New screening methods for probiotics with adhesion properties to sialic acid and sulphate residues in human colonic mucin using the Biacore assay.

AIMS: To determine the relationship between adhesive ability of probiotics and acidic residues in human colonic mucin, we developed a new screening method using Biacore to evaluate adherence of bacteria before and after sialic acid or sulphate residues were blocked or removed from mucin. METHODS AND RESULTS: Ten strains of lactobacilli and three strains of bifidobacteria isolated from human faeces were evaluated for their adhesive properties to soluble human colonic mucin (sHCM) using the Biacore binding assay. Three strains (Lactobacillus strain ME-522, Lact. gasseri ME-527 and Bifidobacterium bifidum MCC1092) showing significant adherence were selected. Decreased binding activities were observed after removing sialic acid of sHCM using sialidase. However, after removing the sulphate residue using sulphatase, the adhesion of ME-527 decreased; whereas the remaining two strains had increased adhesion. The adhesion of three probiotics significantly decreased after the sulphate residue was blocked by elution with barium chloride. CONCLUSIONS: A new evaluation method using the Biacore assay was developed to observe binding properties to the acidic residues of sHCM. Results indicated that there was a strong relationship between probiotic adhesion and acidic residues of sHCM. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing a screening method that quantitatively measures the binding between bacteria and acidic residues in sHCM using the Biacore binding assay; and provides a new method for the selection of probiotics in the future.

Inhibition of interleukin-1-induced uveitis and corneal fibroblast proliferation by interleukin-1 blockers.

Interleukin-1 (IL-1) is known to trigger induction of inducible nitric oxide synthase (iNOS) to persistently mass produce nitric oxide (NO) to induce various diseases such as cancer, inflammation, Alzheimer's disease and eye diseases, including uveitis, retinopathy, age-related macular degeneration, glaucoma and myopia. Therefore, IL-1 blockers could become an important class of drugs to fight numerous diseases. Among the many compounds studied so far, 1-methyl hydrazino analogs are among the most promising agents to be developed. A minor structural change of 1-methyl hydrazino group into 1-methyl thiosemicarbazido group enhanced their anti-inflammatory activity but reduced their antiproliferation activity on corneal fibroblast cell growth.

[Recent endoscopic treatment in gastroenterology].

Endoscopic diagnosis and treatment became to be necessary in gastroenterology for last two decades. Indication of endoscopic treatment is amazingly expanding because of developing new techniques and easy-to-use devices. Nowadays, the indication for endoscopic treatment includes the removal of foreign bodies in alimentary tract, the dilatation of stricture lesions by balloon or expandable metallic stent, the resection of polypoid and superficial tumors by polypectomy and EMR (endoscopic mucosal resection) techniques, the injection sclerotherapy and ligation method for gastroesophageal varices, the hemostasis of gastrointestinal hemorrhages by injection method or heat-burn method. In biliary and pancreatic area, there are some endoscopic treatments that are removal and destruction of stones in common bile duct, biliary and pancreatic drainage by tubing method. Our results of endoscopic resection for esophageal and gastric tumors are shown in this papers. In conclusion, complete resection that means histologically tumor negative of lateral and vertical margin of resected specimen is important to prevent recurrence of tumors after resection.

Inhibitory effects of flavonoids on rabbit heart carbonyl reductase.

The inhibitory effects of flavonoids (galangin, kaempferol, quercetin, myricetin, morin, and taxifolin) on rabbit heart carbonyl reductase (RHCR) were investigated using 4-benzoylpyridine (4BP) as the substrate. The stereochemical characteristics of the flavonoids were found to be a factor determining their inhibitory potencies toward RHCR. Furthermore, the lipophilicity, and the scavenging or antioxidative effects of the flavonoids were likely to complicate the structure-activity relationship of their inhibitory effects on RHCR. Quercetin inhibited RHCR uncompetitively with respect to NADPH and competitively with respect to 4BP, suggesting that it competes with 4BP at the substrate-binding site of RHCR. RHCR efficiently reduced benzoquinones (1,4-benzoquinone and 2-methyl-1, 4-benzoquinone) and naphthoquinones (1,4-naphthoquinone and menadione). Galangin was a potent inhibitor of RHCR when menadione was used as the substrate, and prevented the formation of the superoxide anion radical in the presence of RHCR, NADPH, and menadione. Flavonoids may be useful compounds for suppressing the cardiotoxicity of quinones by inhibiting RHCR.

Effects of interleukin-1 blockers on corneal fibroblast proliferation in vitro and ocular inflammation in vivo.

