Interaction between hepatic membrane type 1 matrix metalloproteinase and acireductone dioxygenase 1 regulates hepatitis C virus infection.

Membrane type 1 matrix metalloproteinase (MT1-MMP) binds to and regulates the function of tetraspanin-enriched microdomains. It also physically interacts with claudin-1 and acireductone dioxygenase 1 (ADI1), both associated with hepatitis C virus (HCV) cell entry. Here, we examined hepatic expression of MT1-MMP, ADI1 and claudin-1 as well as their physical interaction in relation to serum or intrahepatic HCV-RNA levels. A total of 104 liver biopsies obtained from chronic hepatitis C patients and 84 liver tissues obtained from noncancerous parts of surgically removed HCV-related hepatocellular carcinoma were analysed. Positive cytoplasmic ADI1 in liver biopsies was associated with higher serum HCV-RNA levels (P = 0.009). Positive MT1-MMP and ADI1 interaction assessed by co-immunoprecipitation was associated with lower tissue HCV-RNA levels (P = 0.009). Hepatic HCV-RNA levels were positively associated with ADI1 levels in the MT1-MMP and ADI1 co-immunoprecipitates (P = 0.030). Overexpression of MT1-MMP in Huh7.5 cells suppressed cell entry of HCV pseudoparticles as well as HCVcc infection. The suppression effect could be reversed by co-expression of ADI1 in a dose-dependent manner. In summary, clinical and cell-based experiments suggested that physical interaction between MT1-MMP and ADI1 led to suppression of HCV infection. This inhibitory effect could be reversed by ADI1 overexpression.

Hepatic expression of MxA and OAS1 in an ex vivo liver slice assay independently predicts treatment outcomes in chronic hepatitis C.

Antiviral effect of interferon is mediated by the expression of interferon-stimulated genes (ISGs). However, because of the difficulty in obtaining paired liver biopsies before and after interferon treatment, the key ISGs expressed in human hepatocytes and responsible for interferon-based antiviral activities in chronic hepatitis C remain illusive. Prior to a standard course of peginterferon plus ribavirin therapy, 104 patients underwent a liver biopsy. A small piece of the liver biopsy sample from each patient was submitted for ex vivo tissue culture in the presence or absence of interferon. Hepatic expression of 8 ISGs was detected by RT-PCR. The ISG expression patterns and clinicopathological variables were correlated with subsequent treatment outcomes. Multivariate logistic regression analysis showed that hepatic MxA expression (P = 0.008) and leucocyte count (P = 0.040) independently predicted the end of therapeutic virological response, while hepatic OAS1 expression (P = 0.003), genotype 1 (P = 0.002), HCV-RNA level (P = 0.007), AST/ALT ratio (P = 0.004) and leucocyte count (P = 0.034) independently predicted the sustained virological response. Immunohistochemistry analysis showed that interferon-induced OAS1 expression localized to the hepatocytes. In conclusion, hepatic MxA and OAS1 expression predicted, respectively, the end of therapeutic and sustained virological responses in interferon-based treatment of chronic hepatitis C.

A liver slice culture-based ex vivo assay to predict the outcome of antiviral therapy for chronic hepatitis C.

