Conditional deficiency of Rho-associated kinases disrupts endothelial cell junctions and impairs respiratory function in adult mice.

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, ß-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/ß-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion.

Effective concentration of intravenous immunoglobulin for neutralizing Panton-Valentine leukocidin in human blood.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infects healthy individuals, although the precise cause remains unclear. CA-MRSA produces Panton-Valentine leukocidin (PVL), which often causes severe invasive infection; however, antitoxin drugs against PVL are limited. Intravenous immunoglobulin (IVIg) possesses antitoxin activity, but unfortunately, the optimal dose is unknown. Here, we measured the PVL neutralizing antibody titer in the plasma of Japanese individuals and sera of American donors. Next, we compared the cytotoxic effects of PVL on neutrophils in phosphate buffered saline (PBS) or whole blood to determine the effect of the neutralizing antibody. Finally, we evaluated the effective concentration of IVIg required to neutralize PVL in PBS and whole blood. We observed that the titer of PVL neutralizing antibody in healthy individuals polarized as high and low/none group. Additionally, the PVL neutralizing antibody titer considerably affected the concentration at which IVIg elicited its effect. This suggests that PVL-producing CA-MRSA might be involved in determining the severity of infection in healthy individuals without neutralizing antibody against PVL. The neutralizing effect of IVIg was observed in both PBS and whole blood. However, the optimal concentration of IVIg required for neutralizing PVL varied between PBS and whole blood. In addition, since the PVL-neutralizing activity of IVIg also largely depends on blood composition, such as neutralizing antibody concentration, the optimal dosage of IVIg as an antitoxin drug should be decided in a timely manner after considering the patient's medical background.

Helicobacter pylori activates gastric epithelial cells to produce interleukin-8 via protease-activated receptor 2.

BACKGROUND/AIM: Recently, it has been shown that serine proteases derived from microorganisms stimulate epithelial cells to produce inflammatory mediators through protease-activated receptor (PAR). We investigated the involvement of PAR2 in the interleukin (IL)-8 production by Helicobacter pylori-infected gastric epithelial cells. METHODS AND RESULTS: Human gastric epithelial cells, MKN45 cells, were used. The expression of PAR2 was assessed by real-time PCR and immunocytochemistry, and IL-8 protein was measured by an enzyme-linked immunosorbent assay. PAR2 mRNA and protein were constitutively expressed on unstimulated MKN45 cells. The treatment of cells with H. pylori resulted in a significant increase in PAR2 expression. In addition, trypsin (a natural PAR2 agonist), SLIGKV amide (a synthetic PAR2 agonist), H. pylori live bacteria or H. pylori culture supernatant significantly induced IL-8 production from MKN45 cells. H. pylori-induced IL-8 production was inhibited by nafamostat mesilate (a serine protease inhibitor), neutralizing antibody to PAR2 and in PAR2-deficient cells treated with siRNA. CONCLUSIONS: These results reveal that H. pylori-derived protease activates gastric epithelial cells to produce inflammatory cytokines through PAR2, suggesting an important role for PAR2 in the modulation of gastric inflammation associated with H. pylori.

Recombinant human antithrombin expressed in Chinese hamster ovary cells shows in vivo efficacy on rat DIC model similarly to plasma-derived antithrombin regardless of different N-glycosylation.

Plasma-derived human antithrombin (pAT) is used for the treatments of disseminated intravascular coagulation (DIC) and hereditary antithrombin deficiencies. We expressed recombinant human antithrombin (rAT) in Chinese hamster ovary (CHO) cells. The purified rAT is composed of 55% alpha-isoform and 45% beta-isoform. The structure of the N-linked oligosaccharides of rAT is the same biantennary complex type as previously found in pAT with less sialylated on the non-reducing ends. Most of the oligosaccharides of rAT are fucosylated at the reducing ends of N-acetylglucosamine, while those of pAT are not fucosylated. Despite of the difference in sialylation and fucosylation of the oligosaccharide units, rAT and pAT showed indistinguishable heparin cofactor and progressive activities, and they bound to thrombin in a one-to-one stoichiometric manner. In lipopolysaccharide (LPS)-induced and thromboplastin-induced DIC rat models, rAT reduced fibrinogen and platelet consumption to a similar extent with pAT. In LPS-induced DIC model, both ATs similarly restrained the increase of alanine aminotransferase and aspartate aminotransferase activities. Finally, pharmacokinetic analysis showed that both ATs had similar half-lives in the circulation of normal rats. Together, the present study demonstrated that rAT prepared in CHO cells has potential for a substitute of pAT in therapeutic use.

A new interferon, limitin, displays equivalent immunomodulatory and antitumor activities without myelosuppressive properties as compared with interferon-alpha.

