Study On The Black Pigment Gallstone Formation Caused By Nanobacteria Infection Of Bile In Rabbit

IntroductionGallstone disease is still one of the most common digestive diseases and remains a serious health concern affecting millions throughout the world. There are three major types of gallstones: cholesterol gallstone, brown pigment gallstone and black pigment gallstone. Generally, it is believed that cholesterol gallstones may originate mainly from the disturbances of cholesterol metabolism and the imbalance between biliary pro-nucleating and anti-nucleating proteins. Brown pigment gallstones are caused by bacterial infection. The bacterial β-glucuronidase deconjugates bilirubin diglucuronide, resulting in the release of free bilirubin and glucuronic acid. The former precipitates with calcium ion to form calcium bilirubinate, which is the major component of brown pigment stones. At the same time, bacteria can produce lipidase that lysate lipid into palmitate, and palmitate precipitates with calcium ion to form calcium palmitate, thus brown pigment gallstones formed. Black pigment gallstones are black, amorphous and hard stones. They are believed to consist of polymers of bilirubin, with large amounts of mucin glycoproteins. The pathogenesis of black pigment gallstones is not clear and the stones are more common in patients with haemolytic diseases and cirrhosis.Nanobacteria were discovered in a mammal cell culture by Kajander, a Finnish scientist. They are supper microorganisms and are filterable through 100nm pore-size sterile filtration. They are named as Nanobacterium sanguineum (the shortened form is Nanobacteria), referring to their small size and their blood habitat. Nanobacteria are very archaic super-micro bacteria. They can utilize aminophenol but glucose as energy source. And their metabolism rate is 10,000-fold than that of common bacteria. In disadvantage environment, they can make use of the calcium and phosphorus to form hydroxyapatite biofilms, in which they can escape from damage. Normally, nanobacteria live in the blood serum of mammal animals. Because they are filterable through 100nm pore-size sterile filter, there is nanobacteria contamination in 80%cattle blood products. Nanobacteria contamination is a major cause of the death of culture cells. Normal bacteria methods fail to detect them, but phase contrast microscope and electron microscope provide a better procedure for detecting them. After internalizing byreceptor-mediated endocytosis, nanobacteria are digested incompletely in the lysosomes, then cytotoxic component(s) are released, and the cultured cells lysis (apoptotic). Some studies demonstrate that nanobacteria might be associated with many diseases such as nephrolith, gallstone, prostatitis, polycystic kidney disease and coronary artery disease. It has been confirmed that nanobacteria infection can cause the formation of kidney stone. In addition, the positive rate of nanobacteria infection in gallstone patients is 4 times of that in normal people. And the positive rate of nanobacteria is about 61.3% in the bile of gallstone patients. These data suggest that nanobacteria injection may induce the formation of gallstones. So, we design the study to observe the effects of nanobacteria infection on rabbit gallstone formation. There are two major parts in our study: First, we culture nanobacteria from the bile of gallbladder patients, then observe nanobacteria's biologic characteristic and detect them by the methods mentioned by literatures; Second, we inject nanobacteria into rabbits' gallbladder, and then observe the formation of gallstone, examine the gallbladder, bile and gallstones.Methods1 Seven gallbladder bile samples were obtained from gallstone patients without acute cholecystitis symptoms and without antibiotic therapy before LC. Then, the bile samples were cultured for aerobic bacteria, anaerobic bacteria and L form of bacteria. Also, the bile samples were cultured for nanobacteria under cell culture conditions.2 The sub-cultured nanobacteia were observed by electron microscopy. And then they were analyzed with EDX.3 Nanobacteria were embedded in Epon resin, and ultrathin sections were cut using a diamond knife, then immuno-electron microscopy was carried out by using mouse anti-nanobacteria mAb 8D10 and colloid-gold-goat-anti-mouse mAb.4 A drop of nanobacteria suspension was precipitated, fixed on a sterilized glass slide as small discrete round aggregates, then the nanobacteria slides were made. Hoechst 33258 staining, indirect immunofluorescence staining (IIFS) and VON KOSSA were performed separately as described by literature.5 Sixty rabbits were randomly assigned to following 5 groups. Ten rabbits (test 1 group) wereinjected with nanobacteria solution and were fed with 1.2% cholesterol diet; Ten rabbits (control 1 group) were injected with DMEM solution and were fed with 1.2% cholesterol diet; Twenty rabbits (test 2 group) were injected with nanobacteria solution and were fed with normal diet; Ten rabbits (control 2 group) were injected with DMEM solution and were fed with normal diet; Ten rabbits (control 3 group) were injected with hydroxyapatite solution and were fed with normal diet.6 After anesthetizing the rabbits, surgery was performed with a top-right incision as the protocol described bellow: briefly, sought the gallbladder, extracted gallbladder bile, injected the prepared solution, then conglutinated the hole in the gallbladder using fibrin adhesive and closed the incision at last. All the rabbits were injected with Penicillin intramuscular at a dose of 400,000 IU once a day for three days postoperatively. After being fed in separate cages for 2 weeks, rabbits were