Development of an isogenic human cell trio that models polyglutamine disease.

Polyglutamine (polyQ) diseases are rare autosomal-dominant neurodegenerative diseases associated with the expansion of glutamine-encoding triplet repeats in certain genes. To investigate the functional influence of repeat expansion on disease mechanisms, we applied a biallelic genome-engineering platform that we recently established, called Universal Knock-in System or UKiS, to develop a human cell trio, a set of three isogenic cell lines that are homozygous for two different numbers of repeats (first and second lines) or heterozygous for the two repeat numbers (third line). As an example of a polyQ disease, we chose spinocerebellar ataxia type 2 (SCA2). In a pseudodiploid human cell line, both alleles of the glutamine-encoding triplet repeat in the SCA2-causing gene, ataxin 2 or ATXN2, were first knocked in with a donor sequence encoding both thymidine kinase and either puromycin or blasticidin resistance proteins under dual drug selection. The knocked-in donor alleles were then substituted with a payload having either 22 or 76 triplet repeats in ATXN2 by ganciclovir negative selection. The two-step substitution and subsequent SNP typing and genomic sequencing confirmed that the SCA2-modeling isogenic cell trio was obtained: three clones of 22-repeat homozygotes, two clones of 22/76-repeat heterozygotes and two clones of 76-repeat homozygotes. Finally, RT-PCR and immunoblotting using the obtained clones showed that, consistent with previous observations, glutamine tract expansion reduced transcriptional and translational expression of ATXN2. The cell clones with homozygous long-repeat alleles, which are rarely obtained from patients with SCA2, showed more drastic reduction of ATXN2 expression than the heterozygous clones. This study thus demonstrates the potential of UKiS, which is a beneficial platform for the efficient development of cell models not only for polyQ diseases but also for any other genetic diseases, which may accelerate our deeper understanding of disease mechanisms and cell-based screening for therapeutic drugs.

Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells.

Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we establish a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of ~100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a standard platform with which to pursue the design of noncoding regions for genome writing in human cells.

Eosinophilic Cholangitis with Poor Prognosis after Corticosteroid- and Ursodeoxycholic Acid-Related Remission of Peripheral and Peribiliary Eosinophilia.

A 79-year-old man presented with high fever, marked eosinophilia, altered biochemical liver function tests (LFT) with predominance of biliary enzymes, and severe wall thickening of the gallbladder. Magnetic resonance cholangiopancreatography (MRCP) suggested cholecystitis, without signs of biliary strictures. Laparoscopic cholecystectomy and exploratory liver excision revealed eosinophilic cholangitis and cholecystitis, complicated with hepatitis and portal phlebitis. Prednisolone monotherapy rapidly improved peripheral eosinophilia, but not LFT. Liver biopsy showed that infiltrating eosinophils were replaced by lymphocytes and plasma cells. Treatment with ursodeoxycholic acid improved LFT abnormalities. Nevertheless, after 2 months, transaminase-dominant LFT abnormalities appeared. Transient prednisolone dose increase improved LFT, but biliary enzymes' levels re-elevated and jaundice progressed. The second and third MRCP within a 7-month interval showed rapid progression of biliary stricture. The repeated liver biopsy showed lymphocytic, not eosinophilic, peribiliary infiltration and hepatocellular reaction to cholestasis. Eighteen months after the first visit, the patient died of hepatic failure. Autopsy specimen of the liver showed lymphocyte-dominant peribiliary infiltration and bridging fibrosis due to cholestasis. Though eosinophil-induced biliary damage was an initial trigger, repeated biopsy suggested that lymphocytes played a key role in progression of the disease. Further studies are needed to elucidate the relationship between eosinophils and lymphocytes in eosinophilic cholangitis.

Virtual digital subtraction angiography using multizone patch-based U-Net.

