Schwartz, Maria K.

Biologic nanoparticles (NPs) can be isolated from bovine serum, human kidney stones, and human arterial calcifications. In addition, it is possible to detect both antigen and antibody for NPs in human blood, suggesting that perhaps NPs represent a novel infectious agent participating in human disease. However, it is not known when and how persons might become exposed to NPs and if exposure produces sensitization to subsequent exposures. Experiments were designed to determine whether exposure to nanoparticles would induce an immune response in rabbits that can be detected by a delayed‐type hypersensitivity test. Rabbits were injected intravenously with bovine‐derived NPs (1ml of 1000NTU solution). Plasma was collected prior to injection and on days, 3, 7, 14, 21, 28, 35 post‐injection. At this time, NPs (1ml of 1000NTU solution) were injected subcutaneously in the ear of exposed and naïve rabbits. Areas of injection were observed daily and after three days, blood and ears were collected for analysis. Injection sites showed leukocyte infiltration only in ears of rabbits previously exposed to NPs. These data indicate that biologic NPs induce immune sensitization in rabbits.

Biologic nanoparticles (NPs) isolated from human arterial calcifications and kidney stones form an adherent film on the bottom of flasks when placed in standard cell culture conditions. Since NPs have certain features consistent with microorganisms, we hypothesized that antibiotics and/or RNAse might inhibit this biofilm formation. NPs isolated from calcified human arterial tissue were cultured onto glass coverslips in 24‐well plates containing DMEM with 10% fetal calf serum, plus either no treatment (control), RNase, tetracycline or gentamicin. At 1, 7 and 14 days after inoculation, coverslips were fixed, mounted, and analyzed using a CytoViva darkfield scope and KS‐400 imaging software. In control wells, the percent coverslip covered by biofilm increased by 120% from day 1 to day 14, compared to 47% and 35% in wells treated with RNase and gentamicin, respectively. Wells treated with tetracycline showed a 3% decrease in biofilm by day 14. Since RNase should not enter intact cells, these results indicate that extracellular RNA may play a role in biofilm formation by NPs. Although tetracycline can chelate calcium, it also inhibits ribosomal function, as does gentamicin. Therefore these results also suggest that intact ribosomal function is required for NP‐associated biofilm growth. These experiments suggest that biofilm formation by NPs consists of biological as well as physicochemical processes.

Calcifying nanoparticles (NPs) have been implicated in many diseases including kidney stones and vascular calcification. The chemical composition of these NPs is unknown and advances in understanding their contribution to disease etiology has been hampered by the lack of specific antibodies. Therefore, a monoclonal antibody (MoAb) was developed using decalcified NPs isolated from a human kidney stone as an antigen. After clone screening, a monoclonal antibody was produced that recognized an approximately 125kDa band in the kidney stone NP isolate. Immunoprecipitation of NP lysate followed by proteomics was used to further characterize the protein(s) recognized by the antibody. Consecutive tissue sections from a human atherosclerotic plaque were also stained with the MoAb, which recognized punctuate structures at the media/plaque border and around cholesterol clefts. The staining pattern was similar to that observed with a commercially available antibody in our previous studies. Therefore, we have developed a monoclonal antibody against human kidney stone‐derived NPs that recognizes structures in human atherosclerotic plaques. This MoAB has great potential for use in NP detection in other diverse human tissues and in experimental protocols. Supported by NIH HL88988 and Division of Experimental Surgery, Mayo Clinic.