euthanized under anesthesia.7 Gallbladder bile was aspirated. Drops of the fresh bile were examined for cholesterol monohydrate crystals using a phase contrast microscope. After centrifuged at 1000 g for 10 min, the supernatant bile was kept for subsequent analysis at -20 °C avoiding light. Bile samples were bleached with white light at 4°C for 12h. The bile was diluted with normal saline. And then lecithin, cholesterol and the total bile acid concentrations were measured by enzymatic methods. The cholesterol saturation index (CSI) was calculated according to the method described by Carey.8 Each gallbladder was opened and gallstones were evaluated. Stone specimens were washed with deionized water until the supernatant became clear, then protected from light and air-dried. The FTIR measurements of gallstones were performed by Potassium Bromide method using an AVATAR 360 FTIR spectrometer.9 The gallbladder samples were fixed in 10% paraformaldehyde, dehydrated, embedded in paraffin, and cut using standard techniques. Deparaffinized sections were stained using standard H&E staining, VON KOSSA staining and indirect immunofluorescence staining (IIFS) methods, respectively.10 The numerical data were shown as means ± SD. Statistically significant differences were assessed by Student's /-test or Chi-square test. Statistical analysis of those results was carriedout using SPSS software. Results1 Results of culture for aerobic bacteria, anaerobic bacteria and L form of bacteria were negative in all of the seven bile samples. The nanobacteria were positive in 3/7 of bile samples.2 Nanobacteria were successfully cultured in DMEM with 10% y-FBS under mammalian cell culture conditions (37°C; 5% CO2). Their growth was very slowly with their doubling time of 3~6 days. After about a two-weeks culture period, tiny particles could be observed making Brownian movement near the bottom of the culture vessel by using phase contrast microscope. After four weeks, many were in clumps and started to attach to the bottom of the culture vessel, and white-colored precipitates could be seen in the cultures by the naked eye. When we increased the concentration of y-FBS to 20%~30%, the biofilm formation of nanobacteria was decreased. While, when they were transferred to serum free DMEM, the hydroxyapatite biofilms formed quickly and hardly. During the cause of culture, the changing of the pH value and concentration of glucose were not obvious. The sub-cultured nanobacteria take the same characteristic as the original.3 Negative staining of nanobacteria revealed 80~350 nm particles. They appeared bacilli or coccoid in shape, either as single particles or predominantly as clusters by attaching to each other with biofilm-like material. Elements of calcium, phosphor, aluminium, silicon and sulfur could be detected by EDX microanalysis, and the calcium-phosphate ratio of nanobacteria was 1.58, which was similar to that of hydroxyapatite.4 Calcified nanobacteria (in biofilms) could be stained as black dots by VON COSSA method. Both the new born nanobacteria and calcified nanobacteria (in biofilms) showed the special reaction with mouse anti-nanobacteria monoclonal antibody 8D10, as showed by immuno-electron microscopy. Stained with Hoechst 33258, nanobacteria showed reaction to the dye by producing the characteristic blue fluorescence, which indicted that DNA exist in nanobacteria.5 During the animal experiments, the health of the rabbits was well and no rabbit died. Therewere no significant differences in the change of the body weight among the five groups.6 Light microscopic examination of the rabbits' gallbladder bile showed no evidence of cholesterol monohydrate crystal in any of the five groups before the injection. While, 2 weeks latter the injection, cholesterol monohydrate crystals could be observed in the two cholesterol diets groups. Also, the concentration of cholesterol and the CSI in these two groups were significantly higher than that in the three normal diets groups.7 Between the two cholesterol diets groups, the gallstone incidence was significantly greater in nanobacteria-injected group (10/10) than that in DMEM-injected group (1/10). And among the three normal diets groups, the gallstone incidence was significantly greater in nanobacteria-injected group (8/10) than that in DMEM-injected group (2/10) and hydroxyapatite-injected group (2/10). No significant difference of the gallstone incidence rates was observed between the two control groups. All gallstones appeared as black amorphous hard stones. All the gallstones appeared similar FTIR spectra, which indicted that mucin glycoproteins, calcium bilirubinate and phosphate are the major components. A little cholesterol but without palmitate were also detected in the stones. These data demonstrate they were black pigment gallstones.8 Neither peritonitis nor acute cholecystitis was observed in all the rabbits when the rabbits were killed in the end of the experiment. H&E staining results showed little pathologic changes in the cell shape and tissue structure of rabbit's cholecyst. VON KOSSA staining results showed small calcified particles distributed in epithelial cells and smooth muscle cells of nanobacteria-injected gallbladders, while DMEM-injected gallbladders did not. Nanobacteria with bright green color were clearly detected in epithelial cells and smooth muscle cells of nanobacteria-injected gallbladders by IIFS method. But they were not detected in DMEM-injected or hydroxyapatite-injected gallbladders.Conclusions1 Nanobacteria do exist in gallstone patients' gallbladder bile. Normally, the patients show no symptoms of acute cholecystitis. Nanobacteria can not be detected by normal bacteria methods.