Prognostic Value of Electrical Impedance Spectroscopy (EIS) When Used as an Adjunct to Colposcopy - A Longitudinal Study.

OBJECTIVE: Colposcopy can be used with Electrical Impedance Spectroscopy (EIS) as an adjunct, to assess the presence of High Grade Cervical Intra-epithelial Neoplasia (CIN2+). This analysis of longitudinal data has used the results from women with a negative colposcopy, in order to see if the initial (index) EIS results were able to predict the women who subsequently developed CIN2+. A further objective was to investigate what tissue structural changes might be reflected in the electrical impedance spectra. METHODS: 847 patients were referred with low grade cytologly. EIS measurements were made around the transformation zone of the cervix during colposcopy. Every EIS spectrum was matched to a template representing CIN2+ and the result was positive if the match exceeded a probability index threshold. The colposcopic impression was also recorded. All the women who developed biopsy proven CIN2+ within three years of the index colposcopy were identified. RESULTS: The median follow-up was 30.5 months. Where both CI and EIS were initially positive, there was an increased prevalence (8.13%) of CIN2+ developing as opposed to 3.45% in the remaining patients (p=0.0159). In addition, if three or more EIS spectra were positive there was a higher prevalence (9.62% as opposed to 3.56% p=0.0132) of CIN2+ at three years. The index spectra recorded from the women who developed CIN2+ showed EIS changes consistent with increases in the extracellular volume and in cell size inhomogeneity. CONCLUSION: EIS does offer prognostic information on the risk of CIN2+ developing over the three-year period following the EIS measurements. The changes in EIS spectra are consistent with an increase in cell size diversity as pre-malignancy develops. These changes may be a consequence of increased genetic diversity as neoplasia develops.

Diagnostic reagents for hepatitis C virus.

The development of diagnostic methods for hepatitis C virus is presented. Special attention is paid to the selection of antigenic markers, the type of assay selected and the interpretation of results. A few of the pitfalls and ambiguities of various assays are discussed and possible future methods are described.

The expression in Escherichia coli of sequences coding for the p18 protein of human immunodeficiency virus and the use of the recombinant protein in characterizing a panel of monoclonal antibodies against the viral p18 protein.

The sequences coding for the p18 protein of CBL-1, a British human immunodeficiency virus (HIV) type 1 isolate, were expressed in Escherichia coli as beta-galactosidase fusion proteins. The recombinant proteins were used to screen a panel of five monoclonal antibodies (MAbs) raised against p18 expressed in CBL-1-infected cells. The regions containing the epitopes for four of the MAbs were located using carboxy deletion mutants and synthetic peptides. The epitope of one of the MAbs (1D9) was reconstructed as part of an unfused, E. coli-expressed p18 protein using the polymerase chain reaction technique. Four different HIV strains and one lymphadenopathy virus type 2 strain were analysed by fluorescence-activated cell sorting of live infected cells using the p18-reactive MAbs.

Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein.

Synthetic vaccines for viral diseases can use defined regions of viral proteins as immunogens: the peptide sequence of amino acids 141-160 of the VP1 protein of foot and mouth disease virus (FMDV) elicits virus-neutralizing antibodies to protect guinea pigs, cattle and pigs either when coupled to a carrier protein or when administered in liposomes or in incomplete Freund's adjuvant. The immune response to these peptides is much lower than that to complete virus particles and the same sequence fused to the N terminus of beta-galactosidase did not produce a more potent immunogen than synthetic peptide alone. We report here an expression system for immunogenic epitopes linked to a carrier protein, hepatitis B core antigen, to form part of a virus-like complex which can present these epitopes to the immune system at high density. The immunogenicity of these structures approaches that of FMDV particles.

Molecular cloning of foot and mouth disease virus genome and nucleotide sequences in the structural protein genes.

Foot and mouth disease virus (FMDV), of the family Picornaviridae, consists of a single-stranded RNA (approximately 8,000 nucleotides), the translation of which is initiated on the 3' side of a 150-nucleotide poly(C) tract and yields a single polyprotein which is processed by host cell proteases into four primary products (Fig. 1). One or more virus-specified proteases further cleave these into the final products, the capsid proteins (VP1-4) being derived from the precursor p88 (for review see ref. 5). There are seven serotypes of the virus and as it has been shown that the immunizing activity of FMDV particles is associated primarily with VP1 (refs 6, 7), it seems likely that antigenic variation in FMDV is a result of changes in the structure of this protein. To further our understanding of this variation and as a first step in the possible development of FMDV vaccines from genetically manipulated microorganisms, we report here the construction and analysis of recombinant plasmids containing cDNA copies of the RNA. Comparison of the deduced amino acid sequence with the known polypeptide sequences shows that the NH2-termini of VP2 and VP3 are conserved between the A and O serotypes whereas that of VP1 (the immunizing antigen) varies by as much as 42% between serotypes.

