Acute exacerbations of chronic type B hepatitis are accompanied by increased T cell responses to hepatitis B core and e antigens. Implications for hepatitis B e antigen seroconversion.

T cell proliferative responses to hepatitis B virus-encoded envelope antigen (S + preS2 + preS1), recombinant core antigen (HBcAg), and natural hepatitis B e antigen (HBeAg) were examined in 22 HBeAg-positive patients with chronic type B hepatitis and 17 healthy hepatitis B surface antigen (HBsAg) carriers. The results showed that HBeAg-positive patients had (a) higher levels of T cell responses to HBcAg/HBeAg than those of healthy HBsAg carriers (P less than 0.001 and P less than 0.01, respectively); (b) a further increase in these T cell responses during acute exacerbations (P less than 0.05 and P less than 0.05, respectively); (c) subsidence in the T cell responses to HBcAg/HBeAg after recovery from acute exacerbations and HBeAg seroconversion, whereas the responses would persist at high levels if the patients did not enter a clinical remission; and (d) low levels of T cell responses to S + preS2 + preS1 either before or after HBeAg seroconversion. The appearance of increasing T cell responses to HBcAg/HBeAg usually occurred in the early phase of acute exacerbations. These findings imply that HBcAg/HBeAg-specific T cells play an important role in the exacerbations of chronic hepatitis B and in HBeAg seroconversion. HBcAg/HBeAg-specific precursor T cell frequencies were serially studied in selected cases by limiting dilution assay. Elevation (two- to fourfold) of HBcAg/HBeAg-specific precursor T cell frequencies contributed to the increase of HBcAg/HBeAg-specific T cell proliferation during acute exacerbations.

Molecular basis of an autoantibody-associated restriction fragment length polymorphism that confers susceptibility to autoimmune diseases.

Recently, combined serological and molecular studies of autoantibodies have revealed that these antibodies play an important role in the normal function of the immune system and in the development of the B cell repertoire. Accordingly, we hypothesized that a homozygous deletion of a critical autoantibody-associated Ig variable (V) gene may alter the immune system and thus predispose the host to autoimmune disorders. Initial experiments revealed several restriction fragment length polymorphisms (RFLP) of the Humhv3005 gene, that is likely to encode heavy chains of rheumatoid factors, and the closely related 1.9III gene. By probing EcoR1-digested DNA with the Humhv3005/P1 probe, we found that one of the four major hybridizing bands was missing in approximately 20% of patients with either rheumatoid arthritis or systemic lupus erythematosus, but only 2% of normal subjects. To delineate the genetic basis of this polymorphism, we have now employed the PCR to amplify and analyze hv3005, 1.9III, and homologous genes in individuals with characteristic RFLP genotypes. Our results indicate that the human Vh gene repertoire contains several hv3005- and 1.9III-like genes, and that a complete deletion of the hv3005-like genes is relatively restricted to a subset of autoimmune patients. These findings provide initial evidence for deletion of developmentally regulated autoreactive V genes in autoimmune diseases.

Mutation of p53 gene in hepatocellular carcinoma in Taiwan.

To elucidate the role of p53 mutation in hepatocarcinogenesis in Taiwan, a hepatitis B viral infection hyperendemic area, exons 5 to 8 of the p53 gene in the tumor tissue of 61 hepatocellular carcinomas were amplified and sequenced. A total of 20 cases (32.8%) were found to have mutations; 36.6% (15 of 41) for the hepatitis B surface antigen positive group and 25.0% (5 of 20) for the hepatitis B surface antigen negative group. The corresponding normal liver showed no mutation. The mutation is widely distributed throughout exons 5 to 8. Only 4 cases (6.6%), all positive for hepatitis B surface antigen, had a specific hot spot mutation at codon 249 with G to T transversion. Our results show that scattered point mutations in p53 are not uncommon in hepatocellular carcinoma samples from Taiwan and may be important in the development of this cancer. However, the aflatoxin related specific mutation seems much less related to the genesis of hepatocellular carcinoma in Taiwan.

Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2.