The success of keratorefractive surgical procedures is limited by the wound healing process in the corneal stroma. The proliferation and matrix synthesis of corneal stromal fibroblasts is the central element of the wound healing process that is triggered by an initial inflammation. In order to develop new therapeutic strategies to reduce wound healing intensity, we investigated the effect of newly synthesized interleukin-1 (IL-1) blockers on the proliferation of cultured rabbit corneal fibroblasts and the ocular inflammation induced by IL-1. It was found that the addition of IL-1 blockers, such as CK-135 to CK-145, led to a dose-dependent inhibition of cell proliferation after 24, 48 and 72 hr of incubation. The isotope incorporation study showed that the syntheses ofDNA and mRNA were suppressed whereas that of protein was enhanced or unaffected. These compounds also demonstrated a potent anti-inflammation action in the rat uveitis model. Our results indicate that CK (Chiou-Kumamoto) compounds may be valuable therapeutic agents for the prevention of postoperative complications after corneal keratorefractive surgical procedures.

Suppression of interleukin-1alpha-induced uveitis and inhibition of fibroblast-like cell proliferation by synthetic interleukin-1 blockers.

To treat uveitis and prolong the functional life of filtration surgery on glaucomatous eyes, some interleukin-1 (IL-1) blockers were used to inhibit IL-1-induced uveitis in rat eyes and to suppress proliferation of fibroblast-like corneal and conjunctival cells in the cell cultures, respectively. It was found in this research that the blood-aqueous barrier can be broken by IL-1alpha to allow fluorescein to enter the eyeballs and to be detected by fluorotron. The uveitis was effectively blocked by the IL-1 blockers studied in this research. It was also noted that the proliferation of fibroblast-like corneal and conjunctival cells was effectively inhibited by IL-1 blockers. The inhibition of cell growth seems to be caused primarily by the inhibition of RNA synthesis. There was a significant difference in the potency of IL-1 blockers to inhibit corneal vs. conjunctival cells. It was noted that conjunctival cells were more easily inhibited by IL-1 blockers than corneal cells. These results indicate that IL-1 blockers can suppress the proliferation of conjunctival cells at dose levels which do not affect the normal cell growth of corneal cells.

Anti-inflammation and antifibroblastic actions of interleukin-1 blockers.

Interleukin-1 (IL-1) blockers, CK 127 and CK 129, were found to inhibit IL-1-induced posterior uveitis very effectively at 3-10 mg/kg i.p. and were more potent than prednisolone which required at least 20 mg/kg i.p. to achieve the same level of anti-uveitis action. CK 127 and CK 129 were also found to be effective in inhibiting fibroblast-like corneal cells at 30-300 micrograms/ml and conjunctival cells at 0.3-10 micrograms/ml. These results indicate that IL-1 blockers are more potent in inhibiting the cell growth of conjunctival cells than that of corneal cells. From in vitro cell culture experiments, it was found that inhibition of cell growth could be due primarily to the inhibition of DNA. Although the inhibition of cell growth was due mainly to the inhibition of DNA synthesis, mRNA synthesis was also markedly inhibited. In both cells, the protein synthesis was unaffected in a few cases and markedly stimulated in most cases.

Antiuveitis and inhibition of fibroblast-like corneal and conjunctival cell cultures by interleukin-1 blockers, CK 125, CK 126 and CK 128.

Three new interleukin-1 (IL-1) blockers, CK 125, CK 126 and CK 128, were studied for their effects on IL-1-induced uveitis in rat eyes. They were more potent (at 3-10 mg/kg t.i.d.) than prednisolone (20 mg/kg t.i.d.) in effectively inhibiting posterior uveitis. They were also found to inhibit fibroblast-like corneal cells at 10-300 micrograms/ml concentrations and conjunctival cells at 1-30 micrograms/ml levels. The incorporation of leucine into corneal and conjunctival cells was either stimulated or unaffected by CK 126, indicating that the inhibition of cell growth has nothing to do with the protein synthesis. However, the incorporation of uridine into corneal and conjunctival cells was markedly inhibited by CK 126 at 3-30 micrograms/ml concentrations whereas the incorporation of thymidine into the cells was inhibited at a lesser extent than that of uridine. These results indicate that cell inhibition by CK 126 could be related mainly to the synthesis of mRNA and, to a lesser extent, to DNA synthesis.

Effective treatment of experimental uveitis with interleukin-1 blockers, CK 123 and CK 124.

