An Animal Model of Black Pigment Gallstones Caused by Nanobacteria

Abstract

Black pigment gallstones are often founded in gallbladder, but their pathogenesis is unclear. The present study was undertaken to study the role of nanobacteria in pigment gallstone formation in Japanese white rabbits. Nanobacteria were successfully cultured from 3 of 7 cholecystolithiasis patients gallbladder (without acute cholecystitis) bile samples and affirmed by Hoechst 33258 staining and specific immunostaining using monoclonal antibody. Nanobacteria were injected into rabbits' gallbladder. After 2 weeks follow-up, the incidence of black pigment gallstones in rabbits was significantly greater in nanobacteria-injected group (8/10) than that in DMEM-injected group (2/10) and that in hydroxyapatite-injected group (2/10). This study indicates that nanobacteria exists in gallbladder bile of cholelithiasis patients and causes the formation of black pigment gallstones in rabbits. For the first time, we successfully established an animal model of black pigment gallstones caused by nanobacteria. This will be helpful in further delineating the pathogenesis of black pigment gallstones.

The role of calcifying nanoparticles in biology and medicine

“…Bacterioscopic (DNA-specific dyes Hoechst 33258, propidium iodide, PicoGreen, staining after a demineralization is optimal) using scanning and transmission electron microscopy, [16][17][18][19][20][21][22][23][24] von Kossa staining, which is specific to calcium compounds, 16 staining by 2% uranyl acetate (possibly with lead citrate) to detect specific mucus on the hydroxyapatite shell, 1 staining by alizarin red S in the mineralized state, 26 staining by phosphotungstic acid, 16 after the long-term cultivation light microscopy with von Kossa staining is possible to be used for detection. 1,16 Bacteriological (cultivation in DMEM or RPMI-1640 without serum under 37°C for 4-6 weeks after filtration through 0.10-0.22 μm pores), 1,16 replication can be assessed by spectrophotometry (650 nm wavelength).…”

“…Finally, CNPs may cause pathological processes in blood vessels even without a mineral shell; 31 this may indicate the presence of specific immunogenic bacterial protein antigens in their structure, and also, these proteins may have toxin-like activity. The ability of hydroxyapatite crystals to cause the formation of black pigment gallstones after an injection into rabbit gallbladder was several times lower than with the injection of CNPs, 23 which points to additional CNP pathogenic mechanisms and contradicts the hypothesis of an inorganic nature and indicates a biological origin.…”

“…[16][17][18][19][20][21][22][23][24] Furthermore, Hudelist et al 19 and Kumar et al 22 were successful in extracting DNA and RNA from CNPs, indicating the presence of nucleic acids. The suggestion that nucleic acids are only bound to highly charged molecules of mineralo-protein complexes outside and are not synthesized in CNPs 12 can be disproved by the results of Ciftçioglu et al, 17 Khullar et al, 20 Miller et al, 21 and Kumar et al, 22 who observed DNA-specific staining of already demineralized CNPs, which could not have been possible in the case of a simple binding of nucleic acids outside by a mineralo-protein shell.…”

“…47 Of interest, 42.5% of patients had CNP antigens in their bile but not their gallbladder mucosa, which suggests that gallbladder mucosa has the ability to prevent CNP infiltration, but when CNPs invade the mucosa and submucosa, they may form calcifications and cause gallbladder fibrosis. 47 Wang et al 23 observed that CNPs, extracted from the bile of patients with cholecystolithiasis, may cause formation of black pigment gallstones in rabbits after the intravesical injection in 80% of cases (eight out of ten), whilst hydroxyapatite crystals and culture media for CNPs led to the occurrence of such gallstones only in 20% of cases (two out of ten). These authors therefore concluded that CNPs can be a cause of cholecystolithiasis (particularly in the case with black pigment gallstones), and inorganic hydroxyapatite crystals are not equivalent to CNPs in the ability to induce this pathological condition, 23 which is consistent with the results of Schwartz et al, 31 who have also shown in an animal model that hydroxyapatite cannot be fully responsible for pathogenic activity of CNPs.…”

“…Many already performed basic investigations (for instance, those where the presence of DNA in CNPs was proved by positive DNA-specific staining, but DNA was not extracted) 17,18,20,21,23,24 are close to this group, and they can be represented as "first generation" studies. Investigations by Hudelist et al 19 and Kumar et al 22 may be represented as prototypes for further investigation (in another words, they can be represented as "second generation" studies), since CNP nucleic acids were successfully extracted, although accurate sequencing was not conducted.…”