Akira Nishizono

Gastric Ulcer, Atrophic Gastritis, and Intestinal Metaplasia Caused by Helicobacter pylori Infection in Mongolian Gerbils

BACKGROUND Helicobacter pylori infection is associated with gastroduodenal disease in humans. In this study we aimed to show this relationship directly in Mongolian gerbils. METHODS The animals were challenged orally with H. pylori and killed 1, 2, 3, and 6 months after inoculation for histologic and anti-H. pylori antibody titer examination. RESULTS The spiral bacteria were observed in the mucus and gastric pits of all infected animals. A severe infiltration of the lamina propria by polymorphonuclear and mononuclear cells was seen 1 month after H. pylori inoculation. The submucosa was infiltrated by mainly mononuclear cells with formation of lymphoid follicles. Erosion of the gastric mucosa appeared soon after inoculation, whereas gastric ulcers, gastritis cystica profunda, and atrophy with goblet cell metaplasia occurred between 3 and 6 months after inoculation. In the duodenal mucosa a mild inflammatory cell infiltration with ballooning and diminished number of duodenal glands was seen. The IgG anti-H. pylori antibody titer increased gradually after 2 months of inoculation. CONCLUSIONS Since the gastritis, gastric ulcers, atrophic gastritis, and intestinal metaplasia that developed in Mongolian gerbils were similar to those observed in humans, this model may be useful to study the therapy of gastric ulcer and, with a longer observation period, to confirm a possible relationship between H. pylori and malignancy.

TNF-α-inducing protein, a carcinogenic factor secreted from H. pylori, enters gastric cancer cells

TNF‐α inducing protein (Tipα) is secreted from Helicobacter pylori (H. pylori): it is a potent inducer of TNF‐α and chemokine genes, mediated through NF‐κB activation, and it also induces tumor‐promoting activity in Bhas 42 cells. To investigate the carcinogenic mechanisms of H. pylori with Tipα, we first examined how Tipα acts on gastric epithelial cells. We found that fluorescent‐Tipα specifically bound to, and then entered, the cells in a dose‐ and temperature‐dependent manner, whereas deletion mutant of Tipα (del‐Tipα), an inactive form, neither bound to nor entered the cells, suggesting the presence of a specific binding molecule. Mutagenesis analysis of Tipα revealed that a dimer formation of Tipα with a disulfide bond is required for both specific binding and induction of TNF‐α gene expression. A confocal laser scanning microscope revealed some Tipα in the nuclei, but del‐Tipα was not present, which indicated that an active form of Tipα can penetrate the nucleus and may be involved in the induction of TNF‐α gene expression. Examination of Tipα production and secretion in 28 clinical isolates revealed that H. pylori obtained from gastric cancer patients secreted Tipα in significantly higher amounts than did H. pylori from patients with chronic gastritis, suggesting that Tipα is an essential factor in H. pylori inflammation and cancer microenvironment in the human stomach. Tipα is thus a new carcinogenic factor of H. pylori that can enter the nucleus through a specific binding molecule, and its mechanism of action is completely different from that of CagA.

Therapeutic Oral Vaccination Induces Mucosal Immune Response Sufficient to Eliminate Long‐Term Helicobacter pylori Infection

We examined the efficacy of therapeutic oral vaccination using Helicobacter pylori‐whole cell sonicate and cholera toxin (CT) in mice persistently infected with H. pylori. Efficacy was determined by bacterial culture and microscopic examination of gastric tissues for the persistence of bacteria at 6 weeks after the last vaccination. Vaccination of H. pylori‐whole cell sonicate combined with CT eradicated bacteria in 10/16 mice (62.5%). Interestingly, oral vaccination with CT alone also eliminated the bacteria in 8/17 mice (47.1%). However, a therapeutic intraperitoneally administered vaccine failed to eradicate H. pylori from the stomach (1/17 mice, 5.9%). Identification of the type of immunity involved in the eradication process showed that oral vaccination enhanced the antigen‐specific IgA in the feces and saliva. The efficacy of eradication of H. pylori correlated well with increases in IgA secretion in mucosal tissue and a higher labeling index of IgA‐positive lumina of pyloric glands. Moreover, the expression of IL‐4 mRNA in the stomach of mice with eradicated bacteria was higher than in the uneradicated group. Our results suggest that the efficacy of vaccination depends on the mucosal IgA response in the gastrointestinal tract against H. pylori via Th2 cell activation and that therapeutic oral vaccination induces a mucosal immune response sufficient to eradicate long‐term infection with H. pylori.

