Pathogenesis Of Fulminant Hepatitis Research Articles

Meteorin-like protein overexpression ameliorates fulminant hepatitis in mice by inhibiting chemokine-dependent immune cell infiltration.

Myokines, which are recently identified cytokines secreted by skeletal muscle in response to stimulation, are crucial for the maintenance of liver function. Fulminant hepatitis (FH) is a life-threatening pathological condition with severe hepatic dysfunction. In this study, we investigated the role of meteorin-like (METRNL), a new myokine, in the pathogenesis of FH. We compared serum samples and liver tissues from FH patients and healthy controls and found that hepatic and serum METRNL levels were significantly increased in FH patients, and serum METRNL levels were related to disease severity in FH patients. We then established a concanavalin A-induced FH model in METRNL-overexpressing and control mice. We found that hepatic METRNL levels in FH mice were significantly increased, and METRNL in the liver was mainly derived from macrophages. In the cultured mouse macrophage line (RAW264.7 cells) and mouse primary peritoneal macrophages (PMs), METRNL overexpression significantly inhibited the release of the proinflammatory cytokines TNF and IL-1β. In METRNL-overexpressing mice, concanavalin A-induced liver injury was significantly ameliorated. Moreover, METRNL overexpression significantly reduced chemokine-dependent inflammatory cell infiltration into the liver. METRNL overexpression also suppressed liver CD4+ T cell differentiation into Th 1 cells and inhibited the secretion of Th 1 cytokines. Taken together, these data suggest that METRNL overexpression effectively ameliorates FH. Therefore, METRNL may serve as a potential biomarker and therapeutic target for FH.

Macrophage scavenger receptor 1 contributes to pathogenesis of fulminant hepatitis via neutrophil-mediated complement activation

The macrophage scavenger receptor 1 (Msr1, also called SRA) is a pattern recognition receptor primarily expressed on myeloid cells, which plays an important role in the maintenance of immune homeostasis. Since MSR1 expression was upregulated in the livers of patients with fulminant hepatitis (FH), we investigated the functional mechanism of Msr1 in FH pathogenesis. Msr1-deficient (Msr1-/-) mice and their wild-type (WT) littermates were infected with mouse hepatitis virus strain-A59 (MHV-A59) to induce FH, and the levels of tissue damage, serum alanine aminotransferase, inflammatory cytokines and complement component 5a (C5a) were measured and compared. Liver injury was studied after MHV infection with or without neutrophil depletion. Our results showed that Msr1-/- mice were resistant to MHV-induced hepatitis. Treatment with the C5a receptor antagonist (C5aRa) diminished the differences in inflammatory responses and liver injury between MHV-infected wild-type and Msr1-/- mice, suggesting that C5a-induced pro-inflammatory response plays a critical role in the Msr1-mediated regulation of FH pathogenesis. We demonstrated that Msr1 efficiently enhanced transforming growth factor-activated kinase-1 phosphorylation in neutrophils upon MHV-A59 stimulation, thereby promoting the activation of the extracellular signal-regulated kinase pathway and subsequent NETosis formation. Moreover, we provided evidence that blockage of Msr1 attenuated the liver damage caused by MHV-A59 infection. Msr1 promotes the pathogenesis of virus-induced FH by enhancing induction of neutrophil NETosis and subsequent complement activation. Targeting Msr1 may be employed as a new immunotherapeutic strategy for FH. Virus-induced fulminant hepatitis (FH) is a disease with a high mortality worldwide. Enhanced levels of macrophage scavenger receptor 1 (Msr1) in the liver of patients with FH and of murine experimental FH indicated Msr1 plays a role in the pathogenesis of FH. Herein, we demonstrate that mice deficient in Msr1 are resistant to FH induced by MHV-A59, and the Msr1 inhibitor fucoidan suppresses the progression of FH in mice. Our study suggests that use of drugs inhibiting MSR1 function could be beneficial to patients with FH.

