There are several sites on IgG Fc that have been reported to be the epitopes for binding rheumato... more There are several sites on IgG Fc that have been reported to be the epitopes for binding rheumatoid factors (RF). It is now established that there are alterations in the oligosaccharides on IgG from patients with rheumatoid arthritis and it has been suggested that these changes may enhance immune complex and cryoglobulin formation. We have used a series of IgG preparations differing in their content of oligosaccharide chains lacking galactose from 18 to 86% to determine whether changes in sugar content affect the binding of rheumatoid factor. Five of 16 monoclonal rheumatoid factors prepared from synovial tissue, from patients with juvenile or adult rheumatoid arthritis, bound better to IgG which was deficient in galactose. Six of the 16 rheumatoid factors from the same patients bound independently of the galactose content. Four of the 16 rheumatoid factors could not be absolutely grouped in this manner but seemed to demonstrate a preference for agalactosyl IgG. One rheumatoid factor bound better to fully galactosylated IgG. There was an association between enhanced binding to galactose-deficient IgG and monoreactivity and a very strong association between the functional affinity of the rheumatoid factors and the dependent binding.
Several different chromatographic methods and a lectin-based assay have been compared for the qua... more Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of Ž. oligosaccharides released from immunoglobulin G IgG. The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 Ž. Ž. Ž. galactose residues G0, G1 and G2 in order to a identify the method that can be most widely used for quantitation, b Ž. accurately define the range of G0 values found in patients with rheumatoid arthritis, and c make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on Ž. reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases G0 mix and Biogel P4 Ž. chromatography of 2-aminobenzamide 2-AB derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolia
In this paper, we report on our year-long experience with the magnetic cell sorter (MACS), and pr... more 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 immedately following separation. We used MACS for the positive and negative selection of CD45RO cells: the enriched fraction was of high purity (> 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.
Serum IgG from rheumatoid arthritis patients contains a decreased number of oligosaccharide struc... more Serum IgG from rheumatoid arthritis patients contains a decreased number of oligosaccharide structures ending in galactose and thus there is an increase in N-acetylglucosamine as the terminal sugar, compared with healthy individuals. The relationship between these two sugars varies depending on the disease examined: IgG from patients with rheumatoid arthritis, juvenile onset chronic arthritis and Crohn's disease are at one extreme, and exhibit a reciprocal galactose:N-acetylglucosamine relationship, while Sjögren's syndrome and osteoarthritis IgG are at the other extreme, exhibiting a parallel increase in the expression of both galactose and N-acetylglucosamine. These results may occur as a consequence of more than one glycosylation site which is differentially glycosylated, but more likely by changes in the level of bisecting N-acetylglucosamine.
Page 1. Glycosylation & Disease, 1994, 1, 105-110 Changes in the glycosylation of IgG in the ... more Page 1. Glycosylation & Disease, 1994, 1, 105-110 Changes in the glycosylation of IgG in the collagen-induced model of arthritis Meinir G. Jones, Susan A. Dilly*, Angela Bond and Frank C. Hay Immunology Division and *Histopathology ...
The MRL-lprllpr (MRLllpr) mouse spontaneously develops a disease syndrome which, in many respects... more The MRL-lprllpr (MRLllpr) mouse spontaneously develops a disease syndrome which, in many respects, is similar to human rheumatoid arthritis. These mice develop 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 MRLllpr 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 significiantly 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 IgG2JIgG3 fraction from protein A IgG subclass chromatography. Our results suggest that the IgG glycosylation defect seen in rheumatoid arthritis is apparent in the MRLllpr mouse and may contribute, through complex formation, to the pathological processes in the rheumatoid syndrome.
Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient's synovia a... more Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient's synovia are restricted in their light chains, using predominantly the xIIIb 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.
