Search PubMed⌕ Search

Biomedical subjects

R Jefferis

Publications and source records attributed to R Jefferis.

At least 37 records · Page 2Linked to original sources
PubMed1998

The frequent expansion of a subpopulation of B cells that express RF-associated cross-reactive idiotypes: evidence from analysis of a panel autoreactive monoclonal antibodies.

Preferential expression of VH gene segments is evident within the adult human primary B-cell repertoire. The repertoire may be influenced by genetic factors, e.g. VH gene segment polymorphisms, or in a temporal manner due to the exposure to environmental antigens. The molecular characteristics of 15 autoreactive human monoclonal antibodies (MoAbs) are reported. All antibodies were of the IgM isotype, and 12 of the 15 were polyreactive and included rheumatoid factor type specificity, i.e. reactivity with IgG. Nine of the 15 MoAbs are products of VH3 gene segments, as evidenced by staphylococcal protein A binding; four of these express the cross-reactive idiotype recognized by the mouse MoAb 3H7 and are thus products of the VH26 gene segment. One of the five remaining VH3 gene products expresses the cross-reactive idiotypes recognized by the mouse MoAbs B6 and D12. V-gene family usage, determined by polymerase chain reaction (PCR) amplification of cDNA and further hybridization with family-specific oligonucleotide probes, confirmed the cross-reactive idiotype studies and showed that only VH3-gene-encoded proteins bound staphylococcal protein A. Five of the six non-VH3 gene segment products express the cross-reactive idiotype recognized by the mouse MoAb LC1 and could be assumed to be products of the VH4.21 gene segment; however, one human MoAb is shown to be the product of a VH2 gene segment. This is interesting because it turns LC1 from being an anti-cross-reactive idiotype antibody into an anticlan reagent.

Animals↗
PubMed1998

Human antibodies elicited by a pneumococcal vaccine express idiotypic determinants indicative of V(H)3 gene segment usage.

Human immunodeficiency virus (HIV)-infected persons manifest decreased antibody responses to pneumococcal polysaccharide vaccines. Since human antibody responses to polysaccharides are often restricted, the molecular structure of antibodies elicited by a 23-valent pneumococcal vaccine was analyzed. Anti-idiotypic reagents were used to detect V(H)1, V(H)3, and V(H)4 gene usage by antibodies to pneumococcal capsular polysaccharides in HIV-uninfected and HIV-infected subjects by ELISA. HIV-uninfected persons generated beta-mercaptoethanol-sensitive and -resistant antibodies to pneumococcal capsular polysaccharides expressing V(H)3 determinants recognized by the D12, 16.84, and B6 monoclonal antibodies; antibodies expressing V(H)1 determinants were not detected, and V(H)4 determinants were expressed by beta-mercaptoethanol-sensitive antibodies only; and HIV-infected subjects had significantly lower capsular polysaccharide-specific and V(H)3-positive antibody responses. These findings confirm decreased antibody responses to pneumococcal vaccination in HIV-infected persons and suggest that their poor responses may result from HIV-associated depletion of restricted B cell subsets.

Adult↗
PubMed1998

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

Animals↗
PubMed1997

Glycosylation of polyclonal and paraprotein IgG in multiple myeloma.

It has previously been shown that in multiple myeloma (MM) each IgG paraprotein exhibits a unique oligosaccharide profile. It has been assumed that this results from a clone specific glycosylation machinery. However, the abnormal physiological environment of the bone marrow in this disease may also affect normal plasma cells producing polyclonal IgG. We present data to show that this is so and that, in two cases, the oligosaccharide profile of the polyclonal IgG reflected that of the paraprotein from the same patient rather than that of normal polyclonal IgG.

Chromatography, DEAE-Cellulose↗
PubMed1997

Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction.

Rheumatoid factors are the characteristic autoantibodies of rheumatoid arthritis, which bind to the Fc regions of IgG molecules. Here we report the crystal structure of the Fab fragment of a patient-derived IgM rheumatoid factor (RF-AN) complexed with human IgG4 Fc, at 3.2 A resolution. This is the first structure of an autoantibody-autoantigen complex. The epitope recognised in IgG Fc includes the C gamma 2/C gamma 3 cleft region, and overlaps the binding sites of bacterial Fc-binding proteins. The antibody residues involved in autorecognition are all located at the edge of the conventional combining site surface, leaving much of the latter available, potentially, for recognition of a different antigen. Since an important contact residue is somatic mutation, the structure implicates antigen-driven selection, following somatic mutation of germline genes, in the production of pathogenic rheumatoid factors.

Antigen-Antibody Reactions↗
PubMed1996

Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fc gamma receptor I and influence the synthesis of its oligosaccharide chains.

Glycosylation at Asn297 within the CH2 domains of IgG is important for recognition by the effector ligands Fc gammaR and C. Protein engineering has been used to replace amino acid residues within the extensive oligosaccharide interaction site that contact the core hexasaccharide (GlcNAc2Man3GlcNAc). Replacement of residues Phe241, Val264, or Asp265, in particular, results in reduced recognition of human chimeric anti-nitroiodophenacetyl IgG3 produced in Chinese hamster ovary cells, by guinea pig C and human C1q. Replacement of residues Val264 or Asp265, in particular, results in reduced superoxide production triggered through human Fc gammaRI expressed on U937 cells. These results suggest that noncovalent interactions of multiple amino acid residues of IgG with oligosaccharide residues that include the primary and secondary GlcNAc are necessary for optimal recognition of IgG by human Fc gammaRI and C1q. Replacement of residues 241, 243, 264, 265, or 301 with alanine in each case resulted in increased galactosylation and sialylation relative to the wild-type oligosaccharide chains. In particular, for the mutant FA243 there was much increased sialylation of its oligosaccharide chains (73%) relative to the wild-type (4%). Thus, even single residue replacements within the oligosaccharide interaction site of the C region can influence galactosylation and sialylation of its oligosaccharide chains. These data suggest a protein engineering route to the production of more homogeneously glycosylated IgG molecules with or without compromised biologic activities.