Ursodeoxycholyl Lysophosphatidylethanolamide Protects Against CD95/FAS-Induced Fulminant Hepatitis.

Increased activation of CD95/Fas by Fas ligand in viral hepatitis and autoimmunity is involved in pathogenesis of fulminant hepatitis and liver failure. We designed a bile-acid phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE with LPE containing oleate at the sn-1) as a hepatoprotectant that was shown to protect against fulminant hepatitis induced by endotoxin. We herein further assessed the ability of UDCA-LPE to prevent death receptor CD95/Fas-induced fulminant hepatitis. C57BL/6 mice were intravenously administered with CD95/Fas agonistic monoclonal antibody (Jo-2) with or without 1 h pretreatment with 50 mg/kg UDCA-LPE. Jo-2 administration caused massive hepatocyte damage as seen by histology, and this was associated with a significant decrease in hepatic phosphatidylcholine (PC), lysoPC, and lysophosphatidylethanolamine levels. By histology, UDCA-LPE pretreatment improved hepatocyte damage and restored the loss of these phospholipids in part by a mechanism involving an inhibition of cytosolic phospholipaseA2 expression. Accordingly, Jo-2 treatment increased hepatic expression of cleaved caspase 8, caspase 3, and poly (ADP-Ribose) polymerase-1, and on the other hand decreased that of anti-apoptotic cellular FLICE-inhibitory protein. UDCA-LPE pretreatment was able to reverse all these changes. Moreover, UDCA-LPE attenuated inflammatory response by lowering the levels of Jo-2-induced proinflammatory cytokines TNF-α, IL-6, and IL-1β in liver and serum. UDCA-LPE was also able to decrease the levels of stimulated Th1/Th17 cytokines in Jo-2-primed isolated splenocytes. Taken together, UDCA-LPE exhibited potent anti-inflammatory effects against CD95/Fas-induced fulminant hepatitis.

IL-25 prevents and cures fulminant hepatitis in mice through a myeloid-derived suppressor cell-dependent mechanism

Fulminant hepatitis (FH) is a disease characterized by massive destruction of hepatocytes with severe impairment of liver function. The pathogenesis of FH is not fully understood, but hyperactivity of T cells and macrophages with excessive production of cytokines are important hallmarks of the condition. In this study, we investigated the role of interleukin (IL)-25 in FH. IL-25 expression was evaluated in patients with FH and in livers of mice with FH induced by D-galactosamine (D-Gal) and lipopolysaccharide (LPS). Mice were treated with IL-25 before D-Gal/LPS-induced FH and before or after concanavalin A (ConA)-induced FH. Mononuclear cells were isolated from livers of mice treated with or without IL-25 and analyzed for GR1(+) CD11b(+) cells. CFSE-labeled T cells were cocultured with GR1(+) CD11b(+) cells and their proliferation was evaluated by flow cytometry. Mice were also treated with a depleting anti-GR1 antibody before IL-25 and D-Gal/LPS administration. IL-25 was constitutively expressed in mouse and human liver and down-regulated during FH. IL-25 prevented D-Gal/LPS-induced FH and this effect was associated with increased infiltration of the liver with cells coexpressing GR1 and CD11b. In vitro studies showed that GR1(+) CD11b(+) cells isolated from mice given IL-25 inhibited T-cell proliferation. Consistently, in vivo depletion of GR1(+) cells abrogated the protective effect of IL-25 in experimental D-Gal/LPS-induced FH. IL-25 was both preventive and therapeutic in ConA-induced FH. IL-25 expression is markedly reduced during human and experimental FH. IL-25 promotes liver accumulation of GR1(+) CD11b(+) cells with immunoregulatory properties.

Expansion of Liver-Infiltrating CTL Clone In Concert with the Development of Hepatitis-Associated AA.

