[Malignant pleural mesothelioma].

Malignant pleural mesothelioma carries a poor prognosis, for which no standard therapy has been established. We report 15 cases of malignant pleural mesothelioma experienced since 2000 focusing on their clinical features. They included 14 male and 1 female aged 38 to 81 (62.8 on average) years. All patients were diagnosed by pleural biopsy under thoracoscopic guidance. Histology of the pleural biopsy specimen showed epithelial mesothelioma in 8 patients, biphasic mesothelioma in 3, sarcomatous mesothelioma in 2 and desmoplastic malignant mesothelioma (DMM) in 2. Twelve patients received chemotherapy. Of these, 3 were followed by surgery. In addition to 2 of these 3 patients, 2 underwent extrapleural pneumonectomy (EPP) without adjuvant treatment. Remaining 1 received palliative treatment only. As a result, 6 patients are surviving, 7 died of primary diseases and 2 died of other diseases. The longest survival time with chemotherapy is 41 months in a surviving patient with epithelial mesothelioma and that with EPP is 25 months in a surviving patient with DMM. The 2-year survival rate of the 14 patients was 44.4% and the median survival time in patients with epithelial mesothelioma was 30.6 months.

Real-time detection system for quantitation of hepatitis C virus RNA: a comparison with the other three methods.

Real-time detection (RTD) system for quantitation of hepatitis C virus (HCV) was developed. Its sensitivity and usefulness were compared with the other three commercially available methods for quantitation of HCV. The sera of 166 patients positive for serum HCV RNA by Amplicor HCV test were assessed. HCV was detected in 78.5% (128/163) by branched DNA assay, in 88.8% (111/125) by HCV core protein assay, in 94.5% (156/165) by Amplicor HCV Monitor test, and in 97.0% (161/166) by the RTD system. The values of viral load by the RTD system were significantly well correlated with those obtained by the other three methods. In the 50 patients treated by interferons (IFNs), the range which predicts the highest sustained response rate was less than 0.5 Meq/ml for branched DNA assay (sustained response rate: 57.9% (11/19)), less than 1 kcopies/ml for Amplicor HCV Monitor test (85.7% (6/7)), and less than 10(4) copies/ml for RTD system (100% (7/7)). None of the patients with greater than or equal to 2.8 Meq/ml by branched DNA assay (n=14), greater than or equal to 250 kcopies/ml by Amplicor HCV Monitor test (n=19), or greater than or equal to 2x10(6) copies/ml by RTD system (n=16) obtained sustained response. In conclusion, RTD system was demonstrated to be the most sensitive method for quantitation of HCV, and useful for the prediction of sustained response to IFN therapy.

Clinical implications of TT virus superinfection in patients with chronic hepatitis C.

OBJECTIVE: TT virus (TTV) has been identified as a candidate agent of non-A-E hepatitis virus. We investigated superinfection of TTV in patients with chronic hepatitis C and studied the susceptibility to interferon (IFN) treatment and its association with liver disease caused by hepatitis C virus (HCV). METHODS: TTV DNA was examined using the seminested polymerase chain reaction (PCR), and its virus level was measured by the real-time fluorometric PCR. RESULTS: TTV DNA was detected in 20 of 102 (19.6%) patients examined. There was no significant difference in the alanine aminotransferase (ALT) level between patients with or without TTV DNA. Quantitative analysis of HCV RNA and TTV DNA revealed no correlation between virus levels in HCV/TTV-coinfected patients. Both TTV and HCV were sensitive to IFN therapy. Complete response to IFN with a sustained loss of viremia for 24 wk after completion of IFN treatment was found in 11 of 20 (55%) patients with respect to TTV DNA and in five of 20 (25%) patients with respect to HCV RNA. The mean pretreatment HCV RNA level was significantly lower in the complete-response cases than in the no-response cases, but there was no significant difference in the pretreatment TTV DNA levels between them. ALT normalization resulting from IFN therapy was not attributable to the eradication of TTV DNA but was attributable to that of HCV RNA. Superinfection by TTV did not influence the effect of IFN against HCV. No specific TTV genotype correlating with IFN sensitivity was found. CONCLUSIONS: These results suggest that TTV infection stands independent of HCV infection, with no influence on liver injury as a result of HCV infection.

