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Biomedical subjects

D J Rowlands

Publications and source records attributed to D J Rowlands.

At least 55 records · Page 3Linked to original sources
PubMed1989

Diagnosis and assessment of mitral and aortic valve disease by cine-flow magnetic resonance imaging.

Seventy-six aortic and mitral valves, in 44 patients and 5 normal volunteers, were studied by Cine-Flow MRI (on a 0.26-T superconducting magnet system), utilizing compound oblique imaging planes and a Field Echo Even Rephasing sequence. All patients had had cardiac catheterization and echocardiography. All patients with valvular stenosis and aortic sclerosis (n = 45) showed complete signal loss distal to the respective valve. Length of signal loss distal to the aortic valve in those in whom it was measured (n = 15) allowed differentiation of aortic stenosis (n = 9) from sclerosis (n = 6). This also permitted grading of stenosis with highly significant correlation (T = 0.86; P less than 0.002) with pressure gradient measurement. In mitral stenosis (n = 12) calculation of the area of signal loss distal to the mitral valve as a percentage of left ventricular cross-sectional area showed a highly significant correlation (T = 0.77; P = 0.001) with pressure gradient measurement. Clinically significant valvular regurgitation was graded by size and duration of signal loss proximal to the value with concordance with angiocardiography. It is concluded that Cine-Flow MRI has a clinical role in the diagnosis and assessment of valvular heart disease.

Adult↗
PubMed1989

Host cell selection of antigenic variants of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) A22 Iraq 24/64 adapted to grow in BHK monolayer cells induced antibodies which neutralized many isolates belonging to the A serotype. Plaque-purified virus isolated from this stock also induced broadly reactive antibodies, showing that this property is not due to the combined response to a mixture of variants in the original stock virus. However, viruses obtained by passage in suspension BHK cells of either the monolayer cell-adapted virus or a virus cloned from this stock resulted in the selection of virus which induced antibodies with highly specific neutralizing activity. In addition to their antigenic properties the monolayer and suspension cell-adapted viruses could be distinguished by plaque morphology, tendency to aggregate and ability to attach to BHK cells. Monoclonal antibodies (MAbs) induced with the plaque-purified monolayer-adapted virus had neutralizing activity almost as broad as polyclonal serum, showing that this property can be represented by a single epitope on the virus. These neutralizing MAbs recognize a trypsin-sensitive epitope on the virus. Surprisingly, sequence analysis of the structural protein-coding regions of the genomic RNAs of monolayer and suspension cell-adapted viruses showed no amino acid differences in VP1, the protein known to contain the major neutralization epitope in FMDV and to be the only protein susceptible to cleavage by trypsin in the virus particle. Although three coding differences were found in the capsid protein these were all located in VP2.

Amino Acid Sequence↗
PubMed1989

Epitope mapping of foot-and-mouth disease virus with neutralizing monoclonal antibodies.

Epitopes of strain A22 Iraq 24/64 of foot-and-mouth disease virus have been mapped with monoclonal antibodies (MAbs). Three methods were used: (i) an indirect ELISA using an overlapping set of peptides, (ii) production of neutralization escape variants against each MAb and (iii) sequencing of neutralization escape variants. The study has shown that the virus has at least three overlapping liner neutralizing epitopes within a major antigenic site on VP1. The presence of a second, conformational site was demonstrated but its position on the virus particle was not located. Synthetic peptides with sequences representing the major site elicit antibodies which have similar broad cross-neutralizing activity to polyclonal serum or neutralizing MAbs produced with the virus against a range of field isolates.

Amino Acid Sequence↗
PubMed1989

Serological prospects for peptide vaccines against foot-and-mouth disease virus.

Antibodies to a synthetic peptide corresponding to the 141 to 160 amino acid sequence of the protein VP1 of type O foot-and-mouth disease virus (FMDV) neutralize a wider range of type O isolates than anti-virion serum. Extending this peptide at the amino terminus reduced the number of strains neutralized by the antipeptide sera. Reactions with antisera to peptides representing non-contiguous native sequences showed that it was also possible to increase the number of strains effectively neutralized. Selected substitutions of a single amino acid at position 148 markedly altered the neutralizing specificity of antibodies elicited by the 141 to 160 peptide. In particular, a peptide with an L----S substitution at this position induced antibodies which neutralized a type O and a type A virus equally, and guinea-pigs inoculated with it were protected from challenge with either virus. Attempts to isolate variant viruses resistant to neutralization with anti-peptide antibody indicated that these occurred at low frequency, and there was some evidence that resistance may be partially conferred by mutations outside the peptide sequence.

Amino Acid Sequence↗
PubMed1989

The cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD (arginine-glycine-aspartic acid).

The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.

Amino Acid Sequence↗
PubMed1989

Neutralizing epitopes of type O foot-and-mouth disease virus. I. Identification and characterization of three functionally independent, conformational sites.

