Search PubMed⌕ Search

Biomedical subjects

C R Howard

Publications and source records attributed to C R Howard.

At least 73 records · Page 4Linked to original sources
PubMed1986

HBsAg: anti-HBs immune complexes. A method for separating the constituent components and assessment of the affinity of the antibody.

A number of chemical disruption agents were assessed for their ability to dissociate HBsAg:anti-HBs immune complexes and to release both the antibody and antigen component in immunologically active forms. The most appropriate reagent was 0.1 M diethylamine which could elute up to 81% of anti-HBs antibody bound to solid-phase HBsAg and retained 93% of its antigen-combining activity. Complexes formed at various degrees of antigen excess and pre-exposed to 0.1 M diethylamine at room temperature for 18 h before ultracentrifugation on sucrose density gradients were effectively dissociated. The released antibody and antigen banded at their expected densities. However, the affinity of the isolated antibody for the detergent-solubilized polypeptide complex from purified HBsAg (gp30/p25) and cyclical peptides representing amino acids 124-137 and 139-147 of HBsAg were found to be considerably lower than that of the original pooled anti-HBs immunoglobulin used to form the immune complexes. These results suggest that the highest affinity antibody subpopulation may not be completely dissociated from the complex. Care should thus be exercised in the interpretation of the significance of the observed affinity of the antibody isolated by this and other similar dissociation procedures.

Antibody Affinity↗
PubMed1986

A surrogate hepatitis B virus antigenic epitope represented by a synthetic peptide and an internal image antiidiotype antibody.

The use of molecules that represent single, defined epitopes able to substitute for antigen (i.e. surrogate antigens) offers considerable advantages over the use of native antigen for the precise manipulation of the immune response. We have investigated the immunochemical characteristics of two types of surrogate hepatitis B surface antigen (HBsAg) epitopes: (a) linear and cyclical synthetic peptides representing amino acid residues 139-147, a hydrophilic region corresponding to part of the a determinant of the HBsAg, and (b) four monoclonal antiidiotypes raised against anti-HBs mAb, two of which behave as an internal image of an a determinant. Polyclonal anti-HBs antisera bound the monoclonal antiidiotypes with affinities of the order of 10(8)/M, and to the peptides with greater than 10-fold lower affinities. However, the levels of antibody in the polyclonal antisera for the peptides was greater than for the antiidiotypes. In inhibition RIA, the surrogate antigens show concordance in that the internal image antiidiotypes inhibit the binding of both monoclonal and polyclonal anti-HBs to the linear and cyclical 139-147 peptides. These results imply that surrogate antigens could indeed be useful as potential hepatitis vaccines, but while the antiidiotypes may stimulate B cells of higher affinity, they would react with a more restricted range of B cell reactivities than would the peptides. A future HBV vaccine may thus comprise a synthetic peptide such as cyclical 139-147 or a cluster of monoclonal internal image antiidiotypes.

Animals↗
PubMed1986

Antibody responses to recombinant and plasma derived hepatitis B vaccines.

The antibody response to hepatitis B surface antigen (anti-HBs) induced in 25 recipients of a recombinant hepatitis B vaccine derived from yeast was compared with that induced in 25 recipients of a vaccine prepared from hepatitis B surface antigen (HBsAg) derived from plasma. Anti-HBs affinity and specificity were compared using assays of antibody affinity with two different antigens, a complex of the major polypeptide of HBsAg (p25; molecular weight 25 000 daltons) covalently linked to its glycosylated form (gp30) prepared from native purified HBsAg, and a cyclical synthetic peptide representing amino acid residues 139-147 of the major polypeptide of HBsAg and known to represent a major part of an a determinant. There was no difference in anti-HBs affinity or molar antigen binding sites of the antibody measured with either antigen between the two groups. All subjects in both groups produced antibody that bound to the gp30/p25 complex antigen, whereas 22 of the recipients of the plasma derived vaccine compared with 24 of those receiving the yeast derived vaccine produced antibodies that bound to the cyclical synthetic peptide 139-147. These results support the finding of similar levels of anti-HBs, measured by commercial solid phase radioimmunoassay, in the two vaccine groups after three doses of vaccine. These results show no significant difference in the quantity, quality, or specificity of the anti-HBs response induced by the recombinant hepatitis B vaccine and the plasma derived hepatitis B vaccine.

