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F C Hay

Publications and source records attributed to F C Hay.

At least 19 recordsLinked to original sources
PubMed1992

A critical evaluation of the magnetic cell sorter and its use in the positive and negative selection of CD45RO+ cells.

In this paper, we report on our year-long experience with the magnetic cell sorter (MACS), and present a critical evaluation of its pitfalls and benefits. Satisfactory separation of lymphocytes into subsets with preservation of function can be achieved, but there are several drawbacks: in comparison with Dynal beads, MACS results in a higher cell loss due to the increased number of separation steps and requires depletion of plastic-adherent cells as these will engulf microbeads and contaminate the enriched fraction, and is more expensive. The advantage of MACS over Dynal beads is that the microbeads are biodegradable and do not interfere with proliferation assays: both the depleted and enriched fractions of cells can therefore be used in culture immediately following separation. We used MACS for the positive and negative selection of CD45RO cells: the enriched fraction was of high purity (greater than 98%), but a depleted fraction of greater than 90% purity could not be obtained even after running the same sample over three separating columns. Dynabeads, on the other hand, achieved 98% pure CD45RO-depleted fractions after three separation runs.

Antigens, CD↗
PubMed1992

Lymphocytes from patients with rheumatoid arthritis produce agalactosylated IgG in vitro.

The percentage of oligosaccharide chains lacking galactose was measured in IgG obtained from pokeweed mitogen-activated cultures of blood lymphocytes from patients with rheumatoid arthritis and controls. Secreted IgG from rheumatoid arthritis lymphocytes was deficient in galactose compared with IgG from the lymphocytes of controls. This confirms that agalactosylation is a significant feature of the disease and demonstrates that it can occur at the B cell level and is not merely a post-secretory event.

Adult↗
PubMed1991

Framework peptides from kappa IIIb rheumatoid factor light chains with binding activity for aggregated IgG.

Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient's synovia are restricted in their light chains, using predominantly the kappa IIIb subfamily. Very few sequence differences are found between these light chains. Light chains with similar variable region framework sequences are also found in some mouse monoclonal RF derived from mice stimulated with lipopolysaccharide or secondarily immunized with protein antigens. There are two likely explanations for this restriction in framework sequences between the two species: (a) the sequences are important for the immunoregulation of RF production or (b) the sequences are concerned with the antibody binding specificity of the RF. We have examined overlapping octapeptides from the kappa IIIb light chain variable region and show that some framework peptides have the ability to bind aggregated IgG. Replacement of amino acids within the peak binding peptide have indicated the critical amino acids necessary for binding.

Amino Acid Sequence↗
PubMed1991

Epitope mapping.

The immune response to extrinsic and intrinsic antigens involves specific antigen receptors on T and B cells. The precise antigenic determinants, or epitopes, recognized by these receptors are discrete sequences within the native antigen. The ability to identify and manufacture key epitopes in the immune response has important implications for disease diagnosis and immunointervention. Consequently, increasingly sophisticated technologies are being applied to epitope mapping. This report from a recent workshop gives a balanced view of progress to date and the challenges ahead.

B-Lymphocytes↗
PubMed1991

Abnormalities in the glycosylation of IgG in spouses of patients with rheumatoid arthritis. A family study.

Forty-seven members of eight families with a rheumatoid proband were analysed for abnormal glycosylation of IgG. The results (%G(o) which is the percentage of oligosaccharide chains lacking galactose) were corrected for age and expressed as SD units about the mean for the normal population. Seven of 8 probands, 3/8 spouses, 3/5 RA relatives and 4/26 non-RA relatives had %G(o) values greater than 1SD above the age corrected mean for the normal control population (P less than 0.001, less than 0.01, less than 0.005 and greater than 0.5 respectively). A further 13 spouse pairs were studied. Ten of 13 probands and 8/13 spouses had %G(o) values greater than 1SD above the mean (P less than 0.001 and less than 0.001 respectively). Thus in total, a strikingly high number of unaffected spouses had high %G(o) values (11/21). IgM, IgA and IgG rheumatoid factors were studied. While RA patients' sera showed a correlation between IgM and IgA rheumatoid factors and %G(o), (IgM, r = 0.41 0.05 greater than P greater than 0.02, IgA, r = 0.36, P = 0.05), no correlation between IgG RF and %G(o) was noted in the RA patients. No correlation was found between any of the RF classes and %G(o) in spouses and non-RA relatives.