Digital subtraction angiography (DSA) is a powerful technique for visualizing blood vessels from X-ray images. However, the subtraction images obtained with this technique suffer from artifacts caused by patient motion. To avoid these artifacts, a new method called "Virtual DSA" is proposed, which generates DSA images directly from a single live image without using a mask image. The proposed Virtual DSA method was developed using the U-Net deep learning architecture. In the proposed method, a virtual DSA image only containing the extracted blood vessels was generated by inputting a single live image into U-Net. To extract the blood vessels more accurately, U-Net operates on each small area via a patch-based process. In addition, a different network was used for each zone to use the local information. The evaluation of the live images of the head confirmed accurate blood vessel extraction without artifacts in the virtual DSA image generated with the proposed method. In this study, the NMSE, PSNR, and SSIM indices were 8.58%, 33.86 dB, and 0.829, respectively. These results indicate that the proposed method can visualize blood vessels without motion artifacts from a single live image.

Helicobacter pylori infection might have a potential role in hepatocyte ballooning in nonalcoholic fatty liver disease.

BACKGROUND: Clinical data regarding Helicobacter pylori (H. pylori) infection in nonalcoholic fatty liver disease (NAFLD) are limited. The aim was to evaluate H. pylori infection in patients with NAFLD and its association with disease severity. METHODS: One hundred and thirty patients with biopsy-proven NAFLD [43 with nonalcoholic fatty liver (NAFL) and 87 with nonalcoholic steatohepatitis (NASH)] were recruited for blood samples for anti-H. pylori immunoglobulin G (IgG) and standard biochemical tests were obtained after overnight fasting. Glucose tolerance was evaluated by 75-g oral glucose tolerance test. Liver biopsies were scored for NAFLD activity score (NAS), fibrosis and iron deposits. RESULTS: H. pylori IgG seropositivity was found in 40 % of patients overall. The prevalence of NASH was significantly higher in the patients with H. pylori IgG seropositivity (81 %) than in those without (58 %, p = 0.008). Glucose intolerance was similar between the two groups. The total NAS and the grade of hepatocyte ballooning were higher in the patients with H. pylori IgG seropositivity than in those without, while the hepatic iron grade was lower in the patients with H. pylori IgG seropositivity than in those without. H. pylori infection (p = 0.030), female gender (p = 0.029), and NAFIC score ≥ 2 points (p < 0.001) could independently predict NASH in logistic regression analysis, independent of age, obesity and glucose tolerance. CONCLUSION: The association of H. pylori seropositivity with hepatocyte ballooning suggests that H. pylori infection may represent another contributing factor in the progression from NAFL to NASH. Eradicating H. pylori infection may have therapeutic prospects in NASH treatment.

Human placental extract treatment for non-alcoholic steatohepatitis non-responsive to lifestyle intervention: A pilot study.

AIM: No pharmacological therapies have been established for non-alcoholic steatohepatitis (NASH), which can lead to liver-related mortality. Human placental extract (HPE), which has anti-inflammatory effects, has been expected to be a promising treatment for chronic liver disease. This pilot study was conducted to evaluate the efficacy of HPE for biopsy-diagnosed NASH. METHODS: After a lifestyle intervention for 12 weeks, 10 subjects with abnormal alanine aminotransferase (≥30 IU/L) and biopsy-proven NASH (Non-Alcoholic Fatty Liver Disease Activity Score [NAS], ≥4) received i.m. injections of HPE (Laennec) at a dose of 4 mL/day twice per week for 24 weeks, and seven of them underwent a second liver biopsy after the treatment. Liver biopsies were scored for NAS and fibrosis. Histological response was defined as a decrease of 2 points or more in NAS and no increase in fibrosis. RESULTS: Serum transaminase activities were significantly lower at 8 weeks compared with pretreatment levels in nine patients who continued treatment for 24 weeks. One patient refused to continue the treatment soon after starting therapies. In seven patients undergoing post-treatment biopsies, NAS (mean [standard deviation]) mildly decreased from 5.29 (0.95) to 4.00 (1.83) without reaching statistical significance (P = 0.078). Histological response was observed in all three obese patients and in only one of four non-obese ones. No significant changes were observed in body mass index, lipid profiles and diabetic control/insulin resistance. CONCLUSION: In NASH patients who received HPE treatment, significant reductions in serum liver enzymes were obtained after 8 weeks. Histological efficacy may be better in obese patients than in non-obese ones.

Polymorphic L1 retrotransposons are frequently in strong linkage disequilibrium with neighboring SNPs.

