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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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Western Blotting of bacterial lysates and on bacterial OMPs with Fab 7816.A) Western blotting of bacterial lysates with Fab7816. 1 µg of each bacterial lysate was loaded in each lane. Fab 7816 clearly reacted with Proteus mirabilis and Klebsiella pneumoniae lysates. B) Western blotting on induced/non induced BL21(DE3) bacterial cells transformed with pET28b vector expressing OmpK36. Two pET vectors were constructed: the NcoI/XhoI wild type OmpK36 vector or the HIS-tagged Ompk36 BamHI/XhoI vector. In both cases Fab 7816 recognized only IPTG induced bacteria.
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pone-0042283-g008: Western Blotting of bacterial lysates and on bacterial OMPs with Fab 7816.A) Western blotting of bacterial lysates with Fab7816. 1 µg of each bacterial lysate was loaded in each lane. Fab 7816 clearly reacted with Proteus mirabilis and Klebsiella pneumoniae lysates. B) Western blotting on induced/non induced BL21(DE3) bacterial cells transformed with pET28b vector expressing OmpK36. Two pET vectors were constructed: the NcoI/XhoI wild type OmpK36 vector or the HIS-tagged Ompk36 BamHI/XhoI vector. In both cases Fab 7816 recognized only IPTG induced bacteria.

Mentions: Purified Fab7816 was also tested for its capability to bind and to identify potentially exogenous antigens by screening bacterial lysates in western blot (WB). Fab7816 recognized a protein band of about 35 kD in Proteus mirabilis and Klebsiella pneumoniae lysates. (Figure 8) Among the proteins of Klebsiella pneumoniae with this molecular weight, we cloned and purified the major outer membrane protein (OmpK36). OmpK36 was expressed in E. coli BL21(DE3) strain (Figure 8A) and Fab7816 staining of induced E. coli BL21(DE3) demonstrated specific binding of Fab7816 to OmpK36. Binding experiments on cloned and purified outer membrane protein F (OmpF) of P.mirabilis confirmed that Fab 7816 is able to recognize also the homologous target in P.mirabilis lysate (OmpF) (Figure 8b).


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)

Western Blotting of bacterial lysates and on bacterial OMPs with Fab 7816.A) Western blotting of bacterial lysates with Fab7816. 1 µg of each bacterial lysate was loaded in each lane. Fab 7816 clearly reacted with Proteus mirabilis and Klebsiella pneumoniae lysates. B) Western blotting on induced/non induced BL21(DE3) bacterial cells transformed with pET28b vector expressing OmpK36. Two pET vectors were constructed: the NcoI/XhoI wild type OmpK36 vector or the HIS-tagged Ompk36 BamHI/XhoI vector. In both cases Fab 7816 recognized only IPTG induced bacteria.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042283-g008: Western Blotting of bacterial lysates and on bacterial OMPs with Fab 7816.A) Western blotting of bacterial lysates with Fab7816. 1 µg of each bacterial lysate was loaded in each lane. Fab 7816 clearly reacted with Proteus mirabilis and Klebsiella pneumoniae lysates. B) Western blotting on induced/non induced BL21(DE3) bacterial cells transformed with pET28b vector expressing OmpK36. Two pET vectors were constructed: the NcoI/XhoI wild type OmpK36 vector or the HIS-tagged Ompk36 BamHI/XhoI vector. In both cases Fab 7816 recognized only IPTG induced bacteria.
Mentions: Purified Fab7816 was also tested for its capability to bind and to identify potentially exogenous antigens by screening bacterial lysates in western blot (WB). Fab7816 recognized a protein band of about 35 kD in Proteus mirabilis and Klebsiella pneumoniae lysates. (Figure 8) Among the proteins of Klebsiella pneumoniae with this molecular weight, we cloned and purified the major outer membrane protein (OmpK36). OmpK36 was expressed in E. coli BL21(DE3) strain (Figure 8A) and Fab7816 staining of induced E. coli BL21(DE3) demonstrated specific binding of Fab7816 to OmpK36. Binding experiments on cloned and purified outer membrane protein F (OmpF) of P.mirabilis confirmed that Fab 7816 is able to recognize also the homologous target in P.mirabilis lysate (OmpF) (Figure 8b).

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