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Altered functionality of anti-bacterial antibodies in patients with chronic hepatitis C virus infection.

Lamontagne A, Long RE, Comunale MA, Hafner J, Rodemich-Betesh L, Wang M, Marrero J, Di Bisceglie AM, Block T, Mehta A - PLoS ONE (2013)

Bottom Line: Since the alpha gal epitope is found on gut enterobacteria, it has been hypothesized that anti-gal antibodies are generated as a result of increased bacterial exposure in patients with liver disease.In addition, markers of microbial exposure were determined.In an analysis of serum from more than 100 patients with liver disease, we have shown that those with increased levels of this modified anti-gal antibody had increased levels of markers of bacterial exposure.

View Article: PubMed Central - PubMed

Affiliation: Drexel University College of Medicine, and Department of Microbiology and Immunology and Drexel Institute for Biotechnology and Virology, Doylestown, Pennsylvania, USA. anne.lamontagne@drexelmed.edu

ABSTRACT

Background: Using comparative glycoproteomics, we have previously identified a glycoprotein that is altered in both amount and glycosylation as a function of liver cirrhosis. The altered glycoprotein is an agalactosylated (G0) immunoglobulin G molecule (IgG) that recognizes the heterophilic alpha-gal epitope. Since the alpha gal epitope is found on gut enterobacteria, it has been hypothesized that anti-gal antibodies are generated as a result of increased bacterial exposure in patients with liver disease.

Methods: The N-linked glycosylation of anti-gal IgG molecules from patients with fibrosis and cirrhosis was determined and the effector function of anti-bacterial antibodies from over 100 patients examined. In addition, markers of microbial exposure were determined.

Results: Surprisingly, the subset of agalactosylated anti-gal antibodies described here, was impaired in their ability to mediate complement mediated lysis and inhibited the complement-mediated destruction of common gut bacteria. In an analysis of serum from more than 100 patients with liver disease, we have shown that those with increased levels of this modified anti-gal antibody had increased levels of markers of bacterial exposure.

Conclusions: Anti-gal antibodies in patients with liver cirrhosis were reduced in their ability to mediate complement mediated lysis of target cells. As bacterial infection is a major complication in patients with cirrhosis and bacterial products such as LPS are thought to play a major role in the development and progression of liver fibrosis, this finding has many clinical implications in the etiology, prognosis and treatment of liver disease.

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Increased lectin reactive anti-gal IgG from patients with increasing levels of liver fibrosis.A) Compared to commercially purchased normal human sera, patients with limited liver fibrosis (stage 1–2) have a 3.5 (±1.4) fold increase in lectin reactive anti-gal IgG (LRAGG). More advanced (Stage 5–6) fibrosis patients have a 7.9 (±2.4) fold increase in LRAGG. The fold increase in LRAGG is statistically significant (P<0.001). When serum from more advanced fibrotic patients is used on plates coated with HSA alone and not HSA-alpha-gal, no signal is observed (HSA lane). B) The level of anti-gal IgA, IgM and IgG bound to target rabbit red blood cells as a function of liver fibrosis. There is a statistically significant increase in anti-gal IgA from commercially purchased normal human serum to serum from patients with advanced fibrosis (P = 0.0048); Anti-gal IgA also significantly increases from limited to advanced fibrosis (P = 0.0133). Anti-gal IgM significantly increases from control to limited fibrosis (P = 0.02) and from control to advanced fibrosis (P = 0.002); there is also a significant increase in anti-gal IgM from limited to advanced fibrosis (P = 0.0018). Anti-gal IgG is significantly elevated in advanced fibrosis compared to control (P = 0.0075) and also from limited to advanced fibrosis (P = 0.0217). 50 mM of BSA-alpha-gal can prevent binding of antibodies to RBCC. See text for more details. For panels A & B, samples size is Normal, n = 21; Stage 1–2, n = 22; and Cirrhosis, n = 39.
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pone-0064992-g002: Increased lectin reactive anti-gal IgG from patients with increasing levels of liver fibrosis.A) Compared to commercially purchased normal human sera, patients with limited liver fibrosis (stage 1–2) have a 3.5 (±1.4) fold increase in lectin reactive anti-gal IgG (LRAGG). More advanced (Stage 5–6) fibrosis patients have a 7.9 (±2.4) fold increase in LRAGG. The fold increase in LRAGG is statistically significant (P<0.001). When serum from more advanced fibrotic patients is used on plates coated with HSA alone and not HSA-alpha-gal, no signal is observed (HSA lane). B) The level of anti-gal IgA, IgM and IgG bound to target rabbit red blood cells as a function of liver fibrosis. There is a statistically significant increase in anti-gal IgA from commercially purchased normal human serum to serum from patients with advanced fibrosis (P = 0.0048); Anti-gal IgA also significantly increases from limited to advanced fibrosis (P = 0.0133). Anti-gal IgM significantly increases from control to limited fibrosis (P = 0.02) and from control to advanced fibrosis (P = 0.002); there is also a significant increase in anti-gal IgM from limited to advanced fibrosis (P = 0.0018). Anti-gal IgG is significantly elevated in advanced fibrosis compared to control (P = 0.0075) and also from limited to advanced fibrosis (P = 0.0217). 50 mM of BSA-alpha-gal can prevent binding of antibodies to RBCC. See text for more details. For panels A & B, samples size is Normal, n = 21; Stage 1–2, n = 22; and Cirrhosis, n = 39.