L1 retrotransposons have been the major driver of structural variation of the human genome. L1 insertion polymorphism (LIP)-mediated genomic variation can alter the transcriptome and contribute to the divergence of human phenotypes. To assess this possibility, a genome-wide association study (GWAS) including LIPs is required. Toward this ultimate goal, the present study examined linkage disequilibrium between six LIPs and their neighboring single nucleotide polymorphisms (SNPs). Genomic PCR and sequencing of L1-plus and -minus alleles from different donors revealed that all six LIPs were in strong linkage disequilibrium with at least one SNP. In addition, comparison of syntenic regions containing the identified SNP nucleotides was performed among modern humans (L1-plus and -minus alleles), archaic humans and non-human primates, revealing two different evolutionary schemes that might have resulted in the observed strong SNP-LIP linkage disequilibria. This study provides an experimental framework and guidance for a future SNP-LIP integrative GWAS.

Photocatalytic water splitting using modified GaN:ZnO solid solution under visible light: long-time operation and regeneration of activity.

Overall water splitting using GaN:ZnO solid solution photocatalyst modified with Rh(2-y)Cr(y)O(3) nanoparticles as H(2) evolution cocatalysts under visible light (400 < λ < 500 nm) was examined with respect to long-term durability and regeneration of photocatalytic activity. The rate of visible light water splitting remained unchanged for 3 months (2160 h), producing H(2) and O(2) continuously at a stoichiometric amount. After 6 months of operation, a 50% loss of the initial activity occurred. Regeneration treatment of deactivated catalysts was attempted by reloading the Rh(2-y)Cr(y)O(3) cocatalyst. The degree of activity regeneration depended on the reloading amount. Up to 80% of the initial activity for H(2) evolution could be recovered under optimal treatment conditions. It was also found that deactivation of GaN:ZnO was suppressed to some extent by prior coloading of an O(2) evolution cocatalyst, which helped to suppress oxidative decomposition of GaN:ZnO by valence band holes, thereby improving the durability.

[Development of a cone-beam CT system for radiological technologist education].

For radiological technologists, it is very important to understand the principle of computed tomography (CT) and CT artifacts derived from mechanical and electrical failure. In this study, a CT system for educating radiological technologists was developed. The system consisted of a cone-beam CT scanner and educational software. The cone-beam CT scanner has a simple structure, using a micro-focus X-ray tube and an indirect-conversion flat panel detector. For the educational software, we developed various educational functions of image reconstruction and reconstruction parameters as well as CT artifacts. In the experiments, the capabilities of the system were evaluated using an acrylic phantom. We verified that the system produced the expected results.

Expression of angiotensin II type 1 and type 2 receptor mRNAs in the gastric mucosa of Helicobacter pylori-infected Mongolian gerbils.

BACKGROUND: The renin-angiotensin system (RAS) plays an important role in normal homeostasis, carcinogenesis-related angiogenesis, and cell proliferation. Helicobacter pylori infection causes infiltration of inflammatory cells into the gastric mucosa and is considered the major cause of gastric cancer. Whether RAS plays a role in H. pylori infection-related gastric diseases remains unclear. We investigated the changes in gastric mucosal angiotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R) mRNA levels throughout the time course of H. pylori infection in Mongolian gerbils. METHODS: Mongolian gerbils were infected with wild-type H. pylori (for 12 months) or with its isogenic oipA mutant (for 3 months). Gastric mucosal AT1R and AT2R mRNA levels were assessed using real-time reverse transcription-polymerase chain reaction. RESULTS: The gastric mucosal AT1R mRNA level was significantly associated with the severity of inflammatory cell infiltration into the gastric mucosa that reached maximal levels at 12 months after infection in both the antrum and body. Inflammatory cell infiltration scores and AT1R and AT2R mRNA levels were significantly lower in oipA mutant than wild-type infections. Mucosal AT1R and AT2R mRNA expressions in wild-type H. pylori-infected gerbils with gastric ulcers were significantly higher than in those without ulcers (P < 0.01). CONCLUSIONS: Gastric mucosal ATR expression gradually increases during the course of H. pylori infection. Up-regulation of the RAS in association with progressive gastric inflammation suggests a potential role of the RAS in gastric carcinogenesis. OipA appears to play a role in AT1R and AT2R expression and the resulting inflammation.

