Nakanobu Hayashi

Detection of Hepatitis C Virus Antibody in the Absence of Viral RNA in Patients with Autoimmune Hepatitis

OBJECTIVE To determine whether laboratory findings showing antibodies to hepatitis C virus (HCV) in patients with autoimmune hepatitis represent false-positive results and to identify possible explanations for true-positive results in these patients. DESIGN Cross-sectional. SETTING University-based hospital. PATIENTS Fifty-two patients with non-A, non-B chronic hepatitis as a control group and 26 patients with classic chronic active autoimmune hepatitis. MEASUREMENTS Comparison of the results of five kinds of assays of HCV antibodies and HCV RNA. MAIN RESULTS Of 52 patients with non-A, non-B chronic hepatitis, HCV antibodies (anti-HCV) were detected in 42 patients (81%; 95% CI, 67% to 90%) by a first-generation enzyme-linked immunosorbent assay (ELISA-I), in 39 patients (75%) by Sp42 ELISA, in 37 patients (71%) by RIA-I, in 49 patients (94%) by ELISA-II, and in 48 patients (92%) by RIBA-II. We found HCV RNA in 47 patients (90%; CI, 79% to 97%). Of the 26 patients with autoimmune hepatitis, anti-HCV were detected in 23 patients (88%; CI, 70% to 98%) by ELISA-I, in 12 (46%) by both RIA-I and Sp42 ELISA, in 20 (77%) by ELISA-II, and in 9 (35%) by RIBA-II. However, HCV RNA was found in only five of these patients (19%; CI, 7% to 39%). None of our patients, including controls, had antibodies to superoxide dismutase. Of the 21 patients who had autoimmune hepatitis that was completely responsive to steroid therapy, 18 had anti-HCV by ELISA-I, but 13 of these patients had negative results by RIBA-II, and only two patients had HCV RNA. Of the five patients who did not respond to steroid treatment, all had anti-HCV by ELISA-I, four had negative results by RIBA-II, and three had HCV RNA. CONCLUSIONS Testing for HCV antibodies in patients with autoimmune hepatitis frequently elicits positive results when the ELISA-I or ELISA-II tests are used. Most of these appear to represent false-positive results because HCV RNA is usually absent from the serum. Such false positivity may result from previous infection with HCV or from cross-reaction of an epitope of HCV. Other patients with apparent autoimmune hepatitis who fail to respond to corticosteroid therapy may actually have chronic hepatitis C (or other non-A, non-B hepatitis) infection.

Induction of Ubiquitin-Conjugating Enzyme by Aggregated Low Density Lipoprotein in Human Macrophages and Its Implications for Atherosclerosis

Recently, we have found that aggregated low density lipoprotein (agLDL) inhibits apoptosis of lipid-bearing macrophages, thereby facilitating foam cell formation and atherosclerosis. To clarify the mechanisms by which agLDL inhibits apoptosis of macrophages, we isolated the genes specifically induced by agLDL by using a subtraction-based cloning strategy. One of the cloned genes, termed low density lipoprotein (LDL)-inducible gene (LIG), encodes a human homologue of bovine ubiquitin-conjugating enzyme E2-25K. Although LIG mRNA was ubiquitously expressed among human tissues, including hematopoietic cells, the abundance of transcripts was markedly increased by agLDL treatment in activated monocytes. LIG mRNA expression was not enhanced by nonatherogenic lipoproteins such as native LDL and high density lipoprotein, suggesting a role in atherosclerosis. Polyubiquitination of intracellular proteins was observed in monocytes cultured with agLDL, which coincided with upregulation of LIG. Furthermore, ubiquitin-dependent degradation of p53, an inducer of apoptosis, was accompanied by LIG induction in agLDL-treated monocytes. The antiapoptotic effect of agLDL was abrogated by a specific proteasome inhibitor, which also increased the half-life of p53 in monocytes. These results suggest that LIG contributes to foam cell formation by the suppression of apoptosis of lipid-bearing macrophages through ubiquitination and subsequent degradation of p53.

Antibody responses to the hepatitis C virus E2 protein: relationship to viraemia and prevalence in anti-HCV seronegative subjects.