OBJECTIVE: Limitin is a new member of type I interferon (IFN) identified with an expression cloning based on the growth suppression of a myelomonocytic leukemia cell line WEHI3. Although limitin uses the IFN-alpha/beta receptor, its signal transduction pathways to express the antiviral effects are different from those of IFN-alpha. To clarify the characteristics of limitin, we compared the biological activities of limitin, such as the antiviral, immunomodulatory, antitumor, and myelosuppressive effects, with IFN-alpha. MATERIALS AND METHODS: Limitin and IFN-alpha were titered with a cytopathic effect dye binding assay. Induction of MHC class I on a keratinocyte cell line PAM212 was estimated with flow cytometry. Induction of OVA-restricted cytotoxic T lymphocyte (CTL) activity was analyzed with 51Cr release assay. Antiproliferative effects were evaluated with 3H-thymidine incorporation assay using WEHI3 and a lymphoblast cell line L1210. Myelosuppresive effects were evaluated with colony assay. In vivo side effects were estimated after the injection of limitin or IFN-alpha. RESULTS: Limitin had relatively higher antiviral activity than IFN-alpha. Limitin induced the surface expression of MHC class I, the enhancement of CTL activity, and the growth inhibition of lymphohematopoietic cell lines as strong as IFN-alpha. Nevertheless, the treatment of mice with limitin showed neither myelosuppression nor fever that are common adverse effects of IFN-alpha. CONCLUSIONS: Strong immunomodulatory, antitumor, and antiviral effects with weak myelosuppressive and weak acute toxic effects of limitin indicate that it may be useful as a new therapeutic drug for virus-hepatitis and cancers.

Potent antibacterial activity of Y-754, a novel benzimidazole compound with selective action against Helicobacter pylori.

Y-754, a novel benzimidazole compound, was investigated for in vitro and in vivo antibacterial activity. Unlike amoxicillin, clarithromycin, and metronidazole, the compound had no activity against common aerobic and anaerobic bacteria other than Helicobacter pylori. The minimum inhibitory concentration of Y-754 against H. pylori, at 0.025 microg/ml, was nearly equal to that of amoxicillin and clarithromycin. The respective concentrations of Y-754, amoxicillin, clarithromycin, and metronidazole required to inhibit 90% of 39 isolates of H. pylori were 0.05, 0.39, 6.25, and 25 microg/ml, indicating the potent activity of Y-754, including activity against clarithromycin- and metronidazole-resistant strains. The anti-H. pylori activity of Y-754 was potent even at pH 5.5 and was bactericidal at concentrations of 0.1 microg/ml and above. Exposure of H. pylori to Y-754 did not result in the induction of drug-resistant mutation. Oral administration (10 mg/kg twice a day for 7 days) to Mongolian gerbils infected with strain ATCC 43504 demonstrated that Y-754 was effective in H. pylori eradication and that its eradication efficacy increased in line with the progress of damage to the gastric mucosa caused by H. pylori infection. Y-754 was also efficacious in the treatment of infection by the clarithromycin-resistant strain OIT-36. The results obtained lead to the expectation that the new benzimidazole Y-754 will, in the near future, be used for H. pylori eradication therapy in peptic ulcer patients.

In vitro and in vivo anti- Helicobacter pylori activity of Y-904, a new fluoroquinolone.

Y-904 is a new fluoroquinolone with a broad antimicrobial spectrum. In particular, it has anti- Helicobacter pylori activity superior to that of existing fluoroquinolones. In the present study it was examined for its in vitro antibacterial activity against 51 clinical isolates of H. pylori, including clarithromycin- and metronidazole-resistant strains. The minimum inhibitory concentration of Y-904 at which 90% of isolates were inhibited was close to that of amoxicillin and clarithromycin and lower than that of levofloxacin and metronidazole (0.1, 0.1, 0.2, 3.13, and 12.5 micrograms/ml, respectively). Y-904 showed equally strong activity at pH 5.5 as at pH 7.0. At 10 times the minimum inhibitory concentration, Y-904 decreased the viable count of H. pylori to below 10(-5) within 2 h after exposure. No significant change in the minimum inhibitory concentration was observed when H. pylori, Staphylococcus aureus, and Escherichia coli were successively subcultured in medium containing subinhibitory concentrations of Y-904. Y-904 also strongly inhibited the supercoiling activity of DNA gyrase from H. pylori ATCC43504 (IC(50), 1.48 micrograms/ml). A study of Y-904 treatment in H. pylori-infected Mongolian gerbils using twice-daily oral administration for 7 days demonstrated that the complete clearance dose of Y-904 was 1 mg/kg and that its potency was around 10, 30, and 30 times that of amoxicillin, clarithromycin, and levofloxacin, respectively. These results indicate that Y-904 is a promising candidate for the eradication of H. pylori infection.