A liver slice culture-based, ex vivo drug suppression assay was developed as a pre-therapeutic predictor for the outcome of antiviral therapy. To investigate its clinical application, 106 consecutive patients with chronic hepatitis C virus (HCV) infection were evaluated. Ex vivo drug suppression assay was performed before administrating a standard course of peginterferon plus ribavirin combination therapy. Stepwise logistic regression model was used to estimate sustained virological response (SVR) on the presence of various clinicopathological parameters. Suppression of HCV replication in the ex vivo assay was present in 32 patients, 29 (90.6%) of whom achieved SVR. Stepwise logistic regression analysis indicated that the presence of interferon suppression effect in the ex vivo assay (odds ratio [OR], 5.552; 95% confidence interval [CI], 1.114-27.673; P = 0.036), genotype 1 (OR; 0.045, 95% CI, 0.008-0.259; P = 0.001), HCV-RNA level (OR, 0.739; 95% CI, 0.617-0.885; P = 0.001), the presence of fatty metamorphosis (OR, 0.205; 95% CI, 0.053-0.793; P = 0.022), and albumin (OR, 9.687; 95% CI, 2.237-41.940; P = 0.002) were independent determinants of SVR. Categorical analysis revealed that 17 of 17 (100%) patients with genotype non-1 and positive ex vivo suppression test achieved SVR, while 20 of 40 (50%) with genotype 1 and negative ex vivo suppression test achieved SVR. In conclusion, the ex vivo drug suppression assay may serve as an independent pre-therapeutic predictor for the SVR in interferon-based antiviral therapy.

Diverse immune responses to HBV surface epitope variants after vaccine booster in adolescents immunized in infancy.

OBJECTIVES: We aimed to investigate immunity against hepatitis B surface antigen (HBsAg) mutants before and after boosters in adolescents who had lost antibodies against HBsAg (anti-HBs) despite neonatal vaccination. METHODS: We recruited 142 participants from 15 to 21 years old who had received complete vaccination in infancy but became anti-HBs-negative. Cellular immunity to HBsAg and T-cell epitope variants was assessed before and after boosters. Antibody affinity to variants was assessed after boosters. RESULTS: After one dose of booster, 12 out of 140 (8.6%) participants remained anti-HBs-negative. Anti-HBs titres were higher in those participants <17 (geometric mean, 337.9 ± 10.9 vs. 157.4 ± 16.6 mIU/mL, p = 0.012). Before the booster, HBsAg-specific cell proliferation was present in 58 out of 64 (90.6%) participants. The proliferation response rates to T-cell epitopes were 37.8% and 72.6% (p < 0.001) before and after the booster, respectively. The stimulation index improved from 1.25 ± 1.70 to 2.53 ± 2.32 (p < 0.001) for various T-cell epitopes. HBsAg-specific interleukin (IL)-5- and interferon (IFN)-γ-secreting T-cells were enhanced (45 ± 10 and 50 ± 4 to 420 ± 328 and 355 ± 424 spot-forming cells/106 cells, respectively, p < 0.001). IFN-γ-secreting T cells to epitope 16-33 containing R24K and the antibody affinity to sG145R were still significantly lower than to the wild type. CONCLUSIONS: In immunized adolescents who lost anti-HBs, around 10% also lost immune memory. Cellular immunity to some T-cell epitope variants improved after the booster. Antibody affinity to sG145R and the IFN-γ-secreting cell response to some epitope 16-33 variants were still impaired even after booster administration.

Expression of hepatitis B virus nuclear core antigen in young cirrhotic patients is associated with an unfavourable long-term outcome.

The aim of this case control study was to investigate the clinical significance of hepatitis B virus nuclear core antigen (HBcAg) in young cirrhotic patients. Fifteen cirrhotic patients with nuclear HBcAg in the liver biopsies were included. Their clinicopathological parameters as well as the core gene sequences were compared with those of a sex- and age-matched (1 to 2) control group. The mean follow-up periods were 124 +/- 80 and 102 +/- 43 months, respectively. Expression of nuclear HBcAg in cirrhotic liver was significantly associated with higher aspartate aminotransferase levels (P = 0.001), alanine aminotransferase levels (P < 0.001), and alpha-fetoprotein levels (P = 0.002), as well as a shorter duration to develop hepatocellular carcinoma or liver decompensation (Kaplan-Meier method, P = 0.044). Sequence analysis revealed mutations on the nuclear localization signal (NLS) of core protein in five cirrhotic patients with nuclear HBcAg (Q171K in four and Q179K in one patients). Site-directed mutagenesis experiments demonstrated that both the Q171K and Q179K mutation enhanced nuclear localization of the core protein. In conclusion, expression of nuclear HBcAg in young cirrhotic patients was associated with more severe hepatitis activities as well as an unfavourable long-term outcome. Mutations on the NLS of core protein were selected in some patients with nuclear HBcAg.

