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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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Immunofluorescence on human coronary plaqes and Immunohistochemistry on human carotid sections with Fab7816-FLAG.a) Representative section stained by Movat’s pentchrome (left panel) showed the morphology of a portion of the coronary plaque tissue (plaque ID-A) displaying slightly damaged media rich in smooth muscle cells. Confocal microscopy (right panels) showed the presence of CD45+ cells (red) labelled by Fab7816-FLAG revealed by MAb M2 anti-FLAG-FITC (green) in the coronary plaque tissue region indicated by symbol (#). DAPI stained the nuclei (blue). b) Immunoperoxidase on carotid plaque samples demonstrated the presence of several cells reacting with Fab7816-FLAG in an area close to the lumen (left panels) as revealed by MAb M2 anti-FLAG-HRP developed with DAB (brown). The signal is absent in a serial section where the Fab7816-FLAG is omitted (ctrl-, right panels). Magnified images in the bottom panels evidence the spindle shape of Fab7816-FLAG+ cells and their localization in between a clusters of altered cells, possibly foam cell. Haematoxylin (blue) stains nuclei. Scale bars indicate the magnification.
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pone-0042283-g004: Immunofluorescence on human coronary plaqes and Immunohistochemistry on human carotid sections with Fab7816-FLAG.a) Representative section stained by Movat’s pentchrome (left panel) showed the morphology of a portion of the coronary plaque tissue (plaque ID-A) displaying slightly damaged media rich in smooth muscle cells. Confocal microscopy (right panels) showed the presence of CD45+ cells (red) labelled by Fab7816-FLAG revealed by MAb M2 anti-FLAG-FITC (green) in the coronary plaque tissue region indicated by symbol (#). DAPI stained the nuclei (blue). b) Immunoperoxidase on carotid plaque samples demonstrated the presence of several cells reacting with Fab7816-FLAG in an area close to the lumen (left panels) as revealed by MAb M2 anti-FLAG-HRP developed with DAB (brown). The signal is absent in a serial section where the Fab7816-FLAG is omitted (ctrl-, right panels). Magnified images in the bottom panels evidence the spindle shape of Fab7816-FLAG+ cells and their localization in between a clusters of altered cells, possibly foam cell. Haematoxylin (blue) stains nuclei. Scale bars indicate the magnification.

Mentions: Confocal microscopy analysis on human coronary plaque sections showed specific interaction of Fab7816-FLAG with cellular antigens present in the tissue from its originary coronary plaque (plaque ID-A) (Figure 4a), as well as with cells localized under the fibrous cap into a carotid plaques (Figure 4b). Anti Hepatitis C virus human monoclonal E8Fab-FLAG [25], used as a negative control, failed to show any specific signal (Figure S1). A subset of TAGLN+/CD45+ cells was recognized by Fab7816-FLAG in human coronary samples (Figure 4a). To further characterize the binding capability of Fab7816-FLAG to atherosclerotic tissue antigens, carotid plaque samples were used (Figure 4b, 5,6 and 7). Specific interaction of Fab7816-FLAG but not of E8Fab-FLAG was observed. Fab7816-FLAG labelled carotid plaque antigens localized in the intima (Figure 4,5,6) in 19 patients out of 31 (table S3), and more rarely in the sub-adventitia (not shown). In our limited series of patients no correlation between the presence of an mmunoreactivity vs. Fab7816-FLAG and the histological features of carotid plaque was found (table S3). Double and multiple staining were performed. Double staining with Fab7816-FLAG and anti- TAGLN antibodies (either monoclonal or polyclonal) demonstrated the existence of Fab7816-FLAG+/TAGLN+, (figures S1, S2, S3 and negative controls figure S4) and of Fab7816-FLAG+/CD68+ cells into the carotid plaque (figure S2). By multiple staining the Fab7816-FLAG+ appeared TAGLN+/CD68+ (Figure 5 and negative samples figure S4), and Coll type I+/CD45+ (Figure 6). Moreover triple labelling showed that Fab7816-FLAG+ cells are TAGLN+/CD45+, thus demonstrating that Fab7816-FLAG recognized in the atherosclerotic lesions a subset of cells of monocytoid origin and with a fibrocyte phenotype. In vitro culture of fibrocytes differentiated from CD14+ circulating monocytes from healthy donors (figure 7a) and displaying the main phenotypic markers (figure S5A,B) demonstrated the presence Fab7816-FLAG specific binding in CD45+ cells (Figure 7b). further confirming the specificity of Fab7816-FLAG for fibrocytes.


