The C gene of hepatitis B virus (HBV) codes for a nucleocapsid protein made of 183 amino acid residues and is preceded in phase by the precore (pre-C) region, encoding 29 residues. The pre-C-region product is required for the synthesis and secretion of hepatitis B e antigen (HBeAg), which is made of the C-terminal 10 amino acid residues of the pre-C-region product and the N-terminal 149 residues of the C-gene product. HBV mutants with pre-C-region defects prevailed in the circulation of three asymptomatic carriers as they seroconverted from HBeAg to the corresponding antibody (anti-HBe), and these mutants finally replaced nondefective HBV. HBV DNA clones were propagated from sera of an additional 15 carriers with anti-HBe and sequenced for the pre-C region. Essentially all HBV DNA clones (56 of 57 [98%]) revealed mutations that prohibited the translation of a functional pre-C-region product. A point mutation from G to A at nucleotide 83, converting Trp-28 (TGG) to a stop codon (TAG), was by far the commonest and was observed in HBV DNA clones from 16 (89%) of 18 carriers seropositive for anti-HBe. In addition, there were point mutations involving ATG codon to abort the translation initiation of the pre-C region, as well as deletion and insertion to induce frameshifts. Such mutations leading to pre-C-region defects were rarely observed in persistently infected individuals positive for HBeAg or in patients with type B acute hepatitis after they had seroconverted to anti-HBe. These results would indicate a selection of pre-C-defective mutants in persistently infected hosts, along with seroconversion to anti-HBe, by immune elimination of hepatocytes harboring nondefective HBV with the expression of HBeAg.

This study was designed to investigate possible involvement of type IV collagenolytic matrix metalloproteinases (MMPs; 72-kDa type IV collagenase [MMP-2], 92-kDa type IV collagenase [MMP-9]), and the respective specific tissue inhibitors of these MMPs (TIMP-2 and TIMP-1) in the development of adult respiratory distress syndrome (ARDS). We determined the concentrations of these enzymes in the bronchoalveolar lavage fluid (BALF) from patients with ARDS using newly developed sensitive one-step sandwich enzyme immunoassay methods. BALF obtained from the 17 patients and eight healthy volunteer control subjects were also used for the analysis of the number of the cellular component. Concentrations of the 7S portion of type IV collagen and laminin in the BALF were measured as markers of basement membrane disruption. In the BALF from the ARDS patients, the concentrations of MMP-2 (66.7 +/- 57.0 ng/ml versus < 7.0 ng/ml for controls, p < 0.01) and MMP-9 (118.0 +/- 309.3 ng/ml versus 9.0 +/- 9.5 ng/ml for controls, p < 0.05), and the specific inhibitor of MMP-9 (TIMP-1) (161.0 +/- 145.0 ng/ml versus < 50 ng/ml for controls, p < 0.01) were significantly higher compared with those for healthy control subjects. In the ARDS patients, the concentrations of MMP-2 correlated both with those of 7S collagen and laminin; MMP-9 with the concentration of 7S collagen and the number of neutrophils. These findings suggest that the increased concentration of collagenolytic MMPs in lung plays a role in the pathogenesis of ARDS.

We examined whether prostaglandin (PG) H2, as an endothelium-dependent contracting factor, or the disturbed production of endothelium-derived relaxing factor, impairs endothelium-dependent relaxation and whether long-term inhibition of nitric oxide (NO) synthesis aggravates atherosclerosis in hypercholesterolemic rabbits. Male New Zealand White rabbits were fed one of the following diets: (1) standard chow; (2) 2% cholesterol-supplemented chow; (3) standard chow with 80 micrograms/mL N omega-nitro-L-arginine methylester (L-NAME), an NO synthetase inhibitor, in their drinking water; or (4) 2% cholesterol-supplemented chow with 80 or 160 micrograms/mL L-NAME in their drinking water. The rabbits were fed these diets for 8 or 12 weeks. Then aortic rings were obtained, and changes in isometric tension were recorded. Intimal atherosclerotic areas of the thoracic aortas were subsequently measured by planimetry. The cholesterol-supplemented diet significantly impaired endothelium-dependent aortic relaxation to acetylcholine. Pretreatment with the thromboxane A2/PGH2 receptor antagonist ONO-3708 did not reverse this impaired response. Vessels from both normocholesterolemic and hypercholesterolemic rabbits given L-NAME showed more impaired endothelium-dependent relaxation than those from their dietary counterparts not given L-NAME. Morphometric analysis revealed marked enlargement of intimal atherosclerotic areas in aortas from L-NAME-treated hypercholesterolemic rabbits compared with those from untreated hypercholesterolemic rabbits. These findings suggest that PGH2 does not contribute to impaired endothelium-dependent relaxation and that long-term administration of L-NAME promotes atherosclerosis by inhibition of NO synthesis in the hypercholesterolemic rabbit thoracic aorta.