Purification and characterization of a naturally processed hepatitis B virus peptide recognized by CD8+ cytotoxic T lymphocytes.

In vitro studies in patients with hepatitis B virus (HBV) infection have suggested that hepatocytolysis induced by CD8+ cytotoxic T lymphocytes (CTLs) is the most important effector pathway in eliminating infected cells. The recognition is implicated in the endogenously processed HBV antigens in the context of HLA class I molecules presented on the liver cell membrane. However, the naturally occurring HBV peptide antigens have not yet been demonstrated. We report here that a naturally processed peptide antigen P2 was isolated from HLA class I molecules of HBV-infected liver cell membrane. The P2 peptide exhibited the activity of sensitizing target cells for lysis by CD8+ CTLs. The P2 sequence (YVNVNMGLK) purified from liver tissue was in concordance with that encoded by the viral genome for the HBV nucleocapsid antigen or HBcAg 88-96. P2 peptide could also be isolated from the EBV-transformed B cells that were transfected by HBcAg-expressing vector. The P2 epitope, sharing the HLA-A11 binding motifs, was recognized by HLA-A11-restricted CD8+ CTLs. The data provided direct evidence that, in hepatitis B patients, antigenic peptides of HBV were processed by hepatocytes, presented with the class I MHC molecules, and recognized by CD8+ CTLs.

Acetylation is essential for nuclear heme oxygenase-1-enhanced tumor growth and invasiveness.

Overexpression of heme oxygenase-1 (HO-1), an endoplasmic reticulum-anchored enzyme, is observed in many cancers. HO-1 nuclear translocation has been shown to correlate with progression of several cancers. We recently reported that HO-1 is susceptible to intramembrane proteolysis and translocates to the nucleus to promote cancer growth and invasiveness without depending on its enzymatic activity. In the present study, we show that the HO-1 lacking C-terminal transmembrane segment (t-HO-1) was susceptible to acetylation by p300 and CREB-binding protein (CBP) histone acetyltransferase in the nucleus. Mass spectrometry analysis of HO-1 isolated from human embryonic kidney cells 293T (HEK293T) cells overexpressing CBP and t-HO-1 revealed two acetylation sites located at K243 and K256. Mutation of both lysine residues to arginine (R) abolished t-HO-1-enhanced tumor cell growth, migration and invasion. However, mutation of the lysine residues to glutamine (Q), a mimic of acetylated lysine, had no significant effect on t-HO-1-mediated tumorigenicity. Mechanistic studies demonstrated that transcriptional factor JunD interacted with wild-type (WT) t-HO-1 and mutant carrying K243/256Q but not K243/256 R mutation. Moreover, JunD-induced AP-1 transcriptional activity was significantly enhanced by coexpression with WT and acetylation-mimic but not acetylation-defective t-HO-1. Consistent with the in vitro observations, the implication of K243/256 acetylation in t-HO-1-enhanced tumorigenicity was also demonstrated in xenograft models. Immunohistochemistry performed with a specific antibody against acetyl-HO-1 showed the positive acetyl-HO-1 nuclear staining in human lung cancer tissues but not in the corresponding non-tumor tissues, supporting its clinical significance. Collectively, our findings highlight the importance of nuclear HO-1 post-translational modification in the induction of cancer progression.

Thyroid hormone protects hepatocytes from HBx-induced carcinogenesis by enhancing mitochondrial turnover.

