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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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Anti-gal specific antibodies have poor complement mediated killing ability and are unable to induce phagocytosis of opsonized target cells.A) Results from a hemoglobin release assay using serum from control patients and patients with cirrhosis. Compared to normal serum, serum from cirrhosis patients has an over 60% decrease in the capacity to induce complement mediated killing of target rRBCs. For panel A, sample size is: Normal, n = 20 and Cirrhotics, n = 20. As a control, complement alone was used to indicate the level of the alternative pathway (complement alone). In addition, if normal samples were heat inactivated or not treated with serum or complement, no lysis was observed. B) Results from a bactericidal assay using human serum show the growth pattern of bacteria alone (–), bacteria incubated with functional complement (+), bacteria with functional complement and normal human serum (NHS), or bacteria with functional complement and serum from a pool of 20 cirrhosis patients (Cirr). Error bars are indicated. C) Bacteria incubated with serum from a pool of 20 cirrhosis patients, in the presence of functional complement, show a significantly increased survival rate compared to those exposed to normal human serum (P = 0.013). Data normalized to those that did not receive serum addition. D) Results from an opsonization/phagocytosis assay. Bottom panel show target cells opsonized with serum from cirrhosis patients are not phagocytosed by monocytes, while top panel shows target cells opsonized with purchased normal serum are phagocytosed. Black peak represents monocytes alone, blue peak represents monocytes incubated with non-opsonized target cells, and gray filled peak represents opsonized target cells incubated with monocytes.
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pone-0064992-g003: Anti-gal specific antibodies have poor complement mediated killing ability and are unable to induce phagocytosis of opsonized target cells.A) Results from a hemoglobin release assay using serum from control patients and patients with cirrhosis. Compared to normal serum, serum from cirrhosis patients has an over 60% decrease in the capacity to induce complement mediated killing of target rRBCs. For panel A, sample size is: Normal, n = 20 and Cirrhotics, n = 20. As a control, complement alone was used to indicate the level of the alternative pathway (complement alone). In addition, if normal samples were heat inactivated or not treated with serum or complement, no lysis was observed. B) Results from a bactericidal assay using human serum show the growth pattern of bacteria alone (–), bacteria incubated with functional complement (+), bacteria with functional complement and normal human serum (NHS), or bacteria with functional complement and serum from a pool of 20 cirrhosis patients (Cirr). Error bars are indicated. C) Bacteria incubated with serum from a pool of 20 cirrhosis patients, in the presence of functional complement, show a significantly increased survival rate compared to those exposed to normal human serum (P = 0.013). Data normalized to those that did not receive serum addition. D) Results from an opsonization/phagocytosis assay. Bottom panel show target cells opsonized with serum from cirrhosis patients are not phagocytosed by monocytes, while top panel shows target cells opsonized with purchased normal serum are phagocytosed. Black peak represents monocytes alone, blue peak represents monocytes incubated with non-opsonized target cells, and gray filled peak represents opsonized target cells incubated with monocytes.

Mentions: As recent reports have indicated that the ability of IgG to bind and activate complement molecules is dependent upon the type of N-linked glycan attached [22], we examined the ability of anti-gal antibodies from patients with liver cirrhosis to bind and activate exogenously added complement. It is important to note that this assay is designed to determine how well the antibodies with different glycosylation can bind and activate exogenously (experimentally) added complement and is not affected by the levels of complement that may be present (or not) in each individual. Briefly, rabbit red blood cells (rRBC) were incubated with heat inactivated serum (to destroy any residual complement activity) from patients with varying levels of liver disease and supplemented with functional human complement as described previously [23]. Thus, each sample receives an equal amount of complement and the difference in anti-gal immunoglobulin is the only variable in the assay. Cell lysis is measured by the release of hemoglobin. Figure 3A shows the results of the rRBC lysis assay using serum from control patients (n = 20) and from patients with liver cirrhosis (n = 20). Heat inactivated patient serum without the addition of complement had no killing activity (See figure 3A). In contrast, when functional human complement was added to heat inactivated serum, a clear and statistically relevant pattern emerges. As shown in Figure 3A, alpha gal specific immunoglobulin from control patients had similar levels of killing (mean of 100.9% ±13.63%) as compared to the commercially normal human serum (normalized to 100%). In contrast, the level of complement mediated killing was decreased in patients with liver cirrhosis with a mean of 39.8% killing (±29.3%) as compared to the level observed in commercially normal human serum (p<0.0001). As a control, complement alone was used to indicate the level of killing via the alternative pathway (complement alone). In addition, when normal samples were heat inactivated or left untreated with serum or complement (blank), no lysis was observed.