In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35°C for 24 h in paper disks. At an inoculum level of 10 7 CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 10 3 to 10 4 CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 10 2 CFU/disk). After 22 to 24 months of subsequent storage at 4°C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (10 3 to 10 4 CFU/disk). In contrast to the case for storage at 4°C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25°C and 35°C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70°C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25°C.A nationwide outbreak of gastroenteritis due to consumption of dried squid chips (water activity, 0.5 to 0.6) contaminated with Salmonella enterica serovars Oranienburg and Chester occurred in Japan in 1999, in which a total of 1,634 cases were reported from all over the country (46/47 prefectures) (25). Even though this outbreak was the first one caused by contaminated dry foods in Japan, a number of outbreaks of gastroenteritis caused by dry foods such as dried milk, chocolate, potato chips, and almonds contaminated mainly by salmonellae have been reported in the United States and Europe since the 1960s (5,12,16,18). Since the middle of the 1990s, outbreaks of Shiga toxin-producing Escherichia coli (STEC) O157 infections have also been reported to be associated with dry foods such as deer jerky and salami in the United States (8,14,19,24). Many outbreaks of gastroenteritis associated with dry foods contaminated with salmonellae and STEC strains have been reported all over the world, even though the bacterial growth was inhibited by low water activity (a w ) in these dry foods. Accordingly, we tried to investigate the ability of these desiccated strains to survive under dried conditions, using salmonellae and STEC O157, O26, and O111 strains in paper disks with a w s of 0.5 to 0.6 and in selected dry foods. In order to study the effect of bacterial environments on the survival of bacteria during a process of being made "desiccated" bacteria and of storage conditions on the survival of desiccated bacteria produced after 24 h of drying, we examined t...

Aichi viruses isolated in Vero cells from seven patients in five gastroenteritis outbreaks in Japan, five Japanese returning from Southeast Asian countries, and five local children in Pakistan with gastroenteritis were examined for differentiation based on their reactivities with a monoclonal antibody to a standard strain (A846/88) and a reverse transcription-PCR (RT-PCR) of three genomic regions. The RNA sequences were determined for 519 bases of these 17 isolates at the putative junction between the C terminus of 3C and the N terminus of 3D. The analyses revealed an approximately 90% homology between these isolates, which were then divided into two groups: group 1 (genotype A) included six isolates from four outbreaks and one isolate from a traveler and group 2 (genotype B) included one isolate from the other outbreak, four isolates from returning travelers, and all of the isolates from the Pakistani children. Based on the isolate sequences, a primer pair and a biotin-labeled probe were designed for amplification and detection of 223 bases at the 3C-3D junction of Aichi virus RNA in fecal specimens. The Aichi virus RNA was detected in 54 (55%) of 99 fecal specimens from the patients in 12 (32%) of 37 outbreaks of gastroenteritis in Japan. Of the 12 outbreaks, 11 were suspected to be due to genotype A. These results indicated that RT-PCR can be a useful tool to detect Aichi virus in stool samples and that a sequence analysis of PCR products can be employed to identify the prevalent strain in each incident.