Infection by hepatitis B virus (HBV) accounts for 50-80% of hepatocellular carcinoma (HCC) development worldwide, in which the HBV-encoded X protein (HBx) has critical role in the induction of carcinogenesis. Several studies have shown that thyroid hormone (TH) suppresses HCC development and protects hepatocytes from HBx-induced damage, thus it is of interest to examine whether TH can protect hepatocytes from HBx-induced carcinogenesis. By treating HBx- transgenic mice with or without TH, we confirmed the protective effects of TH on HBx-induced hepatocarcinogenesis, which was achieved via reduction of reactive oxygen species (ROS) inflicted DNA damage. We further found that TH induced biogenesis of mitochondria (MITO) and autophagy of HBx-targeted MITO simultaneously, consequently leading to suppression of HBx-promoted ROS and carcinogenesis. Using microarray data analysis, this protective effect of TH was found to be mediated via activation of PTEN-induced kinase 1 (PINK1) in hepatocytes. PINK1, in turn, activated and recruited Parkin, an E3 ligase, to ubiquitinate MITO-associated HBx protein and trigger selective mitophagy. The pathological significance of the TH/PINK1 pathway in liver protection was confirmed by the concomitant decrease in expression of both TR and PINK1 in matched HCC tumor tissues and negatively correlated with aggressive progression of cancer and poor prognosis. Our data indicate that TH/PINK1/Parkin pathway has a critical role in protecting hepatocytes from HBx-induced carcinogenesis. Notably, several liver-targeting therapeutic derivatives of TH facilitating prevention or therapy of steatosis have been identified. Furthermore, our proof-of-concept experiments suggest that application of T3 constitutes an effective novel therapeutic or preventive option for HCC. Thus, the utilization of the agonists of TRs could be the meaningful strategy in liver relative diseases, ranging from simple hepatic steatosis to HCC.

Aberrant transcripts of the cyclin-dependent kinase-associated protein phosphatase in hepatocellular carcinoma.

The cyclin-dependent kinase (Cdk)-associated protein phosphatase (KAP) is a human dual specificity protein phosphatase that dephosphorylates Cdk2 on threonine 160 in a cyclin-dependent manner. To investigate whether mutations of this enzyme occur in hepatocellular carcinoma (HCC), KAP mRNA was analyzed by reverse transcription-PCR (RT-PCR), followed by cloning and sequencing. Eight of 14 biopsy tissues obtained from advanced HCC, 6 of 13 surgically removed HCC tissues, and 2 of the adjacent noncancerous tissues contained aberrant KAP transcripts. Using the yeast two-hybrid system, five of seven representative KAP mutants were shown to be defective in interacting with Cdk2. These data suggest a possible role of KAP mutations in multiple-step hepatocarcinogenesis.

Nm23H1 mediates tumor invasion in esophageal squamous cell carcinoma by regulation of CLDN1 through the AKT signaling.

Esophageal cancer is a lethal malignancy worldwide. Previously, low expression of metastasis suppressor Nm23H1 and tight junction (TJ) protein claudin-1 (CLDN1) have been known to correlate with poor prognosis in esophageal squamous cell carcinoma (ESCC). However, the molecular interaction between them has not been clarified. In the present study, we first examined the expression of Nm23H1 and CLDN1 in 74 surgical ESCC samples by immunohistochemistry (IHC) to verify their clinicopathologic significance. The biologic effects of Nm23H1 gene silencing or overexpression in ESCC cell lines were then studied by migration and invasion studies, and its regulation on CLDN1 expression was also investigated by western blot analysis. Moreover, the expression of Nm23H1 and CLDN1 at the same invasion front of ESCC tumors was verified by immunofluorescence. The results showed a significantly positive correlation between the expression of Nm23H1 and CLDN1 (γ=0.296, P=0.011) in surgical specimens, especially for the 34 tumors with lymph-node metastasis (γ=0.455, P=0.007). In ESCC cell lines, silencing of Nm23H1 expression markedly enhanced cell invasiveness, accompanied by increased Akt phosphorylation and decreased CLDN1 expression. Conversely, Nm23H1-expressed transfectants exhibited reduced invasiveness, decreased Akt phosphorylation and correspondingly increased CLDN1 expression. Regain of CLDN1 expression in ESCC cells significantly suppressed invasiveness, but did not influence the Akt phosphorylation. Moreover, treating Nm23H1-depleted cells with the AKT inhibitor MK2206 recovered CLDN1 expression, and diminished the invasiveness of ESCC cells. Finally, decreased expressions of both CLDN1 and E-cadherin were observed at the invasive front of the Nm23H1-negative tumors. Overall, our current study documented that reduced Nm23H1 expression activates the AKT signaling pathway, results in diminished CLDN1 expression and potentiates invasiveness of ESCC cells. Enhancement of Nm23H1 expression, inhibition of the AKT signaling pathway, or combined, might be a potential treatment strategy in selective ESCC patients.

