[Significance of hepatitis C virus antibodies in asymptomatic blood donors]. / Bedeutung von Hepatitis-C-Virus-Antikörpern bei asymptomatischen Blutspendern.

The aim of the study was to determine the frequency and course of hepatitis C viremia in clinically healthy, anti-HCV positive test subjects, and to ascertain whether the HCV antibodies of the IgM type differed between viremia and immunity. In 21 anti-HCV positive blood donors (test subjects) with normal transaminase activity, two serum samples, taken at an interval of 25 +/- 10 months, have been investigated for HCV-RNA and HCV-IgM antibodies. In a total of 16 test subjects (76%) HCV-RNA was found during the first test and/or the follow-up: 14 of them were positive on both occasions, and one test subject each was HCV-RNA positive exclusively at the first test and the follow-up respectively. At the time of the follow-up the serum transaminase level was elevated in 4 test subjects. 3 of these 4 were HCV-RNA positive also. On the other hand, the results of the HCV-PCR were nonuniform in HCV-IgM antibody negative test subjects. The above results demonstrate that in the majority of clinically healthy, anti-HCV positive test subjects with normal transaminase activity, a viremia exists which persists and the course of which may include inflammatory phases. The proof of HCV-IgM antibodies correlates with a viremia. On the other hand, the lack of HCV-IgM antibodies does not exclude viremia.

Detection of HBs antigen in "anti-HBc alone" positive sera.

The immunoserological finding "anti-HBc alone" is often observed in defined groups of individuals, such as patients with inflammatory hepatopathies, patients on hemodialyses or with organ transplants, i.v. drug users and homosexuals, but it also occurs in up to 1% of Swiss blood-donors. In order to gain further information about whether "anti-HBc alone" reflects late immunity or points to an ongoing or a recently passed hepatitis B virus infection, 153 serum samples were tested for immune-complex-dissociated HBs-antigen, using acid treatment for complex dissociation. Of the samples tested 31% contained complexed HBsAg, the highest rates being found in individuals with hepatopathies (up to 80%), in i.v. drug users (up to 63%) and in hemodialysis patients (40%). The 153 sera were also tested for HBV-DNA by nested PCR. Sixty (39%) probes yielded positive results, comprising 29 (48%) of 60 sera with immune-complexed HBsAg but only 18 (19%) of 93 probes without complexed HBsAg. The results point to the possibility that at least some of the individuals with "anti-HBc alone" still have an ongoing HBV-infection.

[Hepatitis serological finding "anti-HBc alone", circulating viral DNA and interpretation of findings]. / Der Hepatitis-serologische Befund "Anti-HBc allein", zirkulierende virale DNS und Befund-Interpretation.

The immunological finding "anti-HBc alone" (without HBsAg, without anti-HBs) leaves open the question of the state of HBV infection it reflects: a transitory stage of an uncomplicated, eventually prolonged but resolving infection, a chronic or a late state of immunity. A finding of this kind is often observed in immunocompromised individuals (e.g. patients on hemodialysis, drug addicts) but also occurs in up to 1% of the Swiss blood donor population. Of 8800 sera tested for HBV marker in a diagnostic laboratory, 153 individuals showed "anti-HBc alone". They were investigated for circulating hepatitis B desoxyribonucleic acid (HBV-DNA) by polymerase chain reaction (PCR). 60 individuals (39%) showed a positive result. Also taking into consideration anamnestic measurements of conventional HBV markers in 95 individuals and consecutive testing for HBV-DNA in 50 individuals, the following conclusions emerged: 1. A positive finding of HBV-DNS by PCR does not necessarily prove an ongoing HBV infection, hence a negative result does not rule it out. Therefore, the indication to test for this parameter is limited for routine use. 2. The finding of "anti-HBc alone" implies that a HBV-infection is still going on until proven otherwise. This not only might be of consequence for the individual involved, but also raises the question of screening of blood donors and of pregnant women for anti-HBc.

[Polymerase chain reaction in hepatitis C: increased sensitivity via amplification of shorter cDNA fragments]. / Polymerase-Kettenreaktion bei Hepatitis C: höhere Sensitivität durch Amplifikation von kürzeren cDNS-Fragmenten.

The aim of our study was to compare the sensitivity of hepatitis C virus polymerase chain reaction (HCV-PCR) by use of two different primer sets which amplify PCR products of different length. Serum samples of 70 patients with chronic hepatitis C were tested by "nested primer" PCR, using either "NCR primers" that amplify cDNA-fragments of 340 basepairs (bp), or by "PT primers" which amplify fragments of 59 bp only. HCV-RNA was detected in 40 patients (57%) by "NCR primers" and in 69 patients (90%) by "PT primers" (p < 0.001). 23 of 70 patients (33%), which were HCV-RNA negative by "NCR primers", were positive by "PT primers", but no patient negative by "PT primers" was found to be positive by "NCR primers". 20 healthy controls tested by both primer sets were all HCV-RNA negative. We conclude that the sensitivity of HCV-PCR is significantly improved by use of primers that amplify "short" PCR products and recommend the use of "PT primers" for HCV-PCR.