We characterized the carbohydrate-fermenting ability of 31 strains of Shiga toxin-producing Escherichia coli (STEC) O26 isolated from diarrhea patients in Aichi Prefecture, Japan, in order to establish selective isolation media for these strains. None of the 31 STEC O26 strains (24 O26:H11, 7 O26:H؊) fermented rhamnose, whereas all of the other 108 STEC strains (100 O157, 8 O111) and all of the non-STEC strains except one (i.e., 58 of 59) fermented rhamnose. The great majority of the STEC O26 strains (96.8% [30 of 31]) showed very high resistance to potassium tellurite (MIC > 50 g/ml), whereas the majority of the non-STEC strains (72.9% [43 of 59]) showed very high sensitivity (MIC < 1.56 g/ml) to this compound. Accordingly, we developed a rhamnose-MacConkey (RMAC) medium in which the lactose in MacConkey medium was replaced by rhamnose, and cefixime-tellurite-RMAC (CT-RMAC) medium in which potassium tellurite (2.5 mg/liter) and cefixime (0.05 mg/liter) were added to RMAC. All of the STEC O26 strains generated colorless (rhamnosenonfermented) colonies on both media; the vast majority of selected E. coli strains (95.7% [89 of 93; including 26 STEC O157, 8 STEC O111]), other than STEC O26, generated red colonies on RMAC, and most of the non-STEC strains (84.7% [50 of 59]) did not grow on CT-RMAC. We demonstrate that both the RMAC and the CT-RMAC media can be used for the isolation of STEC O26 and that CT-RMAC has better specificity for the routine isolation for STEC O26 in a laboratory.Since the first recognized outbreak in Oregon and Michigan in the United States in 1982, Shiga toxin-producing Escherichia coli (STEC) O157 has emerged as a food-borne pathogen of a significant public health concern in the United States, Canada, and Europe (8,10,12).In Japan, the first outbreak of STEC O157 was reported in 1990, in which two kindergartners among 319 patients died of hemolytic-uremic syndrome. After this outbreak, the reported number of STEC strains isolated was ca. 100 annually between 1991 and 1995. The number of STEC strains isolated increased abruptly to 3,021 in 1996, and ca. 2,000 strains have been isolated since 1997. STEC O157 has been the predominant O serotype among the isolated STEC strains, comprising 90.7% (476 of 525) of the total from 1991 to 1995 and 72.1% of the total in 1999; however, this predominance has been decreasing gradually in recent years (5). Accordingly, the number of STEC strains isolated other than O157 has been increasing gradually. Among these non-O157 STEC isolates, STEC O26 has been the most common serotype, comprising 18.0% (1,066 of 5,913) of the total number of STEC isolates reported from 1997 to 1999 (5). However, effective and selective isolation media for STEC O26 have not been established, whereas isolation media for STEC O157 are widely used in routine laboratory examination in many facilities.We sought to find useful markers for detecting STEC O26 by investigating its carbohydrate-fermenting ability and to develop and evaluate selective media for STEC O26 isolation on the b...

While asthma is an inflammatory disorder of the airways involving mediators released from mast cells and eosinophils, inflammation alone is insufficient to explain the chronic nature of the disease. Recent progress in the understanding of disease pathogenesis has revealed that airway remodeling, which is at least in part due to an excess of extracellular matrix (ECM) deposition in the airway wall, plays a significant role in airflow obstruction. Matrix metalloproteinases (MMPs) have been suggested to be the major proteolytic enzymes to induce airway remodeling in asthma and COPD. It has been widely accepted that different inflammatory processes are involved in asthma and COPD with different inflammatory cells, mediators, and responses to treatments. Despite these different processes, airflow obstruction and airway remodeling characterize these two diseases. MMP-2 and -9 have been reported to be involved in the pathogenesis of airway remodeling in both diseases and MMP-12, in addition to these MMPs, in the pathogenesis of COPD. In this review, we discuss the current views on the role of MMPs in the pathogenesis of bronchial asthma and COPD. Anti-MMP therapy could theoretically be useful to prevent airway remodeling in asthma and COPD. However, to date no clinical data are available regarding the efficacy of anti-MMP therapies in the treatment of patients with asthma and COPD.

To examine a possibility that matrix metalloproteinases (MMPs) participate in the pathogenesis of asthma and/or the development of asthma attack, we measured the concentrations of MMP-2, MMP-9, and their respective tissue inhibitors of metalloproteinases (TIMP)-2 and TIMP-1, in induced sputa collected from 28 patients with moderate to severe bronchial asthma. Specimens were collected during both the attack and the remission from 15 age- and sex-matched healthy control subjects. The concentration of MMP-9 was significantly (p < 0.05) higher in the patients, even during the remission, as compared to that in healthy controls. The concentrations of MMP-9 (p < 0.05) and its specific inhibitor TIMP-1 (p < 0.01), and MMP-2 (p < 0.01) in these patients during the attack were significantly higher than those in healthy controls. In these patients, the MMP-9 concentration was significantly higher (p < 0.05) during the attack than during the remission. These results suggest that MMPs and TIMPs may be involved in the pathogenesis of bronchial asthma, and that the increased MMP-9 might be involved in the development of attack in patients with chronic asthma.