Animals↗
PubMed1996

Evaluation of monoclonal antibodies with specificity for human IgA, IgA subclasses and allotypes and secretory component. Results of an IUIS/WHO collaborative study.

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN (n = 24), IgA1 (n = 7), IgA2 (n = 3), IgA2m(2) (n = 2), non-IgA2m(2) (n = 4) and SC or secretory IgA (n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, kappa and lambda by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.

Animals↗
PubMed1996

Crystallization of a complex between the Fab fragment of a human immunoglobulin M (IgM) rheumatoid factor (RF-AN) and the Fc fragment of human IgG4.

Rheumatoid factors (RF) are the characteristic autoantibodies found in patients with rheumatoid arthritis. They recognize epitopes in the Fc region of immunoglobulin G (IgG) and are often of the IgM isotype. In order to analyse the nature of RF-Fc interactions, we have crystallized a complex between the Fab fragment of a human monoclonal IgM rheumatoid factor (RF-AN) and the Fc fragment of human IgG4. The stoichiometry of the complex within the crystals was found to be 2:1 Fab:Fc. The crystals diffracted X-rays to 0.3 nm resolution, and the space group was C2, with cell dimensions a = 16.03 nm, b = 8.19 nm, c = 6.42 nm, beta = 98.3 degrees. We have also determined the sequence of the variable region of the RF-AN light chain, not hitherto reported. This belongs to the V lambda III-a subgroup and is closely related to the germline gene Humlv318, from which it differs in three amino acid residues. This is the first reported crystallized complex between a human autoantibody and its autoantigen.

Amino Acid Sequence↗
PubMed1995

Anti-CD40 plus interleukin-4-activated human naive B cell lines express unmutated immunoglobulin genes with intraclonal heavy chain isotype variability.

Combination of anti-CD40 antibody and interleukin-4 (IL-4) induces B cell clonal expansion reminiscent of the T-dependent proliferation following antigenic challenge in vivo. We have analyzed the usage of CH genes and the presence or absence of somatic mutations within the progeny of a single human naive B cell activated with anti-CD40 + IL-4. To address this issue, single-cell cultures of naive (sIgD+) tonsillar B lymphocytes expressing the VH1-restricted G8 idiotype were set up. After culture and RNA extraction, VH1+ Ig mRNA were reverse-transcribed, amplified by polymerase chain reaction and sequenced. A single sIgD+ B cell could generate clones expressing mu, gamma 1, gamma 3, or epsilon, illustrating that the progeny of a single cell can express different isotypes in response to the same stimulus in vitro. The rate of somatic mutations affecting the immunoglobulin variable heavy chain gene was indistinguishable from the background of errors introduced by Taq polymerase.

Antibodies, Anti-Idiotypic↗
PubMed1995

Recognition sites on human IgG for Fc gamma receptors: the role of glycosylation.

Human IgG subclass proteins exhibit more than 95% primary amino acid sequence homology in their Fc regions, but each has a unique profile for recognition by the 3 human Fc gamma receptors. The Fc gamma Rs are themselves highly homologous members of the immunoglobulin supergene family. Consistent with these data we have proposed that Fc gamma RI, Fc gamma RII and Fc gamma RIII recognise overlapping non-identical interaction sites in the lower hinge region of the CH2 domain of the IgG molecule. Evidence in support was provided by protein engineering effecting single amino acid replacements in the proposed site. Alternatively, we have demonstrated that the primary amino acid sequence alone is not sufficient for IgG molecules to fold with the generation of Fc gamma R interaction sites and that glycosylation of Asn 297 of the CH2 domain is essential. We have further defined a 'core' oligosaccharide structure that provides for the generation of Fc gamma R interaction sites which suggests that the addition of outer-arm sugar residues does not affect this primary activity; although in vivo it could influence other essential biological activities. These findings have opened up a new approach to engineering antibody function--by protein engineering of amino acid residues that form contacts with the oligosaccharide moiety. In the present report we demonstrate that replacement of contact residues for galactose on the alpha(1-6) arm does not affect Fc gamma RI and Fc gamma RII recognition while replacement of Asp 265, a contact for a 'core' N-acetylglucosamine residue, results in a loss of Fc gamma RI and Fc gamma RII recognition.

Binding Sites, Antibody↗
PubMed1995

Rheumatoid factors, B cells and immunoglobulin genes.

The paradigm of self, non-self discrimination in the immune system is under review as autoreactive B or T cells are increasingly delineated within normal individuals. The products of autoreactive B cells are, mostly, polyspecific IgM antibodies of low affinity. These 'natural' antibodies include rheumatoid factors (RF) encoded by unmutated germline immunoglobulin genes. In rheumatoid arthritis (RA) the RF may be of the IgM, IgG or IgA isotype, show evidence of somatic mutation and have increased affinity; consistent with maturation of an antigen driven immune response. This response could be initiated or driven by an auto-immunogenic form of IgG or an exogenous cross-reactive antigen. Changes in galactosylation of IgG have been reported to be a valuable diagnostic and prognostic indicator in RA. Speculation that these changes may precipitate some of the disease processes is critically reviewed.

Autoantibodies↗