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Heterogeneity of IgG Glycosylation in Adult Periodontal Disease
J. Novak1,*,
M. Tomana2,
G.R. Shah3,
R. Brown1 and
J. Mestecky1,2
1 Departments of Microbiology-Box 1, 2 Medicine, and 3 Oral Diagnostics, University of Alabama at Birmingham, 845 19th Street South, Birmingham, AL 35294-2170;
Correspondence: * corresponding author, jannovak{at}uab.edu
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ABSTRACT
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Periodontal disease is a chronic inflammatory disease of bacterial etiology. In many other chronic inflammatory diseases, IgG glycans are galactose-deficient and thus capable of complement activation through the lectin pathway. In this study, we examined whether IgG in serum and gingival crevicular fluid, and IgG locally produced by plasma cells in gingiva of periodontal disease patients, display altered glycosylation. We developed a lectin-ELISA to measure levels of galactose-deficient IgG in the fluids and immunofluorescence staining to detect galactose-deficient IgG-producing cells in gingiva. Our results indicated higher levels of galactose-deficient IgG in sera and gingival crevicular fluid from periodontal disease patients, compared with levels in healthy controls. Furthermore, gingivae from periodontal disease patients exhibited infiltration of IgG-producing plasma cells; many of them contained galactose-deficient IgG in the cytoplasm. Analysis of our data suggests that IgG secreted by B-cells was aberrantly glycosylated, which resulted in the production of pro-inflammatory galactose-deficient IgG.
Key Words: IgG • N-glycans • periodontal disease • galactose deficiency.
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INTRODUCTION
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Antigens and bacterial products of Gram-negative bacteria associated with periodontal disease (Socransky et al., 1998) induce chronic inflammatory responses in the periodontium, ultimately resulting in the progressive loss of tooth support (for review, see Fujihashi et al., 1993; Kinane et al., 1999).
In many respects, periodontal disease shares some common features with other chronic inflammatory diseases, particularly with rheumatoid arthritis, including: production of IgM and IgA rheumatoid factor (Hirsch et al., 1989); infiltration of inflamed tissue with mainly IgG-secreting plasma cells (Ogawa et al., 1989); production of auto-antibodies to tissue components (collagen II in rheumatoid arthritis; collagens I and III in periodontal disease; Hirsch et al., 1988); similar cytokine profiles (Fujihashi et al., 1993; Pistoia, 1997); and increased levels of IL-6 (Fujihashi et al., 1993; Pistoia, 1997).
Extensive structural studies of IgG molecules produced at the site of chronic inflammation and detectable in the circulation revealed profound alterations in the glycan moieties. Specifically, deficiencies of some monosaccharides, especially galactose on IgG, have been described in human chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, tuberculosis, and infection with HIV (Mullinax and Mullinax, 1975; Parekh et al., 1985, 1988; Tomana et al., 1988; Bodman et al., 1992; Tsuchiya et al., 1993; Rademacher et al., 1994; Tomana, 1996; Moore et al., 2005). IgG contains one complex-type oligosaccharide (Fig. 1A ) per each heavy chain linked to the conserved glycosylation site on asparagine 297 within constant region domain 2, which presumably has a role in maintaining the three-dimensional structure of the Fc portion of IgG. In IgG with galactose-deficient glycans, the terminally exposed sugar molecule is N-acetylglucosamine. Analysis of experimental data has indicated that galactose-deficient IgG is pathogenic (Rademacher et al., 1994): Galactose-deficient IgG binds to the mannan-binding lectin and thus activates the complement cascade by the lectin pathway (Malhotra et al., 1995). Furthermore, glycans on IgG molecules affect binding and internalization by Fc receptors expressed on phagocytic cells (Krapp et al., 2003) and, thus, influence opsonization of antigens by phagocytosis.