Eleven neutralizing monoclonal antibodies (MAbs) were produced to the O1BFS 1860/67 strain of foot-and-mouth disease virus (FMDV), and were characterized for their ability to bind viral and subviral antigens in different ELISA tests and to neutralize heterologous type O isolates. Neutralization escape variants of the homologous virus, isolated under pressure from five of these MAbs, were used in cross-neutralization tests with all of the 11 antibodies. These studies identified three functionally independent, conformational, neutralizing sites. The most conformationally dependent site bound antibody which neutralized a range of type O virus isolates. A second site was less dependent on conformation and was recognized by antibody that was strain-specific. The least conformational site bound MAbs which showed limited cross-neutralization of other type O strains. This latter site appeared to be immunodominant and contained several overlapping epitopes which showed some differences in their specificities. Isoelectrofocusing and sequencing studies of the variants strongly suggested that polypeptide VP2 contributes to the immunodominant site.

Animals↗
PubMed1989

Neutralizing epitopes of type O foot-and-mouth disease virus. II. Mapping three conformational sites with synthetic peptide reagents.

Four neutralizing monoclonal antibodies (MAbs), recognizing three functionally independent, conformational sites on type O foot-and-mouth disease virus (FMDV) failed to react with immobilized structural proteins or synthetic peptides but bound to the isolated capsid protein VP1 and peptides in solution. Inhibition ELISA techniques were, therefore, applied using peptide antigens and anti-peptide sera to block MAb binding to virus particles, permitting the identification of those portions of the VP1 protein contributing to the epitopes. The binding site of one MAb, which neutralized a range of type O FMDV isolates, was shown to have components within regions 146 to 150 and 200 to 213 of VP1 with a critical involvement of the amino acids at positions 146 and 206 or 207. The determinants recognized by two other MAbs which were directed at similar, but not identical, epitopes from a second site included components from the 200 to 213 and 143 to 146 regions with amino acids 143 and 144, respectively, appearing critical for the inhibition of the virus binding of the two antibodies. These results demonstrate that the two previously identified immunogenic tracts of VP1 are brought into proximity in the quaternary structure of the virion to form an antigenic domain containing several conformational epitopes, some of which are functionally independent. A fourth, strain-specific MAb was effectively blocked from reacting with virus by peptides corresponding to residues 161 to 180 and 200 to 213.

Amino Acid Sequence↗
PubMed1988

Qualitative and quantitative differences in the immune response to foot-and-mouth disease virus antigens and synthetic peptides.

In cross-immunization studies using foot-and-mouth disease virus (FMDV) antigens and a synthetic peptide, from a region within virus coat protein VP1, it has been shown that intact virus will prime the immune system for intact virus, virus subunits and synthetic peptide but not for disrupted virus. In contrast, peptide will prime for a response to peptide and virus subunits but not to intact virus or disrupted virus. Furthermore, studies on antibody populations in anti-virus and anti-peptide antisera demonstrated clear differences in the nature of the antibody response to the two antigens. This result is reflected in protection studies carried out on animals immunized with virus particles or peptides where there is a clearer correlation between in vitro neutralization and protection in vivo following peptide immunization. Thus, it has been shown that there are major qualitative and quantitative differences in the immune response to the FMDV particle and synthetic peptide.

Animals↗
PubMed1988

Modification of the leader protein (Lb) of foot-and-mouth disease virus.

Translation of foot-and-mouth disease virus RNA for extended periods in rabbit reticulocyte lysates results in the appearance of a previously undescribed protein. A protein with similar properties can also be detected in BHK cells at late times after virus infection. Specific immunoprecipitation has shown that this protein (Lb') is closely related to the smaller of the two leader proteins, Lb, although it migrates with an apparently higher Mr in SDS-polyacrylamide gels. The conversion of Lb to Lb' can be mimicked by treatment with carboxypeptidase B. It is suggested that C-terminal trimming of Lb to produce Lb' results in an increase in negative charge and is responsible for its slower migration in SDS-PAGE.

Aphthovirus↗
PubMed1988

Detection of coronary artery bypass graft patency as assessed by magnetic resonance imaging.

In a study of 22 patients with 60 coronary artery bypass grafts, magnetic resonance imaging (MRI) correctly assessed graft patency or occlusion in 90% of cases when compared with selective coronary graft angiography and computed tomography. It is concluded that MRI can detect a normally functioning coronary artery bypass graft and could be used as a non-invasive technique to assess graft patency in patients presenting with post-operative angina.

Adult↗
PubMed1988

A high proportion of anti-peptide antibodies recognize foot-and-mouth disease virus particles.

Synthetic peptides representing the amino acid sequence 141-160 of the structural protein VP1 of foot-and-mouth disease virus (FMDV) elicit virus-neutralizing antibody. Absorption of anti-peptide sera with purified virus particles removed all detectable virus-binding and neutralizing activity, and reduced the ELISA titres against the homologous peptide by 31-41%. The proportion of anti-peptide antibodies that also recognized virus was unaffected by whether the peptide had been inoculated free, carrier-linked or as part of a fusion protein. The majority of these antibodies reacted with sites composed of residues 142-150. Peptides extended at the amino terminus, into regions shown to be poorly antigenic on the intact virus, induced greater neutralizing responses by increasing the proportion of virus-binding antibodies recognizing region 141-150 from 35% to 70%. However, the total proportion of activity against the longer homologous peptide removed by virus absorption remained within the range 31-41%.

