Characterization of small hepatitis B surface antigen epitopes involved in binding to human annexin V.

Previously, we have shown that small hepatitis B surface antigen (SHBsAg) binds specifically to human annexin V (hAV) and that hAV plays a key role in the initial steps of hepatitis B virus (HBV) infection. We have also demonstrated the spontaneous development of anti-idiotypic antibodies (antibodies to HBsAg Ab2) in rabbits immunized with hAV. As Ab2 is able to inhibit the binding of hAV to SHBsAg, Ab2 might contain epitope(s) mimicking a region of hAV for binding to SHBsAg. Identification of this epitope will therefore reveal a SHBsAg sequence involved in hAV binding. Using a panel of synthetic peptides covering the region of SHBsAg located on the outer surface of the virus, binding studies showed that the region incorporating amino acids (aa) 125-131 of SHBsAg is important for binding to Ab2 and consequently also for binding to hAV. Further experiments revealed that not only this region, but also the region incorporating aa 158-169, is involved in the binding of SHBsAg to hAV. As these regions are located in the structural vicinity according to the topological model of HBsAg proposed by Chen et al., our findings suggest that these regions are parts of a conformational epitope of SHBsAg for binding to hAV. Because of the crucial role of hAV in HBV infection, further studies on the HBsAg epitopes for hAV binding may lead to the development of a new generation of vaccines or molecules for prevention and for treatment of patients with chronic hepatitis B.

Metallothioneins in human kidneys and associated tumors.

Human kidneys and their associated tumors (nonneoplastic kidney tissues from patients with a transitional cell carcinoma or an adenocarcinoma and the adenocarcinomas themselves) were evaluated for their Zn, Cd, and Cu contents as well as for their metallothionein (MT) level. The total Cd content was correlated with the MT content, and both values were significantly decreased in the adenocarcinomas in comparison with the other tissues. After extraction and separation by anion-exchange chromatography, MT-0 was identified in the nonneoplastic tissues from both the adenocarcinomas as well as the transitional cell carcinomas. Since until now MT-0 protein was only found in human fetal liver and in Zn-stimulated human monocytes, a possible role for this isoform as an oncofetal marker is hypothesized. Separation of the isoforms of MT by reversed-phase high-performance liquid chromatography and sequence analysis showed besides MT-1e and MT-1l the isoform-MT-1g, which is not expressed in the healthy kidney, and MT-1k, an isoform which is not yet demonstrated in renal tissues. We conclude that the expression profile of the MT isoforms in the kidney changes due to the presence of a tumor.

Influences of myogenin genotypes on birth weight, growth rate, carcass weight, backfat thickness, and lean weight of pigs.

Lean weight is related to muscle fiber number. Muscle fiber formation (myogenesis) occurs only during embryonic development when it is under the control of the MyoD gene family consisting of myogenin, MyoD1, myf-5, and myf-6. Myogenin has a central position within the MyoD gene family because myogenin expression abrogates myoblast proliferation potential and regulates the differentiation of single nucleated myoblasts into multinucleated myofibers. Thus, myogenin genotype could be related to variation in the number of muscle fibers formed, leading to variation in muscle mass and, thus, lean weight. A polymorphism at the porcine myogenin locus was associated with birth weight, growth rate, lean weight at 200 d, and backfat thickness. Yorkshire pigs from two commercial lines were genotyped, and crosses between heterozygous pigs and heterozygous and homozygous pigs were made. Resulting litters were genotyped, and phenotypic data were collected. Significant differences were found between the two homozygous myogenin genotypes for birth weight, growth rate, and lean weight, but not for backfat thickness. Variation at the myogenin locus explained 4% of the total phenotypic variation in birth weight, growth rate, and carcass weight, and 5.8% of the total variation in lean weight. We conclude that myogenin genotype influences porcine growth rate and muscle mass.

Genetic variation at the porcine MYF-5 gene locus. Lack Of association with meat production traits.

The number of muscle fibers at birth appears to determine the maximal lean meat growth capacity in pigs and in cattle. Development of muscle fibers is regulated by the MyoD gene family consisting of MyoD1, myf-5, myf-6, and myogenin. Myf-5 is expressed in proliferating myoblasts. Here we report the genomic sequence of the porcine myf-5 gene with three microsatellites and two RFLPs located close to the coding sequences. Two of the microsatellites are located in the promoter region. The allelic distribution differs between breeds and selection lines. In two GY selection lines, 1216 pigs of two-generation families were genotyped for the HinfI RFLP, which was segregating in the GY breed. The other polymorphic loci are physically linked to this RFLP locus, and therefore the results can be extrapolated to these loci. Statistical analysis revealed no association with birth weight, growth rate, weight at slaughter age, carcass meat weight, and backfat thickness. Thus, in this study myf-5 did not explain genetic variation in meat (muscle) development in pigs.