AbstractAbstract 1159Hepatitis-associated aplastic anemia (HAA) is a variant of acquired aplastic anemia (AA) in which an episode of hepatitis precedes AA, namely hepatitis/aplastic anemia syndrome (HAAS). In AA, cytotoxic T-lymphocytes (CTL) play a central role in bone marrow (BM) destruction, and oligoclonal expansion of CTL correlates with disease activity. Immune mediated mechanisms have also been postulated in the pathogenesis of fulminant hepatitis (FH). Therefore we hypothesized that common CTLs recognizing similar target antigens against both liver and BM cells exist during the early period of FH, and subsequently selective proliferation of clones that are highly tropic to BM cause HAA at later stages. To this end, T cell phenotypes infiltrating liver, BM and peripheral blood (PB) samples obtained from 1-year-old boy with HAAS over time were determined by complementarity-determing region 3 (CDR3) size distribution patterns by polymerase chain reaction. Subsequently, CDR3 nucleotide sequences of predominant products were determined. Extremely skewed pattern was observed in Vb23 in BM and PB specimens at the onset of HAA (3 months after FH). A Vb23 clone with identical CDR3 nucleotide sequence predominated (clone 1) in BM and PB (34% and 60% within Vb23, respectively). This clone constituted a minor, but definitive clone in liver at the onset of FH (16% within Vb23). In contrast, initially predominant clones in liver (clone 3; 47% within Vb23) contracted to a minor subpopulation in BM and PB when the patient developed HAA (17% and 10% within Vb23, respectively).This patient had a very good response to immunosuppressive therapy (IST) using antithymocyte globulin, cyclosporine, and granulocyte-colony stimulating factor. Two months after starting IST, the pattern of CDR3 size distribution was just recovering to normal and initially predominant clones in both liver and BM almost disappeared in Vb23 in PB. However, extremely skewed pattern of CDR3 region was still observed and abnormal clone 1 had still occupied 71% within Vb23 in BM. As of 8 months after starting IST, CDR3 size distribution recovered to normal patterns (Gaussian distribution) and CTL clones against both liver and BM almost disappeared in both BM and PB. These suggest that it would take a relatively long time to recover immunological parameters completely even in IST good responders.In conclusion, our data is the first report to suggest that oligoclonal CTL clones simultaneously directed against liver and BM are involved in the pathogenesis of HAAS. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Nucleotide Substitutions of Hepatitis E Virus Genomes Associated with Fulminant Hepatitis and Disease Severity

Hepatitis E virus (HEV) is one of the causative agents of acute or fulminant hepatitis. Viral factors may play a role in the pathogenesis of fulminant hepatitis E. We aimed to investigate the nucleotide substitutions of the HEV genome affecting the severity of hepatitis E. The comparison of 28 reported full-length nucleotide sequences of genotype 4 HEV showed that the substitution of C at nucleotide 5907 (C5907) was most closely associated with fulminant hepatitis (fulminant hepatitis, 100%; acute hepatitis, 39.1%; p = 0.0204). Analyzing the full-length sequences of 28 genotype-4 and 11 genotype-3 HEV retrievable from DNA databases and 35 partial sequences recovered from patients with acute or fulminant hepatitis, we show that the presence of both U3148 and C5907 is associated with fulminant hepatitis in patients with HEV of genotype 4 (p = 0.0042) and genotype 3 or 4 (p = 0.0009), and that the prothrombin activity is significantly lower in patients infected with HEV carrying U3148 and C5907 than in those without the substitutions (p = 0.0069). U3148 and C5907 are silent substitutions that do not change amino acid. However, since U3148 is located at the RNA helicase domain and C5907 is located within the capsid gene, the secondary structure of the HEV RNA genome carrying U3148 and C5907 may be favorable for translation of the viral proteins. C5907 was associated with high HEV load (> or = 10(5) copies/ml) at initial examination (p = 0.0427). We propose that U3148 and C5907 are associated with the severity of hepatitis E.

