Analyses of amino acid sequences in hypervariable region-1 of hepatitis C virus (HCV) in sera from chimpanzees infected three times with the same HCV strain.

BACKGROUND: To investigate whether or not the same strain of hepatitis C virus (HCV) can twice re-infect the same chimpanzee, we analyzed nucleic and amino acid sequences in HCV hypervariable region-1 (HVR1). Two chimpanzees were inoculated, three times each, with the same HCV strain during the 1983-1991. After each inoculation, chimpanzees developed acute hepatitis C, and then recovered. METHODS: Using sera, HVR1 cloning and antibody to HVR1 major clone measurement were performed. RESULTS: Clones from the first inoculum were divisible into major and minor types. Clones from the second and third inocula, as well as all post-inoculation sera, were essentially identical to the major type. Titers of antibody to HVR1 major clone were consistently low in pre- and post-inoculation sera. CONCLUSIONS: Both chimpanzees were re-infected twice with the same strain of HCV. The sequences from the second and third infections were similar to the major sequences in the first inoculum.

p53/p21(CIP1) cooperate in enforcing rapamycin-induced G(1) arrest and determine the cellular response to rapamycin.

The relationship between G(1) checkpoint function and rapamycininduced apoptosis was examined using two human rhabdomyosarcoma cell lines, Rh1 and Rh30, that express mutated p53 alleles. Serum-starved tumor cells became apoptotic when exposed to rapamycin, but were completely protected by expression of a rapamycin-resistant mutant mTOR. Exposure to rapamycin (100 ng/ml) for 24 h significantly increased the proportion of Rh1 and Rh30 cells in G(1) phase, although there were no significant changes in expression of cyclins D1, E, or A in drug-treated cells. To determine whether apoptosis was associated with continued slow progression through G(1) to S phase, cells were exposed to rapamycin for 24 h, then labeled with bromodeoxyuridine (BrdUrd). Histochemical analysis showed that >90% of cells with morphological signs of apoptosis had incorporated BRDURD: To determine whether restoration of G(1) arrest could protect cells from rapamycin-induced apoptosis, cells were infected with replication-defective adenovirus expressing either p53 or p21(CIP1). Infection of Rh30 cells with either Ad-p53 or Ad-p21, but not control virus (Ad-beta-gal), induced G(1) accumulation, up-regulation of p21(CIP1), and complete protection of cells from rapamycin-induced apoptosis. Within 24 h of infection of Rh1 cells with Ad-p21, expression of cyclin A was reduced by >90%. Similar results were obtained after Ad-p53 infection of Rh30 cells. Consistent with these data, incorporation of [(3)H]thymidine or BrdUrd into DNA was significantly inhibited, as was cyclin-dependent kinase 2 activity. These data indicate that rapamycin-induced apoptosis in tumor cells is a consequence of continued G(1) progression during mTOR inhibition and that arresting cells in G(1) phase, by overexpression of p53 or p21(CIP1), protects against apoptosis. The response to rapamycin was next examined in wild-type or murine embryo fibroblasts nullizygous for p53or p21(CIP1). Under serum-free conditions, rapamycin-treated wild-type MEFs showed no increase in apoptosis compared to controls. In contrast, rapamycin significantly induced apoptosis in cells deficient in p53 ( approximately 2.4-fold) or p21(CIP1) ( approximately 5.5-fold). Infection of p53(-/-) MEFs with Ad-p53 or Ad-p21 completely protected against rapamycin-induced apoptosis. Under serum-containing conditions, rapamycin inhibited incorporation of BrdUrd significantly more in wild-type murine embryo fibroblasts (MEFs) than in those lacking p53 or p21(CIP1). When BrdUrd was added 24 h after rapamycin, almost 90% and 70% of cells lacking p53 or p21(CIP1), respectively, incorporated nucleoside. In contrast, only 19% of wild-type cells incorporated BrdUrd in the presence of rapamycin. Western blot analysis of cyclin levels showed that rapamycin had little effect on levels of cyclins D1 or E in any MEF strain. However, cyclin A was reduced to very low levels by rapamycin in wild-type cells, but remained high in cells lacking p53 or p21(CIP1). Taken together, the data suggest that p53 cooperates in enforcing G(1) cell cycle arrest, leading to a cytostatic response to rapamycin. In contrast, in tumor cells, or MEFs, having deficient p53 function the response to this agent may be cell cycle progression and apoptosis.