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Figure 1. Structure of N-linked glycans of IgG and analyses of IgG galactose deficiency. (A) Example of complex type N-linked glycans. Abbreviations: N-acetylglucosamine (GlcNAc), galactose (Gal), sialic acid (SA), mannose (Man), fucose (Fuc), asparagine (Asn). Variably present saccharides are in parentheses. Fc region-associated glycans of IgG are usually not sialylated, and only some of them are fully galactosylated or with bisecting N-acetylglucosamine. Galactose-deficient IgG glycans are terminated by N-acetylglucosamine residues that react with PVL lectin, while fully galactosylated glycans do not react with PVL. (B) Correlation between PVL reactivity (OD 405 nm, 30 min) and gas-liquid monosaccharide composition (mannose/galactose ratio) of differently galactosylated purified IgGs. Pierson’s correlation coefficient, r = 0.73 (N = 18).
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In this study, we examined whether IgG in the serum, gingival crevicular fluid, or IgG produced locally by plasma cells that infiltrate gingiva in periodontal disease display altered glycan moieties.
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MATERIALS & METHODS
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Patients and Controls
Serum samples were obtained from 14 adult periodontal patients (average age, 55 yrs; range, 38–70 yrs; five males and nine females) and 10 healthy controls (average age, 47 yrs; range, 34–54 yrs; six males and four females). The periodontal disease patients exhibited attachment loss and periodontal pockets of > 3.5 mm (ranging from 4 to 9 mm), and bleeding on probing. The control group consisted of patients with pocket depths between 0 and 3 mm, and no attachment loss or bleeding on probing. Samples of gingival crevicular fluid were collected with the use of Periopaper as described previously (Grbic et al., 1995). Gingiva- and periodontium-containing tissues removed from periodontal disease patients for therapeutic purposes were fixed (as described in Kilian et al., 1989) and used for staining. The study was approved by the Institutional Review Board; an informed written consent was obtained before any samples were collected.
ELISA
We used binding of Psathyrella velutina lectin (PVL) to examine galactose-deficiency of N-linked glycans on IgG molecules. PVL detects galactose-deficient N-linked glycans through their binding to terminal N-acetylglucosamine (Tsuchiya et al., 1993). IgG (or serum diluted 1:1000) was reduced, alkylated, and captured on protein-G-coated plates, and the presence of terminal N-acetylglucosamine was probed with biotin-labeled PVL. Simultaneously, IgG levels were also measured by ELISA; the relative N-acetylglucosamine content of IgG-containing samples was expressed as PVL/IgG ratio.
Gas-liquid Chromatography Analysis of IgG Monosaccharide Composition
The monosaccharides from purified IgG proteins were determined as trifluoroacetates of methyl-glycosides by gas chromatography, as described previously (Tomana et al., 1984). The analyses were performed with a Hewlett-Packard model 5890 series II gas chromatograph equipped with a 25-m fused silica (0.22-mm inner diameter) OV-1701 WCOT column and electron capture detector; GC ChemStation software (Agilent Technologies, Palo Alto, CA, USA) was used for acquisition of chromatographic data and peak integration. The galactose content in IgG was determined relative to that of mannose, since the latter was shown to be constant in patients with various autoimmune diseases and in normal healthy controls (Tomana et al., 1988). About 50 µg of each IgG was used for the analyses.
Immunofluorescence
Gingiva- and periodontium-containing tissues removed from periodontal disease patients for therapeutic purposes were examined by immunofluorescence microscopy. The tissue was stained with fluorescein isothiocyanate- (FITC) or tetramethylrhodamine isothiocyanate- (TRITC) labeled F(ab')2 fragments of antibodies recognizing human IgG, and with biotin-labeled PVL followed by avidin conjugated with 7-amino-4-methyl-coumarin-3-acetate (AMCA) for determination of the presence of IgG-producing cells and the production of galactose-deficient IgG. Tissue fixation and processing were conducted as previously described (Kilian et al., 1989). Control tissues included gingivae from patients who underwent crown-lengthening with no evidence of periodontal disease. The staining performed on such tissues showed the absence or scarcity of IgG-producing cells in healthy gingivae. In addition, all tissues were stained by hematoxylin-eosin so that the cell types present could be determined. Seven gingivae samples from periodontal disease patients and six gingivae samples from normal controls were used.
Statistical Analysis
Correlation coefficients (r) were calculated by means of the Microsoft Excel statistical package. Comparisons between group means were performed by Student’s t test. P values equal to or less than 0.05 were considered statistically significant.