Genetic studies of four highly homologous rheumatoid factor-associated Vk genes in rheumatoid arthritis patients and normal individuals.

Rheumatoid factors (RFs) are autoantibodies directed against IgG molecules. They are present in increased quantity in most patients with rheumatoid arthritis (RA), and are implicated in tissue damage in this disease. Paradoxically, recent studies of RFs have revealed that these autoantibodies are likely a physiological component of the immune system, and may play a role in the development and function of the B cell repertoire. Previously, we found that a significant fraction of RA patients express RF bearing the 6B6.6 cross-reactive idiotype, which is a phenotypic marker of the Humkv328-like genes. In order to elucidate the possible genetic factors that may contribute to the abnormal production of RFs in RA patients, we studied restriction fragment length polymorphisms (RFLP) of four highly homologous RF-related kappa light chain variable region (Vk) genes (i.e. Humkv328, Humkv328h2, Humkv328h5 and Humkv329) in RA patients and normal controls. The results show that kv328, kv328h2 and kv329 are likely to be alleles of the kv328 locus, while kv328h5 is a highly homologous Vk gene residing in a separate locus; and that deletion in one copy of either the kv328 or the kv328h5 loci, but not both loci, occurs in several individuals. However, the frequencies of various RFLP patterns of these two Vk gene loci are similar in patients and normals.

Mutual confounding and interactive effects between hepatitis C and hepatitis B viral infections in hepatocellular carcinogenesis: a population-based case-control study in Taiwan.

A population-based case-control study was conducted in Taiwan to determine the hepatitis C virus (HCV)-associated risk of hepatocellular carcinoma (HCC) in a hyperendemic area for hepatitis B virus (HBV) infection. A total of 58 recently diagnosed HCC patients and 225 matched community controls, who participated in a community-based liver cancer screening program, were recruited between March 1991 and March 1994. Control subjects were matched to HCC patients by age (+/- 5 years), sex, residence, and date of serum sample collection (+/- 3 months). Serum samples from all study subjects were tested for hepatitis B surface antigen (HBsAg) and antibodies to HCV (anti-HCVs) by enzyme immunoassays, as well as HCV RNA by reverse transcription-PCR assays. Accordingly, patients with HCC were more likely than were controls to be positive for HBsAg (82.8% versus 12.9%, with an odds ratio (OR) of 22.9), anti-HCVs (13.8% versus 4.4%, with an OR of 3.9), and HCV RNA (13.8% versus 5.8%, with an OR of 2.7). After adjustment for anti-HCVs and HCV RNA positivities, the matched ORs associated with HBsAg increased to 27.6 and 28.1, respectively, whereas the corresponding adjusted ORs for anti-HCVs and HCV RNA after controlling for HBsAg status were increased to 8.8 and 6.2, respectively. In the meantime, interactive effects between HCV and HBV on risk were also observed. Compared with those who were negative for both anti-HCVs and HBsAg, the matched ORs associated with the sole positivity of anti-HCVs and HBsAg were 4.0 (95% confidence interval = 0.7-24.0) and 24.6 (95% confidence interval = 9.5-64.1), respectively, whereas 6 HCC cases but none of control subjects were positive for both anti-HCVs and HBsAg. These results indicate that there are mutual confounding and interactive effects between HCV and HBV with respect to their links to HCC in this endemic area of chronic HBV infections.

Proto-oncogene expression in differentiating and non-differentiating chronic myelogenous leukaemia cells.

Despite the profound differences between the chronic and blastic phases of chronic myelogenous leukaemia, no differences between chronic and blastic phase cells have been described at the molecular level. Differences have been found in the levels of expression of c-myc, c-myb and p53, which fell when chronic phase cells were cultured, while the levels of expression of the genes were stable when blastic crisis cells were cultured. In contrast c-fms expression increased and MRS expression decreased after culture of chronic or blastic phase cells. The data suggest that the regulation of expression of some genes in blastic crisis cells is unaltered while that of others is disrupted. It is not known whether the failure of c-myc, c-myb and p53 expression to fall during the culture of blastic phase cells is the cause of or a reflection of the failure of these cells to differentiate.