Possible involvement of cytokine gene polymorphisms in fulminant hepatitis

Host genetic factors have been reported as influencing the progress to fulminant hepatitis (FH). Our previous data showed the serum level of tumor necrosis factor (TNF)-alpha influenced by gene polymorphisms to be markedly increased. It was investigated whether polymorphisms in the IL-10 gene, in addition to TNF-alpha and -beta gene polymorphisms, might contribute to the pathogenesis of FH. We analyzed 42 patients with FH, 78 patients with acute hepatitis (AH), and 149 healthy subjects (control). IL-10 polymorphism sites at promoter regions -1028, -819, -592; TNF-alpha polymorphism sites at promoter regions -1031, -863, -857, -308, -238; and TNF-beta first intron Nco1 sites were studied. IL-10 gene polymorphisms were classified into three groups: low IL-10-producing haplotypes (ATA/ATA), intermediate haplotypes (ATA or CCA/CCA), and high haplotypes (ATA/ATG or CCG). The allelic frequency of B2 in the TNF-beta gene was significantly higher in FH patients compared with the control group. The three groups showed no differences in polymorphisms of positions -1031, -863, -857, -308 and -238 in the TNF-alpha gene. The frequency of low IL-10-producing haplotypes tended to be higher in FH patients compared with control and that of high IL-10-producing haplotype tended to be lower in FH patients compared with control. The carrier rate with both the IL-10 haplotype and the TNF-beta gene B2/B2 was significantly higher than control. Variations of cytokine polymorphisms including IL-10 and TNF-beta genes may be attributable to the pathogenesis of FH.

CD154-CD40 interactions drive hepatocyte apoptosis in murine fulminant hepatitis

The CD154-CD40 interaction is a critical costimulatory pathway modulating the cellular immune response. Moreover, fulminant hepatitis of various etiologies is characterized by a hepatic influx of CD154-expressing T cells and an upregulation of CD40 expression on Kupffer cells and hepatocytes, implicating this pathway in the pathogenesis of fulminant hepatitis. In this study, we used a murine model of fulminant hepatitis induced by concanavalin A (con A) and documented a significant influx of CD154-expressing T cells into the livers of mice treated with con A, in association with markedly increased expression of CD40 restricted mainly to hepatocytes in damaged areas of the liver. Furthermore, con A hepatitis in CD154-deficient mice was significantly attenuated compared with that in wild-type controls and was associated with a decrease in hepatic tumor necrosis factor alpha (TNF-alpha) levels and hepatocyte death. We next determined the role of the CD154-CD40 pathway in hepatocyte death in vitro. These in vitro studies demonstrated that TNF-alpha induces CD40 expression in hepatocytes and that subsequent activation of CD40 results in hepatocyte apoptosis mediated at least in part by enhanced hepatocyte expression of FasL. In conclusion, CD154 stimulation of CD40 plays a central role in hepatocyte death in fulminant hepatitis through direct and indirect pathways that may have direct therapeutic implications in humans. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Hepatitis B virus mutations associated with fulminant hepatitis induce apoptosis in primaryTupaiahepatocytes

Hepatitis B virus (HBV) core promoter mutations have been implicated in the pathogenesis of fulminant hepatitis B. Due to the limited availability of primary human hepatocytes, the functional characterization of HBV mutants has been performed predominantly in transformed cells, which may not represent ideal model systems for studying virus-cell interactions. We and others have shown that primary hepatocytes of the tree shrew Tupaia belangeri support HBV infection and replication. In this study, we used primary Tupaia hepatocytes to analyze the phenotype of two HBV core promoter mutations that have been associated with a clinical outbreak of fatal fulminant hepatitis. Similar to previous findings in human hepatoma cells, the HBV core promoter mutations resulted in enhanced viral replication and core expression. Surprisingly, however, the presence of the mutations had a marked effect on hepatocyte viability not previously observed in hepatoma cells. Reduced cell viability was found to be due to the induction of apoptosis, as evidenced by caspase-3 activation and nuclear fragmentation. In conclusion, HBV mutants exhibit a novel phenotype in primary hepatocytes distinctly different from previous findings in hepatoma cell lines. This phenotype may have important implications for the understanding of the fulminant clinical course associated with HBV mutations.