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RESULTS
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Initially, we verified the ELISA method for detection of galactose-deficient IgG using biotin-labeled PVL (Tsuchiya et al., 1993). We used our collection of IgG preparations purified from sera of patients with rheumatoid arthritis, inflammatory bowel disease, and normal controls (Tomana, 1996; Tomana et al., 1988). These IgG preparations were analyzed by gas-liquid chromatography (galactose content expressed as mannose/galactose ratio) and by PVL-ELISA. Results of both of these methods correlated (correlation coefficient, r = 0.73; Fig. 1B ).
PVL reactivity of IgG in sera from 14 periodontal disease patients and 10 healthy controls had mean OD values ± SD of 0.63 ± 0.32 and 0.25 ± 0.22, respectively (P = 0.0046; Fig. 2A). This result indicated that sera from periodontal disease patients contained more galactose-deficient IgG compared with that from normal healthy controls.
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Figure 2. Galactose deficiency of IgG in serum and gingival crevicular fluid of periodontal disease patients and controls. (A) PVL reactivity (OD at 405 nm after 2 hrs of incubation) of serum IgG from patients with periodontal disease (N = 14) and from healthy controls (N = 10). P = 0.0046. (B) IgG glycosylation (PVL index) in serum and gingival crevicular fluid (GCF) from periodontal patients (N = 7).
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Furthermore, we determined the PVL reactivity of IgG present in gingival crevicular fluid and in the matching samples of sera. To compensate for different levels of IgG in the samples of gingival crevicular fluid, we expressed all values relative to the IgG amount as a PVL/IgG index (Fig. 2B). The results indicated the presence of galactose-deficient IgG in gingival crevicular fluid of the patients. When the gingival crevicular fluid and the matched serum samples were compared, two patterns emerged: One group of patients exhibited higher levels of galactose-deficient IgG in serum but lower levels of galactose-deficient IgG in gingival crevicular fluid, while the second group exhibited the reverse pattern. It has been shown that the humoral immune responses in the periodontium have contributions from both the mucosal and systemic compartments (Kinane et al., 1999). This is consistent with the study indicating a possible protective role of IgA in gingival crevicular fluid (Grbic et al., 1995). The latter report showed that the concentration of IgA negatively correlated with mean attachment level, mean probing depth, and mean bleeding on probing, the three major clinical measures of periodontal disease. Using the same samples in which we measured IgG and PVL/IgG index, we also determined the levels of IgA. When the results were analyzed, we found that the probing depth (ranging from 4 to 9 mm) correlated with the levels of the abnormally glycosylated IgG in gingival crevicular fluid (r = 0.52). Furthermore, we found a negative correlation between IgA levels and PVL/IgG index (r = 0.45) and a positive correlation between IgG:IgA ratio and PVL/IgG index (r = 0.46). Even with the small number of samples, these results together support the notion that the abnormally glycosylated IgG is present in gingival crevicular fluid from the periodontal disease patients with more severe disease.
Gingivae from periodontal disease patients with well-developed and uncontrolled disease contained infiltration of plasma cells, producing mostly IgG. To examine whether these IgG-producing cells secrete galactose-deficient IgG, we have used double-immunofluorescence with anti-IgG antibody and PVL. From the results of immunohistochemical staining, we concluded that a proportion of IgG-producing cells contained galactose-deficient IgG in the cytoplasm (Fig. 3).
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Figure 3. Paired immunofluorescence staining for IgG with FITC-conjugated (green) F(ab')2 fragment of anti-human IgG and galactose-deficient N-linked glycans disclosed by biotinylated PVL and AMCA-labeled avidin (blue) of gingivae from patients with advanced periodontal disease. The picture shows a representative example of inflamed periodontal disease tissue (N = 7). Most of the IgG-containing cells (green) co-stain for galactose-deficiency (blue). Arrows point to IgG-containing cells that do not stain with PVL. Bar size, 8 µm.
