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Biomedical subjects

D J Rowlands

Publications and source records attributed to D J Rowlands.

At least 19 recordsLinked to original sources
PubMed1995

Induction of T-helper cell response to hepatitis B core antigen in chronic hepatitis B: a major factor in activation of the host immune response to the hepatitis B virus.

The T helper (Th) cell response to hepatitis B core antigen (HBcAg) was analyzed in 76 chronic hepatitis B virus (HBV) carriers with varying degrees of hepatic inflammation and HBV replication. Fifty-five patients had active viral replication, 28 with minimal histological changes and normal alanine transaminase (ALT) and 27 with active hepatic inflammation and elevated ALT. The remaining 21 chronic hepatitis B surface antigen (HBsAg) carriers had undetectable HBV replication, minimal histological activity, and normal ALT. In addition, 34 chronic HBV carriers were studied prospectively during treatment with alpha-interferon. The HBcAg-specific Th cell response was evaluated by a proliferative assay using 3H-thymidine uptake and gamma-interferon production by peripheral blood mononuclear cells. The proliferative response and gamma-interferon production of patients with active hepatic inflammation were significantly higher than in patients with minimal histological changes and in controls. In the longitudinal analysis during alpha-interferon treatment, 22 of 34 patients sustained an ALT flare accompanied by a parallel, significant Th cell response, which preceded or coincided with the ALT flare. The elevation in the Th cell response and the ALT flare were followed by a significant rise in the serum immunoglobulin (Ig) M anti-HBc index. Ten of twenty-two patients with an enhanced Th cell response and an ALT flare seroconverted after alpha-interferon treatment. The Th cell activity in the 10 responders rapidly subsided after hepatitis B e antigen (HBeAg) to anti-HBe seroconversion, whereas in the 12 nonresponders it remained elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine Transaminase↗
PubMed1995

Rhinoviruses and cells: molecular aspects.

Recent developments in our understanding of the molecular structure and functioning of the human rhinoviruses are briefly reviewed. The determination of virus structure at near atomic resolution has provided a wealth of information that can be used to help interpret dynamic properties of the virus. The mechanisms involved in capsid assembly, attachment to cell surfaces, uncoating, and the initiation of infection and antibody-mediated neutralization of infectivity can all be examined afresh in the light of the structural data. In addition, molecular techniques for manipulating and analyzing the biological properties of nucleic acids and proteins are providing a wealth of information on details of the replication machinery of the viruses. Probably one of the least understood aspects of viral replication is the precise nature of the interactions between host and viral components during infection. Unfortunately, despite all of these impressive scientific advances the cure for the common cold is still elusive.

Capsid↗
PubMed1992

How can peptide vaccines work?

Peptide antigens frequently induce antibodies which recognise the denatured form of a protein from which their sequences are derived. However, the ability to induce antibodies which crossreact with the native, fully folded form of the protein is less commonly observed. Although there is a growing number of examples in which this is the case, the ability to predict peptides having this property is extremely limited. Given the large surface areas involved in antibody/antigen interaction it is surprising that peptides could ever induce antibodies which would recognise the native protein well enough to have biological activity, such as the neutralization of infectivity. A mechanism is proposed to explain such observations which is compatible with many of the properties of antipeptide antibodies.

Antibodies, Viral↗
PubMed1992

Comparison between in vitro neutralization titres and in vivo protection against homologous and heterologous challenge induced by vaccines prepared from two serologically distinct variants of foot-and-mouth disease virus, serotype A22.

Guinea-pigs were challenged with homologous or heterologous strains of foot-and-mouth disease virus (FMDV) following vaccination with baby hamster kidney (BHK) monolayer cell-adapted or BHK suspension cell-adapted strains of FMDV serotype A22 Iraq 24/64. The protection afforded by these vaccines was analysed as a function of antigen dose and the in vitro serum virus neutralization titres achieved. The results show that the level of neutralizing antibody induced that afforded 50% protection was similar for both vaccines in homologous or heterologous challenge situations. However, although the dose of antigen required to achieve this titre against homologous virus was similar for the two vaccines, approximately 20-fold more of the suspension cell-adapted virus was required to elicit a protective titre against heterologous challenge compared to the dose of monolayer cell-adapted virus required. A synthetic peptide representing the amino acid sequence 135 to 167 of VP1, which is identical in the A22 Iraq 24/64 variant viruses, was shown to induce protection against both homologous and heterologous virus challenge.

Animals↗
PubMed1991

Foreign epitopes in immunodominant regions of hepatitis B core particles are highly immunogenic and conformationally restricted.

The presentation of heterologous amino acid sequences on the surface of hepatitis B core antigen (HBcAg) particles has been studied using a defined linear neutralization site from human rhinovirus (HRV). Previous work has shown that fusion particles, in which the HRV peptide sequence is linked to the amino terminus of the HBcAg protein, induce excellent immune responses in experimental animals. Using predictive models of HBcAg particulate structure and the approximate location of the major immunogenic regions we have designed and constructed bacterial expression vectors which direct synthesis of chimeric particles in which heterologous sequences are presented within an immunodominant area on the particle. Immunological responses to the heterologous peptide sequence are improved by at least tenfold when compared with amino terminal fusions of the same peptide sequence to HBcAg. Moreover, the restriction placed on the heterologous peptide by its linkage at both ends within the HBcAg protein results in a more constrained structure. In the case of the rhinovirus peptide sequence this results in an antigenic conformation more closely resembling that on the native virus particle. Such a system lends itself well as a general approach to the induction of high titre antibodies against defined epitopes.