Helicobacter pylori outer membrane proteins on gastric mucosal interleukin 6 and 11 expression in Mongolian gerbils.

BACKGROUND AND AIM: The levels of interleukin (IL)-6 and IL-11 in the gastric mucosa are related to mucosal inflammation; however, the chronological changes in cytokine expression during different phases of Helicobacter pylori infection and the effects of H. pylori virulence factors, particularly those of outer membrane proteins, remain obscure. The aim of this study was to clarify the chronological changes in cytokine levels in relation to several H. pylori outer membrane proteins. METHODS: We studied Mongolian gerbils inoculated with wild-type H. pylori 7.13 for up to 48 weeks and then examined animals infected with oipA, babA, or alpAB isogenic mutants for 12 weeks. Mucosal IL-6 and IL-11 mRNA levels were measured using real-time reverse transcription-polymerase chain reactions. RESULTS: High levels of gastric mucosal IL-6 and IL-11 mRNA in gerbils infected with wild-type H. pylori were observed during the chronic phase of infection, reaching maximums at 12 and 6 months, respectively. Infection with oipA and babA mutants resulted in significantly reduced cytokine levels and inflammatory cell infiltrations compared to gerbils infected with wild-type strains, and this persisted throughout the observation period. The alpAB mutants did not infect gerbils. Mucosal IL-6 and IL-11 levels were significantly associated with the grade of inflammatory cell infiltration. CONCLUSIONS: OipA and BabA result in more severe H. pylori infection and increased IL-6 and IL-11 levels, which in turn may increase the risk of developing H. pylori-induced gastrointestinal diseases.

Effects of blood group antigen-binding adhesin expression during Helicobacter pylori infection of Mongolian gerbils.

Helicobacter pylori outer membrane proteins, such as the blood group antigen-binding adhesin (BabA), are associated with severe pathological outcomes. However, the in vivo role of BabA during long-term infection is not clear. In this study, Mongolian gerbils were infected with H. pylori and necropsied continuously during 18 months. Bacterial clones were recovered and analyzed for BabA expression, Leb-binding activity, and adhesion to gastric mucosa. BabA expression was completely absent by 6 months post-infection. Loss of BabA expression was attributable to nucleotide changes within the babA gene that resulted in a truncated BabA. In response to the infection, changes in the epithelial glycosylation pattern were observed that were similar to responses observed in humans and monkeys. Furthermore, infections with BabA-expressing and BabA-nonexpressing H. pylori showed no differences in colonization, but infection with the BabA-expressing strain exhibited histological changes and increased inflammatory cell infiltration. This suggests that BabA expression contributes to severe mucosal injury.

Role of deletion located between the intermediate and middle regions of the Helicobacter pylori vacA gene in cases of gastroduodenal diseases.

The vacuolating cytotoxin gene of Helicobacter pylori, vacA, induces cytoplasmic vacuolation in gastric epithelial cells. Recently, the vacA intermediate (i) region, which is located between the signal (s) and middle (m) regions, was identified as a third polymorphic determinant of vacA activity. In vacA, there are approximately 81-bp deletions between the vacA i and m regions (denoted the d region). The aim was to clarify the roles of the vacA d region in relation to H. pylori-related diseases and histopathological gastric mucosal changes. We assessed the vacA signal s-, m-, i-, and d-region genotypes and cagA status in H. pylori isolates recovered from Western countries (n = 266) and East Asian countries (n = 244) by PCR. In East Asian countries, there were no relationships between the vacA genotypes and the clinical outcomes and histopathological changes. In Western countries, strains with the vacA s1, m1, i1, or d1 (no deletion) genotype significantly increased the risk for the development of gastric cancer compared with the risk from strains with the s2, m2, i2, or d2 genotype (adjusted odd ratios, 3.17 [95% confidence interval {CI}, 1.07 to 9.45] for s1, 10.65 [95% CI, 3.36 to 31.35] for m1, 8.57 [95% CI, 2.85 to 25.81] for i1, and 8.04 [95% CI, 2.67 to 24.16] for d1). The highly virulent vacA genotypes significantly enhanced neutrophil infiltration and gastric atrophy in univariant analysis, whereas only the vacA d-region genotype was significantly associated with neutrophil infiltration and gastric atrophy in both the antrum and the corpus by multiple linear regression analysis. The presence of the vacA d1 genotype in H. pylori strains could be an improved predictor of histological inflammation and the potential for atrophy compared with the presence of the vacA s-, m-, and i-region genotypes in Western countries.

