Akira Ohtsuru

Impact of Helicobacter pylori infection on gastric and plasma ghrelin dynamics in humans.

OBJECTIVES:There are contradictory reports on the relationship between Helicobacter pylori and circulating ghrelin. We sought to clarify the influence of H. pylori infection on gastric and plasma ghrelin dynamics in humans.METHODS:Using endoscopic biopsies from the corpus of 56 H. pylori-infected patients and 25 uninfected subjects, ghrelin mRNA expression levels and gastric ghrelin peptide contents were measured by real-time polymerase chain reaction and radioimmunoassay, respectively. We also measured plasma ghrelin concentrations and analyzed the numbers of ghrelin immunoreactive cells in the fundic gland area. Fifty-one patients with H. pylori infection were treated with a 7-day triple therapy consisting of lansoprazole, clarithromycin, and amoxicillin.RESULTS:The gastric ghrelin mRNA expression level of H. pylori-positive patients (1.64 ± 1.27 in arbitrary units) was significantly lower than in H. pylori-negative subjects (4.87 ± 4.1, p < 0.0001). A similar trend was noted for ghrelin peptide contents (31.2 ± 27.5 vs 81.2 ± 64.1 ng/mg protein, respectively, p < 0.0001). There was no significant difference in the number of ghrelin immunoreactive cells/mm2 in terms of H. plyori status. Plasma ghrelin concentrations in H. pylori-infected patients (144.6 ± 7.8.8 fmol/ml) were significantly lower than in uninfected subjects (196.1 ± 97.2, p < 0.05) and increased following cure of the infection. Plasma ghrelin levels correlated positively with the expression levels of ghrelin mRNA (r = 0.583, p < 0.0001) and peptide products (r = 0.574, p < 0.0001). There was a significant stepwise decrease in gastric ghrelin mRNA expression (p < 0.05), peptide contents (p < 0.01) and density of ghrelin immunoreactive cells (p < 0.05) with progression of histological severity of glandular atrophy in the corpus. The histological severity of chronic inflammation also negatively influenced the ghrelin mRNA expression (p < 0.001) and peptide production (p < 0.005).CONCLUSIONS:H. pylori infection has a negative impact on gastric and plasma ghrelin dynamics. Chronic inflammatory and atrophic changes associated with the infection may affect gastric ghrelin biosynthesis and contribute to the low circulating levels.

Enhanced Expression of Interleukin-8 and Activation of Nuclear Factor Kappa-B in Endoscopy-negative Gastroesophageal Reflux Disease

OBJECTIVE:Interleukin-8 (IL-8) mediates neutrophil trafficking via its receptors. Recent studies have shown that IL-8 is likely involved in the development and progression of erosive reflux esophagitis (RE), yet little is known about its implication in endoscopy-negative gastroesophageal reflux disease (GERD). The purpose of this study was to determine IL-8 messenger ribonucleic acid (mRNA) expression levels in endoscopy-negative GERD, along with assessment of nuclear factor kappaB (NF-κB) activation, which upregulates IL-8 expression.METHODS:We studied 31 patients with endoscopy-negative GERD, 15 patients with erosive RE, and 15 asymptomatic controls. Paired biopsy samples were taken from the esophagus 3 cm above the gastroesophageal junction; one biopsy was snap-frozen for measurement of IL-8 mRNA levels by real-time quantitative polymerase chain reaction, and another was formalin-fixed for histopathological evaluation. In nine endoscopy-negative GERD patients, the IL-8 mRNA expression levels were measured before and 8 wk after treatment with lansoprazole. We also sampled additional specimens for NF-κB-DNA binding assay and immunohistochemical analyses of NF-κB p65 and p50 subunits, IL-8 and specific IL-8 receptor, CXCR-1.RESULTS:The relative IL-8 mRNA expression levels were significantly higher in esophageal mucosa of patients with endoscopy-negative GERD than those of the controls. The presence of basal zone hyperplasia and intraepithelial neutrophils, histopathological hallmarks of GERD, were associated with higher levels of IL-8 mRNA. Lansoprazole treatment significantly reduced the IL-8 mRNA expression levels. The esophageal epithelium of patients with GERD showed intense immunoreactivity for IL-8, and expressed CXCR-1 antigen. We found NF-κB activation in esophageal mucosa in GERD patients and the NF-κB subunits were localized predominantly in the nuclei of IL-8-expressing cells.CONCLUSIONS:Our results demonstrate enhanced mucosal expression of IL-8 in incipient GERD even without mucosal breaks. NF-κB activation may be implicated in the pathogenesis in GERD.

