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Cross-reacting antibacterial auto-antibodies are produced within coronary atherosclerotic plaques of acute coronary syndrome patients.

Canducci F, Saita D, Foglieni C, Piscopiello MR, Chiesa R, Colombo A, Cianflone D, Maseri A, Clementi M, Burioni R - PLoS ONE (2012)

Bottom Line: In vitro fibrocytes obtained by differentiating CD14+ cells isolated from peripheral blood mononuclear cells also interacted with Fab7816, thus supporting the hypothesis of a specific recognition of fibrocytes into the atherosclerotic lesions.Interestingly, the same antibody, cross-reacted with the outer membrane proteins of Proteus mirabilis and Klebsiella pneumoniae (and possibly with homologous proteins of other enterobacteriaceae present in the microbiota).From all the other three libraries, we were able to clone, by immunoaffinity selection, human monoclonal antibodies cross-reacting with bacterial outer membrane proteins and with transgelin.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology and Virology, Ospedale San Raffaele, Milan, Italy. Canducci.filippo@hsr.it

ABSTRACT
Coronary atherosclerosis, the main condition predisposing to acute myocardial infarction, has an inflammatory component caused by stimuli that are yet unknown. We molecularly investigated the nature of the immune response within human coronary lesion in four coronary plaques obtained by endoluminal atherectomy from four patients. We constructed phage-display libraries containing the IgG1/kappa antibody fragments produced by B-lymphocytes present in each plaque. By immunoaffinity, we selected from these libraries a monoclonal antibody, arbitrarily named Fab7816, able to react both with coronary and carotid atherosclerotic tissue samples. We also demonstrated by confocal microscopy that this monoclonal antibody recognized human transgelin type 1, a cytoskeleton protein involved in atherogenesis, and that it co-localized with fibrocyte-like cells transgelin+, CD68+, CD45+ in human sections of coronary and carotid plaques. In vitro fibrocytes obtained by differentiating CD14+ cells isolated from peripheral blood mononuclear cells also interacted with Fab7816, thus supporting the hypothesis of a specific recognition of fibrocytes into the atherosclerotic lesions. Interestingly, the same antibody, cross-reacted with the outer membrane proteins of Proteus mirabilis and Klebsiella pneumoniae (and possibly with homologous proteins of other enterobacteriaceae present in the microbiota). From all the other three libraries, we were able to clone, by immunoaffinity selection, human monoclonal antibodies cross-reacting with bacterial outer membrane proteins and with transgelin. These findings demonstrated that in human atherosclerotic plaques a local cross-reactive immune response takes place.

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Cross reactivity of representative Fabs from all patients with TAGLN and OMPs.A) WB of purified human TAGLN (400 ng) with all representative Fabs (10 µg/mL). Unrelated human e8Fab-FLAGwas used as negative control. Anti-MYC-tag (C-terminal tag) and commercial anti-TAGLN were used as positive controls. While the commercial anti-TAGLN recognize selectively only one form of TAGLN, cloned human Fabs recognized both forms. B) WB of OmpK36 (500 ng) with all representative Fabs (10 ng/mL). C) ELISA with all representative Fabs on bacterial OMPs. Reactivity against bovine serum albumin (BSA) used as blocking antigen, is also shown.
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pone-0042283-g010: Cross reactivity of representative Fabs from all patients with TAGLN and OMPs.A) WB of purified human TAGLN (400 ng) with all representative Fabs (10 µg/mL). Unrelated human e8Fab-FLAGwas used as negative control. Anti-MYC-tag (C-terminal tag) and commercial anti-TAGLN were used as positive controls. While the commercial anti-TAGLN recognize selectively only one form of TAGLN, cloned human Fabs recognized both forms. B) WB of OmpK36 (500 ng) with all representative Fabs (10 ng/mL). C) ELISA with all representative Fabs on bacterial OMPs. Reactivity against bovine serum albumin (BSA) used as blocking antigen, is also shown.

Mentions: All representative Fabs displayed a strong binding and cross-reacting capability against both OMPs and human TAGLN (Figure 10). In particular, ELISA and WB experiments, conducted using purified commercial human TAGLN preparations showed that all Fabs bind to TAGLN in WB (FIG. 10a) but not in ELISA (Figure S6) as initially observed with Fab7816 (figure S6). Since electrophoresis was conducted under denaturing conditions, it is very likely that Fabs recognized a linear epitope of the TAGLN protein that is poorly exposed in these ELISA conditions, whilst fully accessible in WB under denaturing conditions (see also Figure 3b). Reactivity was also verified with Fab7816 expressed and purified as full IgG (IgG7816) (protocol S1) (figure 10).