Hepatocarcinogenesis in transgenic mice carrying hepatitis B virus pre-S/S gene with the sW172* mutation.

Hepatitis B virus (HBV) carrying the rtA181T/sW172* mutation conferred cross-resistance to adefovir and lamivudine. Cell-based and clinical studies indicated that HBV carrying this mutation had an increased oncogenic potential. Herein, we created transgenic mouse models to study the oncogenicity of the HBV pre-S/S gene containing this mutation. Transgenic mice were generated by transfer of the HBV pre-S/S gene together with its own promoter into C57B6 mice. Four lines of mice were created. Two of them carried wild-type gene and produced high and low levels of HBV surface antigen (HBsAg) (TgWT-H and L). The other two carried the sW172* mutation with high and low intrahepatic expression levels (TgSW172*-H and L). When sacrificed 18 months after birth, none of the TgWT mice developed hepatocellular carcinoma (HCC), whereas 6/26 (23.1%) TgSW172*-H and 2/24 (8.3%) TgSW172*-L mice developed HCC (TgWT vs TgSW172*; P=0.0021). Molecular analysis of liver tissues revealed significantly increased expression of glucose-regulated protein 78 and phosphorylated extracellular signal-regulated kinases 1 in TgSW172* mice, and decreased expression of B-cell lymphoma-extra large in TgSW172*-H mice. Higher proportion of apoptotic cells was found in TgSW172*-H mice, accompanied by increased cyclin E levels, suggesting increased hepatocyte turnover. Combined analysis of complimentary DNA microarray and microRNA array identified microRNA-873-mediated reduced expression of the CUB and Sushi multiple domains 3 (CSMD3) protein, a putative tumor suppressor, in TgSW172* mice. Our transgenic mice experiments confirmed that HBV pre-S/S gene carrying the sW172* mutation had an increased oncogenic potential. Increased endoplasmic reticulum stress response, more rapid hepatocyte turnover and decreased CSMD3 expression contributed to the hepatocarcinogenesis.

Identification and characterization of a prevalent hepatitis B virus X protein mutant in Taiwanese patients with hepatocellular carcinoma.

The aim of this study was to investigate whether there was a particular hepatitis B virus (HBV) X protein (HBx) mutant associated with Taiwanese patients with hepatocellular carcinoma (HCC). Initially, the entire coding region of HBx gene from the serum samples of 14 Taiwanese patients were sequenced. A novel mutant, HBx-A31, was preferentially found in patients with HCC. Sera from 67 patients with HCC and 100 patients with chronic hepatitis B were thus subjected for codon 31 analysis using a dual amplification created restriction site method. HBx-A31 was detected more frequently in patients with HCC (52% versus 12%; P<0.001) and in patients with liver cirrhosis (44% versus 6%; P<0.001). Site directed mutagenesis experiment revealed that HBx-A31 was less effective in transactivating HBV enhancer I-X promoter complex, less efficient in supporting HBV replication, and less potent in enhancing TNF-alpha induced increment of CPP32/caspase 3 activities in HepG2 cells. In conclusion, a prevalent HBx mutant was identified in Taiwanese patients with hepatocellular carcinoma. Development of this mutant might represent a strategy of the virus to escape immune surveillance and thus contribute to the process of multiple-step hepatocarcinogenesis.