Expressions of cancer-testis antigens in human hepatocellular carcinomas.

Though regular sonographic examination can early detect small hepatocellular carcinoma, the therapeutic results remains unsatisfactory. Antigen-specific immunotherapy is an alternative approach for controlling tumors. The prerequisite for antigen-specific cancer immunotherapy is the identification of appropriate tumor antigens. Recently, a new category of tumor-specific shared antigens, called cancer-testis antigens, has been identified. The cancer-testis antigens have been found in a variety of cancers. However, the expression of cancer-testis antigens in human hepatocellular carcinomas is unknown. The aim of this current study is to investigate the expression of cancer-testis antigens in human hepatocellular carcinomas. Reverse-transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of the SSX-1,-2,-4,-5, SCP-1, NY-ESO-1 genes in tumorous and corresponding non-tumorous liver tissues. In the 30 hepatocellular carcinomas studied, SSX-1,-2,-4,-5, SCP-1, and NY-ESO-1 mRNA expressions were detected in 24 (80%), 14 (46.7%), 22 (73.3%), 10 (33.3%), 2 (6.7%), and 11 (36.7%), respectively. Expressions of these genes were detected in few non-tumor liver tissues. The cancer-testis antigens are expressed in a high percentage of hepatocellular carcinomas. These cancer-testis antigen gene products are potential targets for antigen-specific immunotherapy of hepatocellular carcinoma.

Evaluation of third-generation hepatitis C antibody assay in chronic hepatitis C and chronic non-B, non-C hepatitis.

Second-generation hepatitis C virus (HCV) antibody assays are more sensitive and specific than first-generation assays in the detection of HCV infection; such tests are widely used. However, there are still HCV-infected patients who test seronegative for anti-HCV even by second-generation assays. In this study we evaluated the performance of the new third-generation HCV assay (HCV 3.0) in 230 individuals with different second-generation anti-HCV (HCV 2.0) and HCV RNA patterns. Our results showed the followings: only one healthy adult had a discrepant result in 200 subjects negative (group I) or positive (group II) for HCV 2.0 and HCV RNA; 7 of 13 (54%) HCV 2.0-negative but HCV RNA-positive patients (group III) were HCV 3.0-positive; two of 17 (12%) so-called chronic non-B, non-C hepatitis patients (group IV) were positive for HCV 3.0. We conclude that third-generation anti-HCV assays are more sensitive and specific than second-generation assays in the detection of chronic HCV infection; however false-positive results may be observed among low-risk healthy persons.

Assessment of c-myc expression in individual leukemic cells.

The expression of the proto-oncogene c-myc was studied at the protein level in cells obtained from patients with AML and CML. In florid AML and during the blastic phase of CML the majority of cells contain c-myc protein with the amount of protein differing widely among the cells of individual patients. In contrast, during complete remission in AML and during the chronic phase of CML cells containing c-myc protein are rare. Several studies demonstrated a discordance in the amount of c-myc transcript and the amount of c-myc protein present in cell populations thereby suggesting the presence of translational or post-translational regulation of c-myc expression. Further, the data suggest that high levels of c-myc protein in the leukemic cells of AML patients are associated with a poor response to therapy and that high levels in AML patients in CR or in the peripheral blood of chronic phase CML patients may be indicative of impending acute leukemia.

A human anti-cardiolipin autoantibody is encoded by developementally restricted heavy and light chain variable region genes.

Based on recent structural analyses of monoclonal autoantibodies, it appears that a number of these antibodies express germ-line immunoglobulin variable region (V) genes with little or no somatic mutation. In addition, our group and others have noted the identity or near identity of some autoantibody-associated V genes to V genes apparently expressed preferentially in the fetal pre-B cell repertoire. To extend these data, we now report that the heavy and light chain V genes of an anti-cardiolipin antibody derived from a healthy individual display 99% nucleotide sequence homology with V genes expressed in early B cell ontogeny. Sequence comparisons indicate the likely use of fetal-restricted V genes by this autoantibody. Taken together with other data on autoantibody V gene usage, these findings provide further evidence for overlap between the autoantibody-associated and early ontogeny expressed V gene repertoires and suggest that natural autoreactivity may be instrumental in the development and maintenance of the normal immune repertoire.