Detection of clarithromycin-resistant helicobacter pylori strains by a preferential homoduplex formation assay.

It has been shown that resistance to clarithromycin, a major cause of failure in Helicobacter pylori eradication therapy, is associated with point mutations in the 23S rRNA gene. We sought to apply the preferential homoduplex formation assay (PHFA), a novel technique for the efficient detection of point mutations, to detection of the mutations. PHFA was performed on streptavidin-coated microtiter plates with biotin- and dinitrophenyl-labeled amplicons to detect the wild-type gene or each mutant gene. DNA samples were extracted from gastric juice specimens of 412 patients with H. pylori infection and were applied to the assay. The detection threshold of PHFA was as few as 10 gene copies. The sensitivity of PHFA for the detection of H. pylori infection was higher than those of culture and the rapid urease test. A total of 337 (81.8%) samples had the wild-type gene, 38 (9.2%) had the A2144G mutation, and 37 (9.0%) contained both the wild type and a mutation (A2144G in 30 samples, A2143G in 5 samples, and A2143G plus A2144G in 2 samples). About half the strains isolated from patients with mixed infection were susceptible by the agar dilution method (MIC, <0.1 mg/liter). Therefore, PHFA can detect clarithromycin-resistant H. pylori strains, even in patients with mixed infections with the wild type, that are not detectable by the agar dilution method.

Quantitative DNA analysis of low-level hepatitis B viremia in two patients with serologically negative chronic hepatitis B.

Low-level viremia due to hepatitis B virus (HBV) was demonstrated in the sera of two patients diagnosed previously as having non-B, non-C chronic hepatitis. Both patients had a "silent" HBV infection, because they were negative for both hepatitis B surface antigen (HBsAg) and anti-hepatitis B core antibody. The TaqMan chemistry polymerase chain reaction (PCR) amplified the HBV DNA, enabling quantitation of the virus in their sera. Their serum HBV DNA concentrations were low: the amount of each HBV S or X gene amplified showed there were approximately 10(3) copies/ml and HBV DNA was detected occasionally during clinical follow-up. Positive HBsAg staining in liver tissues was demonstrated by an immunoperoxidase technique. Vertical transmission of silent HBV from one patient to her daughter was confirmed. Direct nucleotide sequencing of the amplified HBV X region revealed several mutations, suggesting reduced viral replication. One patient had a T-to-C mutation at the extreme 5'-terminus of the direct repeat 2 region and the other exhibited a coexisting X region with a 155-nucleotide deletion. These findings suggest that HBV replication is suppressed considerably in patients with silent hepatitis B.

[Virological response to interferon therapy in patients dually infected with TT virus and hepatitis C virus].

In 30 chronic hepatitis C patients co-infected with TTV, TTV DNA in the sera immediately after cessation of IFN and 6 months after the end of IFN was examined. Sustained loss of TTV DNA was observed in 12 of 30 (40.0%) patients. In 7 of 30 (23.3%) patients, TTV DNA re-appeared 6 months after becoming undetectable at the end of IFN therapy. In the remaining 11 patients (36.7%), TTV DNA remained detectable during the entire follow-up period. The ALT values correlated only with the presence of HCV RNA regardless of the effect of IFN on TTV replication. This study indicates that IFN therapy is effective against TTV.

[A phase I trial of adenoviral p53 gene therapy for non-small cell lung cancer].