Signal peptide peptidase-mediated nuclear localization of heme oxygenase-1 promotes cancer cell proliferation and invasion independent of its enzymatic activity.

Heme oxygenase-1 (HO-1) is a heme-degrading enzyme anchored in the endoplasmic reticulum by a carboxyl-terminal transmembrane segment (TMS). HO-1 is highly expressed in various cancers and its nuclear localization is associated with the progression of some cancers. Nevertheless, the mechanism underlying HO-1 nuclear translocation and its pathological significance remain elusive. Here we show that the signal peptide peptidase (SPP) catalyzes the intramembrane cleavage of HO-1. Coexpression of HO-1 with wild-type SPP, but not a dominant-negative SPP, promoted the nuclear localization of HO-1 in cells. Mass spectrometry analysis of cytosolic HO-1 isolated from HeLa cells overexpressing HO-1 and SPP revealed two adjacent intramembrane cleavage sites located after S275 and F276 within the TMS. Mutations of S275F276 to A275L276 significantly hindered SPP-mediated HO-1 cleavage and nuclear localization. Nuclear HO-1 was detected in A549 and DU145 cancer cell lines expressing high levels of endogenous HO-1 and SPP. SPP knockdown or inhibition significantly reduced nuclear HO-1 localization in A549 and DU145 cells. The positive nuclear HO-1 stain was also evident in lung cancer tissues expressing high levels of HO-1 and SPP. Overexpression of a truncated HO-1 (t-HO-1) lacking the TMS in HeLa and H1299 cells promoted cell proliferation and migration/invasion. The effect of t-HO-1 was not affected by a mutation in the catalytic site. However, blockade of t-HO-1 nuclear localization abolished t-HO-1-mediated effect. The tumorigenic effect of t-HO-1 was also demonstrated in the mouse model. These findings disclose that SPP-mediated intramembrane cleavage of HO-1 promotes HO-1 nuclear localization and cancer progression independent of HO-1 enzymatic activity.

Identification of thioridazine, an antipsychotic drug, as an antiglioblastoma and anticancer stem cell agent using public gene expression data.

Glioblastoma (GBM) is a common and malignant tumor with a poor prognosis. Glioblastoma stem cells (GSCs) have been reported to be involved in tumorigenesis, tumor maintenance and therapeutic resistance. Thus, to discover novel candidate therapeutic drugs for anti-GBM and anti-GSCs is an urgent need. We hypothesized that if treatment with a drug could reverse, at least in part, the gene expression signature of GBM and GSCs, this drug may have the potential to inhibit pathways essential in the formation of GBM and thereby treat GBM. Here, we collected 356 GBM gene signatures from public databases and queried the Connectivity Map. We systematically evaluated the in vitro antitumor effects of 79 drugs in GBM cell lines. Of the drugs screened, thioridazine was selected for further characterization because it has potent anti-GBM and anti-GSCs properties. When investigating the mechanisms underlying the cytocidal effects of thioridazine, we found that thioridazine induces autophagy in GBM cell lines, and upregulates AMPK activity. Moreover, LC3-II was upregulated in U87MG sphere cells treated with thioridazine. In addition, thioridazine suppressed GBM tumorigenesis and induced autophagy in vivo. We not only repurposed the antipsychotic drug thioridazine as a potent anti-GBM and anti-GSCs agent, but also provided a new strategy to search for drugs with anticancer and anticancer stem cell properties.

Response of patients with dual hepatitis B virus and C virus infection to interferon therapy.