Detection of hepatitis C viraemia in Caucasian patients with hepatocellular carcinoma.

Potential risk factors for the development of hepatocellular carcinoma were analysed in 40 Caucasian patients with this malignancy. A higher proportion (14 of 40; 35%) had evidence of hepatitis C virus (HCV) infection than had evidence of either hepatitis B virus (HBV) carriage (17.5%) or alcohol abuse (30%). In all 14 patients whose sera were reactive by HCV ELISA (Ortho second generation test), the presence of antibodies to HCV were confirmed by recombinant immunoblot assay (Ortho RIBA-2). Furthermore, two independent laboratories detected HCV-RNA in 10 of the 14 (71%) anti-HCV positive sera. Two additional sera were shown to contain HCV-RNA when reanalysed by a modified PCR using oligonucleotide primers designed to amplify a shorter fragment of the 5' noncoding region of the genome. Seven of the anti-HCV positive patients also had evidence of prior HBV infection and 2 admitted to alcohol abuse. HCV infection was the only identifiable risk factor in 6 patients. These data confirm the association between HCV infection and hepatocellular carcinoma and suggest that persistent viral replication accompanies tumour development in the majority of patients whose serum contains anti-HCV.

[Second generation hepatitis-C virus test and polymerase chain reaction in anti-C 100 negative patients with chronic non-A, non-B hepatitis]. / Hepatitis-C-Virus-Test der zweiten Generation und Polymerase-Kettenreaktion bei anti-C 100-negativen Patienten mit chronischer Non-A-Non-B-Hepatitis.

The aim of our study was to evaluate whether a negative HCV test of the first generation (HCV-ELISA 1) using the antigen C100-3 excludes chronic HCV infection, or whether patients exist who are negative for antibodies to C100-3 in spite of chronic hepatitis C. 27 patients with histologically proven chronic non-A, non-B hepatitis, all of whom were HCV-ELISA 1 negative, were tested by the HCV test systems of the second generation (Ortho-HCV-ELISA 2 and Chiron-HCV-RIBA 2) based on the distinct HCV antigens 5-1-1, C100-3, C33c and C22-3. To determine the presence of viremia, serum samples were also tested for HCV-RNA with "nested" PCR. 10 of 27 patients proved to be persistently negative when tested with the second generation assays. One patient showed low grade reactivity by HCV-ELISA 2, but non-reactivity by HCV-RIBA 2. In none of these 11 patients was HCV-RNA detected. 16 (60%) of 27 patients negative with HCV-ELISA 1 were positive with HCV-ELISA 2. HCV-RIBA 2 detected antibodies to the structural core antigen C22-3 in all of these 16 patients and antibodies to the non-structural antigen C33c in 14 of them, while antibodies to 5-1-1 or C100-3 were not found in any of these cases. 10 (63%) of the 16 HCV-ELISA 1 negative, but HCV-ELISA 2 and HCV-RIBA 2 positive patients were positive for HCV-RNA by "nested" PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

The 5' flanking region and chromosomal localization of the gene encoding human platelet membrane glycoprotein Ib alpha.

The human blood platelet membrane glycoprotein Ib (GPIb) functions as a receptor for von Willebrand factor and thrombin. The gene (gpIb alpha) encoding the GPIb alpha-chain was cloned from a genomic cosmid library. The promoter region of this gene was characterized by sequencing two BamHI fragments including 2.8 kb of the 5' flanking region where several Alu repeated elements and purine-rich sequences were found. Possible cis-regulatory elements were identified by comparing the gpIb alpha gene with established consensus sequences known to function as binding sites for transcription factors. To obtain further information on possible megakaryocyte-specific promoter or enhancer sequences, the gpIb alpha promoter region was compared with other genes expressed in platelets that are known so far. The gpIb alpha gene was found to be located on chromosome 17 in region 17p12-ter, by in situ hybridization.

Isolation and characterization of human blood platelet mRNA and construction of a cDNA library in lambda gt11. Confirmation of the platelet derivation by identification of GPIb coding mRNA and cloning of a GPIb coding cDNA insert.

We have developed a purification method for the isolation of platelet-specific poly (A+) RNA and demonstrated that human blood platelets, despite the absence of a nucleus, contain stable mRNA. The poly (A+) RNA was used to construct a platelet-specific cDNA expression library in lambda gt11. The platelet derivation of the purified mRNA was confirmed by identification of membrane glycoprotein Ib (GPIb) message by immunoprecipitation of rabbit reticulocyte lysate translation products with poly- and monoclonal antibodies against GPIb alpha and by sequencing of a GPIb alpha cDNA clone.

Cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived lambda gt11 expression library.

We report here the cloning of the cDNA coding for platelet connective tissue-activating peptide-III (CTAP-III) from a lambda gt11 expression library prepared using messenger RNA (mRNA) isolated from human platelets. The open reading frame of the clone coded for a protein with 128 amino acid residues. Since the precursor of CTAP-III, platelet basic protein (PBP is 94 amino acids long, the 5'-translated region of the cDNA codes for a leader sequence 34 amino acids long. This leader sequence, like the sequence of mature CTAP-III, shows significant homology to the sequence of platelet factor 4 (PF4), the only other platelet specific alpha-granule protein cloned until now, from a human erythroleukemic (HEL) cell line-derived cDNA library. These leader sequences are probably critical for targeting such proteins to the alpha-granule. Northern blot hybridization with platelet and megakaryocyte mRNA shows a single species mRNA of approximately 0.8 kb, suggesting that the corresponding cDNA is full length. The cloning of platelet specific CTAP-III provides additional evidence for the platelet specificity of the cDNA library used.

Structure of the human blood platelet membrane glycoprotein Ib alpha gene.

The gene for human platelet glycoprotein Ib alpha-chain has been cloned from a genomic cosmid library using a partial cDNA clone as probe. 3530 bp were sequenced including the entire transcribed part, as well as additional 5' and 3' regions. A single intron was found 6 bp upstream of the ATG initiation codon. An exceptionally long exon was identical to the recently published cDNA sequence (1). The 5' upstream promoter region is atypical for eukaryotic genes with only a weak homology to the characteristic promoter consensus sequences. The 3' region contains two repetitive Alu elements, belonging to distinct subfamilies, connected by an oligo(dA) linker.

The glycoprotein Ib complex of human blood platelets.

Human glycoprotein Ib (GPIb) is a major integral membrane protein on human blood platelets responsible for the initial attachment of platelets to the exposed vascular subendothelium. In this report we describe an isolation method for a 'GPIb complex' as well as for its individual components. A three-step procedure involving Triton X-114 phase-partition, affinity chromatography on wheat germ agglutinin and ion-exchange chromatography on DEAE-Sephacel yielded milligram quantities of purified GPIb complex. The single components of the complex were further purified by gel filtration on AcA34 in 0.1% sodium dodecyl sulfate. As well as GPIb, the complex contains GP17, actin-binding protein, actin and a series of unidentified proteins with different molecular masses. In contrast to GPIb alpha, which is very rich in O-linked oligosaccharides, sugar analysis revealed that GPIb beta and GP17 seem to have only N-linked chains of the lactosamine type. The C-terminal alpha-chain remnant, which probably spans the plasma membrane, was identified and isolated for the first time. Western blotting with polyclonal rabbit anti-GPIb antibodies and silver-staining of one- or two-dimensional dodecyl sulfate/polyacrylamide gels revealed that it has an apparent molecular mass of 20 kDa and is linked to GPIb beta by a disulfide bridge close to the membrane. The thrombin-binding site on GPIb is located near the N-terminus on a 40-kDa fragment of GPIb alpha. A disulfide bridge in the N-terminal region is not essential for thrombin binding to GPIb.

Structure and function of platelet membrane glycoproteins Ib and V. Effects of leukocyte elastase and other proteases on platelets response to von Willebrand factor and thrombin.

Treatment of platelets with human leukocyte elastase causes a rapid loss in response to von Willebrand factor and a biphasic loss in response to thrombin, first rapid then more slowly. The rapid phase corresponds to cleavage of a 45-kDa glycopeptide from the extracellular end of membrane glycoprotein GPIb. Longer treatment removes 80-kDa and 90-kDa glycopeptides and a glycopeptide corresponding to the major part of GPV. The 45-kDa and 90-kDa species could be obtained by elastase treatment of glycocalicin, the major proteolytic cleavage product of GPIb. Polyclonal rabbit antibodies against the purified 45-kDa glycopeptide had the same effect on the action of von Willebrand factor and thrombin on platelets as cleavage of GPIb by elastase. These results indicate that both the von Willebrand factor and thrombin binding sites on GPIb are located in the same region on the outside of the molecule. Thrombin activation of platelets involves at least two receptors, one on the 45-kDa glycopeptide, which when occupied causes an increase in the speed of response of the platelets to the cleavage of the second. GPV, a candidate for the second receptor, is a hydrophobic glycoprotein that is cleaved from the platelet membrane by several proteases. Proteases that do not activate platelets but degrade the second receptor remove larger fragments from GPV than do proteases such as thrombin or trypsin which activate platelets.