Molecular cloning of avian sarcoma virus closed circular DNA: structural and biological characterization of three recombinant clones.

Unintegrated, circular viral DNA, isolated from Prague A avian sarcoma virus (PrA-ASV)-infected quail cells (QT6), was cloned in the lambda vector lambda gtWES x lambda B. Three independent lambda-ASV recombinants were identified, and each contained a complete copy of the PrA-ASV genome. The arrangement of the ASV sequences within the recombinants was determined by restriction enzyme analysis and hybridization with labeled ASV-specific complementary DNA. One of the recombinants (lambda RPA101) resulted from cloning at the EcoRI site located within the terminally repeated sequence and therefore was virtually co-linear with PrA-ASV virion RNA. The other two recombinants (lambda RPA102 and 103) resulted from cloning at the EcoRI site located within the viral env gene. By restriction enzyme analysis and by measurement of R-loops formed between lambda RPA101 and PrA-ASV virion 35S RNA, the viral genome was estimated to be 9,100 bases in length. Genome length viral DNA purified from clones lambda RPA102 and 103 was biologically active. Transfection of chicken embryo cells with viral DNA, in the form of either circles or linear dimers, produced foci of transformed cells within 8 to 10 days. Linear DNA was much less efficient at inducing transformation. Viral DNA from the clone lambda RPA101 was unable to cause transformation; the basis for this defect is unknown.

Deletion mutant of the Bratislava-77 strain of Rous sarcoma virus containing a fusion of the group-specific antigen and envelope genes.

The genetic compositions of two independently derived preparations of the Bratislava-77 strain (B77) of Rous sarcoma virus were analyzed after each was passaged seven or more times in duck embryo fibroblasts. RNase, T1-resistant oligonucleotide fingerprint analysis of virion RNA from both preparations of duck-passaged B77 revealed the presence of two large noncontiguous deletions. Approximately 75% of the RNAs contained a deletion which spans oligonucleotides 304 to 4 on the viral genome (about 3,500 nucleotides) and encompasses all of the B77 polymerase gene. More than 90% of the RNAs also contained a deletion which spans src-specific oligonucleotides 6 and 5(about 2,200 nucleotides) and is identical to the deletion observed in transformation-defective B77. Virion RNA from duck-passaged B77 also contained two oligonucleotides (D1 and D2) not observed in the RNA of B77 virus grown on chicken embryo fibroblasts. Analysis of the virion RNA of duck-passaged B77 by denaturing agarose gel electrophoresis revealed four major subunits with molecular weights of 3.40 x 10(6), 2.65 x 10(6), 2.25 x 10(6), and 1.55 x 10(6). Whereas the 3.40- and 2.65-megadalton (Mdal) RNA species comigrated with the nondefective and transformation-defective RNAs of B77 propagated on chicken embryo fibroblasts, no counterparts to the 2.25- and 1.55-Mdal RNAs were observed in the RNA of B77 grown on chicken embryo fibroblasts. Oligonucleotide fingerprint analysis of these RNA species revealed that the 2.65-Mdal RNA contains the src-specific deletion and that 2.25-Mdal RNA contains the polymerase region deletion; both of these deletions were observed in the 1.55-Mdal RNA, which was the major RNA subunit species detected in duck-passaged B77. The new oligonucleotides (D1 and D2) observed in the duck-passaged virus were present in the 2.25- and 1.55-Mdal RNA species in vitro and in vivo and directs the synthesis of a 130,000-dalton protein (p130). p130 contains antigenic determinants specific for p27 (gag gene) and gp85 (env gene) but does not contain sequences which cross-react with antisera directed against the alpha beta form of RNA-dependent DNA polymerase (pol gene). This RNA, therefore, is generated by a fusion of the gag and env genes of Rous sarcoma virus B77.

Protein synthesis in chloroplasts. VII. Initiation of protein synthesis in isolated intact pea chloroplasts.

Isolated intact pea chloroplasts use light energy to synthesise N-formayl [35S]-methionylpuromycin when incubated with L-[35S]methionine and puromycin. Control experiments establish that this synthesis occurs on chloroplast ribosomes, and not on contaminating mitochondrial or bacterial ribosomes. The amount of N-formylmethionylpuromycin formed suggests that each messenger RNA that is being translated in vitro undergoes initiation at least twice. We conclude that isolated, intact pea chloroplasts carry out the initiation of protein synthesis, as well as the elongation and termination of polypeptide chains.