Amino Acid Sequence↗
PubMed1991

Proliferative responses of T cells primed against human rhinovirus to other rhinovirus serotypes.

Lymphocytes from mice immunized with human rhinovirus (HRV) serotypes 1A or 15 proliferated in vitro in response to HRV and the activated cells were shown to be helper T (Th) cells. Lymphocytes from mice primed with HRV-1A responded to seven of eight heterologous virus serotypes, the responses to other minor cell receptor group viruses being greater than to those belonging to the major cell receptor group. A similar bias was seen with cells from mice primed with HRV-15 in that they responded preferentially to other major receptor group viruses. This pattern of cross-serotype recognition was shown to be similar in three inbred mouse strains and was not dependent upon the major histocompatibility complex haplotype. These results have revealed that there are determinants within the viral proteins of a number of serotypes of HRV that are recognized by Th cells primed against a single HRV serotype. Thus, at the level of Th cell recognition of HRV, a cross-serotype reactivity is seen which is not reflected in the B cell antibody response to virus, which is generally highly serotype-specific.

Animals↗
PubMed1991

In vitro translation of hepatitis A virus subgenomic RNA transcripts.

A subgenomic cDNA clone from hepatitis A virus strain HM175, composed of the last eight nucleotides of the 5' non-translated region and the first 2248 nucleotides of the coding sequence (P1 region), was inserted into a vector under the control of the T7 promoter. Restriction enzyme digestion at sites within the structural region and subsequent transcription in vitro yielded RNA products which were translated efficiently in rabbit reticulocyte lysates to produce proteins of the predicted sizes. The translation products were specifically precipitated with antipeptide antisera; these reactions were not affected by denaturation of the antigens by boiling in 1% SDS. The translated proteins were also precipitated by antivirion antisera, but recognition was totally abolished following denaturation. Thus antivirion antisera recognized conformation-dependent epitopes expressed on the translated products exclusively.

Autoradiography↗
PubMed1991

Parameters influencing the attachment of hepatitis A virus to a variety of continuous cell lines.

We have investigated the interactions of purified radiolabelled hepatitis A virus (HAV) with a variety of continuous cell lines. Virus labelled either in vitro with radiolabelled iodine or in vivo with radiolabelled uridine bound to cells with similar efficiency. Attachment to BS-C-1 cells was calcium ion-dependent and this correlated with infectivity assay results. The cell tropism of HAV attachment was examined using cell suspensions and confluent cell monolayers at both 4 degrees C and 37 degrees C. The maximum level of attachment was observed at 4 degrees C with cells in suspension, but was severely inhibited by 2% foetal calf serum; these results again correlated with infectivity assays. The components of serum which inhibit attachment have been characterized by gel filtration chromatography, sucrose density gradient analysis, immunoprecipitation and Western blotting. The data show that such components are of high Mr and that the serum glycoprotein, alpha 2-macroglobulin, can partly mimic the inhibitory effect of whole serum.

Adsorption↗
PubMed1991

A CD strategy for the study of polypeptide folding/unfolding. A synthetic foot-and-mouth disease virus immunogenic peptide.

The circular dichroism spectrum of the 20-residue immunogenic peptide from the foot-and-mouth disease virus (VP1; 141-160 of serotype A, subtype 12) was solvent- and temperature-dependent. Careful solvent titration revealed two isodichroic points and plateaux consistent with stepwise unfolding of specific stable conformations. Variable temperature studies in cryogenic solvents and urea perturbation were consistent with the existence of three conformational moieties, the left-handed extended helix, the alpha-helix, and the 3(10) helix. The number of residues in each helix was confirmed by CD spectral simulations. The strategy described here can be used to determine the components of a conformational equilibrium and their statistical weights, to study peptide folding and unfolding and to determine the bioactive conformation(s) of linear peptides. The conclusions were supported by 2D-NMR studies. A new mechanism for the stabilization of left-handed extended helices and destabilization of alpha-helices by urea is proposed. The structure of the peptide as resolved by CD spectroscopy is of particular significance since the conformation of this antigenic sequence in situ has so far not been solved by X-ray crystallography.

Amino Acid Sequence↗
PubMed1990

Immunological properties of hepatitis B core antigen fusion proteins.

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

Adjuvants, Immunologic↗
PubMed1990

Presentation and immunogenicity of viral epitopes on the surface of hybrid hepatitis B virus core particles produced in bacteria.

We recently reported the enhanced immunogenicity of a peptide epitope when it was presented as a fusion protein with hepatitis B core antigen. In those experiments the fusion protein was expressed in vaccinia virus. We have now refined the system so that large amounts of highly immunogenic particles can be produced using a simple bacterial expression system. We describe the expression of three different viral epitopes as chimeric particles that induce good antibody responses to each epitope after one dose of low amounts of antigen. Finally we demonstrate that the immunogenicity is a reflection of both T helper cell sites within the core protein and also the particulate nature of the immunogens.

Amino Acid Sequence↗
PubMed1990

Neutralizing antibodies to all seven serotypes of foot-and-mouth disease virus elicited by synthetic peptides.

Uncoupled peptides from all seven serotypes of foot-and-mouth disease virus (FMDV) protein VP1 have been used to elicit neutralizing antibody responses in guinea-pigs. The responses were largely serotype specific, although some significant cross-neutralization was observed. Dimeric tandem peptides have also been used to simultaneously elicit neutralizing antibodies to two different FMDV serotypes. The possible existence of structural features common to the B-cell neutralization sites or the guinea-pig helper T-cell sites within all seven peptides are analysed and discussed.

Amino Acid Sequence↗
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↗