New information regarding rheumatoid factors (RFs) indicates that the RFs synthesized in synovium... more 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 R F associated with R.4 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 IgC than for rabbit IgC. They use the human germline heavy-chain variable region (V,) gene V,III more frequently than other V, genes, and light chains from multiple families. (In contrast, monoclonal RFs use predominantly V,l and very commonly the VJIIb germline gene HUMkv325). RA IgC 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 IgC molecule, leaving an empty "pocket" between the Cy2 domains of heavy chains. The IgC RFs self-associate. This may result at least in part when galactose on the F(ab), portion of one IgC 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. Rheumatoid factors (RFs) have provided a fruitful field of study for more than 50
Oligosaccharides can be of fundamental importance to glycoprotein function. Glycosylation abnorma... more Oligosaccharides can be of fundamental importance to glycoprotein function. Glycosylation abnormalities are present in rheumatoid arthritis (RA) and may be associated with disease pathogenesis. To determine whether similar disease mechanisms occur in the MRL-1pr/1pr autoimmune arthritic mouse, studies on B lymphocyte galactosyltransferase (GTase) have been carried out. In MRL mice, a significant reduction in peripheral blood lymphocyte (PBL) GTase activity was found when compared to their paired splenic (SP) GTase activity (-69%, p = 0.002) and histocompatible non-autoimmune control CBA/Ca mice (-67%; p = 0.002). The changes in PBL GTase activity are similar to those found in RA and on further analysis, using mixing experiments in the presence of purified human milk GTase, this reduction was shown not to be due to the presence of a soluble intracellular GTase inhibitor. Furthermore when examining MRL derived hybridoma cells producing IgG, significantly reduced GTase activity was detected in the rheumatoid factor (RF) producing hybridoma cells compared to those secreting an irrelevant antibody (-21%, p < 0.05). Together these findings suggest that the glycosylation changes observed in this study, and those reported previously in RA, are tissue-specific, may result from cells trafficking from centres of disease activity and are not the result of direct enzyme inhibition. It is now important to further understand the mechanisms controlling glycosylation and relate disease associated changes with those occurring as part of normal cellular physiology.
To investigate the association between glycosylation and immune complex formation in various dise... more To investigate the association between glycosylation and immune complex formation in various disease groups. Immune complexes and IgG were isolated from serum and their carbohydrate content evaluated in a dot-blot assay using specifically binding lectins. Significantly more N-acetylglucosamine was detected in complexes from patients with rheumatoid arthritis (RA) than in those from patients with systemic lupus erythematosus, Crohn's disease, or infectious endocarditis, or from normal controls (P < 0.001). The immune complex concentration in the circulation was strongly associated with N-acetylglucosamine levels (P < 0.001 by chi-square analysis). The composition of immune complexes from RA patients is distinct in carbohydrate content from those found in other disease groups.
International Archives of Allergy and Immunology, 1976
A convenient technique suitable for the routine estimation of IgG and IgA anti-thyroglobulin anti... more A convenient technique suitable for the routine estimation of IgG and IgA anti-thyroglobulin antibodies has been devised. The assay involves the binding of anti-thyroglobulin to human thyroglobulin linked to the surface of plastic tubes; the amount of antibody bound is then determined by adding radiolabelled anti-human IgG or IgA. IgG antibody was raised in virtually all patients and significantly elevated levels of IgA antibody were also found in some patients. The test should prove helpful in diagnosis and provide quantitative evaluation of research studies.
Clinical medicine. Arthritis and musculoskeletal disorders, 2008
Epitope mapping allowed the location of antigenic determinants on a protein macromolecule to be i... more Epitope mapping allowed the location of antigenic determinants on a protein macromolecule to be identified. In particular, pepscan techniques that utilize a series of overlapping peptides, help detect key amino acid residues that are important in antibody recognition and binding. In a previous study, we employed 15-mer peptides spanning the entire length of IgG1Fc to ascertain successfully the target epitopes of isotypic/allotypic monoclonal reagents. As an extension to this work we have used these peptides to evaluate the location of epitope targets of five IgM rheumatoid factor antibodies (RFAbs). Overall, 2 antibodies, RFAb TS2 and TS1, detected a similar epitope within the CH3 domain (360-KNQVSLTCLVKGFYP-374), whilst 1 (RFAb SJ1) recognised an epitope in the CH2 domain (294-EQYNSTYRVVSVLTV-308). In contrast, 2 RFAbs, PRSJ2 and PRTS1 detected four and five epitopes respectively within the Fc region. RFAb PRSJ2 recognised epitopes detected by RFAB TS2 and TS1 but also further epit...