Patients with dual infection with hepatitis B virus (HBV) and delta virus (HDV) responded poorly to interferon (IFN) therapy. Little is known about the effect of IFN therapy in patients with HBV and hepatitis C virus (HCV) dual infection. The patients in two randomized controlled trials with chronic HBV infection were retrospectively assayed for HCV markers. The HBV responses to IFN therapy in patients with and without HCV markers were compared. An open trial was conducted in 4 patients who had lost their serum HBV surface antigen (HBsAg) but had continuing HCV viremia and hepatitis. Of the 15 patients seropositive for HCV marker(s), only 1 (6.7%) responded with seroclearance of HBV DNA and HBV e antigen, as compared with 46 (28%) of 164 HCV-negative patients (p = 0.058). Icteric hepatitis developed in 1 patient on emergence of serum HCV RNA in association with seroclearance of HBV DNA. In contrast, good response was demonstrated in 3 of the 4 patients who had lost serum HBsAg before therapy. The results suggest that IFN therapy is not only of limited value in patients with dual infection with HBV and HCV but also has a potential risk of severe hepatitis if the clearance of one virus removes its suppressive effect on and facilitates the emergence of the other. However, patients with continuing HCV hepatitis after termination of the chronic HBsAg carrier state responded well to IFN therapy.

A highly attenuated strain of Japanese encephalitis virus induces a protective immune response in mice.

A pair of virulent (RP-9) and attenuated (RP-2ms) mutants of Japanese encephalitis virus (JEV) were generated from a Taiwanese isolate NT109. The mutants differed in several aspects in vitro and in vivo. RP-2ms exhibited smaller plaque than RP-9 on BHK-21 cells, and when intracerebrally injected, RP-2ms was much less neurovirulent than RP-9. As peripherally inoculated, RP-2ms lost neuroinvasiveness while RP-9 penetrated blood-brain barrier, replicated in mouse brain, and killed all the mice. Single RP-2ms immunization completely protected C3H and ICR mice from a lethal challenge with RP-9; the sera from such mice contained antibodies against JEV envelope and nonstructural 1 proteins, indicating RP-2ms had replicated in the mice Neutralizing activity against NT109 in such sera was further demonstrated by plaque reduction neutralization test. In addition, significant lymphoproliferation was detected in spleen cells from the RP-2ms-immunized mice, and cytotoxic activity in these cells specific for the MHC-matched, JEV-infected cells, but not mock cells, was also observed. Altogether, these results demonstrate that RP-2ms, a highly attenuated JEV strain, can induce a protective immunity in mice.

Quantitative assessment of hepatitis C virus RNA by polymerase chain reaction and a digoxigenin detection system: comparison with branched DNA assay.

A method for quantifying hepatitis C virus (HCV) RNA in serum using reverse transcription-polymerase chain reaction (RT-PCR) followed by slot-blot hybridization with a specific, digoxigenin-labeled probe was developed. Using RNA synthesized from cloned HCV cDNA as a standard, serum concentration of HCV RNA above 10 copies/ml can be quantitatively determined. To compare this method with branched DNA (bDNA) assay, 45 serum samples from 26 patients with newly acquired acute hepatitis C (n = 16) or hepatitis C with acute exacerbation (n = 10) were submitted to both assays. HCV RNA in 30 (67%), 12 (27%) and three (6.7%) samples can be quantitatively determined by both, either and none of the two assays, respectively. Using a standardized qualitative HCV RNA detection test (Amplicor HCV test) as a reference, 1 and 0 false positive results were found by bDNA and this assay, respectively. This quantitative assay using RT-PCR and a digoxigenin detection system was comparable to bDNA assay. Since a false positive result was rarely found, this technique can be used as a first line test to screen a large number of samples rapidly and economically.

Detection of Helicobacter pylori by polymerase chain reaction assay using gastric biopsy specimens taken for CLOtest.