Gastric mucosal interleukin-17 and -18 mRNA expression in Helicobacter pylori-induced Mongolian gerbils.

Helicobacter pylori infection causes characteristic mucosal infiltration of inflammatory cells, resulting in the development of peptic ulcers and gastric cancer in approximately 10% of cases. Different clinical expressions of the infection may reflect different patterns of cytokine expression. Interleukin (IL)-1ss, tumor necrosis factor (TNF)-alpha, IL-17, and IL-18 have been reported to be involved in H. pylori-induced gastric mucosal inflammation, but the details and relation to different patterns of inflammation remain unclear. Moreover, the proinflammatory virulence factor outer inflammatory protein (OipA) was reported to be associated with gastric mucosal inflammatory cytokine levels. To clarify these findings, Mongolian gerbils were infected for up to 12 months with wild-type H. pylori 7.13 or with isogenic oipA mutants for 3 months, and mucosal cytokines (IL-1ss, IL-17, IL-18, and TNF-alpha) mRNA levels were then assessed using real-time RT-PCR. Antral mucosal IL-1beta and IL-18 mRNA levels peaked 1 month after infection, whereas the peak of TNF-alpha mRNA was at 6-12 months; IL-17 levels peaked at 12 months. The inflammatory cell infiltration and mRNA levels of all cytokines studied were significantly lower in oipA mutants than in wild-type-infected gerbils. Mucosal IL-1ss, IL-17, and TNF-alpha expression, but not that of IL-18, were significantly associated with the grade of inflammatory cell infiltration. The pattern of increased inflammatory cytokines differed relative to the phase of the infection and pattern of inflammation. OipA appears to play a role in IL-1ss, IL-17, and TNF-alpha expression and the resulting inflammation.

Relationship between Helicobacter pylori hopQ genotype and clinical outcome in Asian and Western populations.

BACKGROUND AND AIMS: Outer membrane proteins of Helicobacter pylori mediate important pathogen-host interactions such as colonization, adhesion and the inflammatory response. hopQ genotypes have been suggested to be associated with increased risk of peptic ulcer. The aim of this study was to test the relation of hopQ genotype to H. pylori-related disease and histological changes in Asian and Western countries. METHODS: hopQ genotype, cagA status and vacA genotype of H. pylori isolated from patients from Asian and Western countries were determined and the results were compared with the clinical presentation and gastric histology. RESULTS: Most Asian strains possessed virulent genotypes (hopQ type I, vacA s1-m1 and cagA-positive). In Western countries, hopQ type I genotype was significantly linked with vacA s1 and m1 genotypes and cagA-positive status. Inflammatory cell infiltration and atrophy scores were significantly higher in patients with hopQ type I strains than those with type II in Western patients. However, the hopQ type I genotype was not associated with an increased risk for peptic ulcer or gastric cancer, and had no additive effects to vacA genotypes or cagA-positive status. CONCLUSION: The expression of multiple putative virulence factors in Asian strains likely explains the relatively high incidence of clinical outcomes including gastric cancer compared with other parts of the world. Although hopQ genotype did not improve the predictive value above other genotyping for development of H. pylori-related gastroduodenal diseases, the hopQ genotype might be able to add a useful virulence marker for gastroduodenal diseases.

Cardioprotective mechanism of telmisartan via PPAR-gamma-eNOS pathway in dahl salt-sensitive hypertensive rats.