P0337 INNATE AND ADAPTIVE IMMUNITY IN FULMINANT HEPATITIS B

Introduction: Innate and adaptive immune responses are believed to play a key role in the clearance of intracellular viruses, including hepatitis B infection. Experimental studies have proposed a hitherto unsuspected role for NK cells during acute HBV infection. Fulminant hepatitis B is thought to be due to an overwhelming T cell response. Little, however, has been tested in humans, especially at the hepatic level. The aim of the present study was to investigate both T and NK cells in the liver infiltrate of fulminant hepatitis B. Methods: A 9-year old girl developed fulminant hepatitis B (HBsAg and IgM anti-HBc positive, HBV-DNA negative, AST 1550 IU/L, bilirubin 640 μmol/l, INR 3,7) and underwent emergency liver transplantation. The massive mononuclear cellular infiltrate observed in the explanted liver by conventional histology was analysed immunohistochemically on both frozen and formalin fixed sections for the following markers: CD3 (pan-T), CD4 (T-helper), CD8 (T-cytotoxic), CD45RA/RO (naïve/committed cells), CD56 (natural killer cells), CD161 (natural killer T cells), perforin, and granzyme B (effector molecules of cytolysis, present in CD8, NK, and NKT cells). Results: The majority of the infiltrating mononuclear cells expressed CD3, CD8, and CD45RO. CD56 cells were also present, but at lower frequency. Cells marking for perforin were especially abundant. Double-staining showed that approximately half of them belonged to the CD8 T cell population, and though not tested experimentally, the other half can only belong to the NK/NKT cell population. Conclusion: Perforin-expressing cells that participate in both the adaptive and the innate immune response characterize the mononuclear cell infiltrate of fulminant hepatitis B. In view of their lytic potential, it is conceivable that these are involved in the pathogenesis of fulminant hepatitis B.

Prevalence of herpesviridae and hepatitis virus sequences in the livers of patients with fulminant hepatitis of unknown etiology in Japan.

Fulminant non-A, non-B, non-C hepatitis has a high mortality rate, making the identification of its causative agent imperative. Cytomegalovirus, Epstein-Barr virus, human herpesvirus-6, and herpes simplex virus are all members of the herpesviridae family that are associated with fatal hepatic failure. We investigated the involvement of herpesviridae and hepatitis virus in the pathogenesis of fulminant hepatitis. The study participants consisted of 11 patients with fulminant hepatitis and 11 with acute hepatitis negative for known hepatitis viral markers and any other liver diseases. Viral DNA was extracted from liver tissues and amplified. In situ hybridization was then performed for 1 patient to detect viral DNA and RNA, and viral protein was localized by monoclonal antibodies. Human herpesvirus-6 was detected in liver tissues from seven patients, (five children and two adults) with fulminant hepatitis and two patients with acute hepatitis. Two patients with fulminant hepatitis also had cytomegalovirus in the liver. Although Epstein-Barr virus and herpes simplex virus were detected in the patients with fulminant hepatitis, they were not specific to these patients. In situ hybridization in one of the patients localized DNA and RNA of human herpesvirus-6 in hepatocyte nuclei, and an envelope antigen of this virus was detected in hepatocyte cytoplasm. Human herpesvirus-6 was frequently detected in Japanese pediatric patients with fulminant non-A, non-B, non-C hepatitis. Although the significance of human herpesvirus-6 in liver pathogenesis remains unclear, this virus may replicate in hepatocytes in some patients with acute onset hepatitis.