Transfection of a rat hepatoma cell line with a construct expressing human liver annexin V confers susceptibility to hepatitis B virus infection.

Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.

Hepatitis B virus infection in microcarrier-attached immortalized human hepatocytes cultured in molecularporous membrane bags: a model for long-term episomal replication of HBV.

Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12,000-14,000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.

Hepatitis G virus infection in acute fulminant hepatitis: prevalence of HGV infection and sequence analysis of a specific viral strain.

Hepatitis G virus (HGV) is a recently discovered RNA virus, which belongs to the Flaviviridae family. Although HGV infection is usually not associated with elevated serum transaminases, some recent studies have reported that HGV infection is found in a significant number of patients with fulminant hepatitis and may play a role in its etiopathogenesis. In this study the prevalence of HGV infection was determined in 500 healthy blood donors and in 24 patients admitted to hospital because of acute liver failure caused by fulminant hepatitis. The presence of HGV RNA was tested in sera, obtained at admission and before any transfusion was given, by a sensitive seminested reverse transcriptase-polymerase chain reaction (RT-PCR) assay specific for detection of the non-structural (NS)5 region. Nine of the 500 blood donors (1.8%) and two of the 24 patients (8.3%) were found to be HGV RNA positive. One patient was co-infected with HCV and was known to be an intravenous (i.v.) drug user. After intensive supporting treatment, this patient recovered completely. The second patient had no serological markers of known viral hepatitis infection, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV). This patient was successfully transplanted. From both patients, from HGV RNA-positive healthy blood donors and from other patients coinfected with HCV, a part of the HGV NS3 region (nucleotides 4191-4345, EMBL entry U45966) was cloned and sequenced. Sequence comparison revealed that the NS3 region of HGV in patients with fulminant hepatitis contained three nucleotide substitutions as part of the six substitutions described in previous work. These nucleotide substitutions were not found in the tested blood donors or in patients with HCV co-infection. Our findings therefore support the concept of the association of fulminant hepatitis with infection of a specific HGV strain.

Anti-hepatitis G E2 antibody detection and its relation to serum HGV-RNA in patients with clotting disorders: high prevalence of HGV infection and spontaneous remission.

In a previous study, we have determined the prevalence of serum HGV-RNA in patients with congenital clotting disorders. Twenty-six (15%) of 175 patients investigated were serum HGV-RNA positive. In addition, HGV-RNA was detectable in peripheral blood mononuclear cells (PBMC) in ten percent of the cases, three of these patients were serum HGV-RNA negative. In the present study, we have determined the prevalence of anti-HGV-E2 antibodies in the same patient population. Anti-HGV-E2 as determined by ELISA was detected in 45 patients (25.7%). Forty of these patients were serum HGV-RNA negative. Ninety-two percent of the 26 HGV viremic patients and all but one patient (44 patients) with detectable anti-HGV-E2 had coinfection with the hepatitis C virus (HCV). Of these coinfected patients, 62.5% of HGV viremic patients and 53% of anti-HGV-E2 positive patients showed elevated serum ALT levels. Anti-HGV-E2 seroconversion is thus not associated with HCV infection. Two patients who were solely infected with HGV had normal serum ALT levels. In a retrospective longitudinal study, we have observed in 15 patients that serum HGV-RNA persisted during one to 19 years of follow-up, while anti-HGV-E2 was repeatedly negative. Five additional patients who were anti-HGV-E2 positive with concomitant detectable HGV-RNA (4 patients in serum and 1 patient in PBMC) became HGV-RNA negative during follow-up, ranging from 1 to 8 years after the first detection of anti-HGV-E2 antibodies. Two patients had lost anti-HGV-E2 antibodies 3 to 6 years after the seroconversion without the re-appearance of serum HGV-RNA. From these findings, it is clear that the prevalence rate of HGV infection in patients with clotting disorders as determined by PCR assay for HGV-RNA and anti-HGV-E2 by ELISA is actually higher than the prevalence of HGV viremia. Although HGV viremia may persist for longer than 19 years, most of the patients infected with HGV may clear the viremia spontaneously. The clearance of viremia is usually associated with seroconversion to anti-HGV-E2. In addition, anti-HGV-E2 may be lost during years of follow-up without the reappearance of the HGV-RNA. Although HGV infection does not seem to influence the fate of HCV infection and does not induce increased levels of serum ALT, the clinical significance of long-term infection remains to be established.

High prevalence of hepatitis G virus infection compared with hepatitis C virus infection in patients undergoing chronic hemodialysis.