BACKGROUND: Recently, some investigators have shown that telmisartan, an angiotensin II (Ang II)-receptor blocker (ARB), is a partial agonist of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma). We investigate whether telmisartan improves cardiovascular remodeling associated with the production of endothelial nitric oxide synthase (eNOS) through PPAR-gamma, inhibits the Rho-kinase pathway, and suppresses oxidative stress in Dahl salt-sensitive (DS) hypertensive rats. METHODS: Telmisartan (1 mg/kg per day) or telmisartan plus PPAR-gamma inhibitor, GW9662 (1 mg/kg per day) was administered from the age of 6-11 weeks. Age-matched male Dahl salt-resistant (DR) rats served as a control group. RESULTS: The levels of eNOS and PPAR-gamma expression, and eNOS phosphorylation were significantly lower in DS rats than in DR rats. Chronic telmisartan treatment in DS rats significantly increased these parameters, but not telmisartan plus GW9662. Telmisartan effectively inhibited the vascular lesion formation such as medial thickness and perivascular fibrosis, but not telmisartan plus GW9662. Moreover, upregulated RhoA protein, Rho-kinase mRNA, and myosin light-chain phosphorylation in DS rats was decreased by telmisartan to a similar degree as observed after treatment with Y-27632, a selective Rho-kinase inhibitor. In addition, NAD(P)H oxidase p22phox, p47phox, gp91phox expression, and mitogen-activated protein kinase and its downstream effector p70 S6 kinase phosphorylation in DS rats was also inhibited by telmisartan. CONCLUSIONS: These results suggest that the cardioprotective mechanism of telmisartan may be partly due to improvement of endothelial function associated with PPAR-gamma-eNOS, oxidative stress, and Rho-kinase pathway.

Cardioprotective effect of benidipine on cardiac performance and remodeling in failing rat hearts.

BACKGROUND: We have known that endothelial nitric-oxide synthase (eNOS) and oxidative stress may play a key role in cardiac performance in failing rat hearts. However, the interactions between eNOS or oxidative stress and bradykinin (BK) under treatment of calcium channel blockers (CCBs) remain unknown. To elucidate the mechanism underlying the cardioprotective effect of long-acting dihydropyridine CCBs, we evaluated the effect of benidipine on the BK-eNOS and NAD(P)H oxidase pathway in Dahl salt-sensitive (DS) rats with heart failure. METHODS: 11-week-old DS rats were treated with one of the following drug combinations for 7 weeks until the onset of the failing stage: vehicle, BK B2 receptor antagonist (FR172357 (FR)) alone, hydralazine, benidipine, and benidipine plus FR. The left ventricular end-systolic pressure-volume relationship (ESPVR) (contractility: Ees) was evaluated using a conductance catheter. RESULTS: Downregulated Ees and per cent of fractional shortening (%FS) assessed by echocardiography and eNOS expression in the failing stage were both significantly increased by using benidipine; this result was not found, however, when using FR alone or hydralazine or benidipine plus FR. Upregulated expression of NAD(P)H oxidase p22phox and p47phox and lectin-like oxidized low-density lipoprotein receptor-1, and downregulated superoxide dismutase-1 (SOD-1) were significantly ameliorated by benidipine, but not by FR alone or by hydralazine or benidipine plus FR. Benidipine effectively inhibited vascular lesion formation and suppressed atrial natriuretic peptide (ANP) and transforming growth factor-beta1 (TGF-beta1), but this was not the case when using FR alone or hydralazine or benidipine plus FR. CONCLUSIONS: These results suggest that benidipine may be useful for cardioprotective agents in preventing the cardiac dysfunction and remodeling associated with the BK-eNOS and oxidative stress pathway.

Cardioprotective effect of angiotensin II type 1 receptor antagonist associated with bradykinin-endothelial nitric oxide synthase and oxidative stress in Dahl salt-sensitive hypertensive rats.