Detection of Divergent Hepatitis C Virus Envelope Sequences.

The nucleotide sequences of the putative envelope region (E1) and the junction between the E1 and envelope 2/nonstructural 1 (E2/NS1) region of the hepatitis C virus (HCV) genome are divergent among different genotypes. To characterize them, we introduced a set of nested primers that are conserved among four different genotypes (types I-IV) of HCV for polymerase chain reaction (PCR) amplification. The amplified products include the variable full-length E1 region, and the 5' end of the E2/NS1 region, the so-called hypervariable region-1 (HVR-1). Of 53 patients with histologically confirmed chronic liver disease and HCV viremia, type II virus was the most dominant strain as detected by the PCR genotyping method and the envelope region could be amplified in more than half of them irrespective of their genotypes. The specificity was confirmed by subsequent nucleotide sequence analysis. The positivity of envelope region PCR was not correlated with histologic diagnosis and hepatitis activities in these patients. Our results suggest that the nested primers can amplify the variable E1 and hypervariable 5' end of E2/NS1 of the HCV genome with moderate efficiency, and thus will be useful in future studies of HCV infections. Copyright 1994 S. Karger AG, Basel

Characterization of T Cell Clones Specific to a Determinant of Hepatitis B Virus Core and e Antigens in Chronic Type B Hepatitis: Implication for a T Cell Mechanism of HBV Immunopathogenesis.

T cell clones specific for hepatitis B core (HBcAg) and e (HBeAg) antigens of hepatitis B virus (HBV) were generated from liver infiltrates of HBeAg-positive patients. Analyzed with a panel of overlapping synthetic peptides spanning the complete sequences of HBcAg and HBeAg, eight clones responded specifically to the e2 peptide (PAYRPPNAPIL; amino acid residues 130-140 of HBcAg and HBeAg), which was doubly restricted by class I and II molecules. A preferential usage of the T cell receptor (TCR) alpha chain variable (V(alpha)) gene was found: V(alpha)12.1 for five HLA-Cw9(3)-restricted cytotoxic T lymphocyte (CTL) clones, and V(alpha)7.1 for three other HLA-DRw52-restricted type 1 helper T cell (Th1) clones. Although heterogeneous in the usage of TCR alpha chain joining region (J(alpha)) segments, their junctional-region sequences revealed conserved hydrophilic serine residues in seven of the eight e2-specific T cell clones. Single alanine substitution of the centrally located and the only hydrophilic asparagine residue of e2 peptide abrogated T cell responsiveness. The nonstimulatory e2 analogue could competitively inhibit e2-specific responses. These results demonstrate that both CTL and Th1 clones recognizing a determinant of HBcAg and HBeAg are present in the liver of chronic hepatitis B patients. The preferential V(alpha) gene usage and the expression of conserved residues in junctional-region sequences of TCRalpha chains by viral-peptide-specific, intrahepatic T cells may provide a T cell mechanism of HBV immunopathogenesis. Copyright 1994 S. Karger AG, Basel

Intraoperative hepatic ultrasonography--an indispensable procedure in resection of small hepatocellular carcinomas.

Hepatic ultrasonography (US) was performed during laparotomy in 47 patients with hepatocellular carcinomas (HCC) less than 5 cm in size. It detected more tumors than did preoperative US and other imaging modalities. In this series, 45.9% of HCCs smaller than 3 cm and 14.2% of those between 3 and 5 cm were invisible and impalpable during laparotomy, and another 15.5% of the total of HCCs were only partially visible or equivocally palpable; thus they needed intraoperative US to make a three-dimensional localization of the tumors. We concluded that, in the resection of small HCCs, intraoperative US should serve as a routine procedure.

Liver transplantation for acute hepatic failure.