Prevention of hepatitis C virus infection in a chimpanzee by vaccination and epitope mapping of antiserum directed against hypervariable region 1.

We previously reported on a chimpanzee immunized with both putative envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV), strain HCV-N2, and synthetic peptides of hypervariable region 1 (HVR1) of a different isolate, HCV-#6. The chimpanzee showed complete protection against HCV-#6 infection only when the titer of anti-HVR1 increased, suggesting that an immune response to the HVR1 is more essential in protecting a chimpanzee from HCV infection than an immune response to E1 and E2. In this study, we immunized this chimpanzee with only synthetic HVR1 peptides after anti-E1 and anti-E2 antibody levels dropped and then rechallenged with 10 infectious chimpanzee doses of HCV. The immunized animal was protected, and neutralization of HCV with the antiserum from the protected animal was achieved by inoculating another chimpanzee with HCV preneutralized by this antiserum mixture. Epitope analysis of HVR1 by Pin-ELISA using this antiserum seemed to demonstrate that the antibody response was directed mainly against the C terminus of HVR1. Moreover, our results showed that, if a part of the sequences was conserved, a broad cross-reactivity of the antiserum could be observed, even if amino-acid sequences in this epitope were substituted for those of other HCV strains.

[Pathological, cytological and immunohistochemical study of normal-sized ovary carcinoma syndrome].

Normal-sized ovary carcinoma syndrome(NOCS) is characterized by diffuse metastatic malignant disease of the abdominal cavity in females, with normal-sized ovaries, and no origin assigned definitively by intraoperative evaluation. To elucidate the characteristics of NOCS arising from ovarian carcinoma, we investigated the immunohistochemical reactivities of keratin, epithelial membrane antigen(EMA), vimentin, and proliferative cell nuclear antigen(PCNA) in 4 cases of ovarian carcinoma with NOCS(NOCS group), including 3 cases of serous surface papillary adenocarcinoma(SSPC) and 1 case of small ovarian primary serous papillary adenocarcinoma(SPC). These cases were compared with 3 cases of ovarian carcinoma without NOCS(non-NOCS group), including 3 cases of SPC. Cytological and histological testing of the NOCS and non-NOCS groups showed the same findings. Similar reactivities for keratin, EMA and vimentin were observed in the NOCS and non-NOCS groups. No significant change of PCNA labeling index between the groups was seen. However, clinical stage of the NOCS group was higher than that of non-NOCS group. It was hypothesized that exposure of cancer cells to peritoneal cavity in ovarian SSPC leads to the spread. In NOCS case arising from small-sized ovarian carcinoma, another factor was thought to be involved, such as genetic alteration, but it is not known.

Immunoglobulin G and M hepatitis C virus core antibody (JCC.2) response in chimpanzees infected experimentally with hepatitis C virus.

OBJECTIVE: To evaluate changes in serum immunoglobulin M (IgM) and G (IgG) hepatitis C virus (HCV) core antibodies after HCV infection in acute hepatitis C. METHODS: Serum HCV RNA and IgM and IgG HCV core antibodies were investigated using sera sequentially sampled from three chimpanzees experimentally infected with HCV. Serum IgG HCV core antibody titer was measured using a JCC.2 enzyme-linked immunosorbent assay (ELISA) kit (Chemo-Sera-Therapeutic Research Center, Kumamoto, Japan). IgM core antibody titer was measured using horseradish peroxidase-labeled monoclonal anti-human IgM as the secondary antibody for the JCC.2 ELISA kit. Serum HCV RNA was detected using the 5' noncoding region as the primer according to the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and competitive RT-PCR method. RESULTS: IgM JCC.2 antibody was detected when alanine aminotransferase (ALT) peaked, showing the closest correlation with the changes in ALT. A period during which IgM JCC.2 antibody was positive but HCV RNA as determined by RT-nested PCR was negative was observed after the elevation of ALT level. CONCLUSION: These results indicate the usefulness of detection of serum IgM JCC.2 antibody in making a definitive diagnosis of acute hepatitis C and the follow-up observation of hepatitis C.

Isolated dissection of the celiac artery--a case report.