A recently discovered non-A-E hepatitis virus has been designated as hepatitis G virus (HGV) and identified as a new member of the Flaviviridae family. Infection by this virus is thought to be associated with blood-borne hepatitis and usually in the presence of hepatitis C or hepatitis B virus (HBV) infection. In this study, the presence of HGV-RNA in serum or plasma and the prevalence of antibodies against an HGV envelope protein (E2) were investigated in patients undergoing chronic hemodialysis using a sensitive reverse-transcriptase polymerase chain reaction and an enzyme-linked immunosorbent assay, respectively. HGV-RNA was detected in 19 of 112 patients investigated (17%) and anti-E2 antibodies were detected in 15 of 106 patients studied (14.2%). With the exception of two patients, the appearance of anti-E2 is associated with the clearance of serum HGV-RNA. The total prevalence of current (HGV-RNA positivity) and/or past (anti-E2 positivity) HGV infection in this patient population is thus 28.6% (32 of 112 patients were positive for serum HGV-RNA and/or anti-E2 antibodies). In apparently healthy blood donors, serum HGV-RNA was detected in four of 358 individuals (1.12%) and anti-E2 was not detected in 50 individuals investigated. From the 19 patients with serum HGV-RNA positivity, nine were coinfected with other hepatitis viruses (seven with HBV; one with HBV, hepatitis C virus [HCV], and hepatitis D virus; and one with HBV and cytomegalovirus). Thirteen of 15 patients with anti-E2 positivity (10 were positive for only anti-E2 and three were also positive for anti-HBc) had no detectable HGV-RNA. In two patients, both HGV-RNA and anti-E2 antibodies were concomitantly present (both patients were coinfected with HCV or HBV). Of the HGV-infected patients, only three who were coinfected with HBV showed elevated serum alanine aminotransferase levels. The serum HCV-RNA and/or anti-HCV were detected in five (4.5%) of 112 patients. From these findings, we conclude that there is a high prevalence of HGV infection (28.6%) compared with HCV (4.5%) in patients undergoing hemodialysis in our hospital. However, approximately 50% of patients had spontaneously lost the viremia and developed anti-HGV-E2 antibodies. We confirm that HGV infection alone is not associated with elevated serum transaminases, and the appearance of anti-HGV-E2 is usually accompanied with clearance of serum HGV-RNA. In contrast to the results of our previous study, the majority of patients infected with HGV are not coinfected with HCV, indicating that HGV is capable of independent transmission. It is likely that there is a preferential HGV acquisition in the hemodialysis unit. The clinical significance of long-term infection with HGV remains to be established.

Genetic variation in the porcine myogenin gene locus.

The myogenin (MYOG) gene fulfills a key function in muscle differentiation by controlling the onset of myoblast fusion and the establishment of myofibers. In meat-producing animals like pigs and cattle, myofiber numbers have been related to growth capacity. We have characterized the porcine MYOG gene to detect genetic variation at this locus and to relate it to growth characteristics. MYOG gene fragments were isolated by PCR on genomic DNA and by screening a genomic library with a mixture of the four human MyoD cDNA fragments. Both the exons and promoter region were very similar to the human and mouse genes. Southern blot analysis of 105 unrelated pigs revealed three polymorphic MspI sites, located in the promoter region, the second intron, and at the 3' side of the gene. PCR-RFLP tests detecting four MYOG alleles were developed. PCR analysis of a panel of pig-rodent somatic cell hybrids confirmed the genetic localization of MYOG on pig Chromosome (Chr) 9. The PCR-RFLP tests and microsatellite markers on Chr 9 offer the possibility to genotype large numbers of pigs for studies of genetic linkage to meat deposition and growth characteristics.

Hepatitis G viral RNA in serum and in peripheral blood mononuclear cells and its relation to HCV-RNA in patients with clotting disorders.

The hepatitis G virus (HGV) has recently been identified as a new member of the Flaviviridae family. Infection by this virus is thought to be associated with blood borne hepatitis. In this study, the presence of HCV- and HGV-RNAs in serum or plasma (175 patients) and in peripheral blood mononuclear cells (PBMC) (133 patients) was investigated in patients with clotting disorders using a sensitive reverse transcriptase polymerase chain reaction (RT-PCR). HGV-RNA was detected in serum of 26 patients (14.8%). In apparently healthy blood donors, serum HGV-RNA was detected in 4 of 358 individuals investigated (1.12%). Ninety two percent of the 26 serum HGV-RNA positive patients had coinfection with the hepatitis C virus (HCV), especially with HCV genotype 1b, the most common genotype in Belgium. Of these coinfected patients, 15 (62.5%) showed elevated serum ALT levels. Two patients who were solely infected with HGV had normal serum ALT.HGV-RNA in PBMC was found in 18 patients, of whom 3 were negative for serum HGV-RNA. As in case of HCV, HGV-RNA in PBMC is preferentially sensitive to interferon treatment. Nevertheless, rapid reappearance of HGV-RNA in PBMC was observed after cessation of treatment. In one patient, persistent serum ALT elevation seems to be associated with continued HGV viremia, despite the disappearance of serum HCV-RNA.