Molecular analysis of hepatitis B virus genomes isolated from black African patients with fulminant hepatitis B

To investigate further the possible role of mutant hepatitis B viruses in the pathogenesis of fulminant hepatitis B, the genomic sequence of hepatitis B virus isolates from 9 South African blacks with this disease, including 5 entire genomes, was analysed. Seven of the isolates were genotype A. The mutation most often reported in patients with fulminant hepatitis B, the G1896A precore stop-codon substitution, was, as expected, not present in the genotype A isolates with the exception of one in which it was accompanied by a compensatory C1858T substitution. G1896A was, however, present in the one genotype D isolate. No other precore-defective mutants were detected. The other mutation commonly found in patients with fulminant hepatitis B, the paired A1762T, G1764A substitution in the basic core promoter, was present in only one patient and G1764A in one other. The pre-surface initiation-codon mutation documented in a number of patients with fulminant hepatitis B was not found in our isolates. An 18-amino acid deletion present in the pre-surface region of one isolate has not previously been described in fulminant hepatitis B. Variations within the surface region were mainly genotype specific and not previously described. A relatively large number of mutations were present in the middle region of the core gene in those isolates without G1896A or A1762T, G1764A mutations, although the pattern was not consistent with those in published studies. Thus, as in other published series in which the entire genome of hepatitis B virus responsible for fulminant hepatitis was sequenced, we detected many mutations in different genes, but none was common to all the reported isolates.

A dominant hepatitis B virus population defective in virus secretion because of several S-gene mutations from a patient with fulminant hepatitis

There is increasing evidence that certain pathogenic hepatitis B virus (HBV) variants may play a role in the pathogenesis of fulminant hepatitis (FHB). Recently, we isolated from a patient with fulminant recurrent hepatitis B after liver transplantation variants with enhanced replication competence and a possible defect in viral particle secretion. Both viral features may have contributed to the severity of the disease. The aim of this study was to prove the secretion defect of these variants, to analyze the consequences, and to identify the responsible viral mutations. The variant genomes and appropriate wild-type/variant hybrid genomes were functionally characterized after transfection in human hepatoma cells. Two cloned genomes and the polymerase chain reaction (PCR)-amplified mixture of full-length genomes showed a block in viral particle secretion. This was caused by a combination of amino acid changes in the S-protein including the mutation G145R frequently emerging after hyperimmunoglobulin treatment. The mutations induced retention of the surface proteins in an endoplasmic reticulum (ER)-like compartment, but no intracellular accumulation. These data provide evidence for the in vivo existence of a dominant HBV population with a severe defect in viral particle secretion caused by mutations in the S-gene. This viral phenotype in combination with the enhanced replication competence may have contributed to the fulminant clinical course of the infection. (HEPATOLOGY 2001;34:385-394.)

Indispensable role for TNF-alpha and IFN-gamma at the effector phase of liver injury mediated by Th1 cells specific to hepatitis B virus surface antigen.

We report the development and characterization of a novel model of severe hepatitis induced against hepatitis B virus surface Ag (HBsAg). HBsAg was successfully targeted into the liver in soluble form. Using this unique property of HBsAg, we established a liver injury model induced by HBsAg-specific Th1 cells. Severe liver injury was induced in C57BL/6 mice by injection of HBsAg together with HBsAg-specific Th1 cells. Histochemical examination demonstrated extensive necroinflammatory hepatic lesions in these animals. Application of this liver injury model to mutant or gene knockout mice enabled us to define the effector mechanisms of Th1 cells in fulminant hepatitis. When Fas-deficient lpr mice were used as recipients, a similar degree of liver injury was induced as in wild-type mice. Moreover, HBsAg-specific Th1 cells obtained from perforin-/- mice could induce severe liver injury in both wild-type and lpr mice. These results indicated that neither Fas ligand nor perforin are essential for Th1-mediated liver injury in this model. Pretreatment with anti-TNF-alpha mAb prevented liver injury, whereas severe liver injury was induced in TNF-alpha-/- mice. Moreover, IFN-gamma receptor-deficient mice were resistant to Th1-mediated liver injury. Therefore, TNF-alpha and IFN-gamma, which were produced by HBsAg-specific Th1 cells during the effector phase, appeared to be indispensable in the pathogenesis of fulminant hepatitis.

Experimental transmission of hepatitis C virus-associated fulminant hepatitis to a chimpanzee.