Antibodies, Viral↗
PubMed1986

Hepatitis B surface antigen polypeptide micelles from antigen expressed in Saccharomyces cerevisiae.

Hepatitis B micelles containing the p25 component of hepatitis B surface antigen (HBsAg) have been produced by Triton X-100 solubilization followed by ultracentrifugation in linear sucrose gradients. The product was found to resemble micelle forms prepared from plasma-derived HBsAg with the surface being composed of discrete globular and stranded sub-units. The degree of immunochemical relatedness of the micellular preparation was compared to the native 22-nm HBsAg particle present in either plasma or yeast cell extracts. The yeast micelle preparation competed for anti-HBs in a similar manner as intact HBsAg of plasma origin. Enhanced immunogenicity may be expected for micelles containing a recombinant HBsAg protein as has previously been shown for the plasma-derived antigen.

Centrifugation, Density Gradient↗
PubMed1985

Imported epidemic non-A, non-B hepatitis in Qatar.

During one year, 198 patients were admitted to the Hamad General Hospital, Qatar, with acute viral hepatitis. Sera from 126 of these were tested for HBsAg, IgM anti-HBc, IgG anti-HAV, IgM anti-HAV, and delta antibody in those positive for HBsAg. Only 6% of the patients were Qatari nationals and the remainder were immigrants. Of the 126 patients tested, 7 had acute hepatitis A, 29 had acute hepatitis B (none were positive for delta antibody), and the remaining 91 were regarded as having had acute non-A, non-B hepatitis. Of this latter group, 75% were Indian immigrants of whom 59% presented within six weeks of arrival in Qatar and only 2 patients presented later than eight weeks. These patients were thought to have contracted the infection in transit camps in India before immigration to Qatar.

Adolescent↗
PubMed1985

Thermal inactivation of Pichinde virus.

Detailed information regarding the kinetics of thermal inactivation of Pichinde, an arenavirus, is presented. Inactivation of virus infectivity proceeded as a first order reaction over the temperature range 22-53 degrees C. The determined inactivation rates analysed as a function of absolute temperature revealed that two different reactions were involved. Below 37 degrees C, the energy of activation was determined to be compatible with RNA degradation, whereas at higher temperatures a correspondingly greater value suggests that protein inactivation contributes significantly to loss of infectivity. Both inactivation reactions were retarded in the presence of foetal calf serum to a final concentration of 1%. The relatively short half-life of 12-24 h at 22 degrees C suggests transmission in nature via contaminated foodstuffs and soil may be inefficient.

Arenaviridae↗
PubMed1985

Delayed neovascularization in free skin flap transfer to irradiated beds in rats.

A model for the study of neovascularization with a normal epigastric free flap set into an irradiated defect in the Fischer F344 rat is presented. In this model, both the administration of radiation and the flap transfer mimic the clinical situation. Significantly less tissue survives loss of the complete vascular pedicle at the second to fourth days following flap creation in rats with an irradiated bed. Later survival is not different from controls. Delayed neovascularization is proposed as the mechanism responsible for this effect during the period corresponding to the onset of the late phase of the response to skin radiation in rats. That neovascularization does occur, although delayed, suggests that the induced endarteritis may not be as important as previously suggested.

Animals↗
PubMed1985

Properties and characterization of monoclonal antibodies to Tacaribe virus.