Interleukin-1 (IL-1) blockers are known to inhibit IL-1-induced uveitis. CK 123 and CK 124 are new IL-1 blockers which showed potent anti-uveitis actions that are more potent than the classic corticosteroid, prednisolone. In addition to anti-inflammation, CK 123 and CK 124 were also found to inhibit proliferation of fibroblast-like corneal and conjunctival cells, indicating that these compounds could be used not only as anti-uveitis agents but also as useful agents to prolong the functional period of aqueous humor outflow after trabeculectomy. It was also found that DNA and mRNA synthesis were markedly inhibited by CK 123 and CK 124, yet the protein synthesis was either untouched or enhanced.

Antagonism of interleukin-1 (IL-1)-induced uveitis with synthetic IL-1 blockers.

Because of the discovery of potent interleukin-1 (IL-1) blocking effects by CK-103A (4,6-dihydropyridazino[4,5-c]pyridazin-5 (1H)-ones) on rat uveitis induced by IL-1, numerous derivatives of CK-103A have been synthesized and their efficacies on the same animal model studied. The uveitis was induced by injection of 1 ng IL-1/10 microliters intravitreally. The inflammation reached peak at 12 hr after the injection of IL-1. The prevention/blockade of IL-1-induced uveitis was measured at this peak inflammation time point. It was found that 8 out of 12 CK-analogs studied produced an effective blockade of IL-1-induced uveitis. Most of them were at least equipotent or even more potent than prednisolone in blocking IL-1-induced uveitis. It is concluded that most dihydropyridazinopyridazin derivatives are effective anti-uveitis compounds. Some could be found to be safe and useful for the treatment of this dreadful disease.

Prevention of ocular inflammation induced by lens protein, endotoxin, and interleukin-1 with synthetic interleukin-1 blockers.

It is well known that corticosteroids are potent anti-inflammatory agents, yet they produce serious side effects. Although arachidonate metabolite blockers have been developed for the treatment of inflammation, they are much less potent than corticosteroids. Furthermore, they still process serious side effects. In search of potent and safe non-steroidal anti-inflammatory agents (NSAIA), interleukin-1 (IL-1) blockers have been developed. Among 121 CK-analogs studied, CK-17, CK-101A and CK103A have been identified as promising anti-inflammatory agents as potent as prednisolone in inhibiting lens proteins-induced inflammation and twice as potent as prednisolone in inhibiting endotoxin-and IL-1-induced uveitis. No serious side effects could be noticed with the doses of these compounds tested to date. These results indicate that the development of potent NSAIAs is feasible. Moreover, these compounds are not related to arachidonate metabolites.

Prevention and treatment of ocular inflammation with a new class of non-steroidal anti-inflammatory agents.

New non-steroidal anti-inflammatory agents (NSAIAs) were tested on lens protein-, endotoxin- and interleukin-1-induced ocular inflammation. It was found that most NSAIAs, including REV 5901, mefenamic acid, indomethacin, CK-17 and CK-102, inhibited lens protein-induced inflammation. Endotoxin induced inflammation indirectly through the release of IL-1 which was inhibited by fewer agents, including CK-17, CK-102 and prednisolone. However, the direct effect of IL-1 can only be suppressed by CK-17 and prednisolone. Therefore, CK-17 could become an important NSAIA which acts similarly to corticosteroids yet produces no steroidal side effects. CK-17 was different from most NSAIAs as it affected little, if any, arachidonate metabolism. Most importantly, CK-17 was found to be 2-fold more potent than prednisolone in inhibiting IL-1-induced uveitis, while no side effects were noted at doses tested to date.

Effect of some phytogenic agents and synthetic compounds on complement cascade-mediated hemolysis.

A variety of anti-inflammatory compounds obtained from various medicinal plants (phytogenic) as well as some synthetic compounds were tested on the complement cascade in vitro. Bovine erythrocytes were treated with rabbit antibovine red blood cell antibody. Rabbit plasma was diluted with veronal buffer and mixed with erythrocytes in the presence or absence of drugs and incubated. Erythrocytes were pelleted and the absorbance of the supernatant at 412 nm determined. Drugs could be grouped into three categories; (a) those with no effect whatsoever; (b) those which produced definite enhancement of the release of hemoglobin by the complement cascade, and (c) prednisolone, which inhibited the release of hemoglobin by the complement cascade. We suggest that prednisolone and the drugs which had no effect on the complement cascade are safer to use as anti-inflammatory agents, while drugs enhancing the complement cascade may have potential adverse properties.