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

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

Widespread presence of cytomegalovirus DNA in tissues of healthy trauma victims.

AIMS: To determine the localisation of human cytomegalovirus (CMV) DNA in abdominal aorta, spleen, and transplantable organs, such as kidney, pancreas, and liver, obtained from healthy individuals; to characterise the cell type(s) in these tissues that serve as a reservoir for latent CMV. METHODS: CMV DNA was detected by dot blot DNA hybridisation and in situ DNA hybridisation with a probe for CMV major immediate early sequences (UL123) and nested PCR with primers derived from the CMV major immediate early (IE) gene exon 4 (UL123ex4). Samples of liver, abdominal aorta, spleen, kidney, and pancreas were obtained at necropsy or from donor kidneys from healthy subjects. RESULTS: CMV DNA was detected in most tissue samples using dot blot hybridisation and nested PCR. In situ hybridisation demonstrated that, in addition to smooth muscle cells in the arterial wall, hepatocytes, tubular and glomerular kidney cells, splenic red pulp cells, and pancreatic acinar cells also harboured CMV DNA. CMV DNA was detected in seropositive and in some seronegative subjects. CONCLUSION: CMV DNA is widely distributed in organs of healthy subjects. CMV DNA was found in various cell types in several organs, suggesting that during latency, CMV DNA is present thoughout the body.

Detection and sequence analysis of the major immediate early and PP150 gene of latent human cytomegalovirus in spleen, liver, and kidney tissues of trauma victims.

The presence of human cytomegalovirus (HCMV) DNA in liver, spleen, and kidney samples of HCMV-seropositive trauma victims during latency was demonstrated by polymerase chain reaction (PCR), using primers reactive with the major immediate early gene exon 4 and the structural gene pp150. Sequence analysis of the PCR amplificates showed more than 95% homology with the reference HCMV strain AD169. The few mutations observed were mostly distributed randomly. In one subject two types of the MIE-4 gene were detected, and in another subject two types of the pp150 gene were found, suggesting that different strains of HCMV can be found in organs of the same patient during latency.

Cloning and sequence analysis of the genes encoding DNA polymerase, glycoprotein B, ICP18.5 and major DNA-binding protein of rat cytomegalovirus.

In all herpesviruses a block of genes is present which is composed of the genes encoding DNA polymerase, glycoprotein B (gB), ICP18.5 and major DNA-binding protein (MDBP). Here we report the cloning and sequencing of this gene block from rat cytomegalovirus (RCMV). The gene block spans 13.3 kbp and contains the four genes in the order pol, gB, ICP18.5 and MDBP. A similar order of genes has previously been reported for human and murine cytomegaloviruses. The pol, gB, ICP18.5 and MDBP genes contain open reading frames which have the capacity to encode proteins of 1120, 914, 893 and 1281 amino acids, respectively. Comparison of the predicted amino acid sequences of the four RCMV proteins with the corresponding proteins of other herpesviruses revealed a close relationship between RCMV and other cytomegaloviruses, which corroborates the usefulness of the RCMV-rat model for studying cytomegalovirus biology.

Selection of phage-displayed antibodies specific for a cytoskeletal antigen by competitive elution with a monoclonal antibody.

A phage-display combinatorial library of VL and VH sequences of mouse antibodies was constructed, which contained 4.5 x 10(7) independent clones. From this library pools of phage were selected by up to four biopanning rounds on cytoskeletal preparations of ovarian carcinoma cells (OVCAR-3). Phage of these pools were then allowed to bind to a cytoskeleton preparation of bladder carcinoma cells (T24). The binding phage were challenged by a monoclonal antibody (mAb) directed against an epitope on cytokeratin 8. Displaced phage were rescued and screened for anti-cytokeratin immunoreactivity by ELISA, indirect immunofluorescence and Western blotting. About 50% of the phage selected by competition with the cytokeratin mAb reacted with the cytoskeletal preparations of T24 cells in ELISA. In contrast, in non-cytokeratin-containing cells, no reaction was observed. Immunofluorescence and Western blotting studies with a number of these clones showed reactivity against cytokeratin. We conclude that the phage-display competitive elution method can be used as a rapid technique to obtain immunoreactive phages, and eventually single-chain Fv (scFv) antibodies directed against defined epitopes, which were formerly characterized and validated by mAbs.