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)

Anti-gal specific antibodies have poor complement mediated killing ability and are unable to induce phagocytosis of opsonized target cells.A) Results from a hemoglobin release assay using serum from control patients and patients with cirrhosis. Compared to normal serum, serum from cirrhosis patients has an over 60% decrease in the capacity to induce complement mediated killing of target rRBCs. For panel A, sample size is: Normal, n = 20 and Cirrhotics, n = 20. As a control, complement alone was used to indicate the level of the alternative pathway (complement alone). In addition, if normal samples were heat inactivated or not treated with serum or complement, no lysis was observed. B) Results from a bactericidal assay using human serum show the growth pattern of bacteria alone (–), bacteria incubated with functional complement (+), bacteria with functional complement and normal human serum (NHS), or bacteria with functional complement and serum from a pool of 20 cirrhosis patients (Cirr). Error bars are indicated. C) Bacteria incubated with serum from a pool of 20 cirrhosis patients, in the presence of functional complement, show a significantly increased survival rate compared to those exposed to normal human serum (P = 0.013). Data normalized to those that did not receive serum addition. D) Results from an opsonization/phagocytosis assay. Bottom panel show target cells opsonized with serum from cirrhosis patients are not phagocytosed by monocytes, while top panel shows target cells opsonized with purchased normal serum are phagocytosed. Black peak represents monocytes alone, blue peak represents monocytes incubated with non-opsonized target cells, and gray filled peak represents opsonized target cells incubated with monocytes.
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Related In: Results  -  Collection

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pone-0064992-g003: Anti-gal specific antibodies have poor complement mediated killing ability and are unable to induce phagocytosis of opsonized target cells.A) Results from a hemoglobin release assay using serum from control patients and patients with cirrhosis. Compared to normal serum, serum from cirrhosis patients has an over 60% decrease in the capacity to induce complement mediated killing of target rRBCs. For panel A, sample size is: Normal, n = 20 and Cirrhotics, n = 20. As a control, complement alone was used to indicate the level of the alternative pathway (complement alone). In addition, if normal samples were heat inactivated or not treated with serum or complement, no lysis was observed. B) Results from a bactericidal assay using human serum show the growth pattern of bacteria alone (–), bacteria incubated with functional complement (+), bacteria with functional complement and normal human serum (NHS), or bacteria with functional complement and serum from a pool of 20 cirrhosis patients (Cirr). Error bars are indicated. C) Bacteria incubated with serum from a pool of 20 cirrhosis patients, in the presence of functional complement, show a significantly increased survival rate compared to those exposed to normal human serum (P = 0.013). Data normalized to those that did not receive serum addition. D) Results from an opsonization/phagocytosis assay. Bottom panel show target cells opsonized with serum from cirrhosis patients are not phagocytosed by monocytes, while top panel shows target cells opsonized with purchased normal serum are phagocytosed. Black peak represents monocytes alone, blue peak represents monocytes incubated with non-opsonized target cells, and gray filled peak represents opsonized target cells incubated with monocytes.
Mentions: As recent reports have indicated that the ability of IgG to bind and activate complement molecules is dependent upon the type of N-linked glycan attached [22], we examined the ability of anti-gal antibodies from patients with liver cirrhosis to bind and activate exogenously added complement. It is important to note that this assay is designed to determine how well the antibodies with different glycosylation can bind and activate exogenously (experimentally) added complement and is not affected by the levels of complement that may be present (or not) in each individual. Briefly, rabbit red blood cells (rRBC) were incubated with heat inactivated serum (to destroy any residual complement activity) from patients with varying levels of liver disease and supplemented with functional human complement as described previously [23]. Thus, each sample receives an equal amount of complement and the difference in anti-gal immunoglobulin is the only variable in the assay. Cell lysis is measured by the release of hemoglobin. Figure 3A shows the results of the rRBC lysis assay using serum from control patients (n = 20) and from patients with liver cirrhosis (n = 20). Heat inactivated patient serum without the addition of complement had no killing activity (See figure 3A). In contrast, when functional human complement was added to heat inactivated serum, a clear and statistically relevant pattern emerges. As shown in Figure 3A, alpha gal specific immunoglobulin from control patients had similar levels of killing (mean of 100.9% ±13.63%) as compared to the commercially normal human serum (normalized to 100%). In contrast, the level of complement mediated killing was decreased in patients with liver cirrhosis with a mean of 39.8% killing (±29.3%) as compared to the level observed in commercially normal human serum (p<0.0001). As a control, complement alone was used to indicate the level of killing via the alternative pathway (complement alone). In addition, when normal samples were heat inactivated or left untreated with serum or complement (blank), no lysis was observed.

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