There are several sites on IgG Fc that have been reported to be the epitopes for binding rheumato... more There are several sites on IgG Fc that have been reported to be the epitopes for binding rheumatoid factors (RF). It is now established that there are alterations in the oligosaccharides on IgG from patients with rheumatoid arthritis and it has been suggested that these changes may enhance immune complex and cryoglobulin formation. We have used a series of IgG preparations differing in their content of oligosaccharide chains lacking galactose from 18 to 86% to determine whether changes in sugar content affect the binding of rheumatoid factor. Five of 16 monoclonal rheumatoid factors prepared from synovial tissue, from patients with juvenile or adult rheumatoid arthritis, bound better to IgG which was deficient in galactose. Six of the 16 rheumatoid factors from the same patients bound independently of the galactose content. Four of the 16 rheumatoid factors could not be absolutely grouped in this manner but seemed to demonstrate a preference for agalactosyl IgG. One rheumatoid factor bound better to fully galactosylated IgG. There was an association between enhanced binding to galactose-deficient IgG and monoreactivity and a very strong association between the functional affinity of the rheumatoid factors and the dependent binding.
Several different chromatographic methods and a lectin-based assay have been compared for the qua... more Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of Ž. oligosaccharides released from immunoglobulin G IgG. The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 Ž. Ž. Ž. galactose residues G0, G1 and G2 in order to a identify the method that can be most widely used for quantitation, b Ž. accurately define the range of G0 values found in patients with rheumatoid arthritis, and c make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on Ž. reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases G0 mix and Biogel P4 Ž. chromatography of 2-aminobenzamide 2-AB derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolia
In this paper, we report on our year-long experience with the magnetic cell sorter (MACS), and pr... more 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 immedately following separation. We used MACS for the positive and negative selection of CD45RO cells: the enriched fraction was of high purity (> 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.
Serum IgG from rheumatoid arthritis patients contains a decreased number of oligosaccharide struc... more Serum IgG from rheumatoid arthritis patients contains a decreased number of oligosaccharide structures ending in galactose and thus there is an increase in N-acetylglucosamine as the terminal sugar, compared with healthy individuals. The relationship between these two sugars varies depending on the disease examined: IgG from patients with rheumatoid arthritis, juvenile onset chronic arthritis and Crohn's disease are at one extreme, and exhibit a reciprocal galactose:N-acetylglucosamine relationship, while Sjögren's syndrome and osteoarthritis IgG are at the other extreme, exhibiting a parallel increase in the expression of both galactose and N-acetylglucosamine. These results may occur as a consequence of more than one glycosylation site which is differentially glycosylated, but more likely by changes in the level of bisecting N-acetylglucosamine.
Page 1. Glycosylation & Disease, 1994, 1, 105-110 Changes in the glycosylation of IgG in the ... more Page 1. Glycosylation & Disease, 1994, 1, 105-110 Changes in the glycosylation of IgG in the collagen-induced model of arthritis Meinir G. Jones, Susan A. Dilly*, Angela Bond and Frank C. Hay Immunology Division and *Histopathology ...
The MRL-lprllpr (MRLllpr) mouse spontaneously develops a disease syndrome which, in many respects... more The MRL-lprllpr (MRLllpr) mouse spontaneously develops a disease syndrome which, in many respects, is similar to human rheumatoid arthritis. These mice develop 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 MRLllpr 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 significiantly 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 IgG2JIgG3 fraction from protein A IgG subclass chromatography. Our results suggest that the IgG glycosylation defect seen in rheumatoid arthritis is apparent in the MRLllpr mouse and may contribute, through complex formation, to the pathological processes in the rheumatoid syndrome.
Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient's synovia a... more Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient's synovia are restricted in their light chains, using predominantly the xIIIb 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.