Lung cancer is one of the leading causes of cancer deaths worldwide. The high mortality rate for lung cancer results from the absence of standard therapeutic strategies, suggesting that lung cancer may be a suitable target disease for a novel gene-based therapy. Restoration of the function of a single pivotal gene product appears sufficient to mediate antitumor effects that are potentially clinically significant. Preclinical studies in animal models have demonstrated tumor regression following intratumoral administration of an adenovirus vector containing The wild-type tumor suppressor p53 gene. The efficacy and safety of the p53 gene therapy protocol for non-small cell lung cancer are now being evaluated in clinical trials. Although much research needs to be done following trials in the earliest stages, the treatment may offer a unique mechanism of action with a potentially high therapeutic index. This article reviews recent highlights in this rapidly evolving field.

[A case of refractory pulmonary fistula, managed with bronchoscopic therapy using occluding spiral embolus].

We report a successful case of bronchoscopic therapy using occluding spiral embolus and fibrin glue for refractory pulmonary fistula. A 22-year-old female underwent left lower lobectomy for giant bulla of the lung. Air leakage began 6 days after lobectomy. Closing alveolar fistula was performed 12 days after first operation. Relapsing air leakage began 4 days after second operation. Bronchography revealed a fistula from left B1+2c. The insertion of embolus through bronchofiberscope, following administration of fibrin glue, was performed to close the fistula. This method is effective for refractory pulmonary fistula.

Use of a gastric juice-based PCR assay to detect Helicobacter pylori infection in culture-negative patients.

A gastric juice-based PCR assay was compared with culture, microscopy, and a rapid urease test with specimens from 114 subjects. The PCR and conventional tests were positive for 76 and 62% of the subjects, respectively. The prevalence of gastroduodenal disease and seropositivity for anti-Helicobacter pylori immunoglobulin G were similarly high among conventional-test-positive and PCR-only-positive subjects compared to all-negative ones. The PCR assay is recommended to confirm the H. pylori status of culture-negative peptic-ulcer patients.

Comparison of the nucleic acids of helical and coccoid forms of Helicobacter pylori.

The nucleic acids of the helical and coccoid forms of Helicobacter pylori were studied to determine if the coccoid forms are "viable (capable of growing) but nonculturable." Using a reference strain (NCTC 11638) and five clinical strains, the nucleic acid contents, DNA integrity, and results of PCR and reverse transcription-PCR (RT-PCR) were compared for helical H. pylori and coccoid forms induced using glycochenodeoxycholic acid or bismuth citrate. The DNA and RNA contents of the coccoid forms were respectively 6.8- and 8.1-fold lower than those of helical H. pylori after 3 days of induction and 11.5- and 14.7-fold lower after 7 days. Agarose gel electrophoresis of DNA extracted from the coccoid forms after 3 days of induction showed a smear pattern indicating DNA cleavage, whereas DNA from helical H. pylori showed a single band with a high molecular mass. After 12 days of induction, all RNA samples from 100% coccoid cultures were negative for the mRNA of urease A or the 26-kDa species-specific protein by RT-PCR. However, most RNA samples obtained after 3 or 7 days of induction were positive at low levels despite the lack of recovery from these cultures. These results suggest that the coccoid form of H. pylori has impaired genomic DNA and is in the process of cellular degeneration, thus being still alive but nonincreasable.

[Development of a detection system for Helicobacter pylori DNA in gastric juice].

A rapid and sensitive PCR-based microwell plate assay (PCR-MWP) system to detect the 16 S ribosome RNA gene of Helicobacter pylori was developed. Analytical sensitivity, evaluated with purified recombinant plasmid DNA and genomic DNA of H. pylori, was one copy of DNA per PCR. Specificity was validated with a panel of DNA from 75 kinds of microorganisms including Helicobacter showed weak positives, when 1 pg of DNA was input. Other microorganisms gave negative signals even when 100 pg of DNA was used for PCR. When compared with a Nested-PCR system to detect the urease A-subunit gene performed by a commercial reference laboratory, the results obtained (sensitivity 93.3% and specificity 73.3%) was almost equivalent. The PCR-MWP was rapid and easy for the detection of H. pylori DNA in gastric juice specimen.