Arthritis, Rheumatoid↗
PubMed1991

Reduction in IgG galactose in juvenile and adult onset rheumatoid arthritis measured by a lectin binding method and its relation to rheumatoid factor.

Glycosylation changes in patients with juvenile chronic and adult onset rheumatoid arthritis have been studied using a novel binding method. Both these major types of arthritis showed decreased galactosylation of serum IgG, which confirms earlier studies using a different, more complex chemical method. No significant correlation between serum IgG, IgM, and IgA rheumatoid factors and age corrected G(o) (percentage of oligosaccharide chains lacking galactose) was found. The possibility that the less glycosylated IgG is preferentially confined to circulating IgM/IgG immune complexes cannot be excluded, however.

Adolescent↗
PubMed1991

In vivo effects of anti-idiotype on Plasmodium chabaudi infection in mice.

A polyclonal anti-idiotype was raised in rabbits following immunization with a murine monoclonal antibody which recognized a 250,000 MW antigen of Plasmodium chabaudi-infected erythrocytes. The monoclonal antibody, NIMP M23 (clone 3,) has been shown to protect mice against homologous parasite challenge. Following purification, the anti-idiotype was shown to bind only the immunizing idiotype and to recognize antigen-binding site-associated anti-idiotype. Mice primed with anti-idiotype and challenged with live parasites had an altered course of infection, with significant reduction in their peak parasitaemia levels. Anti-idiotype priming did not induce an antigen-reactive antibody response in vivo but a population of T cells capable of proliferating in vitro to P. chaubaudi-infected red cells was stimulated. These data are discussed in the context of possible idiotypic interaction in murine malaria.

Animals↗
PubMed1991

Rheumatoid factors and complex formation. The role of light-chain framework sequences and glycosylation.

New information regarding rheumatoid factors (RFs) indicates that the RFs synthesized in synovium and lymphoid tissues of patients with rheumatoid arthritis (RA) are different from monoclonal and nonspecific RFs associated with other inflammatory states. The characteristics of RF associated with RA are as follows. They are of all Ig isotypes (not just IgM), indicating T-cell participation in antibody maturation. They have higher avidity for human IgG than for rabbit IgG. They use the human germline heavy-chain variable region (VH) gene VHIII more frequently than other VH genes, and light chains from multiple families. (In contrast, monoclonal RFs use predominantly VH1 and very commonly the V kappa IIIb germline gene HUMkv325). RA IgG is somatically mutated. (In contrast, monoclonal RFs use unmutated germline Ig genes). This suggests they are matured by stimulation either with specific antigens or other activation signals such as cytokines. They are abnormally glycosylated. In general, during periods of disease activity in adult and juvenile RA, a galactose is missing from the Fc of the IgG molecule, leaving an empty "pocket" between the C gamma 2 domains of heavy chains. The IgG RFs self-associate. This may result at least in part when galactose on the F(ab')2 portion of one IgG molecule fills the empty pocket in the Fc of another Ig molecule. Self-association forms immune complexes capable of fixing complement and probably of causing joint damage and vasculitis.

Animals↗
PubMed1990

Analysis of glycosylation changes in IgG using lectins.

A simple rapid assay based on the ability of lectins to bind carbohydrates has been developed to analyse changes in the oligosaccharide chains of IgG. Bandeiraea simplicifolia lectin and Ricinus communis agglutinin have been used to detect terminal N-acetylglucosamine and galactose moieties respectively in IgG using immunodot-blotting. IgG samples (approximately 1 micrograms) were dot-blotted onto nitrocellulose followed by boiling of the blots to expose the carbohydrate moieties. The blots were then treated with biotinylated lectins followed by either streptavidin-biotin-hydrogen peroxidase conjugate or 125I-labelled streptavidin. The colour was developed using chloronaphthol and the blots read on a densitometer. The labelled blots were cut and read on a gamma counter. The use of a monoclonal antibody to N-acetylglucosamine is also discussed. The results obtained using this method are comparable to those obtained by structural analysis.