INTRODUCTION: The mortality rate of acute hepatic failure (AHF) with conservative treatment is 40% to 90%, depending on the etiology. Hepatitis B infection is the major cause of AHF in Asia. In this study, we examined the role of liver transplantation for adult patients with AHF. METHODS: Sixteen patients with AHF received liver transplants in the past 6 years. Eight patients received cadaveric donor and another 8 living-related donor grafts. Fifteen patients suffered from hepatitis B-related disease and 1 had drug-induced AHF. Extracorporeal charcoal hemoperfusion was used as a bridge to liver transplantation in the first 2 patients and plasma exchange was used in the following patients. RESULTS: One patient died 1 month after the operation due to primary nonfunction. The other 15 patients are alive with good graft function at 2 months to 6 years follow-up. The success rate is 94%. Postoperative complications included infection in 10 patients (62.5%), acute rejection in 4 patients (25%), and biliary complication in 2 patients (12.5%). No neurological complications were noted. CONCLUSION: Liver transplantation is the most effective treatment for patients with AHF. Living donors may be considered due to the organ shortage and the critical patient disease.

Liver transplantation for patients with hepatocellular carcinoma.

BACKGROUND: Liver transplantation (LT) has been advocated as a salvage treatment for unresectable hepatocellular carcinoma (HCC). Selection criteria still need to be developed in Taiwan. OBJECTIVES: The purpose of our study was to assess the clinical findings and outcome of cirrhotic patients with HCC undergoing liver transplantation. METHODS: Our study consisted of 13 HCC patients who underwent liver transplantation during October 1996 to March 2003. The medical records and pathologic reports were analyzed retrospectively. RESULTS: Overall survival rates at 1 and 3 years were 86% and 61%, respectively. HCC recurrences occurred in three patients, one of whom is still alive with HCC recurrence 2 years after LT. The other two patients died of HCC recurrence 1 and 2 years after LT, respectively. Pretransplant alpha-fetoprotein (AFP) levels of >200 ng/mL were noted in all three patients with HCC recurrence. In contrast, only one of the ten patients without HCC recurrence had pretransplant AFP >200 ng/mL (P = .003). Four patients did not meet Milan criteria, two of whom had HCC recurrence. However, the other two patients with microscopic vascular invasion survived and were free of HCC. The only one patient, who had histologic grade 4 HCC, died of recurrence, although his tumor was AJCC stage 1. CONCLUSIONS: High AFP level is a risk factor for HCC recurrence after LT. In addition to Milan criteria, histologic tumor grading should be considered in patient selection. Microscopic vascular invasion may not affect the outcome of the patients with early HCC.

Surgical complications and outcome of living related liver transplantation.

INTRODUCTION: Living donor liver transplantation (LDLT) is now widely performed for patients to resolve the critical shortage of organs from cadavers. Despite rapid implementation and expansion of the procedure, both outcome and complication analyses of LDLT are still incomplete. OBJECTIVES: To analyze the outcome of LDLT, with particular reference to complications of those in need of surgical or radiological intervention. METHODS: Forty-eight LDLTs performed at National Taiwan University Hospital between December 1997 and April 2003 were reviewed retrospectively. RESULTS: Forty-two (87.5%) patients survived the operation. The 1-year graft and patient survival rate was 81.5%. Seventeen of the 48 LDLT patients had at least one postoperative complication, which needed surgical or radiological intervention. The complications included bile leakage (n = 3), biliary stricture (n = 4), internal bleeding (n = 7), intra-abdominal abscess (n = 2), liver abscess (n = 1), hepatic artery thrombosis (n = 2), duodenal ulcer bleeding (n = 1), jejunal perforation (n = 1), adhesion ileus (n = 1), and intracranial hemorrhage (n = 1). Nine of the 17 patients with complications died. In contrast, only 2 of the other 31 patients died. Seven of the mortalities were related to the complications. All survivors received only one definite intervention early after the complications were diagnosed. However, the others received an average of 1.71 +/- 0.95 (0 to 3) interventions. CONCLUSIONS: Complications requiring surgical or radiological treatment caused major mortality of LDLT. Early and definite treatment of these complications is important to improve the patient's outcome.