Expression of Tie‐1 and 2 receptors, and angiopoietin‐1, 2 and 4 in gastric carcinoma; immunohistochemical analyses and correlation with clinicopathological factors

Aims:u2002 There is strong evidence that tyrosine kinases are involved in the regulation of tumour progression, cellular growth and differentiation. Recently, many kinds of tyrosine kinase receptors have been reported, and among them Tie‐1 and 2 constitute a major class. Angiopoietin (Ang)‐1 is known as a ligand of the Tie‐2 tyrosine kinase receptor. The aim of this study was to determine the expression profile of Tie‐1 and 2 and Ang‐1, 2 and 4 in gastric adenocarcinoma.

Inhibition of ABL Tyrosine Kinase Potentiates Radiation-Induced Terminal Growth Arrest in Anaplastic Thyroid Cancer Cells

Abstract Podtcheko, A., Ohtsuru, A., Namba, H., Saenko, V., Starenki, D., Palona, I., Sedliarou, I., Rogounovitch, T. and Yamashita, S. Inhibition of ABL Tyrosine Kinase Potentiates Radiation-Induced Terminal Growth Arrest in Anaplastic Thyroid Cancer Cells. Radiat. Res. 165, 35–42 (2006). Gleevec®, a selective tyrosine kinase inhibitor, retarded the growth of anaplastic thyroid cancer cell lines in vitro and in vivo through selective inhibition of ABL tyrosine kinase activity. In the present study, we investigated the ability of Gleevec® to modulate the in vitro and in vivo radiation response of anaplastic thyroid cancer cells. Cell growth assays, colony formation assays and xenograft models were used to quantify the radiosensitizing effect of Gleevec® in cells of the anaplastic thyroid cancer cell lines ARO and FRO. FACS, Western blotting and histochemical techniques were employed to study the mechanisms of radiation response after exposure to Gleevec®. Gleevec® (7.0 μM) increased the anti-proliferative effect of radiation on the growth ARO and FRO cells in vitro. Clonogenic analysis demonstrated that Gleevec® reduced cell survival after irradiation. Gleevec® combined with radiation produced an increase in tumor growth inhibition compared to treatment with either modality alone in mice bearing anaplastic thyroid cancer xenografts. The drug suppressed radiation-induced ABL activation and promoted CDKN1A (p21cip1) accumulation in irradiated anaplastic thyroid cancer cells. Gleevec® had an additional effect on radiation-induced apoptosis in cells of both cell lines and potentiated the induction of terminal growth arrest accompanied by the expression of senescence-associated β-galactosidase. The antitumor effect of Gleevec® is potentiated in adjunctive therapy with radiation not only due to inhibition of proliferative cell growth with transient cell cycle arrest and apoptosis, but also due to the terminal growth arrest associated with senescence, suggesting that tumor cell senescence is a mechanism for tumor targeting therapy in combination with ionizing radiation.