A small proportion of patients with chronic hepatitis C virus (HCV) infection show no serological responses to the HCV polypeptides incorporated in commercial III generation immunoassays. To determine whether sera from these subjects contain antibodies to the highly immunoreactive second envelope polypeptide E2, which is not included in current anti‐HCV assays, we studied 59 anti‐HCV negative subjects who were found consistently to be HCV RNA positive by polymerase chain reaction (PCR). Controls included 167 anti‐HCV seropositive patients with or without serum HCV RNA and normal subjects. Antibodies to the E2 region were sought for by ELISA using the following antigens: a full length E2 protein expressed in insect cells using a baculovirus vector and extracted under denaturing conditions (dE2), and a C‐terminal truncated soluble E2 (sE2) protein (a.a. 390–683), also expressed with a baculovirus vector, containing a signal peptide of rabies virus G protein which allows its secretion into the culture supernatant. Sera from only two (3.4%) of the 59 anti‐HCV negative, HCV RNA positive patients recognised sE2 and none dE2. In sharp contrast, 82% of seropositive, viraemic patients recognised sE2 and 60% dE2, the difference in immunoreactivity being statistically significant (P < 0.0003). A significantly lower proportion of sera from anti‐HCV positive, HCV RNA negative subjects recognised either sE2 or dE2 (16% and 13%, respectively, P < 0.000001). Healthy controls were consistently negative. These results indicate that antibody responses to predominantly conformational epitopes on the HCV E2 protein are common in patients with chronic HCV infection and are strictly related to the presence of circulating viral genomes. In contrast, only a minor proportion of HCV RNA positive patients, but anti‐HCV seronegative by commercial immunoassays, have humoral immune responses to the HCV E2 region. J Med Virol 51:1–5, 1997.

Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells

Recombinant baculoviruses that produce a putative non-structural protein 1 (NS1) of hepatitis C virus (HCV), predicted to be the second envelope glycoprotein, were constructed. The recombinant NS1 protein (re-NS1) produced in infected insect cells was localized on the cell surface and was apparently glycosylated, because it was susceptible to treatment with both tunicamycin and N-glycanase. Furthermore, re-NS1 was effectively secreted into the culture supernatant when the putative NS1 signal peptide (SP) was replaced by the SP of rabies virus G protein, and the C-terminal hydrophobic region was eliminated. The secreted re-NS1 was tagged with six His residues at the C terminus and purified simply by native Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) affinity column chromatography. An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of HC using purified re-NS1. Anti-NS1 antibody (Ab) was detected in 55 of 60 patients (92%) with chronic HC liver diseases. Thus, this ELISA for Ab directed against HCV re-NS1 produced in insect cells is useful for the detection of chronic HC patients.

Hepatitis E Virus: cDNA Cloning and Expression

Viral hepatitis E is endemic, frequently provoking epidemic outbreaks in many developing countries. We have attempted to clone the viral genome and to develop an antibody assay system. A lambda gt11 cDNA library was constructed from the bile juice containing putative causative viruses and was immunoscreened by the antisera obtained from patients and monkeys infected with hepatitis E. Three virus‐specific clones were isolated and were revealed to overlap one another in sequence, with 1,459 nucleotides in total length. These clones direct the synthesis of polypeptides probably having common immunological epitope(s). Immunoplaque assay revealed the occurrence of antibodies against this epitope in the sera from experimental monkeys with the convalescent phase and from patients of Myanmar, Nepal and India. The data indicate that the cDNA fragments are useful for immunodiagnosis of hepatitis E.

Expression of a multidrug-resistance gene in human malignant lymphoma and related disorders

The expression of mdr1 gene was measured to determine whether it plays a role in clinical resistance to chemotherapy of human malignant lymphomas. mdr1 expression was found in 4 of 9 cases resistant to chemotherapy. Expression of mdr1 was not detectable in any of 7 chemotherapy-sensitive tumors. The 2 cases of reactive lymphadenitis and the 3 samples of normal mononuclear cells did not show any expression of mdr1 gene, either. These results indicate that expression of the mdr1 gene is not always detectable in cases of malignant lymphoma resistant to chemotherapy, but the detectable expression of mdr1 gene may predict clinical resistance to chemotherapy.

Identification of novel p53-binding proteins by biomolecular interaction analysis combined with tandem mass spectrometry

Electrospray tandem mass spectrometry (ESI-MS/MS) was combined with biomolecular interaction analysis (BIA) to develop a method of direct protein identification after real-time analysis of protein-protein interactions. Using this method, called BIA-MS/MS, we detected multiple p53-interacting proteins in whole tissue extracts from human placenta and liver. Peptide sequencing revealed three proteins whose interaction with p53 had not been previously reported: a cyclin-dependent kinase inhibitor p57/Kip2, a serine/threonine protein phosphatase PP1C, and hemoglobin. Using our system, unambiguous sequence information can be obtained at the femto- to picomole level after repeating the recovery procedure five times. Furthermore, the association and dissociation constants are easily determined by kinetic analysis. This system provides a powerful tool for analyzing complex biological materials in a simple but highly specific and sensitive manner.