Monoclonal antibodies prepared against Tacaribe and Junin viruses have been used to define further the serological relationships between arenaviruses of the Tacaribe complex. A close relationship was found between these two viruses and the heterologous Amapari and Machupo viruses, with Pichinde virus and Parana virus being more distantly related. Among the antibodies specific for Tacaribe virus, five were found to react with viral antigens at the surface of infected cells and to neutralize virus infectivity in vitro. These five antibodies could be differentiated by competitive immunoassay as recognizing at least two antigenically distinct epitopes. The kinetics of reaction between antibody and virus were examined for all five neutralizing antibodies. One antibody (2.25.4) effectively neutralized all infectious virus. The remaining four directed against a second epitope gave significant persistent fractions which could be reduced by addition of complement, anti-mouse immunoglobulin, or antibody 2.25.4. Variants of Tacaribe virus resistant to neutralization by antibody 2.25.4 were obtained by growth in the presence of this antibody and neutralization kinetics were reexamined using the heterologous monoclonal neutralizing antibodies. Several different neutralization profiles were obtained, suggesting that point mutations resulted in conformational changes at topographically selected distinct epitopes recognized by the remaining antibodies.

Animals↗
PubMed1984

Determination of the affinity of antibodies to hepatitis B surface antigen in human sera.

The measurement of the affinity of anti-HBs antibody in human sera using 3 HBsAg-related antigens is described. The antigens used were (i) a synthetic linear peptide corresponding to amino acids 139-147 of the major polypeptide of HBsAg, (ii) a cyclical form of this same peptide and (iii) a polypeptide complex of a 28,000 MW glycoprotein and a 23,000 MW protein from purified HBsAg. The method was established with a pooled human anti-HBs immunoglobulin preparation and a monoclonal anti-HBs antibody reactive to the 'a' determinant of HBsAg. The results indicate that both these antibody preparations effectively bind the 3 antigens with affinity values of between 2 X 10(6) to 9 X 10(7) litres/mole. However, the affinity of both antibody preparations for the cyclical form of the peptide was higher than for the linear form. The level of antibody (expressed as Abt, molar antigen binding sites) in the pooled human immunoglobulin for each of the 3 antigens was similar. Measurements of anti-HBs antibodies in the sera of recovered acute hepatitis B patients and from HBsAg negative chronic liver disease patients showed that the cyclical form of the antigen was bound with a higher affinity than the linear form. Affinity values of antibody in the sera of the latter group of patients was significantly lower (3 X 10(5) to 2.7 X 10(6) litres/mole) than those observed in sera from other individuals. The implication of these results in determining the importance of the measurement of affinity in the assessment of the efficacy of vaccines is discussed.

Amino Acid Sequence↗
PubMed1984

Affinity of antibody responses in man to hepatitis B vaccine determined with synthetic peptides.

The affinity and level of antibody to hepatitis B surface antigen (anti-HBs) in recipients of a plasma-derived hepatitis B vaccine were determined with three different antigens. The first two antigens were prepared by chemical synthesis, to represent linear or cyclical forms of aminoacid sequences 139 to 147 of the major hepatitis B surface antigen (HBsAg) polypeptide. The binding of antibodies to these synthetic peptides was compared with that to a third antigen, prepared by solubilisation of the naturally occurring HBsAg, the basic component of the currently licensed hepatitis B vaccine in the United Kingdom. Antibody levels, expressed as total antibody combining sites (Abt) in fixed volumes of immune sera, increased throughout the course of immunisation and correlated with the development of antibody as measured by a commercially available radioimmunoassay. Abt values were similar for both forms of the synthetic peptide, although higher affinity values were found with the cyclical structure, which illustrates the importance of protein conformation in antibody responses to HBsAg. Antibody affinity for the three antigens increased progressively throughout the immunisation schedule but the pattern of affinity maturation varied according to the peptide used as an antigen probe and between subjects. Most subjects showed a significant rise in antibody affinity after the third (booster) dose of vaccine given at six months. The use of synthetic peptides allowed a quantitative and qualitative assessment of antibody responses to hepatitis B vaccine and confirmed that selected peptides corresponding to relevant HBsAg epitopes may be useful as alternative hepatitis B vaccines.

Antibody Affinity↗