal antibodies is native H. pylori catalase and that the monoclonal antibodies are able to specifically detect the antigen, which exists in an intact form, retaining the catalase activity in human feces.

Production and application of new monoclonal antibodies specific for a fecal Helicobacter pylori antigen.

The aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecal Helicobacter pylori antigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity for H. pylori antigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form of H. pylori, and fecal samples from H. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41 H. pylori clinical isolates and in fecal samples from seven H. pylori-positive subjects. Several kinds of Helicobacter species (Helicobacter felis, Helicobacter hepaticus, Helicobacter mustelae, and Helicobacter cinaedi) except H. pylori, major bacteria in feces (Campylobacter jejuni, Bacteroides vulgatus, Bifidobacterium breve, Bifidobacterium infantis, and Escherichia coli), and fecal samples from six H. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis of H. pylori infection.