HLA-A, B, C and DR antigens in chronic hepatitis B viral infection.

To study the role of genetic factors in hepatitis B virus (HBV)-related liver diseases, HLA typing with 47 specificities of HLA-A, B, C and DR loci using Terasaki's 2-stage microlymphocytotoxicity method was performed in 253 normal subjects and 305 patients with various HBV-related liver diseases, including 95 healthy carries of HBV, 30 with chronic persistent hepatitis (CPH), 74 with chronic active hepatitis (CAH), 51 with liver cirrhosis and 55 with hepatocellular carcinoma (HCC). The frequency of HLA-B17 was significantly higher in patients with HCC than in healthy carriers (27.3% vs 4.2%, Pc less than 0.01). A similar situation was noted for HLA-DR3 in a comparison of patients with CAH and healthy carriers (37% vs 10%, Pc less than 0.05). Comparisons among various groups involving other specificities were statistically nonsignificant. It is concluded that genetic predisposition to the development of CAH, as well as HCC is present in HBsAg carriers, and further clarification of underlying mechanisms is needed.

Reactivation of hepatitis B virus in anti-HBe-positive chronic active type B hepatitis: molecular and immunohistochemical studies.

The causes of acute clinical exacerbations, and the role of reactivation of hepatitis B virus (HBV) in 16 non-cirrhotic patients with chronic active type B hepatitis (CAH-B) negative for serum hepatitis B e antigen (HBeAg) but positive for anti-HBE, were studied by molecular hybridization and immunohistochemical methods. IgM antibody to hepatitis A virus (anti-HAV IgM) and antibody to delta agent (anti-delta) were negative in all. HBeAg reappeared transiently in only two patients. Serum hepatitis B virus (HBV) DNA levels increased during acute exacerbations in 14 patients (88%), and decreased after the episode. Cytoplasmic hepatitis B core antigen (HBcAg) expression was found in 9 out of 13 patients (69%) during acute exacerbation. By Southern blot hybridization, 5 of 6 (83%) liver tissues obtained during clinical exacerbations had free replicative forms of HBV DNA. In 20 control patients with no exacerbation, serum HBV DNA, HBcAg expression in hepatocytes and free replicative forms of HBV DNA were positive in 15% (3/20), 10% (2/20) and 25% (2/8), respectively--figures significantly lower than those of the group studied. We conclude that acute exacerbations sometimes seen in patients with anti-HBe-positive CAH-B in Taiwan are caused mainly by reactivation of HBV.

Intratumor injection of OK-432 for the treatment of small hepatocellular carcinoma.

Intratumor injection of OK-432, a biological response modifier, in the treatment of small HCC was studied in 7 inoperable patients. After evaluation with ultrasound (US), computed tomography (CT), angiography and US-guided biopsy, implantation of a steel coil in the tumor, intratumor injection was performed under US guidance. After completion of the treatment, liver biopsy and image studies were again done to evaluate the extent of tumor necrosis. One patient was alive and well without recurrence 19 months after treatment. Four had recurrent tumors at different site of the liver 4 months, 9 months, 9 months and 8 months later. Two died of progressive malignancy 3 months and 8 months later. In the 6 patients with elevated serum alpha-fetoprotein (AFP) levels, 4 had decreased AFP after treatment, and the 2 mortalities had steadily increased AFP. The most common side effects are fever and chills. Transient abdominal pain with elevated transaminase activities, cough with hemoptysis, and vomiting were seen in 1 case each. After treatment, the biopsy specimens showed total necrosis of HCC. Although the T4/T8 ratio of peripheral blood was increased as compared with that before treatment in 4 cases, peritumoral cytotoxic T lymphocyte and monocyte infiltration were seen in one specimen only, and another 7 examined specimens showed negative staining with monoclonal antibodies of T cells. We conclude that intratumor injection of OK-432 is an alternative treatment for small HCC in inoperable cases. The effectiveness may be due to the direct tumoricidal mechanism of OK-432.