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In conclusion, analysis of our data suggests a possibility that, in periodontal disease, as in other chronic inflammatory diseases, the terminal differentiation of B-cells is affected, resulting in the production of pro-inflammatory galactose-deficient IgG. Such IgG was present in gingival crevicular fluid, as well as in the circulation, of periodontal disease patients.
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DISCUSSION
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The results of these studies indicate that IgG present in the sera and gingivae from periodontal disease patients is deficient in galactose on N-linked glycans. Thus, in this respect, this IgG was analogous to that produced in other chronic inflammatory diseases of autoimmune character (such as rheumatoid arthritis), or induced by chronic microbial infections such as HIV (Mullinax and Mullinax, 1975; Parekh et al., 1985, 1988; Tomana et al., 1988; Bodman et al., 1992; Tsuchiya et al., 1993; Rademacher et al., 1994; Tomana, 1996; Moore et al., 2005).
A growing body of evidence indicates that glycan moieties on glycoproteins, including immunoglobulins, play an essential role in many biological processes and functions (Varki, 1993). For example, glycans on immunoglobulin molecules participate in binding to Fc receptors expressed on phagocytic cells (Monteiro et al., 1990; Leader et al., 1991), opsonization of antigens for phagocytosis, and activation of complement (Tao et al., 1993). These essential functions of immunoglobulin-associated glycans have been demonstrated by studies of immunoglobulin molecules modified by various glycosidases or chemical cleavages, replacement of asparagine with other amino acid residues by site-directed mutagenesis, or inhibitors of glycosylation (e.g., tunicamycin) in immunoglobulin-producing cells (Leader et al., 1991). Polymorphism of IgG glycosylation is associated with variable interactions with Fc RIII receptor and activation of the complement cascade through the lectin pathway (Malhotra et al., 1995). The role of glycans in complement activation indicated that, in such molecules, N-acetylglucosamine, which becomes the terminal glycan in galactose-deficient IgG, binds mannan-binding lectin. In turn, this may result in local complement activation by complexes composed of tissue auto-antigens or microbial antigens (see below) and corresponding antibodies, all with inflammatory consequences. Importantly, IgA and IgG4 that normally do not activate complement become complement-binding isotypes when glycan moieties are altered (Parekh et al., 1985; Garred et al., 1989; Russell et al., 1997; Tao et al., 1993). Galactose-deficient IgG4 may, in tissues, activate complement, with all the resultant inflammatory consequences. Thus, the anti-inflammatory properties of IgA and IgG4, manifested by their ability to interfere with IgG1-, IgG2-, and IgG3-mediated complement activation, are lost due to their altered glycans (Russell et al., 1989, 1997).
In autoimmune diseases, such as rheumatoid arthritis, tissue components and cartilage collagen Type II, in particular, are the autoantigens recognized by galactose-deficient IgG molecules (Rademacher et al., 1994). We have demonstrated, in our earlier studies, that in periodontal disease, collagens Type I and III are recognized by locally produced IgG antibodies (Hirsch et al., 1988). Therefore, in addition to antigens derived from bacteria associated with periodontal disease, tissue autoantigens may also be targets of IgG antibodies that, in turn, activate, in the form of immune complexes, complement cascade with all its inflammatory sequelae and subsequent tissue damage.
Glycosylation of immunoglobulins is mediated by a large family of enzymes—glycosyltransferases—specific for individual carbohydrates (Schachter and Roseman, 1980). These enzymes catalyze the transfer of intracellularly activated monosaccharides (e.g., UDP-galactose) to suitable acceptors. The decreased levels of glycosyltransferases or their functional deficiency results in reduced or absent glycosylation of the acceptor—in this case, IgG molecules. Differentiation of B-cells is regulated by several cytokines, particularly IL-6 (Fujihashi et al., 1993; Pistoia, 1997), whose increased levels may result in decreased glycosylation. In view of the fact that high levels of IL-6 are produced by mononuclear cells isolated from the gingivae of patients with periodontal disease (Fujihashi et al., 1993), it is possible that, in addition to the stimulation of local B-cell differentiation into immunoglobulin-secreting plasma cells abundant in the inflamed gingiva (Kilian et al., 1989; McGhee et al., 1989; Ogawa et al., 1989), decreased galactosylation of IgG further enhances the inflammatory response.