The concordance rate between a rapid urease test (CLOtest) and polymerase chain reaction (PCR) assay for the detection of Helicobacter pylori in gastric biopsy samples was investigated. To avoid the bias produced by patchy distribution of the organism in the stomach, the samples used for these two tests were not obtained from two different sites of the antrum. Instead, the PCR assay was performed with the the same biopsy sample that was taken for the CLOtest. Among 82 biopsy samples included for this study, 56 were positive and 26 were negative by CLOtest. Of the 56 CLOtest-positive samples, 52 (93%) were also positive by PCR assay, and of the 26 CLOtest-negative samples, 20 (78%) were negative by PCR assay. The total concordance rate of these two tests was 87.6%. Of the 4 cases with CLOtest-positive and PCR-negative results, 3 had been treated with long-term H2 blockers. Of the 6 patients with CLOtest-negative and PCR-positive results, 4 suffered from recurrent or poorly healing duodenal ulcers. Interestingly, a significantly lower density of the PCR products was observed during electrophoresis analysis for all the 6 cases, presumably due to a small number of H. pylori in these samples. These results indicated that PCR might be used as a complementary assay for CLOtest. False negative results by CLOtest might occur when only a small amount of H. pylori was present in the samples, which could be detected by subsequent PCR assays using the same biopsy specimens. The clinical significance of such CLOtest-negative and PCR-positive cases requires further study.

Genetic alternations of p73 are infrequent but may occur in early stage hepatocellular carcinoma.

p73, a structural homologue of the tumor suppressor gene, p53, has recently been identified and mapped to chromosome 1p36, where genomic loss of heterozygosity (LOH) often occurs in human hepatocellular carcinoma (HCC). To determine whether p73 is involved in the development of HCC and whether there is an inverse correlation between the mutations of p73 and p53, we examined 22 paired tumors/noncancerous liver tissues for allelic expression, LOH and mutation of p73 and for mutation of p53. p73 was biallelically expressed in noncancerous liver tissues and in 7 out of the 8 informative tumors. One tumor tissue expressed only a single allele. LOH of p73 was found in 2 out of the 11 (18%) informative cases. A tumor-specific five-nucleotide deletion mutation causing a reading frameshift/early truncation of p73 DNA-binding domain was found, in which case no concomitant mutation in the DNA-binding domain of p53 was identified. Nine out of the 22 cases (41%) contained tumor-specific mutations in the DNA-binding domain of p53. Two of the three cases with p73 genetic alternations had a tumor size of less than 2 centimeters. These results suggest that p73 is a biallelically expressed gene in the liver and that allelic loss and mutation of p73 is infrequent and may occur early in HCC. p73 is unlikely to be the putative tumor suppressor gene located at chromosome 1p36 in HCC.

Evaluation of ferric oxalate as an agent for use during surgery to prevent post-operative root hypersensitivity.

The aim of this study was to evaluate the effectiveness of a 6% ferric oxalate solution applied during periodontal surgery to prevent post-operative tooth hypersensitivity. Twenty-five adult patients with similar bilateral periodontal defects participated in this study. Data were collected at baseline (1 week prior to surgery) and 1, 2, 4, and 6 weeks following surgery. Sensitivity level was determined using the visual analog scale (VAS) with the following stimuli: 1) mechanical stimulation with a No. 23 dental explorer; 2) water at 50 degrees C; 3) ice; and 4) electric pulp tester (EPT). Teeth were randomly assigned to either test (6% ferric oxalate in 0.9% saline) or control (0.9% saline) groups. Solutions were applied to the exposed root surfaces for 1 minute during surgery. Data were analyzed by repeated measures ANOVA, paired t-test, and Pearson's correlation test. Results from this study demonstrated statistically significant reduction in the responses to thermal stimuli, especially cold, between groups treated with ferric oxalate as compared to those treated with saline. For the cold test the difference increased with time from baseline to 6 weeks. Statistically significant (P < 0.05) differences in sensitivity to heat between groups were also observed, but only at 2 and 4 weeks following surgery. There were no differences at any time period between the test and control groups when tactile or EPT techniques were used. In addition, there was no correlation between sensitivity and other clinical parameters. It was concluded from this study that 6% ferric oxalate was effective in reducing post-surgical cold sensitivity when applied during periodontal surgical procedures.