Mentions: As shown in Figure 2A this plate-based lectin assay is able to detect changes in glycosylation with the progression of liver disease. Specifically, patients with mild fibrosis (Stage 1–2 ISHAK; n = 21) have a 3.5 (±1.4) fold increase in lectin-reactive anti-gal immunoglobulin (LRAGG) over normal serum (n = 20), while patients with more advanced fibrosis (ISHAK 5–6) n = 39) have a 7.9 (±2.4) increase in LRAGG. In Figure 2B we show that, concurrent with the change in glycosylation, there is a change in the amount of anti-gal immunoglobulin in patients with liver disease. Figure 2B shows an increase in levels of anti-gal IgA, IgM and IgG bound to target rabbit red blood cells as a function of liver fibrosis. As shown, there is 3 fold increase in anti-gal IgG, in patients with Stage 1–2 fibrosis as compared to normal serum and close to a five fold increase in anti-gal IgA, IgG and IgM in patients with Stage 5–6 fibrosis (cirrhosis). To determine the specificity of the interaction we demonstrated that the binding of antibodies to rRBC could be completely abolished by the addition of the sugar Gal α-1-3Galß1-(3)4-GlcNAc, which brought immunoglobulin binding of antibodies to background levels (labeled as BSA-alpha-gal). In contrast addition of the disaccharide lactose had little effect on IgG binding to the rRBC (data not shown).


Altered functionality of anti-bacterial antibodies in patients with chronic hepatitis C virus infection.

Lamontagne A, Long RE, Comunale MA, Hafner J, Rodemich-Betesh L, Wang M, Marrero J, Di Bisceglie AM, Block T, Mehta A - PLoS ONE (2013)

Increased lectin reactive anti-gal IgG from patients with increasing levels of liver fibrosis.A) Compared to commercially purchased normal human sera, patients with limited liver fibrosis (stage 1–2) have a 3.5 (±1.4) fold increase in lectin reactive anti-gal IgG (LRAGG). More advanced (Stage 5–6) fibrosis patients have a 7.9 (±2.4) fold increase in LRAGG. The fold increase in LRAGG is statistically significant (P<0.001). When serum from more advanced fibrotic patients is used on plates coated with HSA alone and not HSA-alpha-gal, no signal is observed (HSA lane). B) The level of anti-gal IgA, IgM and IgG bound to target rabbit red blood cells as a function of liver fibrosis. There is a statistically significant increase in anti-gal IgA from commercially purchased normal human serum to serum from patients with advanced fibrosis (P = 0.0048); Anti-gal IgA also significantly increases from limited to advanced fibrosis (P = 0.0133). Anti-gal IgM significantly increases from control to limited fibrosis (P = 0.02) and from control to advanced fibrosis (P = 0.002); there is also a significant increase in anti-gal IgM from limited to advanced fibrosis (P = 0.0018). Anti-gal IgG is significantly elevated in advanced fibrosis compared to control (P = 0.0075) and also from limited to advanced fibrosis (P = 0.0217). 50 mM of BSA-alpha-gal can prevent binding of antibodies to RBCC. See text for more details. For panels A & B, samples size is Normal, n = 21; Stage 1–2, n = 22; and Cirrhosis, n = 39.
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Related In: Results  -  Collection