Isolated arterial dissection, which occurs with the absence of aortic dissection, has been reported in carotid and renal arteries but rarely in visceral arteries. A case of isolated celiac artery dissection is reported here. A healthy 58-year-old man experienced sudden upper abdominal pain, which continued for several days. A body computed tomogram (CT) showed a multiple low-density wedge-shaped area in the spleen, which was diagnosed as splenic infarction, and an aneurysm with thrombus in the celiac artery. A selective angiogram showed dilatation of the celiac artery with wall irregularity, and proximal occlusion of the hepatic artery. The distal hepatic artery was fed by collateral arteries from the superior mesenteric artery. Splenic infarction was probably due to the embolism from the thrombus in the dissected celiac artery. The absence of other vascular lesions and causes or risks for the arterial dissection would suggest the occurrence of spontaneous dissection. The dissection of visceral arteries should be considered in diagnosing acute abdominal pain.

Neuronal differentiation in human neuroblastoma cells by nerve growth factor following TrkA up-regulation by interferon-gamma.

BACKGROUND: TrkA mRNA expression has been reported to be related to favorable outcome of neuroblastoma (NB). Previously, we found that interferon-gamma (IFN-gamma) can enhance TrkA mRNA expression in NB cell lines. In the present study, we examined the effect of nerve growth factor (NGF) on IFN-gamma-induced TrkA protein to clarify the relationship between TrkA and cell differentiation of NB. PROCEDURE: The effect of IFN-gamma on the TrkA mRNA expression was screened in six human NB cell lines and a freshly prepared sample, SK-rib, from a stage IV patient. Using two of them, we examined their morphological change during simultaneous loading of NGF and IFN-gamma. Tyrosine phosphorylation pattern after 5 min of NGF stimulation was also examined in immunoblot analysis with anti-gp140(trkA) antibody and antiphospho tyrosine antibody. RESULTS: After a 4-day treatment with 500 IU/ml IFN-gamma, TrkA mRNA increased in five cell lines and SK-rib cells in association with growth inhibition. Although the degree of morphological differentiation did not increase in proportion to the TrkA expression induced by IFN-gamma, continuous loading of both IFN-gamma and NGF caused marked morphological differentiation in a cultured KP-N-RT cell line and SK-rib cells during 10 days. Moreover, 5 min of NGF stimulation after IFN-gamma treatment caused the phosphorylation of TrkA protein and downstream proteins. CONCLUSIONS: IFN-gamma could induce the functional NGF receptor even in the aggressive phenotype of NB.

N-Myc induction stimulated by insulin-like growth factor I through mitogen-activated protein kinase signaling pathway in human neuroblastoma cells.

Insulin-like growth factor I (IGF-I) stimulates proliferation, survival, and differentiation in many cell types, including pediatric neuroblastomas. The effect is mediated via the type I IGF-I receptor (IGF-IR), which is essential for growth in these cells. Several lines of evidence indicate that IGF-IR function may be particularly important in the pathogenesis of neuroblastoma. Amplification of the N-myc oncogene or overexpression of N-Myc oncoprotein has been reported to be associated with resistance to therapy and poor prognosis of neuroblastomas. It was therefore of interest to analyze whether IGF-I signaling regulated expression of N-myc in KP-N-RT human neuroblastoma cells as an experimental model that has amplified N-myc. We found that IGF-I induces N-myc mRNA and protein in the KP-N-RT with maximums of four and six times more than the basal level at 2 and 3 h after stimulation, respectively. These effects of IGF-I were blocked by a neutralizing antibody against IGF-IR (alpha-IR3). Exogenous IGF-I induced phosphorylation and activation of extracellular signal-regulated kinases p44/42 (ERK1 and ERK2), with a maximal level 30 min after the stimulation. The MEK1 inhibitor PD98059 reduced IGF-I-mediated p44/42 MAPKs phosphorylation and produced a parallel reduction of IGF-I-stimulated N-Myc induction. Furthermore, both alpha-IR3 and PD98059 inhibited G1-S cell cycle progression stimulated by IGF-I. Our results demonstrate that IGF-I induces N-Myc in the KP-N-RT neuroblastoma cell line at the RNA level and establishes a clear correlation between N-Myc induction and activation of p44/42 MAPK signaling.

Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees.

A chimpanzee was immunized with two recombinant envelope glycoproteins E1 and E2 of hepatitis C virus (HCV), strain HCV-N2, and the hypervariable region 1 (HVR1) peptides of a different isolate, HCV-#6, then received an intravenous inoculation of 10 chimpanzee infectious doses of HCV-#6. With high humoral immune response against E1 and E2 but a low response against HVR1, the vaccinee became infected with the HCV. However, after increasing the titer of anti-HVR1 against HCV-#6, the vaccinee showed protection. Neutralization of HCV-#6 with the antiserum from this protected vaccinee was achieved by inoculation of this mixture into another chimpanzee. These results suggest that vaccination with a peptide-vaccine of homologous HVR1 is effective in the chimpanzee.

Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells.

The mammalian target of rapamycin (mTOR) has been shown to link growth factor signaling and posttranscriptional control of translation of proteins that are frequently involved in cell cycle progression. However, the role of this pathway in cell survival has not been demonstrated. Here, we report that rapamycin, a specific inhibitor of mTOR kinase, induces G1 cell cycle arrest and apoptosis in two rhabdomyosarcoma cell lines (Rh1 and Rh30) under conditions of autocrine cell growth. To examine the kinetics of rapamycin action, we next determined the rapamycin sensitivity of rhabdomyosarcoma cells exposed briefly (1 h) or continuously (6 days). Results demonstrate that Rh1 and Rh30 cells were equally sensitive to rapamycin-induced growth arrest and apoptosis under either condition. Apoptosis was detected between 24 and 144 h of exposure to rapamycin. Both cell lines have mutant p53; hence, rapamycin-induced apoptosis appears to be a p53-independent process. To determine whether induction of apoptosis by rapamycin was specifically due to inhibition of mTOR signaling, we engineered Rh1 and Rh30 clones to stably express a mutant form of mTOR that was resistant to rapamycin (Ser2035-->Ile; designated mTOR-rr). Rh1 and Rh30 mTOR-rr clones were highly resistant (>3000-fold) to both growth inhibition and apoptosis induced by rapamycin. These results are the first to indicate that rapamycin-induced apoptosis is mediated by inhibition of mTOR. Exogenous insulin-like growth factor (IGF)-I protected both Rh1 and Rh30 from apoptosis, without reactivating ribosomal p70 S6 kinase (p70S6K) downstream of mTOR. However, in rapamycin-treated cultures, the response to IGF-I differed between the cell lines: Rh1 cells proliferated normally, whereas Rh30 cells remained arrested in G1 phase but viable. Rapamycin is known to inhibit synthesis of specific proteins but did not inhibit synthesis or alter the levels of mTOR. To examine the rate at which the mTOR pathway recovered, the ability of IGF-I to stimulate p70S6K activity was followed in cells treated for 1 h with rapamycin and then allowed to recover in medium containing > or =100-fold excess of FK506 (to prevent rapamycin from rebinding to its cytosolic receptor FKBP-12). Our results indicate that, in Rh1 cells, rapamycin dissociates relatively slowly from FKBP-12, with a t1/2 of approximately 17.5 h. in the presence of FK506, whereas there was no recovery of p70S6K activity in the absence of this competitor. This was of interest because rapamycin was relatively unstable under conditions of cell culture having a biological t1/2 of approximately 9.9 h. These results help to explain why cells are sensitive following short exposures to rapamycin and may be useful in guiding the use of rapamycin analogues that are entering clinical trials as novel antitumor agents.

Establishment and characterization of a cisplatin-resistant human neuroblastoma cell line.

BACKGROUND: To investigate the mechanisms of cisplatin (CDDP)-resistance in neuroblastoma(NB), we established a CDDP-resistant human NB cell line, BM1R2. MATERIALS AND METHODS: We characterized BM1R2 in terms of the susceptibilities to other anticancer agents, MDR1 and MRP expression, MYCN amplification, intracellular gultathione-S-transferase(GST-pi), metallothionein(MT) and gultathione(GSH) levels, and immunocytochemical and cytogenetic features. RESULTS: When compared to parent BM1 line, BM1R2 exhibited a 17.0-fold resistance to CDDP and cross-resistance to other agents. MRP expression was only observed in BM1R2, whereas MDR1 was expressed in both lines. Notably higher intracellular GST-pi and MT levels were observed in BM1R2 cells. MYCN amplifications were 50 and 6 copies in BM1 and BM1R2, respectively, and additional aberrations were observed in chromosome 1 and 2 in BM1R2. CONCLUSION: It was suggested that GST-pi and MT could exert crucial roles on CDDP-resistance in our system. BM1R2 is of great interest for investigating the mechanisms of CDDP-resistance in NB.

Murine antibodies against E2 and hypervariable region 1 cross-reactively capture hepatitis C virus.

