Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves

Abstract

Mechanisms mediating vascular calcification remain incompletely understood. Nanometer scale objects hypothesized to be a type of bacteria (nanobacteria) are associated with calcified geological specimens, human kidney stones, and psammona bodies in ovarian cancer. Experiments were designed to evaluate human vascular tissue for the presence of similar nanometer-scale objects. Calcified human aneurysms ( n = 8), carotid plaques ( n = 2), femoral arterial plaques ( n = 2), and cardiac valves ( n = 2) and noncalcified aneurysms from patients with bicuspid aortic valve disease ( n = 2) were collected as surgical waste from the Heart Hospital of Austin, Austin, Texas, and Mayo Clinic, Rochester, Minnesota. Whole mounts or adjacent sections from each specimen were examined by electron microscopy, stained for calcium phosphate, or stained with a commercially available antibody (8D10). Filtered (0.2 μm) homogenates of aneurysms were cultured and costained with 8D10 antibody followed by PicoGreen to detect DNA or incubated with [3H]uridine. Staining for calcium phosphate was heterogeneously distributed within all calcified tissues. Immunological staining with 8D10 was also heterogeneously distributed in areas with and without calcium phosphate. Analysis of areas with positive immunostaining identified spheres ranging in size from 30 to 100 nm with a spectral pattern of calcium and phosphorus (high-energy dispersive spectroscopy). Nanosized particles cultured from calcified but not from noncalcified aneurysms were recognized by a DNA-specific dye and incorporated radiolabeled uridine, and, after decalcification, they appeared via electron microscopy to contain cell walls. Therefore, nanometer-scale particles similar to those described as nanobacteria isolated from geological specimens and human kidney stones can be visualized in and cultured from calcified human cardiovascular tissue.

Purported nanobacteria in human blood as calcium carbonate nanoparticles

“…It should be noted that most studies using 8D10 and 5/3 antibodies have actually focused on ELISA (5) and tissue or cell culture immunostaining (7). Miller et al (10) showed that the antibody 8D10 reacts against one single band of 50 kDa from cell pellets prepared under reducing and denaturing conditions comparable with our experiments.…”

“…We were also intrigued by earlier publications describing the use of monoclonal antibodies specific for nanobacteria, now commercially available through Nanobac Oy (3,10). These antibodies were obtained from splenocyte hybridoma cells of mice that were immunized with purified nanobacteria of bovine origin and fused with the myeloma strain P3 ϫ 63-Ag-8.653 (10).…”

“…More importantly, they showed that HAP could be propagated similarly to a bacterial subculture; they also reattributed the published nanobacteria 16S rDNA sequence to an occasional contaminant of PCR reagents. However, the nanobacteria field has largely ignored these important findings and, in recent years, has continued to host a flurry of research (10,20) and review papers (11,24) that purport to find a pathogenic role for nanobacteria in various disease processes.…”

An Overview on Clinical Implications of Nanobacteria

“…The Nanobacterium sanguineum is the main causative organism which is engulfed by phagocytes in the body and due to its low virulence it bypasses the body defences and starts its multiplication. 37 The most peculiar feature of these bacteria is formation of slime-like biofilm, which may contribute to the 'stickiness' of the calcifying nanoparticle complex (cell-like vesicles and associated apatite precipitates) in human tissue. 37 Biofilm links together the cell-like nanoparticles and provides anchoring to fixed structures such as arterial walls or kidney tissue.…”

“…37 The most peculiar feature of these bacteria is formation of slime-like biofilm, which may contribute to the 'stickiness' of the calcifying nanoparticle complex (cell-like vesicles and associated apatite precipitates) in human tissue. 37 Biofilm links together the cell-like nanoparticles and provides anchoring to fixed structures such as arterial walls or kidney tissue. It shields the bacterium against host defences helps in colonization and provides an inherent shelter for the growth of nanobacteria.…”