Hepatitis C virus (HCV) was transmitted from a patient with fulminant hepatitis C to a chimpanzee. The patient had developed two episodes of fulminant hepatitis C, each occurring after a separate liver transplantation. Serial serum and liver samples from the patient and the chimpanzee were analyzed for HCV replication, genotype, quasispecies heterogeneity, and antibodies. In the patient, the levels of HCV replication in serum and liver correlated with the degree of hepatocellular necrosis and the clinical expression of fulminant hepatitis. The same HCV strain, genotype 1a, was recovered from both episodes of fulminant hepatitis. An unusually severe acute hepatitis was also observed in the chimpanzee. The viruses recovered from the patient and the chimpanzee were almost identical and displayed relatively little quasispecies heterogeneity. Thus, the same HCV strain induced two episodes of fulminant hepatitis in a single patient and severe hepatitis in a chimpanzee, suggesting that the pathogenicity or virulence of a specific HCV strain may be important in the pathogenesis of fulminant hepatitis C.

Mutations in the basic core promotor and the precore region of hepatitis B virus and their selection in children with fulminant and chronic hepatitis B.

The involvement of precore stop codon 1896-A and base exchanges in the AT-rich region at positions 1762 and 1764 of the hepatitis B core promotor has been controversely discussed in adults with fulminant hepatitis B. Because no data are currently available on children, we analyzed the basic core promotor (BCP) and precore region in children with chronic and fulminant hepatitis B. The BCP and precore region were sequenced directly and after cloning from mothers and infants. Thirteen children suffered from chronic liver disease, 6 of whom were treated with interferon alfa (IFN-alpha). All 13 patients seroconverted from hepatitis B e antigen (HBeAg) to hepatitis B e antigen antibodies (anti-HBe), and sera were analyzed before and after seroconversion. Nine vertically infected infants developed a fulminant course of hepatitis B. The occurrence of BCP (1762-T/1764-A, 7.7%) and precore (1896-A, 7.7%; 1899-A, 15%) mutations in chronic hepatitis B was rare. A genotype shift from D to A was observed in 3 patients after development of anti-HBe. A high number of base exchanges was detected in those infants with fulminant hepatitis B. Eight of nine showed a G-A exchange at positions 1896/97 (89%), 1899 (56%), and/or mutations at nucleotide (nt) positions 1762 (56%) and 1764 (78%). All virus strains belonged to genotype D, whereas in the only surviving infant, a D-to-A shift was detected. Hepatitis B virus (HBV) DNA clones were examined from 3 babies and 5 mothers. Our results showed a heterogeneous virus population in 4 of 5 mothers. In contrast, a homogeneous virus population emerged in the infants. According to our data, the analysis in children with fulminant and chronic hepatitis B revealed a striking presence of BCP and precore mutants in infants with fulminant hepatitis (FH) when compared with clinically inapparent anti-HBe-positive children (P <.002), which could be one factor in the pathogenesis of fulminant hepatitis B in children.