The absence of readily available animal and cell culture models for hepatitis C virus (HCV) replication has bottlenecked research on protective immunity to HCV infection. Antibodies reactive with HCV virions in vitro are assumed to be candidates for neutralizing or inhibitory antibodies against HCV. To find potentially neutralizing or inhibitory antibody candidates, anti-C, anti-E1, anti-E2, and anti-HVR1 antisera acquired from mice immunized with corresponding recombinant proteins or synthetic peptides were used to capture HCV viral particles in vitro based on antibody-virus interaction assays. Both anti-E2 and anti-HVR1 antibodies effectively captured HCV in vitro. Furthermore, it was found that anti-E2 and anti-HVR1 antibodies could immunoprecipitate an isolate of HCV unrelated to the original antigenic HCV isolate. ELISA confirmed that anti-HVR1 antibodies cross-reactively bind to these unrelated HVR1 peptides. These findings suggest that anti-E2 and anti-HVR1 antibodies induced in mice have the ability to bind with HCV particles in an isolate cross-reactive manner and highlight the possible application of combining several sequences of HVR1 to generate broadly reactive anti-HVR1 antibodies.

Studies on the mechanism of resistance to rapamycin in human cancer cells.

Rapamycin is a potent cytostatic agent that arrests cells in the G1 phase of the cell cycle. The relationships between cellular sensitivity to rapamycin, drug accumulation, expression of mammalian target of rapamycin (mTOR), and inhibition of growth factor activation of ribosomal p70S6 kinase (p70(S6k)) and dephosphorylation of pH acid stable protein I (eukaryotic initiation factor 4E binding protein) were examined. We show that some cell lines derived from childhood tumors are highly sensitive to growth inhibition by rapamycin, whereas others have high intrinsic resistance (>1000-fold). Accumulation and retention of [14C]rapamycin were similar in sensitive and resistant cells, with all cells examined demonstrating a stable tight binding component. Western analysis showed levels of mTOR were similar in each cell line (<2-fold variation). The activity of p70(S6k), activated downstream of mTOR, was similar in four cell lines (range, 11.75-41. 8 pmol/2 x 10(6) cells/30 min), but activity was equally inhibited in cells that were highly resistant to rapamycin-induced growth arrest. Rapamycin equally inhibited serum-induced phosphorylation of pH acid stable protein I in Rh1 (intrinsically resistant) and sensitive Rh30 cells. In serum-fasted Rh30 and Rh1 cells, the addition of serum rapidly induced c-MYC (protein) levels. Rapamycin blocked induction in Rh30 cells but not in Rh1 cells. Serum-fasted Rh30/rapa10K cells, selected for high level acquired resistance to rapamycin, showed >/=10-fold increased c-MYC compared with Rh30. These results suggest that the ability of rapamycin to inhibit c-MYC induction correlates with intrinsic sensitivity, whereas failure of rapamycin to inhibit induction or overexpression of c-MYC correlates with intrinsic and acquired resistance, respectively.

Comparison of amino acid sequences in hypervariable region-1 of hepatitis C virus clones between human inocula and the infected chimpanzee sera.

We analyzed nucleic and amino acid sequences of the hypervariable region (HVR)-1 of the hepatitis C virus (HCV) from inocula and the infected chimpanzee sera. One milliliter each of 10(-5), 10(-3) and 10(-2) dilutions of serum no. 4, and those of 10(-4), 10(-5) and 10(-6) dilutions of serum no. 6 were inoculated to six different chimpanzees. Both inocula nos. 4 and 6 contained 10(7) copies of HCV RNA/ml. The two chimpanzees inoculated with 10(-4) and 10(-5) dilutions of no. 6 were infective, while none of the dilutions from inoculum no. 4 were infective. Our results indicated that HCV RNA titer in sera did not correlate with in vivo infectivity. RNA from both inocula and the infected chimpanzee sera were extracted and transcribed to cDNA. The PCR products amplifying the HCV HVR-1 were incorporated into a vector, followed by transformation. Twenty colonies were picked up randomly from each sample. DNA was extracted and the DNA and amino acid sequences were determined. Genetic variations of 20 clones from inoculum no. 6 were divided into six groups, whereas, 40 clones from infected chimpanzees were completely identical to the major quasispecies of inoculum no. 6 in amino acid sequences. The study suggested that the major quasispecies in the diluted inocula were transmitted and replicated.

Diffuse bilateral thalamic astrocytomas as examined serially by MRI.