Antibodies, Viral↗
PubMed1987

Potential secondary and tertiary structure in the genomic RNA of foot and mouth disease virus.

The nucleotide sequence of the 5' untranslated region of foot and mouth disease virus (FMDV), serotype A10 has been determined. This completes the first total genomic sequence for any one serotype of FMDV. Analysis of the sequence to the 3' side of the poly (C) tract reveals the presence of a 24 nucleotide repeated motif which has homologies with a sequence located upstream of the transcriptional initiation site from several mammalian fibrinogen genes. The function of this element in FMDV is unclear. However, computer analysis of this region predicts the presence of a high degree of secondary and tertiary structure in which these repeats form an important part. The implications of these predictions are discussed.

Aphthovirus↗
PubMed1987

All foot and mouth disease virus serotypes initiate protein synthesis at two separate AUGs.

Translation of the foot and mouth disease virus genome in vitro and in vivo indicated that all seven serotypes initiate protein synthesis at two separate AUGs. Sequence analysis of the region surrounding these AUGs has shown that the efficiency with which the initiating AUG is recognized is dependent on the flanking nucleotides. However, in vitro, the major factor determining which AUG is used is the concentration of Mg2+.

Aphthovirus↗
PubMed1987

A synthetic peptide which elicits neutralizing antibody against human rhinovirus type 2.

Synthetic peptides corresponding to six predicted immunogenic sites on human rhinovirus type 2 (HRV2) have been tested for their reactivity with an anti-virion antibody and for their ability to elicit neutralizing antibody. Four of the peptides reacted with HRV2 antiserum in an indirect ELISA. Rabbit antisera produced to three of these four peptides, one each from VP1, VP2 and VP3, reacted with the virus in an indirect ELISA and with the corresponding proteins by Western blotting. Furthermore, antiserum to one of the peptides, designed to cover the neutralization epitope NIm-II on VP2, not only reacted well in a sandwich ELISA and in an immunoprecipitation test but also neutralized virus infectivity.

Animals↗
PubMed1987

Surface structure and RNA-protein interactions of foot-and-mouth disease virus.

The surface structure of foot-and-mouth disease virus (FMDV) and the interaction of the individual capsid proteins with the virus RNA have been examined using modification reagents. By measuring the extent of modification of the lysine residues of intact and disrupted virus particles and the 12S protein subunit with Bolton & Hunter reagent it was found that 54% of the residues of VP1, 15% of the residues of VP2 and 37% of the residues of VP3, equivalent to five, two and four lysine residues respectively, are on the surface of the intact virus particle. Polypeptide VP4 was not modified in intact virus particles, indicating that it has no lysine residues on the surface of the virus. Modification with sodium metabisulphite, which causes a specific transamination reaction between cytidylic acid residues in ssRNA and closely associated basic amino acids, cross-linked all four structural proteins to the virus RNA. Both fragments of VP1, produced by treatment of the virus particle with trypsin, are also cross-linked to the RNA. These observations have been combined with the evidence that the immunogenic activity of VP1 may be contained in two discontinuous sites, at amino acids 141 to 160 and 200 to 213, in proposing a model for the arrangement of this polypeptide in the virus particle.

Antigens, Viral↗
PubMed1987

Immune response to uncoupled peptides of foot-and-mouth disease virus.

Uncoupled synthetic peptide representing the sequence of amino acids 141-160 of foot-and-mouth disease virus (FMDV) protein VP1 induced a virus-neutralizing antibody response in guinea-pigs. This response required incomplete Freund's adjuvant (IFA) for the primary inoculation and was dependent on the presence of an added cysteine residue with an unblocked sulphydryl group at the carboxy-terminus. Secondary immunization could be carried out in the absence of adjuvant. A study of the relative activities of nested sets of uncoupled peptides from 150-160 to 135-160 and 141-160 to 141-155 indicated that amino acids 146-156 were critical for the induction of virus-neutralizing antibodies and that extension to 137-160 further improved this response. Results of in vitro proliferation studies demonstrated that the carboxy-terminal residues on this peptide may form a T-cell epitope. The significance of these observations in the broader context of synthetic peptide vaccines is discussed.

Adjuvants, Immunologic↗
PubMed1986

The genome-linked proteins of aphthoviruses: specific immunoprecipitation of the three species detected on virus RNA and identification of possible precursors.

Synthetic peptides have been made corresponding to the C-terminal portion of each of the three presumptive genome-linked proteins (VPgs) of foot-and-mouth disease virus type A10. Antisera against each of these peptides efficiently precipitated only the homologous VPg, and the reactions were inhibited by prior absorption with homologous, but not heterologous synthetic peptide. The peptide antisera precipitated a number of proteins from infected cell extracts with mol. wt. of 100, 84, 56, 36, 27, 25 and 20, all X 10(3); all these reactions were inhibited by absorption with homologous peptide, indicating that they were probable precursors of VPg. The relationship between these proteins is at present unclear.

Amino Acid Sequence↗