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)

Immunofluorescence on human coronary plaqes and Immunohistochemistry on human carotid sections with Fab7816-FLAG.a) Representative section stained by Movat’s pentchrome (left panel) showed the morphology of a portion of the coronary plaque tissue (plaque ID-A) displaying slightly damaged media rich in smooth muscle cells. Confocal microscopy (right panels) showed the presence of CD45+ cells (red) labelled by Fab7816-FLAG revealed by MAb M2 anti-FLAG-FITC (green) in the coronary plaque tissue region indicated by symbol (#). DAPI stained the nuclei (blue). b) Immunoperoxidase on carotid plaque samples demonstrated the presence of several cells reacting with Fab7816-FLAG in an area close to the lumen (left panels) as revealed by MAb M2 anti-FLAG-HRP developed with DAB (brown). The signal is absent in a serial section where the Fab7816-FLAG is omitted (ctrl-, right panels). Magnified images in the bottom panels evidence the spindle shape of Fab7816-FLAG+ cells and their localization in between a clusters of altered cells, possibly foam cell. Haematoxylin (blue) stains nuclei. Scale bars indicate the magnification.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412836&req=5

pone-0042283-g004: Immunofluorescence on human coronary plaqes and Immunohistochemistry on human carotid sections with Fab7816-FLAG.a) Representative section stained by Movat’s pentchrome (left panel) showed the morphology of a portion of the coronary plaque tissue (plaque ID-A) displaying slightly damaged media rich in smooth muscle cells. Confocal microscopy (right panels) showed the presence of CD45+ cells (red) labelled by Fab7816-FLAG revealed by MAb M2 anti-FLAG-FITC (green) in the coronary plaque tissue region indicated by symbol (#). DAPI stained the nuclei (blue). b) Immunoperoxidase on carotid plaque samples demonstrated the presence of several cells reacting with Fab7816-FLAG in an area close to the lumen (left panels) as revealed by MAb M2 anti-FLAG-HRP developed with DAB (brown). The signal is absent in a serial section where the Fab7816-FLAG is omitted (ctrl-, right panels). Magnified images in the bottom panels evidence the spindle shape of Fab7816-FLAG+ cells and their localization in between a clusters of altered cells, possibly foam cell. Haematoxylin (blue) stains nuclei. Scale bars indicate the magnification.
Mentions: Confocal microscopy analysis on human coronary plaque sections showed specific interaction of Fab7816-FLAG with cellular antigens present in the tissue from its originary coronary plaque (plaque ID-A) (Figure 4a), as well as with cells localized under the fibrous cap into a carotid plaques (Figure 4b). Anti Hepatitis C virus human monoclonal E8Fab-FLAG [25], used as a negative control, failed to show any specific signal (Figure S1). A subset of TAGLN+/CD45+ cells was recognized by Fab7816-FLAG in human coronary samples (Figure 4a). To further characterize the binding capability of Fab7816-FLAG to atherosclerotic tissue antigens, carotid plaque samples were used (Figure 4b, 5,6 and 7). Specific interaction of Fab7816-FLAG but not of E8Fab-FLAG was observed. Fab7816-FLAG labelled carotid plaque antigens localized in the intima (Figure 4,5,6) in 19 patients out of 31 (table S3), and more rarely in the sub-adventitia (not shown). In our limited series of patients no correlation between the presence of an mmunoreactivity vs. Fab7816-FLAG and the histological features of carotid plaque was found (table S3). Double and multiple staining were performed. Double staining with Fab7816-FLAG and anti- TAGLN antibodies (either monoclonal or polyclonal) demonstrated the existence of Fab7816-FLAG+/TAGLN+, (figures S1, S2, S3 and negative controls figure S4) and of Fab7816-FLAG+/CD68+ cells into the carotid plaque (figure S2). By multiple staining the Fab7816-FLAG+ appeared TAGLN+/CD68+ (Figure 5 and negative samples figure S4), and Coll type I+/CD45+ (Figure 6). Moreover triple labelling showed that Fab7816-FLAG+ cells are TAGLN+/CD45+, thus demonstrating that Fab7816-FLAG recognized in the atherosclerotic lesions a subset of cells of monocytoid origin and with a fibrocyte phenotype. In vitro culture of fibrocytes differentiated from CD14+ circulating monocytes from healthy donors (figure 7a) and displaying the main phenotypic markers (figure S5A,B) demonstrated the presence Fab7816-FLAG specific binding in CD45+ cells (Figure 7b). further confirming the specificity of Fab7816-FLAG for fibrocytes.

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