Overall, these results suggest a possibility that, in periodontal disease, the terminal differentiation of B-cells is affected, resulting in the production of pro-inflammatory galactose-deficient IgG.
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ACKNOWLEDGMENTS
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This work was supported by grant DE 13694 from the National Institutes of Health and by the UAB Center for Research in Oral Biology. We appreciate the assistance of Dr. Jeffcoat in obtaining the gingival tissue samples from periodontal disease patients.
Received for publication December 9, 2004.
Revision received April 15, 2005.
Accepted for publication June 29, 2005.
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REFERENCES
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|---|
- Bodman KB, Sumar N, Mackenzie LE, Isenberg DA, Hay FC, Roitt IM, et al. (1992). Lymphocytes from patients with rheumatoid arthritis produce agalactosylated IgG in vitro. Clin Exp Immunol 88:420–423.[Medline]
[Order article via Infotrieve]
- Fujihashi K, Kono Y, Beagley KW, Yamamoto M, McGhee JR, Mestecky J, et al. (1993). Cytokines and periodontal disease: immunopathological role of interleukins for B cell responses in chronic inflamed gingival tissues. J Periodontol 64:400–406.[Medline]
[Order article via Infotrieve]
- Garred P, Michaelsen TE, Aase A (1989). The IgG subclass pattern of complement activation depends on epitope density and antibody and complement concentration. Scand J Immunol 30:379–382.[CrossRef][Medline]
[Order article via Infotrieve]
- Grbic JT, Singer RE, Jans HH, Celenti RS, Lamster IB (1995). Immunoglobulin isotypes in gingival crevicular fluid: possible protective role of IgA. J Periodontol 66:55–61.[Medline]
[Order article via Infotrieve]
- Hirsch HZ, Tarkowski A, Miller EJ, Gay S, Koopman WJ, Mestecky J (1988). Autoimmunity to collagen in adult periodontal disease. J Oral Pathol 17:456–459.[CrossRef][Medline]
[Order article via Infotrieve]
- Hirsch HZ, Tarkowski A, Koopman WJ, Mestecky J (1989). Local production of IgA- and IgM-rheumatoid factors in adult periodontal disease. J Clin Immunol 9:273–278.[Medline]
[Order article via Infotrieve]
- Kilian M, Ellegaard B, Mestecky J (1989). Distribution of immunoglobulin isotypes including IgA subclasses in adult, juvenile, and rapidly progressive periodontitis. J Clin Periodontol 16:179–184.[Medline]
[Order article via Infotrieve]
- Kinane DF, Lappin DF, Koulouri O, Buckley A (1999). Humoral immune responses in periodontal disease may have mucosal and systemic immune features. Clin Exp Immunol 115:534–541.[CrossRef][Medline]
[Order article via Infotrieve]
- Krapp S, Mimura Y, Jefferis R, Huber R, Sondermann P (2003). Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J Mol Biol 325:979–989.[CrossRef][Medline]
[Order article via Infotrieve]
- Leader KA, Kumpel BM, Hadley AG, Bradley BA (1991). Functional interactions of aglycosylated monoclonal anti-D with Fc gamma RI+ and Fc gamma RIII+ cells. Immunology 72:481–485.[Medline]
[Order article via Infotrieve]
- Malhotra R, Wormald MR, Rudd PM, Fischer PB, Dwek RA, Sim RB (1995). Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nature Med 1:237–243; erratum 1:559.[CrossRef][Medline]
[Order article via Infotrieve]
- McGhee ML, Ogawa T, Pitts AM, Moldoveanu Z, Mestecky J, McGhee JR, et al. (1989). Cellular analysis of functional mononuclear cells from chronically inflamed gingival tissue. Reg Immunol 2:103–110.[Medline]
[Order article via Infotrieve]
- Monteiro RC, Kubagawa H, Cooper MD (1990). Cellular distribution, regulation, and biochemical nature of an Fc alpha receptor in humans. J Exp Med 171:597–613.[Abstract/Free Full Text]
- Moore JS, Wu X, Kulhavy R, Tomana M, Novak J, Moldoveanu Z, et al. (2005). Increased levels of galactose-deficient IgG in sera of HIV-1-infected individuals. AIDS 19:381–389.[Medline]
[Order article via Infotrieve]
- Mullinax F, Mullinax GL (1975). Abnormality of IgG structure in rheumatoid arthritis and systemic lupus erythematosus. Arthritis Rheum 18:417–418.