Analysis of idiotope structure of ovarian cancer antibodies: recognition of the same epitope by two monoclonal antibodies differing mainly in their heavy chain variable sequences.

Two MoAbs, independently raised against ovarian carcinoma cells and referred to as OV-TL3 and OV-TL16, display an identical reaction pattern with a membrane-associated protein in both normal and malignant ovarian cells. Also, a similar binding affinity constant and a similar number of binding sites per cell indicate that both MoAbs bind to the same antigen. Competition assays reveal that OV-TL16 is able to compete with OV-TL3 for binding to OVCAR-3 cells. Epitope mapping using a filamentous phage hexapeptide epitope library showed that both MoAbs are able to select identical phages, suggesting that their epitopes are identical or at least overlapping. However, purified polyclonal and monoclonal anti-idiotypic antibodies directed against OV-TL3 failed to recognize the OV-TL16 idiotype, indicating that the structure of the antigen-binding regions of both antibodies is distinct. This was corroborated by molecular cloning and sequencing of the variable heavy (VH) and light (VL) chain immunoglobulin regions of both MoAbs. The VH regions of both antibodies were found to be distinct, whereas the VL regions are almost identical. Computer modelling of the idiotypes suggests that the complementarity determining regions (CDR), with the exception of VHCDR3, have (almost) identical spatial configurations. Our data indicate that, although structurally different in their VH regions, OV-TL3 and OV-TL16 are able to bind to identical epitopic regions on the antigen, because differences in primary structure do not exclude the formation of sufficient and similar spatial structures for the interaction with an epitope.

Ro ribonucleoprotein assembly in vitro. Identification of RNA-protein and protein-protein interactions.

The human Y RNAs, small RNAs with an unknown function, are complexed with at least three proteins: the 60,000 M(r) Ro protein (Ro60), the 52,000 M(r) Ro protein (Ro52) and the La protein (La). In this study we examined the intermolecular interactions between the components of these so-called Ro ribonucleoprotein (Ro RNP) complexes. Incubation of 32P-labelled hY1 RNA in HeLa S100 extract allows the reconstitution of Ro RNP complexes, which were analysed by immunoprecipitation with monospecific antisera. By immunodepletion of HeLa S100 extracts for either Ro60, Ro52 or La, followed by supplementation with recombinant Ro60 or La, it was demonstrated that both Ro60 and La bind to hY1 RNA directly without being influenced by one of the other proteins. However, binding of Ro52 to hY1 RNA required the presence of Ro60, which strongly suggests that the association of Ro52 with Ro RNPs is mediated by protein-protein interactions between Ro60 and Ro52.

Detection and occurrence of the 60- and 52-kD Ro (SS-A) antigens and of autoantibodies against these proteins.

The simultaneous detection of anti-La, anti-60-kD Ro and anti-52-kD Ro antibodies by immunoblotting is greatly improved by changing the crosslinking level in the gel to an acrylamide/bisacrylamide ratio of 19:1. Using this method for the analysis of a number of systemic lupus erythematosus (SLE) and Sjögren's syndrome patient sera it was observed that antibody to the 52-kD Ro protein without anti-60-kD Ro antibody was restricted to Sjögren's syndrome patients (9/26), whereas antibody to the 60-kD Ro protein without contaminating anti-52-kD Ro antibody was only found in SLE patients (8/38). Moreover, in Sjögren's syndrome patient sera anti-Ro antibody was found only in combination with anti-La antibody (20/26), whereas in SLE patient sera anti-Ro antibody could be found without detectable anti-La specificity (4/38). Double immunofluorescence microscopy revealed that the 52-kD Ro and the 60-kD Ro proteins co-localize in the cytoplasm as well as in the nucleus, whereas immunoprecipitation of [32P]-labelled HeLa cell extract with monospecific anti-52-kD Ro and anti-60-kD Ro sera showed that both proteins are associated with the Ro RNAs. These data suggest the presence of both the 52-kD and the 60-kD Ro proteins in the same ribonucleoprotein complexes. To study the evolutionary conservation of the 52-kD Ro, the 60-kD Ro and the La proteins, extracts of cell lines derived from various mammalian species were analysed on Western blots using monospecific human antibodies. In contrast to the 60-kD Ro and the La antigens which are well conserved in evolution, the 52-kD Ro antigen could be detected in primate cells only by this immunological approach.