A case of sexually transmitted acute hepatitis C: confirmation by analysis of viral genome

Abstract We report a case of acute hepatitis C that appears to have been sexually transmitted. The patient was a 21-year-old female with no history of blood transfusion and no personal or family history related to hepatic decease. The patient first had sexual intercourse with her male partner in November 1994 and she visited our hospital with a chief complaint of general weakness in March 1995. She was admitted because of abnormal liver function test results (ALT 329; AST 152 IU l −1 ). She was negative for serum second-generation anti-hepatitis C virus (HCV) antibody, anti-hepatitis A IgM antibody, anti-hepatitis B core IgM antibody, anti-Epstein–Barr virus IgM antibody, anti-nuclear antibody, anti-DNA antibody and anti-mitochondrial antibody at admission. A serum HCV RNA test gave a positive result on the 7th day after admission; her HCV RNA level was 10 3.5 copies 50 μl −1 serum on the 21st day. Her anti-HCV antibody status became positive on the 30th day after admission. A liver needle biopsy specimen taken on the 35th day showed well-preserved lobular architecture and mild necroinflammation within the parenchyma without accompanying fibrosis. Abdominal ultrasound and computed tomography did not show significant change in the liver or spleen. Taking the above findings together a diagnosis of acute hepatitis C was maden. Treatment by interferon-β succeeded in eradicating the HCV and the patient was free of HCV RNA 1 year later. The patients partner had untreated chronic hepatitis C. The HCVs isolated from the patient and her partner both had the 2b genotype and single-strand conformation polymorphism analysis revealed three virtually identical bands. The nucleotide sequences (E1-E2/NS1 region) of the two HCVs showed 98% homology. These genetic findings suggest that the sexual transmission of HCV occurred in this case.

Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and in vitro DNA amplification-restricted transcription-translation system

Three kinds of hepatitis C virus (HCV) core peptides were produced directly and efficiently in E. coli: 1-120 aa of the C region as NCC, 1-157 aa as NCCT and 1-190 aa as NCCL. These peptides were estimated to be 16, 22 and 24 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The processing to produce p22 core protein observed in insect cells and mammalian systems did not occur in E. coli. These peptides were similarly reactive with serum antibody from patients with hepatitis C. A mutant clone of NCC recombinant plasmid pKNCC4 was obtained, whose product, NCC4, was more stable in the E. coli lysate and was highly immunoreactive with sera of hepatitis C patients. This stable immunoreactive core peptide produced by pKNCC4 is useful for the detection of anti-HCV core antibody. Immunoreactive core peptides were also produced by DNA amplification-restricted transcription-translation. Five kinds of cDNA from C to E1 region were amplified and transcribed in vitro, and these five transcripts were then translated in vitro using rabbit reticulocyte lysate: 1-120 aa as 17 kDa of C1, 1-155 aa as 21 kDa of C2, 1-174 aa as 22 kDa of C3, 1-192 aa as 24 kDa of C4, and 1-213 aa as 26 kDa of C5. Cotranslational processing using microsomal membranes occurred in peptides C4 and C5 to produce p22 the same size as C3. These results indicate that the C-terminus of the mature core protein p22 may be generated at around aa 174 by cleavage with the signal peptidase.

A simple semisolid subtraction method using carbodiimide-coated microplates

In this article, we develop a novel subtraction method using carbodiimide-bound microplates. This method utilizes the high affinity of carbodiimides for both single- and double-stranded nucleic acids. Carbodiimide-mediated end-attachment of driver RNA to microplates allows semisolid phase hybridization between driver RNA and target cDNA, and ensures easy removal of RNA/cDNA hybrids composed of the genes commonly expressed in driver and target. As a result, the target-specific genes are left unhybridized and enriched in the hybridization supernatant. We define the optimal conditions for the method as a target/driver RNA ratio of 1:10 and a period of hybridization of 24 h. There are at least three major advantages with the present method: (1) The entire procedure, which consists of two steps, is very simple; (2) hybridization efficiency can be monitored before further processing of the samples; and (3) rare transcripts can be effectively enriched. This method may be a powerful tool to isolate the genes specifically expressed in particular cell or tissue types, and is easily applicable to many studies in molecular biology and genetics. Isolation of polyploid megakaryocyte-specific genes is shown as an example.