New information regarding rheumatoid factors (RFs) indicates that the RFs synthesized in synovium... more 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 R F associated with R.4 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 IgC than for rabbit IgC. They use the human germline heavy-chain variable region (V,) gene V,III more frequently than other V, genes, and light chains from multiple families. (In contrast, monoclonal RFs use predominantly V,l and very commonly the VJIIb germline gene HUMkv325). RA IgC 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 IgC molecule, leaving an empty "pocket" between the Cy2 domains of heavy chains. The IgC RFs self-associate. This may result at least in part when galactose on the F(ab), portion of one IgC 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. Rheumatoid factors (RFs) have provided a fruitful field of study for more than 50
Oligosaccharides can be of fundamental importance to glycoprotein function. Glycosylation abnorma... more Oligosaccharides can be of fundamental importance to glycoprotein function. Glycosylation abnormalities are present in rheumatoid arthritis (RA) and may be associated with disease pathogenesis. To determine whether similar disease mechanisms occur in the MRL-1pr/1pr autoimmune arthritic mouse, studies on B lymphocyte galactosyltransferase (GTase) have been carried out. In MRL mice, a significant reduction in peripheral blood lymphocyte (PBL) GTase activity was found when compared to their paired splenic (SP) GTase activity (-69%, p = 0.002) and histocompatible non-autoimmune control CBA/Ca mice (-67%; p = 0.002). The changes in PBL GTase activity are similar to those found in RA and on further analysis, using mixing experiments in the presence of purified human milk GTase, this reduction was shown not to be due to the presence of a soluble intracellular GTase inhibitor. Furthermore when examining MRL derived hybridoma cells producing IgG, significantly reduced GTase activity was detected in the rheumatoid factor (RF) producing hybridoma cells compared to those secreting an irrelevant antibody (-21%, p < 0.05). Together these findings suggest that the glycosylation changes observed in this study, and those reported previously in RA, are tissue-specific, may result from cells trafficking from centres of disease activity and are not the result of direct enzyme inhibition. It is now important to further understand the mechanisms controlling glycosylation and relate disease associated changes with those occurring as part of normal cellular physiology.
To investigate the association between glycosylation and immune complex formation in various dise... more To investigate the association between glycosylation and immune complex formation in various disease groups. Immune complexes and IgG were isolated from serum and their carbohydrate content evaluated in a dot-blot assay using specifically binding lectins. Significantly more N-acetylglucosamine was detected in complexes from patients with rheumatoid arthritis (RA) than in those from patients with systemic lupus erythematosus, Crohn's disease, or infectious endocarditis, or from normal controls (P < 0.001). The immune complex concentration in the circulation was strongly associated with N-acetylglucosamine levels (P < 0.001 by chi-square analysis). The composition of immune complexes from RA patients is distinct in carbohydrate content from those found in other disease groups.
International Archives of Allergy and Immunology, 1976
A convenient technique suitable for the routine estimation of IgG and IgA anti-thyroglobulin anti... more A convenient technique suitable for the routine estimation of IgG and IgA anti-thyroglobulin antibodies has been devised. The assay involves the binding of anti-thyroglobulin to human thyroglobulin linked to the surface of plastic tubes; the amount of antibody bound is then determined by adding radiolabelled anti-human IgG or IgA. IgG antibody was raised in virtually all patients and significantly elevated levels of IgA antibody were also found in some patients. The test should prove helpful in diagnosis and provide quantitative evaluation of research studies.
Clinical medicine. Arthritis and musculoskeletal disorders, 2008
Epitope mapping allowed the location of antigenic determinants on a protein macromolecule to be i... more Epitope mapping allowed the location of antigenic determinants on a protein macromolecule to be identified. In particular, pepscan techniques that utilize a series of overlapping peptides, help detect key amino acid residues that are important in antibody recognition and binding. In a previous study, we employed 15-mer peptides spanning the entire length of IgG1Fc to ascertain successfully the target epitopes of isotypic/allotypic monoclonal reagents. As an extension to this work we have used these peptides to evaluate the location of epitope targets of five IgM rheumatoid factor antibodies (RFAbs). Overall, 2 antibodies, RFAb TS2 and TS1, detected a similar epitope within the CH3 domain (360-KNQVSLTCLVKGFYP-374), whilst 1 (RFAb SJ1) recognised an epitope in the CH2 domain (294-EQYNSTYRVVSVLTV-308). In contrast, 2 RFAbs, PRSJ2 and PRTS1 detected four and five epitopes respectively within the Fc region. RFAb PRSJ2 recognised epitopes detected by RFAB TS2 and TS1 but also further epit...
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