Nested-polymerase chain reaction for the detection of Helicobacter pylori infection with novel primers designed by sequence analysis of urease A gene in clinically isolated bacterial strains.

We have established a highly sensitive semi-nested PCR assay for the detection of H. pylori infection using gastric juice samples, which can be aspirated with disposable nasogastric tubes. The primers targeting H. pylori urease A gene were designed based on the sequence conservation analysis of sixteen H. pylori strains isolated from Japanese patients. The efficacy of the PCR assay, designated as the URA-PCR, was confirmed by in vitro and in vivo assessments. Its sensitivity was 97.5% in the gastric juice samples aspirated from forty patients with proven H. pylori infection, and was significantly higher than that obtained with previously described PCR assays.

Hepatitis E virus (87A strain) propagated in A549 cells.

A strain of hepatitis E virus (HEV), the 87A strain isolated in 2BS cells from the feces of a patient with hepatitis E, has been reported previously. In this study, the 87A strain was propagated in A549 cells, and the marked cytopathic effect (CPE) appeared in the infected monolayer cells. The size of this virus is about 30 nm in diameter. Furthermore, HEV-RNA from the supernatants of the virus of different passages was detected by polymerase chain reaction (PCR) amplification using ET1.1 HEV primers. A band of HEV for 239 bp from PCR products was revealed by electrophoresis. PCR products of the fourth passage were sequenced. These results show that the 87A virus replicates in the A549 cell line.

Existing variations on the gene structure of hepatitis E virus strains from some regions of China.

The isolation and identification of the 87A strain of hepatitis E virus (HEV) by means of cell culture have been described previously. This paper reports the nucleotide sequence of a portion of this HEV strain. The RNA extracted from the supernatants of the different passages of the 87A strain cultured in the A549 cell line was reverse-transcribed (RT) to cDNA, and then the polymerase chain reaction (PCR) amplification was carried out using the primers of HEV ET1.1 region. The PCR products from 1) the supernatant of the infected cells at the fourth passage, 2) the virus concentrated by polyethylene glycol (PEG) precipitation at the tenth passage, and 3) the virus purified by a sucrose gradient at the tenth passage were sequenced. In addition, three other PCR products obtained from sera of acute hepatitis E patients in Beijing (B-9) and Guangzhou (G-9 and G-20) were also sequenced. The nucleotide sequences of the above four strains of HEV (located in the genome from positions 4545-4754) were compared to those of some reported HEV strains. The nucleotide sequences of the B-9 strain and the 87A strain were similar to the Burmese strain and may belong to the same branch of HEV. The nucleotide sequences of the G-9 strain and the G-20 strain were a novel and unique branch. The Chinese HEV strains are multiplex and variable in gene structure.

Decrease in protein phosphorylation in central and peripheral nervous tissues of methylmercury-treated rat.

The protein phosphorylation in extracts of nervous tissues of rats acutely exposed to methylmercury chloride (seven daily injections of 10 mg methylmercury chloride/kg body weight) was examined. In the brain, the phosphorylating activity was dependent on cAMP and Mg2+. The effect of methylmercury on the phosphorylation of brain proteins, including tubulin and MAP-2, was hardly discernible. In peripheral nervous tissues such as the dorsal and ventral roots, sciatic nerves and dorsal root ganglia, the phosphorylating activity was dependent on Ca2+, and the maximal activity was obtained when the tissues were extracted in the presence of 1% Triton X-100. SDS-Polyacrylamide gel electrophoresis revealed that the major phosphorylated proteins in the peripheral tissues were myelin proteins. The effects of methylmercury were not uniform regarding protein species and tissues. The most marked changes were observed in sciatic nerves, in which phosphorylation of the 33 kDa, 28 kDa, 19 kDa, 18 kDa and 15 kDa proteins was significantly decreased in the symptomatic phase of intoxication.