ANALYSIS OF THE HEPATITIS B BASIC CORE PROMOTOR AND PRECORE REGION IN CHILDREN WITH FULMINANT AND CHRONIC HEPATITIS B

152 AIMS. The pathogenesis of fulminant and chronic hepatitis B virus(HBV) infection is not well understood. Although the precore stop codon mutation at position 1896 and base exchanges in the T-A rich region at positions 1762 and 1764 of the core promotor have been reported to be more frequently detected in adults with fulminant hepatitis B or arise after seroconversion to anti-HBe, the data are inconsistent and discussed controversely. Since no data are available from children, we analysed core promotor and precore sequences of infants with fulminant and chronic hepatitis B. METHODS. Sera from 22 children and six mothers were analysed. 13 children had chronic liver disease, six of them were treated with alpha-interferon and nine vertically infected newborns had a fulminant course of hepatitis B. The basic core promotor and precore region were amplified by PCR and sequences were identified by direct sequencing. During follow-up all 13 children seroconverted from HBeAg to anti-HBe and the sera were analysed before seroconversion. RESULTS. None of the HBeAg positive children and only one of the anti-HBe positive children had a stop mutant in the precore region. Additionally two anti-HBe positive children showed a mixture of wildtype and 1896-stop mutant. One chronically infected patient developed base exchanges at positions 1762 and 1764 after seroconversion. In contrast eight of nine infants with fulminant hepatitis showed a G-A exchange at positions 1896/97(n=6) and/or mutations at nucleotide positions 1762 (n=5) and 1764 (n=7). Overall the children with fulminant hepatitis displayed significantly more base exchanges in the core promotor region than those with chronic hepatitis B. CONCLUSIONS. Summarizing our data the analysis of core promotor and precore mutations in children with fulminant and chronic hepatitis B revealed a striking presence of core promotor mutants in infants with fulminant hepatitis compared with clinically inapparent anti-HBe positive HBsAg carriers. The combination of a precore stop mutant with core promotor base exchanges at positions 1762 and 1764 might be one factor in the pathogenesis of fulminant hepatitis B in children.

Hepatitis B virus genomes of patients with fulminant hepatitis do not share a specific mutation

The pathogenesis of fulminant hepatitis B virus (HBV) infection is not well understood. The aim of this study was to investigate whether there is an association between specific viral variants and a fulminant disease course. The entire HBV genomes from the serum of eight patients with fulminant HBV infection and one patient with fulminant hepatitis during reinfection after liver transplantation were investigated. After isolation and amplification of viral DNA by polymerase chain reaction (PCR), plus and minus strands were directly sequenced. Sequence data were analyzed by comparative sequence alignments with 35 and 2 complete HBV genome sequences from patients without and with fulminant hepatitis, respectively. Several point mutations were present in all regions of the genomes. Many nucleotide changes had never or rarely been found in the reported HBV isolates from patients without fulminant hepatitis. A distinct mutation present in all genomes was not identified. Clusters of rare and unique mutations were observed in the enhancer II core promoter region. Mutations previously suggested to be associated with fulminant HBV infection were not consistently found. A precore stop codon mutation at nucleotide position 1896 or an A-to-T mutation at nucleotide position 1762 and a G-to-A mutation at nucleotide position 1764 in the core promoter region were present in four and three cases, respectively. Fulminant HBV infection does not appear to be caused by a specific genomic mutation. However, various mutations clustering in the enhancer II core promoter region may contribute to a fulminant disease course. (Hepatology 1996 Aug;24(2):300-6)

Hepatitis B virus genomes of patients with fulminant hepatitis do not share a specific mutation

The pathogenesis of fulminant hepatitis B virus (HBV) infection is not well understood. The aim of this study was to investigate whether there is an association between specific viral variants and a fulminant disease course. The entire HBV genomes from the serum of eight patients with fulminant HBV infection and one patient with fulminant hepatitis during reinfection after liver transplantation were investigated. After isolation and amplification of viral DNA by polymerase chain reaction (PCR), plus and minus strands were directly sequenced. Sequence data were analyzed by comparative sequence alignments with 35 and 2 complete HBV genome sequences from patients without and with fulminant hepatitis, respectively. Several point mutations were present in all regions of the genomes. Many nucleotide changes had never or rarely been found in the reported HBV isolates from patients without fulminant hepatitis. A distinct mutation present in all genomes was not identified. Clusters of rare and unique mutations were observed in the enhancer II core promoter region. Mutations previously suggested to be associated with fulminant HBV infection were not consistently found. A precore stop codon mutation at nucleotide position 1896 or an A-to-T mutation at nucleotide position 1762 and a G-to-A mutation at nucleotide position 1764 in the core promoter region were present in four and three cases, respectively. Fulminant HBV infection does not appear to be caused by a specific genomic mutation. However, various mutations clustering in the enhancer II core promoter region may contribute to a fulminant disease course.

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.