Analysis of p53 gene mutations in Helicobacter pylori-associated gastritis mucosa in endoscopic biopsy specimens.

BACKGROUND No previous report has shown the relationship between Helicobacter pylori infection and a direct sequence analysis of p53 gene mutation in a biopsy sample of human gastric mucosa. METHODS A total of 60 endoscopic biopsies samples (21 patients with H. pylori-positive gastritis and 9 patients with H. pylori-negative gastritis), including antral mucosa and corpus mucosa, were used in this study. Direct DNA sequencing of exons 5, 6, 7, and 8 of the p53 gene was performed by the dyedeoxy terminator method. RESULTS Mutations in the p53 gene were identified in non-hot spot codons in exon 7 and 8 in 11 of 21 samples (52.4%) from H. pylori-positive gastritis patients. There was no mutation in H. pylori-negative gastritis patients. CONCLUSIONS This finding shows that H. pylori infection can induce p53 point mutations and appears to be involved in the pathway leading to dysplasia or carcinoma.

Diversity of human rotavirus G9 among children in Turkey

Between September 2004 and December 2005 a prospective study was conducted to understand the epidemiology of rotavirus infection among children with diarrhea attending two hospitals in Ankara, Turkey. Rotavirus was detected in 39.7% of the 322 stool samples and affected mainly children in the age group of 6–23 months. More than 70% and 39% of these cases occurred in children <2 and <1 year of age, respectively. In the temperate climate of Ankara rotavirus infection was prevalent throughout the year. Serotype G1P[8] was dominant followed by G9P[8]. In 38 samples a total of 5 electropherotypes were detected. All G9P[8] were of long electropherotype except one of short electropherotype. A proportion of G1 and G9 strains were in combination with P[6], P[4] or P nontypable. Mixed serotypes were responsible for 2.4% of the infections. A phylogenetic tree constructed with the deduced amino acid sequences of the VP7 gene showed that 16 Turkish G9 strains clustered with rotaviruses of lineage III. One G9 strain formed a new lineage, lineage IV with the Sri Lankan G9 rotaviruses. In the phylogenetic tree of the VP8* gene, the Turkish G9P[6] rotaviruses clustered with human strains of lineage Ia. Increased diversity of the G/P type combination and the presence of infection throughout the year in Turkey was a situation similar to developing countries. The occurrence of rotavirus infection at later age and low level of mixed infections in Turkey represented the situation of developed countries. This study suggests that diverse G9 rotaviruses are emerging in Turkey. J. Med. Virol. 80:733–740, 2008.

Detection of Human Bocavirus in the Cerebrospinal Fluid of Children With Encephalitis

We report 4 children with encephalitis associated with human bocavirus (HBoV) 1 or 2. All children were severely underweight, and 2 died; 1 of them had a matching HBoV2 nucleotide sequence isolated from serum and bocavirus like particles in the cerebrospinal fluid that were observed with electron microscopy. No further pathogens were detected in the cerebrospinal fluid of these patients.

A simple and rapid immunochromatographic test kit for rabies diagnosis

In rabies endemic countries, funds and infrastructure are often insufficient to employ the approved gold standard for the definitive diagnosis of rabies: the direct fluorescent test. In the present study, two types (type 1 and 2) of an ICT kit were evaluated for detection of rabies. These were developed using monoclonal antibodies which recognize epitope II and III of the nucleoprotein of rabies virus. Both kits specifically detected all rabies virus strains and there was no cross reactivity with Lyssaviruses (Lagos, Mokola and Duvenhage), Rhabdovirus (VSV and Oita 296/1972) and other common canine‐pathogenic viruses. In type 1, a single type of monoclonal antibody was used. It was capable of detecting recombinant nucleoprotein and showed sensitivity of 95.5% (42/44) and specificity of 88.9% (32/36) using brain samples from rabid dogs. In contrast, type 2 which was made of two different monoclonal antibodies had a lower sensitivity of 93.2% (41/44) and higher specificity of 100% (36/36). These ICT kits provide a simple and rapid method for rabies detection. They need neither cold chain for transportation nor complicated training for personnel. This diagnostic test is suitable for rabies screening, particularly in areas with a high prevalence of rabies and where the fluorescent antibody test is not available.