- Ogawa T, McGhee ML, Moldoveanu Z, Hamada S, Mestecky J, McGhee JR, et al. (1989). Bacteroides-specific IgG and IgA subclass antibody-secreting cells isolated from chronically inflamed gingival tissues. Clin Exp Immunol 76:103–110.[Medline]
[Order article via Infotrieve]
- Parekh RB, Dwek RA, Sutton BJ, Fernandes DL, Leung A, Stanworth D, et al. (1985). Association of rheumatoid arthritis and primary osteoarthritis with changes in glycosylation pattern of total serum IgG. Nature 316:452–457.[CrossRef][Medline]
[Order article via Infotrieve]
- Parekh RB, Roitt IM, Isenberg DA, Dwek RA, Ansell BM, Rademacher TW (1988). Galactosylation of IgG associated oligosaccharides: reduction in patients with adult and juvenile onset rheumatoid arthritis and relation to disease activity. Lancet 2(8592):966–969.[Medline]
[Order article via Infotrieve]
- Pistoia V (1997). Production of cytokines by human B cells in health and disease. Immunol Today 18:343–350.[CrossRef][Medline]
[Order article via Infotrieve]
- Rademacher TW, Williams P, Dwek RA (1994). Agalactosyl glycoforms of IgG autoantibodies are pathogenic. Proc Natl Acad Sci USA 91:6123–6127.[Abstract/Free Full Text]
- Russell MW, Reinholdt J, Kilian M (1989). Anti-inflammatory activity of human IgA antibodies and their Fab
 fragments: inhibition of IgG-mediated complement activation. Eur J Immunol 19:2243–2249.[Medline]
[Order article via Infotrieve] - Russell MW, Sibley DA, Nikolova EB, Tomana M, Mestecky J (1997). IgA antibody as a non-inflammatory regulator of immunity. Biochem Soc Trans 25:466–470.[Medline]
[Order article via Infotrieve]
- Schachter H, Roseman S (1980). Mammalian glycosyltransferases. In: The biochemistry of glycoproteins and proteoglycans. Lennarz WJ, editor. New York: Plenum Press, pp. 85–160.
- Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. (1998). Microbial complexes in subgingival plaque. J Clin Periodontol 25:134–144.[CrossRef][Medline]
[Order article via Infotrieve]
- Tao MH, Smith RI, Morrison SL (1993). Structural features of human immunoglobulin G that determine isotype-specific differences in complement activation. J Exp Med 178:661–667.[Abstract/Free Full Text]
- Tomana M (1996). Glycoproteins in inflammatory bowel disease. In: Glycoproteins in disease. Montreuil J, Vliegenhart JFG, Schachter H, editors. Amsterdam: Elsevier, pp. 291–298.
- Tomana M, Prchal JT, Garner LC, Skalka HW, Barker SA (1984). Gas chromatographic analysis of lens monosaccharides. J Lab Clin Med 103:137–142.[Medline]
[Order article via Infotrieve]
- Tomana M, Schrohenloher RE, Koopman WJ, Alarcon G, Paul WA (1988). Abnormal glycosylation of serum IgG from patients with chronic inflammatory diseases. Arthritis Rheum 31:333–338.[Medline]
[Order article via Infotrieve]
- Tsuchiya N, Endo T, Matsuta K, Yoshinoya S, Takeuchi F, Nagano Y, et al. (1993). Detection of glycosylation abnormality in rheumatoid IgG using N-acetylglucosamine-specific Psathyrella velutina lectin. J Immunol 151:1137–1146.[Abstract]
- Varki A (1993). Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3:97–130.[Abstract/Free Full Text]
Journal of Dental Research, Vol. 84, No. 10,
897-901 (2005)
DOI: 10.1177/154405910508401005
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ABSTRACT
INTRODUCTION