We report the case of a 13-year-old girl with diffuse bilateral thalamic astrocytomas. Incoordination was observed at the onset. Cranial computed tomography (CT) showed enlarged thalami, and magnetic resonance imaging (MRI) revealed these lesions to be symmetrically enlarged with high intensity on the T2-weighted image. Owing to these atypical findings in the neuroimaging studies, we had difficulty in making the correct diagnosis of a brain tumor. After the diagnosis of diffuse bilateral thalamic astrocytomas was obtained, we performed hyperfractionated radiotherapy followed by chemotherapy. Radiation therapy was effective for a while, but the girl's condition deteriorated again and she died 8 months after admission. Although diffuse bilateral thalamic astrocytomas are difficult to diagnose because they do not resemble most other neoplasms on neuroimaging studies, pediatricians should keep this entity in mind in order to arrive at a precise and prompt diagnosis.

The dilute-lethal (dl) gene attacks a Ca2+ store in the dendritic spine of Purkinje cells in mice.

The absence of smooth endoplasmic reticulum (SER) in the dendritic spine of Purkinje cells was found in dilute-lethal (dl) mouse cerebella as detected by immunohistochemistry using anti-inositol 1,4,5-triphosphate receptor antibody and electron microscopy. Since SER in the spine has been suggested to play a crucial role for synaptic regulation as an intracellular Ca2+ store (for reviews, see [Miller, R.J., Prog. Neurobiol., 37 (1991) 255-285: Simpson, P.B., Challiss, R.A.J. and Nahorski, S.R., Trends Neurosci., 18 (1995) 299-306]), a neurological defect, characterized by clonic convulsions with opisthotonus and ataxia, in the dilute-lethal mouse with homozygous trait may be attributable to the absence of SER in the dendritic spine of Purkinje cells.

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.

Inactivation of hepatitis C virus cDNA transgene by hypermethylation in transgenic mice.

Transgenic mice were produced by microinjection of a partial hepatitis C virus (HCV) genome sequence including the structural protein region, under the control of the albumin promoter and enhancer into fertilized eggs of C57BL/6 and BDF1 mice. Three founders carrying at least five copies of the transgene but not expressing HCV-specific RNA were generated. Methylation analysis indicated that the transgene was extensively methylated. Mapping of methylated cytosine residues of the transgenic mouse DNA showed that all C residues of a particular part of the HCV genome but not all the CpG island like sequences were methylated. Transiently expressed HCV cDNA in COS7 cells and the active endogenous albumin gene were not methylated. Furthermore, 5-azacytidine, a potent demethylating agent, induced HCV gene expression in a line of these transgenic mice. These results suggest that methylation of HCV cDNA is a cause of its inactive expression in transgenic mice, and that this phenomenon may occur in other stable systems for expression of the HCV genome.

Murine humoral immune response against recombinant structural proteins of hepatitis C virus distinct from those of patients.

We examined the humoral immune response to recombinant structural proteins of hepatitis C virus (HCV) such as C, E1 and E2 in immunized mice. Mice showed high induction of antibodies against these three structural proteins. Conformational and/or linear epitopes of these regions showed high responses in mice. Comparison with patients revealed higher anti-E1 and anti-E2 responses in mice and 15 immunoreactive peptides which are unique to mice, especially 11 peptides from the E2 region. The hydrophilic regions of these proteins were found to be the most immunogenic. Therefore, the murine immune system against recombinant E1 and E2 glycoproteins was distinct from those of patients in natural infection, and may be a target to find protective activity against HCV infection.

Probable implication of mutations of the X open reading frame in the onset of fulminant hepatitis B.

A pathogenic role of precore-defective mutation in the onset of fulminant hepatitis B has been suggested. However, precore-defective mutants do not always cause fulminant hepatitis B and are not always isolated from affected patients. These findings strongly suggest the presence of some additional important mutations outside the precore region in fulminant hepatitis. In the present investigation an attempt was made to sequence the X open reading frame of hepatitis B virus DNA isolated from seven patients with fulminant hepatitis B and five patients with acute hepatitis B. The latter were used as controls. Since the X open reading frame encodes the X protein and contains the core promoter/enhancer II complex, some critical mutations may enhance or disrupt the replication and expression of hepatitis B virus DNA leading to fulminant hepatitis. A C-to-T substitution was found at nucleotide (nt) 1655, an A-to-T substitution at nt 1764 and a G-to-A substitution at nt 1766 in 4, 5 and 5 patients, respectively, out of the seven with fulminant hepatitis. These substitutions were not recognized in the patients with acute hepatitis. These mutations might change the function of the X protein and core promoter/enhancer II complex. It is suggested, therefore, that these mutations, as well as the precore-defective mutation, may play an important role in the pathogenesis of fulminant hepatitis.