Cross-reacting antibacterial auto-antibodies are produced within coronary atherosclerotic plaques of acute coronary syndrome patients.

Canducci F, Saita D, Foglieni C, Piscopiello MR, Chiesa R, Colombo A, Cianflone D, Maseri A, Clementi M, Burioni R - PLoS ONE (2012)

Cross reactivity of representative Fabs from all patients with TAGLN and OMPs.A) WB of purified human TAGLN (400 ng) with all representative Fabs (10 µg/mL). Unrelated human e8Fab-FLAGwas used as negative control. Anti-MYC-tag (C-terminal tag) and commercial anti-TAGLN were used as positive controls. While the commercial anti-TAGLN recognize selectively only one form of TAGLN, cloned human Fabs recognized both forms. B) WB of OmpK36 (500 ng) with all representative Fabs (10 ng/mL). C) ELISA with all representative Fabs on bacterial OMPs. Reactivity against bovine serum albumin (BSA) used as blocking antigen, is also shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412836&req=5

pone-0042283-g010: Cross reactivity of representative Fabs from all patients with TAGLN and OMPs.A) WB of purified human TAGLN (400 ng) with all representative Fabs (10 µg/mL). Unrelated human e8Fab-FLAGwas used as negative control. Anti-MYC-tag (C-terminal tag) and commercial anti-TAGLN were used as positive controls. While the commercial anti-TAGLN recognize selectively only one form of TAGLN, cloned human Fabs recognized both forms. B) WB of OmpK36 (500 ng) with all representative Fabs (10 ng/mL). C) ELISA with all representative Fabs on bacterial OMPs. Reactivity against bovine serum albumin (BSA) used as blocking antigen, is also shown.
Mentions: All representative Fabs displayed a strong binding and cross-reacting capability against both OMPs and human TAGLN (Figure 10). In particular, ELISA and WB experiments, conducted using purified commercial human TAGLN preparations showed that all Fabs bind to TAGLN in WB (FIG. 10a) but not in ELISA (Figure S6) as initially observed with Fab7816 (figure S6). Since electrophoresis was conducted under denaturing conditions, it is very likely that Fabs recognized a linear epitope of the TAGLN protein that is poorly exposed in these ELISA conditions, whilst fully accessible in WB under denaturing conditions (see also Figure 3b). Reactivity was also verified with Fab7816 expressed and purified as full IgG (IgG7816) (protocol S1) (figure 10).

Bottom Line: In vitro fibrocytes obtained by differentiating CD14+ cells isolated from peripheral blood mononuclear cells also interacted with Fab7816, thus supporting the hypothesis of a specific recognition of fibrocytes into the atherosclerotic lesions.Interestingly, the same antibody, cross-reacted with the outer membrane proteins of Proteus mirabilis and Klebsiella pneumoniae (and possibly with homologous proteins of other enterobacteriaceae present in the microbiota).From all the other three libraries, we were able to clone, by immunoaffinity selection, human monoclonal antibodies cross-reacting with bacterial outer membrane proteins and with transgelin.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology and Virology, Ospedale San Raffaele, Milan, Italy. Canducci.filippo@hsr.it

ABSTRACT
Coronary atherosclerosis, the main condition predisposing to acute myocardial infarction, has an inflammatory component caused by stimuli that are yet unknown. We molecularly investigated the nature of the immune response within human coronary lesion in four coronary plaques obtained by endoluminal atherectomy from four patients. We constructed phage-display libraries containing the IgG1/kappa antibody fragments produced by B-lymphocytes present in each plaque. By immunoaffinity, we selected from these libraries a monoclonal antibody, arbitrarily named Fab7816, able to react both with coronary and carotid atherosclerotic tissue samples. We also demonstrated by confocal microscopy that this monoclonal antibody recognized human transgelin type 1, a cytoskeleton protein involved in atherogenesis, and that it co-localized with fibrocyte-like cells transgelin+, CD68+, CD45+ in human sections of coronary and carotid plaques. In vitro fibrocytes obtained by differentiating CD14+ cells isolated from peripheral blood mononuclear cells also interacted with Fab7816, thus supporting the hypothesis of a specific recognition of fibrocytes into the atherosclerotic lesions. Interestingly, the same antibody, cross-reacted with the outer membrane proteins of Proteus mirabilis and Klebsiella pneumoniae (and possibly with homologous proteins of other enterobacteriaceae present in the microbiota). From all the other three libraries, we were able to clone, by immunoaffinity selection, human monoclonal antibodies cross-reacting with bacterial outer membrane proteins and with transgelin. These findings demonstrated that in human atherosclerotic plaques a local cross-reactive immune response takes place.

Show MeSH
Related in: MedlinePlus