Analysis of protein--RNA interactions within Ro ribonucleoprotein complexes.

The interactions between Ro and La proteins and hY RNAs have been analysed. The binding site for the 60 kDa Ro protein on hY RNAs is shown to be the terminal part of the base paired stem structure, which contains the most highly conserved sequence among hY RNAs. The bulged C-residue within this region plays an important role in the recognition by this protein. The same regions of hY RNAs are essential for the association of the 52 kDa Ro protein with the RNAs, strongly suggesting that the 60 kDa Ro protein is required for the 52 kDa Ro protein to bind, presumably via protein-protein interactions, to Ro RNPs. The binding site for the La protein on hY RNAs is shown to be the oligouridylate stretch near the 3'-end of the RNAs, which is also recognized when additional nucleotides flank this motif at the 3'-side. Additional sequence elements in hY3 and hY5, but not in hY1, are bound by the La protein as well. Deletion mutagenesis showed that the RNP motif, previously identified in many ribonucleoprotein (RNP) proteins and in some cases shown to be almost sufficient for the interaction with RNA, of both the 60 kDa Ro and the La protein are not sufficient for the interaction with hY RNAs. Substantial parts of these proteins flanking the RNP motif are needed as well. It is likely that they stabilize the correct conformation of the RNP motif for RNA binding.

The use of adenovirus-infected HeLa cells for the detection of low titer autoantibodies.

Following infection of HeLa cells with adenovirus type 5 the cellular La protein becomes predominantly associated with the virally encoded RNA polymerase III products VAI, and VAII, while most of the host RNA polymerase II (e.g. U1, U2, U4, U5 and mRNA) and RNA polymerase III transcription (e.g. U6 and pre-tRNAs) ceases. Other RNA polymerase III products such as the cellular Ro RNAs continue to be transcribed and assembled into ribonucleoprotein complexes containing the Ro (SS-A) antigens. Using a 32P-pulse chase-labeled, adenovirus-infected HeLa cellular extract as a source of antigen, anti-La (SS-B) and anti-Ro (SS-A) antibodies can be detected simultaneously using an immunoprecipitation assay. In the present study this method was found to be more sensitive in detecting anti-La antibodies then counter immunoelectrophoresis and immunoblotting. In studies of sera from patients suffering from rheumatic diseases the percentage positive for anti-La antibody was significantly elevated using this method, especially in patients with systemic lupus erythematosus.

Upstream regions of the hamster desmin and vimentin genes regulate expression during in vitro myogenesis.

Varying lengths of the hamster desmin and vimentin promoter regions were fused to the bacterial chloramphenicol acetyl-transferase gene. These constructs were transfected into two different myogenic cell lines, T984 and C2C12. In both cell lines an increase in endogenous desmin expression takes place upon myogenesis. A region between -89 and +25 bp relative to the desmin transcription initiation site directs high-level tissue- and stage-specific expression upon in vitro myogenesis. At the myoblast stage, C2C12 cells appeared to express both desmin and vimentin, whereas in T984 myoblasts only vimentin expression was detected. Although vimentin is expressed during all stages of myogenesis, a strong decrease in vimentin expression occurs during differentiation of C2C12 cells. Vimentin--CAT constructs followed the endogenous expression pattern, showing that this down-regulation is mediated by 5' flanking sequences. Vimentin promoter activity is modulated by at least two separate regions, both in myogenic and in non-myogenic cell lines.