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pone-0064992-g002: Increased lectin reactive anti-gal IgG from patients with increasing levels of liver fibrosis.A) Compared to commercially purchased normal human sera, patients with limited liver fibrosis (stage 1–2) have a 3.5 (±1.4) fold increase in lectin reactive anti-gal IgG (LRAGG). More advanced (Stage 5–6) fibrosis patients have a 7.9 (±2.4) fold increase in LRAGG. The fold increase in LRAGG is statistically significant (P<0.001). When serum from more advanced fibrotic patients is used on plates coated with HSA alone and not HSA-alpha-gal, no signal is observed (HSA lane). B) The level of anti-gal IgA, IgM and IgG bound to target rabbit red blood cells as a function of liver fibrosis. There is a statistically significant increase in anti-gal IgA from commercially purchased normal human serum to serum from patients with advanced fibrosis (P = 0.0048); Anti-gal IgA also significantly increases from limited to advanced fibrosis (P = 0.0133). Anti-gal IgM significantly increases from control to limited fibrosis (P = 0.02) and from control to advanced fibrosis (P = 0.002); there is also a significant increase in anti-gal IgM from limited to advanced fibrosis (P = 0.0018). Anti-gal IgG is significantly elevated in advanced fibrosis compared to control (P = 0.0075) and also from limited to advanced fibrosis (P = 0.0217). 50 mM of BSA-alpha-gal can prevent binding of antibodies to RBCC. See text for more details. For panels A & B, samples size is Normal, n = 21; Stage 1–2, n = 22; and Cirrhosis, n = 39.
Mentions: As shown in Figure 2A this plate-based lectin assay is able to detect changes in glycosylation with the progression of liver disease. Specifically, patients with mild fibrosis (Stage 1–2 ISHAK; n = 21) have a 3.5 (±1.4) fold increase in lectin-reactive anti-gal immunoglobulin (LRAGG) over normal serum (n = 20), while patients with more advanced fibrosis (ISHAK 5–6) n = 39) have a 7.9 (±2.4) increase in LRAGG. In Figure 2B we show that, concurrent with the change in glycosylation, there is a change in the amount of anti-gal immunoglobulin in patients with liver disease. Figure 2B shows an increase in levels of anti-gal IgA, IgM and IgG bound to target rabbit red blood cells as a function of liver fibrosis. As shown, there is 3 fold increase in anti-gal IgG, in patients with Stage 1–2 fibrosis as compared to normal serum and close to a five fold increase in anti-gal IgA, IgG and IgM in patients with Stage 5–6 fibrosis (cirrhosis). To determine the specificity of the interaction we demonstrated that the binding of antibodies to rRBC could be completely abolished by the addition of the sugar Gal α-1-3Galß1-(3)4-GlcNAc, which brought immunoglobulin binding of antibodies to background levels (labeled as BSA-alpha-gal). In contrast addition of the disaccharide lactose had little effect on IgG binding to the rRBC (data not shown).

Bottom Line: Since the alpha gal epitope is found on gut enterobacteria, it has been hypothesized that anti-gal antibodies are generated as a result of increased bacterial exposure in patients with liver disease.In addition, markers of microbial exposure were determined.In an analysis of serum from more than 100 patients with liver disease, we have shown that those with increased levels of this modified anti-gal antibody had increased levels of markers of bacterial exposure.

View Article: PubMed Central - PubMed

Affiliation: Drexel University College of Medicine, and Department of Microbiology and Immunology and Drexel Institute for Biotechnology and Virology, Doylestown, Pennsylvania, USA. anne.lamontagne@drexelmed.edu

ABSTRACT

Background: Using comparative glycoproteomics, we have previously identified a glycoprotein that is altered in both amount and glycosylation as a function of liver cirrhosis. The altered glycoprotein is an agalactosylated (G0) immunoglobulin G molecule (IgG) that recognizes the heterophilic alpha-gal epitope. Since the alpha gal epitope is found on gut enterobacteria, it has been hypothesized that anti-gal antibodies are generated as a result of increased bacterial exposure in patients with liver disease.

Methods: The N-linked glycosylation of anti-gal IgG molecules from patients with fibrosis and cirrhosis was determined and the effector function of anti-bacterial antibodies from over 100 patients examined. In addition, markers of microbial exposure were determined.

Results: Surprisingly, the subset of agalactosylated anti-gal antibodies described here, was impaired in their ability to mediate complement mediated lysis and inhibited the complement-mediated destruction of common gut bacteria. In an analysis of serum from more than 100 patients with liver disease, we have shown that those with increased levels of this modified anti-gal antibody had increased levels of markers of bacterial exposure.

Conclusions: Anti-gal antibodies in patients with liver cirrhosis were reduced in their ability to mediate complement mediated lysis of target cells. As bacterial infection is a major complication in patients with cirrhosis and bacterial products such as LPS are thought to play a major role in the development and progression of liver fibrosis, this finding has many clinical implications in the etiology, prognosis and treatment of liver disease.

Show MeSH
Related in: MedlinePlus