GH suppresses TGF-β-mediated fibrosis and retains cardiac diastolic function

Abstract The aims of this study were to elucidate the molecular mechanism by which growth hormone (GH) excess is anti-fibrotic in vitro and in vivo model. The in vivo model GH excess showed a significant increase of relative wall thickness with no concomitant disturbance of cardiac diastolic function. Western blot for extracellular matrix (ECM) structural proteins showed minimal change in the GH treatment group, compared to an Angiotensin II (Ang II) subpressor dose group. In cultured cardiac fibroblasts, we investigated the abundance of ECM proteins, phosphorylation of p38 mitogen-activated protein kinase (MAPK), and transforming growth factor-β (TGF-β)-specific transcriptional activity. GH down-regulated the expression of PAI-1 and fibronectin proteins activated by TGF-β. In reporter assays, GH, but not insulin-like growth factor-1 (IGF-1), reduced TGF-β-specific transcriptional activity. Moreover, GH markedly down-regulated TGF-β-induced phosphorylation of p38 MAPK. These results demonstrated that a chronic excess of GH have an anti-fibrotic effect on cardiac remodeling, probably through a down-regulation of TGF-β signaling via de-phosphorylation of p38 MAPK.

Eradication of hepatocellular carcinoma xenografts by radiolabelled, lipiodol-inducible gene therapy

The promoter region of the early-growth response-1(Egr-1) gene has been shown to be activated by external radiation, thus making a selective tumoricidal effect possible. A previous experiment showed that the Egr-1 promoter can be activated by internal radiation using radioisotopes as well as external radiation. Internal radiation using I-131 lipiodol (I-131-Lip) has been established as one of the most useful therapeutic strategies against hepatoma. We herein linked the Egr-1 promoter to the herpes simplex virus-thymidine kinase (HSV-TK) gene, and investigated its efficacy in hepatoma gene therapy in combination with I-131-Lip. A luciferase assay showed the Egr-1-promoter activity to be markedly increased in hepatoma tissue specimens in an I-131-dose-dependent manner, whereas a less than two-fold increase in this activity was observed in other organs. In addition, the radioactivity derived from I-131 was selectively accumulated in the tumor tissue specimens. To examine the efficacy of EgrTK/ganciclovir (GCV) gene therapy in vivo, subcutaneous hepatoma xenografts in nude mice were transfected using a hemagglutinating virus of Japan (HVJ)-liposome vector. Complete tumor regression was observed in all the EgrTK-transfected tumors following combination treatment with I-131-Lip and GCV 42 days after treatment without any side effects (n=8). In contrast, the tumors continued to grow in all control mice (n=10). Furthermore, the serum α-fetoprotein levels decreased in the combination therapy group, while they increased in the controls. In conclusion, these data indicate that Egr-1 promoter-based gene therapy combined with internal radiation has a selective effect on hepatoma tumors while also showing an improved in vivo efficacy. This combination therapy might, therefore, be an effective human hepatoma gene therapy, even in advanced multiple cases.

The efficacy and safety of gene transfer into the porcine liver in vivo by HVJ (sendai virus) liposome

Background. Gene transfer systems using viral vectors are efficient; however, most viral vectors also tend to evoke immunologic reactions, thereby clinically causing serial side effects. HVJ-liposome vector is a hybrid vector consisting of liposome and an inactivated Sendai virus (Hemmagglutinating Virus of Japan [HVJ]), which has been reported to be less immunogenic and can also be repeatedly administered. We examined the usefulness of this vector for hepatic gene therapy in a pig model. Methods. Genes encoding &bgr;-galactosidase and luciferase were used as reporter genes. The pigs were injected with the reporter gene loaded-HVJ-liposome into the portal vein under total vascular exclusion of the liver. The transfection efficiencies were then assessed by &bgr;-galactosidase staining, a luciferase assay, and RT-PCR for LacZ mRNA. Biochemical and histologic analyses were performed to evaluate tissue toxicity after gene transfer. Results. The luciferase gene expression in the liver reached its highest level at 7 days after transfection. It continued to be detected up to 28 days after transfection, while all pigs remained healthy throughout the observation period. The transfection efficiency was 15% in the hepatocytes according to &bgr;-galactosidase staining. Extrahepatic transgene expression was slightly observed in the lung and kidney, but not in the spleen or ovary. Conclusions. These data suggest for the first time that the use of the HVJ-liposome vector is a safe and feasible modality for liver-directed gene transfer in pigs, and it might therefore be suitable for clinical gene therapy trials.