Organ and species specificity of hepatitis B virus (HBV) infection: a review of literature with a special reference to preferential attachment of HBV to human hepatocytes.

Hepatitis B virus (HBV) infection is still a major public health problem worldwide. Although much information about the molecular biology of HBV has been gained in the last decades, little is known about the mechanism of attachment and penetration of the HBV particle into human hepatocytes. The HBV envelope proteins are important for the interaction between the HBV particle and the hepatocyte plasma membrane. Although initially it was suggested that the preS2 domain could act, via polymerized human serum albumin, as an attachment site to human hepatocytes, in recent years other observations showed that the preS1 domain is probably the most important attachment site to human hepatocytes. However, controversial findings on cellular proteins for binding to the preS1 domain has been described, namely the IgA-, the IL6-, the asialoglycoprotein receptor and GAPD. Although the preS1 attachment site may be important, apo H has been shown to bind specifically to small HBsAg. Recently, we have identified human liver Annexin V as a specific small HBsAg-binding protein. In a preliminary report, the direct involvement of human Annexin V in the initial step of HBV infection has been demonstrated. A rat hepatoma cell line, which does not express human Annexin V and which is not infectable by HBV, gained the ability to become infected by HBV after transfection with human Annexin V. This result may facilitate the progress of HBV receptor research and elucidate the molecular mechanism of the initial step of HBV infection.

Assignment of the porcine loci for MYOD1 to chromosome 2 and MYF5 to chromosome 5.

Porcine-specific polymerase chain reaction (PCR) and a pig-rodent somatic cell hybrid panel were used to map two members of the MyoD gene family. MYOD1 was assigned to pig chromosome 2 and MYF5 to chromosome 5.

Induction by zinc of specific metallothionein isoforms in human monocytes.

A low-molecular-mass zinc-containing protein was isolated by gel permeation and anion-exchange chromatography of lysates of human monocytes induced with zinc acetate. Characterization by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and amino acid sequencing identified the two major charge-separable fractions and an occasionally occurring third fraction as metallothionein-1, metallothionein-2 and metallothionein-0, respectively. Metallothionein-1 was shown to consist of a mixture of isoforms, confirmed as metallothionein-1e, metallothionein-1g and metallothionein-1l by comparison with cDNA sequences obtained by screening a human monocyte cDNA library. We can find no previous observation in the literature of metallothionein-1g at both the protein and RNA level in a non-tumour cell, and of metallothionein-0 in a non-fetal cell or tissue. Since isoform-specific polymerase-chain-reaction amplification showed the presence of metallothionein-0 mRNA in zinc-induced but not in untreated monocytes, these cells can be used as an in vitro system to investigate the expression of this previously considered fetal isoform.

Cloning and specific polymerised-chain-reaction amplification of a third charge-separable human metallothionein isoform.

Metallothionein (MT) fractions isolated from human adult liver tissue are readily separated by anion-exchange chromatography in two isoforms, MT-1 and MT-2, which differ from each other in the nature of the amino acid residue at position 11. In fetal liver tissue, the presence of a third charge-separable MT isoform has been previously reported. We determined its partial amino acid sequence and the sequence of a cDNA clone encoding this MT form. This confirmed the existence of another human MT isoform, hereafter named MT-0, which is characterized by the presence of a negatively charged amino acid at position 8, and by a Glu23 to Lys substitution in a strictly conserved region of the protein. Taking into account these substitutions, we are able to classify human MT isoforms into three instead of two charge-separable groups, based on the nature of three amino acid residues. The unique presence of Glu8 in MT-0 enabled us to develop an MT-0-specific amplification by the polymerase chain reaction, which revealed the presence of MT-0 mRNA in adult liver RNA samples, in spite of the total absence of this isoform at the protein level. This suggests the involvement of post-transcriptional regulatory mechanisms in the expression of this fetal MT form.

Human urinary protein 1: evidence for identity with the Clara cell protein and occurrence in respiratory tract and urogenital secretions.

Protein 1 (P1), a low mol mass urinary protein of unknown function, has been purified, sequenced and quantified in human biological fluids. The molecular size, subunit composition and partial amino acid sequence of P1 are similar to those of the 10 kDa Clara cell protein (CC10), a lung secretory protein. P1 is found in high concentrations in sputum, bronchoalveolar lavages, urine and semen of healthy individuals and in urine of some pregnant women. Contrary to what is claimed, P1 or CC10 is not a specific and unique product of the lung, but like its homologue in rabbits (uteroglobulin) it is also present in urogenital secretions. P1 or CC10 may act as a natural immunosuppressor protecting the respiratory and urogenital tracts from unwanted inflammatory reactions.