OBJECTIVES: The interactions between eNOS or oxidative stress and bradykinin under long-term treatment of angiotensin II type 1 receptor antagonists (ATRA) remain unknown. To elucidate the molecular mechanisms of the cardioprotective effect of ATRA, we evaluated whether valsartan affects the bradykinin-eNOS and nicotinamide adenine dinucleotide (NAD(P)H) oxidase pathway. METHODS: After 5 weeks of feeding an 8% NaCl diet to 6-week-old Dahl salt-sensitive hypertensive (DS) rats, a distinct stage of concentric left ventricular hypertrophy (LVH) was noted. Six-week-old DS rats were treated with one of the following drug combinations for 5 weeks until the onset of LVH: vehicle; bradykinin B2 receptor antagonist FR172,357 alone; high-dose hydralazine; low-dose hydralazine; high-dose valsartan; low-dose valsartan; high and low-dose valsartan plus FR172,357. Age-matched Dahl salt-resistant rats fed the same diet served as controls. RESULTS: eNOS expression and activity, which was decreased in hypertrophy, was increased by high or low-dose valsartan, but not by high and low-dose valsartan plus FR172,357 or FR172,357 alone or high and low-dose hydralazine. The increased expression of NAD(P)H oxidase p22phox, p47phox, p67phox, and gp91phox in DS rats was suppressed by high or low-dose valsartan, but not by high or low-dose valsartan plus FR172,357 or FR172,357 alone or high and low-dose hydralazine. Valsartan effectively inhibited vascular lesion formation and suppressed the expression of transforming growth factor-beta1, connective tissue growth factor, and type I collagen, but not valsartan plus FR172,357 or FR172,357 alone or high and low-dose hydralazine. CONCLUSION: These findings suggest that valsartan may have cardioprotective effects in this model, partly associated with the bradykinin-eNOS and oxidative stress pathway.

Pattern of transcription factor activation in Helicobacter pylori-infected Mongolian gerbils.

BACKGROUND AND AIMS: Helicobacter pylori interact with epithelial cells resulting in activation of cellular signaling pathways leading to an inflammatory response. The pattern and timing of transcription factor activation in H pylori-infected gastric mucosa remain unclear. We investigated the roles of transcription factors in the gastric mucosa of H pylori-infected gerbils over the course of the infection. METHODS: Six-week-old male Mongolian gerbils were inoculated orally with H pylori TN2GF4 or isogenic cagE mutants and examined at 1, 3, 9, and 18 months. We examined the expression of 54 transcription factors using DNA/protein arrays and electrophoretic mobility shift assays. Phosphorylation status of mitogen-activated protein kinases and IkappaB were evaluated by immunoblot and immunohistochemistry. RESULTS: Ten transcription factors were up-regulated by H pylori infection. Six of these factors, including activator protein-1 (AP-1) and cAMP responsive element binding protein (CREB), reached maximal levels at 3 months and were strongly correlated with cellular inflammation and ulceration. Phosphorylation of extracellular signal-regulated kinase correlated with activation of AP-1 and CREB. Levels of nuclear factor-kappaB and interferon-stimulated responsive element (ISRE) peaked at 18 months and correlated with the presence of severe atrophy and with phosphorylation of Jun-N-terminal kinase (JNK), p38, and IkappaB. CONCLUSIONS: The gastric mucosal transcription factors induced by H pylori infection differed according to the phase and outcome of infection; AP-1 and CREB levels were early responders related to inflammation and ulceration, whereas NF-kappaB and ISRE were late responders related to atrophy.

Functional and intracellular signaling differences associated with the Helicobacter pylori AlpAB adhesin from Western and East Asian strains.

Following adhesion of Helicobacter pylori to gastric epithelial cells, intracellular signaling leads to cytokine production, which causes H. pylori-related gastric injury. Two adjacent homologous genes (alpA and alpB), which encode H. pylori outer membrane proteins, are thought to be associated with adhesion and cytokine induction. We co-cultured gastric epithelial cells with wild type H. pylori strains and their corresponding alpA/alpB-deleted mutants (DeltaalpAB). Results were confirmed by complementation. Flow cytometry confirmed that AlpAB was involved in cellular adhesion. Deletion of alpAB reduced interleukin (IL)-6 induction in gastric epithelial cells. Deletion of alpAB reduced IL-8 induction with East Asian but not with Western strains. All AlpAB-positive strains tested activated the extracellular signal-regulated kinase, c-Fos, and cAMP-responsive element-binding protein. Activation of the Jun-N-terminal kinase, c-Jun, and NF-kappaB was exclusive to AlpAB from East Asian strains. DeltaalpAB mutants poorly colonized the stomachs of C57BL/6 mice and were associated with lower mucosal levels of KC and IL-6. Our results suggest that AlpAB may induce gastric injury by mediating adherence to gastric epithelial cells and by modulating proinflammatory intracellular signaling cascades. Known geographical differences in H. pylori-related clinical outcomes may relate to differential effects of East Asian and Western types of AlpAB on NF-kappaB-related proinflammatory signaling pathways.