Molecular cloning of p53 cDNA of Mongolian gerbil and establishment of yeast p53 functional assay system.

BACKGROUND: Epidemiological studies have shown a correlation between Helicobacter pylori infection and human gastric carcinogenesis. A Mongolian gerbil model has demonstrated that H. pylori infection induced gastric carcinoma. However, the disadvantage of this animal model is a lack of information regarding the cellular genes involved in oncogenesis. Mutation of the p53 gene is one of the most common steps in gastric carcinogenesis. In this study, we aimed to clone the p53 gene of the Mongolian gerbil and detect the functional mutations in H. pylori-infected animals. MATERIALS AND METHODS: The p53 complementary DNA (cDNA) of Mongolian gerbil was cloned by the methods of reverse-transcribed polymerase chain reaction and rapid amplification of cDNA ends. RESULTS: The p53 cDNA of Mongolian gerbil has a 78.8% homology to that of humans. A novel yeast p53 assay system was established and enabled to detect the functional mutations of the p53 gene in the stomach of the Mongolian gerbil. CONCLUSIONS: This is the first report of the complete sequence of wild-type p53 cDNA of the Mongolian gerbil. This genetic information and an assay system designed to detect the functional mutations of the p53 gene are useful for further investigations of gastric oncogenesis in this animal model.

Long-term effects of Helicobacter pylori eradication in Mongolian gerbils.

BACKGROUND: In this study, to clarify whether Helicobacter pylori eradication alters the course of the development of gastric mucosal changes in the stomach, we examined the long-term effects of H. pylori eradication on H. pylori-inoculated gerbils. METHODS: A total of 40 H. pylori-inoculated gerbils were randomized and subjected, at 22 months after inoculation, to eradication treatment with dual therapy of omeprazole plus clarithromycin, or with therapy with a novel quinolone compound, Y-34867, alone. The animals were killed at the start of administration (control group) or at 8 months after the completion of therapy (vehicle or eradication-treatment groups). RESULTS: Severe histopathological changes in the gastric mucosa were observed in all H. pylori-inoculated gerbils at the start of administration. At 8 months after completion of therapy, the frequency of gastritis, erosion, intestinal metaplasia, and gastric carcinoid in the eradication therapy groups was markedly reduced compared with that in the control and vehicle groups. Values for anti- H. pylori IgG titer, bacterial counts, and gastrin also decreased significantly. CONCLUSIONS: These results suggest that H. pylori eradication may have had a therapeutic effect not only on gastritis, erosion, and gastric ulcer but also on glandular atrophy, intestinal metaplasia, and gastric carcinoid.

[Neutralizing activity of human immunoglobulin preparation against toxic shock syndrome toxin-1].

Human intravenous immunoglobulin (IVIG) preparation containing a variety of antibodies is widely used against severe infectious diseases. Although IVIG is supposed to promote phagocytosis of opsonized bacteria and neutralize several bacterial toxins, it is unclear what antibodies are responsible for the effect in clinical use. In this study, we found that IVIG showed neutralizing activity against toxic shock syndrome toxin-1 (TSST-1), produced by methicillin-resistant Staphylococcus aureus (MRSA). Whereas intravenous inoculation with culture medium of MRSA 1945 strain into ICR mice causes immediate death, all of the mice survived in case of previous administration of IVIG. Such effect might be attributed to neutralization of TSST-1. Murine splenocytes incubated with TSST-1 (1.0 ng/mL) for 48 hours produced IFN-gamma. By addition of IVIG at 100 micrograms/mL into culture medium, production of IFN-gamma was completely inhibited. From IVIG, anti-TSST-1 antibody was purified by affinity chromatography as one of the effective antibodies. MRSA 1945 mixed with dextran-based microcarrier beads and injected subcutaneously into mice resulted in the formation of abscess and excretion of TSST-1 in serum for 14 days. IVIG and anti-TSST-1 antibody neutralized TSST-1 in blood 24 hours after infection, although bacterial count was kept constant. Experimental rabbit TSS model was established by synergism with lipopolysaccharide (LPS) of Escherichia coli. Anti-TSST-1 antibody protected NZW rabbits from lethal challenge with TSST-1 (1 microgram/kg, i.v.) 4 hours before LPS in a dose-dependent manner (10 micrograms/kg, i.v.), whereas all of the NZW rabbits died in the control group. Thus, IVIG may be a useful tool in the prevention and perhaps therapy of staphylococcal infections and TSS.