Arthritis, Rheumatoid↗
PubMed1990

Glycosylation of IgG, immune complexes and IgG subclasses in the MRL-lpr/lpr mouse model of rheumatoid arthritis.

The MRL-lpr/lpr (MRL/lpr) mouse spontaneously develops a disease syndrome which, in many respects, is similar to human rheumatoid arthritis. These mice developed joint inflammation, circulating rheumatoid factors and immune complexes. We now show that the parallel with human disease extends to a glycosylation defect which is observed on IgG from rheumatoid arthritis patients. Using the lectins ricin and Bandeiraea simplicifolia II we have found that terminal N-acetylglucosamine is clearly raised in MRL/lpr IgG. Increased exposure of galactose was also detected, indicating that a second glycosylation site must be present on these molecules. Polyethylene glycol-precipitated IgG complexes bound significantly more of each lectin than did free IgG, indicating that the changes in glycosylation were associated with complex formation. The sugar abnormality was most marked in the IgG2a/IgG3 fraction from protein A IgG subclass chromatography. Our results suggest that the IgG glycosylation defect seen in rheumatoid arthritis is apparent in the MRL/lpr mouse and may contribute, through complex formation, to the pathological processes in the rheumatoid syndrome.

Acetylglucosamine↗
PubMed1990

The antibody repertoire of early human B cells. III. Expression of cross-reactive idiotopes characteristic of certain rheumatoid factors and identifying V kappa III, VHI, and VHIII gene family products.

Our previous studies have shown that a high frequency of Epstein-Barr virus (EBV)-immortalized cord blood (CB) and fetal liver (FL) clones produce IgM antibodies which display extensive autoreactivity for IgG Fc (rheumatoid factor, RF). To investigate further the repertoire of these early B cells, we have examined the expression of CRI associated with RF paraproteins in relation to antibody specificity and polyreactivity. CRI were detected by ELISA and/or flow cytometry using a panel of well-characterized monoclonal antibodies defining idiotopes associated with particular V kappa and VH gene family products and raised against Fc-specific paraproteins. Many of the CRI were expressed by these clones, suggesting that they may be markers of early B cells. The presence of the CRI was not always associated with Fc specificity. Three of eight CB/FL clones expressed the V kappa III subgroup of light chains, and two of these expressed the V kappa III sub-subgroup associated CRI, 17-109. These two clones reacted with IgG Fc, and one also bound to single-stranded DNA. The VHIII-associated idiotope D12 was expressed on IgM from 4 out of 9 FL and 5 out of 12 CB clones. D12 and B6 (also a VHIII-associated CRI) were coexpressed in 4 out of 5 CB clones but not in the four FL clones. Seven out of nine clones expressing these idiotopes were polyreactive, and five had Fc-binding activity. Three of the 12 CB clones expressed the VHI-associated conformational idiotope G8. One of 20 CLL clones expressed both B6 and D12, and another expressed both 17-109 and the VHI-associated G6 and G8 idiotopes. Taken together, these data provide evidence for the frequent usage, in early B cells, of V kappa subgroups and VH-associated idiotopes of RF paraproteins. The expression of these CRI was not a prerequisite for binding to IgG Fc, but there was a frequent association of these idiotopes with it. Differences in expression of CRI between CLL and early B-cell clones may suggest differences in the pattern of VH usage between these subsets of B cells.

B-Lymphocytes↗
PubMed1990

Specificity of rheumatoid factors in relation to the disease state in rheumatoid arthritis.

Rheumatoid factors found in patients with rheumatoid arthritis react with human IgG and with IgG from some other species. The levels of rheumatoid factor give some indication of prognosis, albeit a rather poor one in this highly variable disease. The high degree of variability may, in part, be due to differences in the fine specificity of the rheumatoid factor in each individual patient, leading to differences in the types of immune complex formed. To study this hypothesis the fine specificity of rheumatoid factors of the IgM, IgA, and IgG classes for IgG from human, baboon, orangutan, macaque, owl monkey, gorilla, marmoset, cow, pig, sheep, goat, horse, mouse, and chicken was examined. Differential reactivity for these species was found and associations between the presence of rheumatoid factor and the development of moderate or severe erosions.

