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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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Identification of putative natural self antigen.A) Bidimensional electrophoresis gel stained with colloidal Coomassie Brilliant Blue. Sixty five µg of purified carotid atherosclerotic plaque proteins were loaded on strip pH 3-10NL, 7 cm. The 2nd dimension was carried out using 12.5% SDS-PAGE. B) 2D electrophoresis Western Blotting. Similarly, 70 µg of proteins were loaded on strip pH 3-10NL, 7 cm. The 2nd dimension was carried out using 12.5% SDS-PAGE. After transfer, proteins were probed with Fab7816-FLAG (10 µg/mL). Protein spots of interest (red box) were excised from the gel, digested with trypsin and analysed by MALDI-ToF mass spectrometry. In both spots human TAGLN was identified with almost complete sequence coverage.
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pone-0042283-g003: Identification of putative natural self antigen.A) Bidimensional electrophoresis gel stained with colloidal Coomassie Brilliant Blue. Sixty five µg of purified carotid atherosclerotic plaque proteins were loaded on strip pH 3-10NL, 7 cm. The 2nd dimension was carried out using 12.5% SDS-PAGE. B) 2D electrophoresis Western Blotting. Similarly, 70 µg of proteins were loaded on strip pH 3-10NL, 7 cm. The 2nd dimension was carried out using 12.5% SDS-PAGE. After transfer, proteins were probed with Fab7816-FLAG (10 µg/mL). Protein spots of interest (red box) were excised from the gel, digested with trypsin and analysed by MALDI-ToF mass spectrometry. In both spots human TAGLN was identified with almost complete sequence coverage.

Mentions: The protein present in human atherosclerotic carotid preparations that was recognized by Fab7816 was identified by Bidimensional electrophoresis (2DE) of carotid plaque lysates (Figure 3) and by mass spectrometry analysis on proteins sampled from two distinct spots in the 2D gel. These experiment unequivocally identified TAGLN as the putative antigen recognized by Fab7816 with almost complete protein coverage of TAGLN in mass spectrometry analysis. 2D gel showed that possibly more than one TAGLN isoforms, with different Ip, were recognized by Fab7816. In fact in both spots of the 2D gel, TAGLN was recognized by Fab7816 (Figure 3B).


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)

Identification of putative natural self antigen.A) Bidimensional electrophoresis gel stained with colloidal Coomassie Brilliant Blue. Sixty five µg of purified carotid atherosclerotic plaque proteins were loaded on strip pH 3-10NL, 7 cm. The 2nd dimension was carried out using 12.5% SDS-PAGE. B) 2D electrophoresis Western Blotting. Similarly, 70 µg of proteins were loaded on strip pH 3-10NL, 7 cm. The 2nd dimension was carried out using 12.5% SDS-PAGE. After transfer, proteins were probed with Fab7816-FLAG (10 µg/mL). Protein spots of interest (red box) were excised from the gel, digested with trypsin and analysed by MALDI-ToF mass spectrometry. In both spots human TAGLN was identified with almost complete sequence coverage.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042283-g003: Identification of putative natural self antigen.A) Bidimensional electrophoresis gel stained with colloidal Coomassie Brilliant Blue. Sixty five µg of purified carotid atherosclerotic plaque proteins were loaded on strip pH 3-10NL, 7 cm. The 2nd dimension was carried out using 12.5% SDS-PAGE. B) 2D electrophoresis Western Blotting. Similarly, 70 µg of proteins were loaded on strip pH 3-10NL, 7 cm. The 2nd dimension was carried out using 12.5% SDS-PAGE. After transfer, proteins were probed with Fab7816-FLAG (10 µg/mL). Protein spots of interest (red box) were excised from the gel, digested with trypsin and analysed by MALDI-ToF mass spectrometry. In both spots human TAGLN was identified with almost complete sequence coverage.
Mentions: The protein present in human atherosclerotic carotid preparations that was recognized by Fab7816 was identified by Bidimensional electrophoresis (2DE) of carotid plaque lysates (Figure 3) and by mass spectrometry analysis on proteins sampled from two distinct spots in the 2D gel. These experiment unequivocally identified TAGLN as the putative antigen recognized by Fab7816 with almost complete protein coverage of TAGLN in mass spectrometry analysis. 2D gel showed that possibly more than one TAGLN isoforms, with different Ip, were recognized by Fab7816. In fact in both spots of the 2D gel, TAGLN was recognized by Fab7816 (Figure 3B).

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