Rapid desensitization of serotonin 5-HT2C receptor-stimulated intracellular calcium mobilization in CHO cells transfected with cloned human 5-HT2C receptors

Abstract: Serotonin 5‐HT2C receptor‐mediated intracellular Ca2+ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5‐HT2C receptors. Fura‐2 acetoxymethyl ester was used to investigate the regulation of 5‐HT2C receptor function. CHO cells, transfected with a cDNA clone for the 5‐HT2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin‐sensitive [3H]mesulergine binding (KD = 0.49 nM). The addition of 5‐HT mobilized intracellular Ca2+ in a dose‐dependent fashion, ranging from a basal level of 99 ± 1.8 up to 379 ± 18 nM, with an EC50 value for 5‐HT of 0.029 µM. Exposure to 5‐HT, 1‐(3‐chlorophenyl)piperazine dihydrochloride (a 5‐HT2C agonist), and 1‐(4‐iodo‐2,5‐dimethoxyphenyl)‐2‐aminopropane (a 5‐HT2C and 5‐HT2A agonist) resulted in increased intracellular Ca2+ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5‐HT‐mediated intracellular Ca2+ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5‐HT in a time‐ and concentration‐dependent manner. Mezerein and phorbol 12‐myristate 13‐acetate, protein kinase C activators, weakly inhibited the intracellular Ca2+ mobilization induced by 10 µM 5‐HT. Furthermore, the protein kinase C inhibitor H‐7 partially prevented the protein kinase C activator‐induced inhibition of the 5‐HT‐mediated increase in intracellular Ca2+ concentration. The desensitization induced by pretreatment with 5‐HT was blocked by W‐7, added in conjunction with 5‐HT, and partially inhibited by W‐5, a nonselective inhibitor of protein kinases and weak analogue of W‐7. Therefore, the 5‐HT2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5‐HT2C receptor activation mobilizes Ca2+ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase‐mediated feedback.

Sequential analyses of the mutations in the core upstream and precore regions of hepatitis B virus genome in anti-HBe positive-carriers developing acute exacerbation.

The nucleotide sequences of the core upstream and precore regions (371 nucleotide length, nt. 1604‐1974) of hepatitis B virus (HBV) were analysed sequentially in three subjects who were positive serorogically for anti‐HBe and had acute clinical exacerbation after immunosuppressive treatment. These patients were asymptomatic HBV carriers before therapy. The results revealed that the mutant with an 8‐bp deletion (nt. 1768–1775) located in the basic core promoter region was dominant in the asymptomatic HBV carrier phase in two of three subjects. After exacerbation, however, such mutant clones possessing 8‐bp deletion disappeared or decreased in number and were replaced by the clones possessing a precore stop codon mutation G to A (nt. 1896) or by the clones possessing additional contiguous point mutations A to T (nt. 1762) and G to A (nt. 1764) and a new point mutation C to T (nt. 1653). Possible relationships between acute exacerbation of liver function accompanied by mutation and the transition of the dominant clones were discussed. J. Med. Virol. 53:266–272, 1997.

Co-dominance of G1 and emerging G3 rotaviruses in Hong Kong: A three-year surveillance in three major hospitals

BACKGROUND The World Health Organization recommends rotavirus vaccines be included in all national immunization programs as part of a strategy to control diarrhoeal diseases. Sentinel surveillance is advised to monitor impact post-vaccine introduction and to document changes in genotype distribution. OBJECTIVES To determine the molecular epidemiology of circulating rotaviruses in Hong Kong prior to implementation of universal rotavirus vaccination. STUDY DESIGN From December 2004 through December 2007, 830 rotavirus-positive stool samples from subjects admitted for acute diarrhea to three major hospitals in Hong Kong were examined. The electropherotypes, and the G and P genotypes of these rotaviruses were determined. Phylogenetic analysis of the VP7 gene was performed. RESULTS G3P[8] was the dominant genotype (46.1%), followed by G1P[8] (36.5%) and G9P[8] (9.2%). A total of 35 electropherotypes were identified. The G3 and G1 strains had high sequence similarities among themselves and were clustered with strains from Asia particularly mainland China. The G9 strains were clustered with the globally spreading strains. G12 and G4 were not found. The prevalence of rotavirus infection peaked in winter season when temperature was low, atmospheric pressure was high, relative humidity was low and rainfall was negligible. CONCLUSIONS Genotype G3 and G1 were the dominant rotaviruses circulating in Hong Kong between 2004 and 2007. Strains were mainly related with those from mainland China. Ongoing surveillance of circulating genotypes should continue in anticipation of universal rotavirus vaccine introduction.