Animals↗
PubMed1989

Analysis of autoantibody reactivity and common idiotype PR4 expression of myeloma proteins.

Sera from 75 patients with monoclonal gammopathies and with no clinical evidence of autoimmune disease have been screened for a wide range of autoreactivity including binding to DNA, cardiolipin, extractable nuclear antigen (ENA), rheumatoid factor activity and the presence of the common anti-DNA antibody idiotype PR4. The sera of 17/75 (23%) patients possessed autoreactivity: six were positive for anti-DNA activity, two had anticardiolipin activity and the PR4 ID was found in two sera (both of which possessed anti-DNA activity). Antibodies to ENA were found in one serum (anti-Ro) and anti-organ-specific antibodies in five. Using iso-electric focusing and immunoblotting we have shown that the PR4 ID and DNA binding activity are carried on the paraprotein and not on some other serum constituent. The IgG subclass distribution of 55 IgG paraproteins has also been investigated. The majority of IgG paraproteins belong to IgG1 subclass (55%), with the others, being IgG2 (4%), IgG3 (9%) and IgG4 (27%). In this study we have shown that sera from myeloma patients frequently possess autoreactivity, and that in many cases this can be attributed to the paraprotein.

Antibodies, Antinuclear↗
PubMed1989

Elevated IgG antibody levels to the mycobacterial 65-kDa heat shock protein are characteristic of patients with rheumatoid arthritis.

We have previously demonstrated raised levels of IgG and IgA antibody to the mycobacterial 65-kDa heat shock protein (hsp) in the sera of patients with rheumatoid arthritis (RA). We have now attempted to determine whether this phenomenon is specific for RA, and whether it is seen only with the mycobacterial homologue of this particular hsp gene family. We therefore screened antibody levels to the mycobacterial and Escherichia coli hsp 65, and the mycobacterial, E. coli, and human hsp70, in sera from RA, systemic lupus erythematosus (SLE), tuberculosis (TB), ankylosing spondylitis (AS), Crohn's disease, and control donors. RA sera show the greatest increase in IgA binding to the mycobacterial hsp65, but no increase in IgA binding to the E. coli homologue. Similarly, only RA and TB sera show increased IgG binding to the mycobacterial hsp65, and we have shown previously that the titre is greater in RA. In contrast, the use of mycobacterial and E. coli hsp70 preparations as control bacterial hsp gene products has shown that RA patients do not differ from TB or SLE patients in their antibody binding to these proteins. Moreover, neither IgA nor IgG antibody to the human hsp70 in RA sera were higher than in TB, and the IgA binding was not higher than in SLE. These findings suggest that elevated IgG antibody levels to the mycobacterial hsp65 shows some disease specificity, and further studies with the human homologue and at the T-cell level are required.

Adult↗
PubMed1989

Raised serum IgG and IgA antibodies to mycobacterial antigens in rheumatoid arthritis.

Autoantigens cross reactive with mycobacteria are implicated in the pathogenesis of adjuvant arthritis in the rat, and there are reports of changes in the immune response to mycobacteria in human rheumatoid arthritis (RA). We have therefore examined the IgM, IgG, and IgA antibody levels to crude mycobacterial antigens and to two recombinant mycobacterial heat shock/stress proteins (65 kD and 71 kD) in sera from patients with RA, systemic lupus erythematosus (SLE), and Crohn's disease, and from healthy controls. IgA binding to the crude mycobacterial antigens was significantly raised in RA sera, though IgG and IgM binding tended to be lower than in controls. Both IgA and IgG binding to the heat shock proteins were significantly raised in the RA sera. Smaller significant rises in both classes were seen in sera from patients with SLE, and in the IgA class only to the 65 kD protein in Crohn's disease. The rises in IgG and IgA antibodies to the 65 kD protein in RA were significantly higher than in the other diseases, however. It is interesting that this protein is the one responsible for adjuvant arthritis in the rat.

Adult↗