[Ultrastructural changes of hepatocyte fibrogenesis in cholelithiasis].

OBJECTIVE: To explore the ultrastructural changes of hepatocyte fibrogenesis in cholelithiasis in biliary tract. METHODS: l0 liver biopsies were taken from the patients suffered from gallstone and choledocholithiasis during surgical treatment and the ultrastructural changes were observed under electromicroscope. RESULTS: There were plentiful collagenous microfibrils (CMFs) grown within some hepatocytes. These CMFs distributed locally or diffusely in cytoplasm even extended into nucleus. In 7 cases numerous megamitochondrias appeared in several hepatocytes, the inclusions mimicking fibrils could be frequently seen and grew beyond the envelope. Furthermore, typical CMFs could be seen in the large microbodies, and several vesicular or cystic structures similar as fibroblast were presented in marginal areas of the hepatocytes. CONCLUSIONS: We deduce that the fibrosed hepatocytes may be remained and take part in the hyperplasia of hepatic fibrous tissue.

Rokitansky-Aschoff sinuses of the gallbladder are associated with black pigment gallstone formation: a scanning electron microscopy study.

Rokitansky-Aschoff sinuses are the result of hyperplasia and herniation of epithelial cells through the fibromuscular layer of the gallbladder wall and are usually referred to as adenomyomatosis. The role of this study is to demonstrate that Rokitansky-Aschoff sinuses of the gallbladder are a risk factor for the formation of black pigment gallstones. A total of 179 removed gallbladders, were hystologically examined. Sixty-four of the 179 consecutive cholecystomized patients had typical adenomyomatosis. Thirty-eight of the 64 patients with adenomyomatosis had black pigment gallstones, alone (n=22) or in association with single (n=12) or multiple (n=4) cholesterol gallstones in the same gallbladder. Twelve of these patients did not have the typical risk factors for black stones (hemolysis, cirrhoses, gastrectomy, etc). Gallstones were examined by infrared spectroscopy and X-ray diffractometry. In addition, in a subset of 14 patients, the gallstones and the gallbladder wall were examined by scanning electron microscopy. At least in the initial phases of formation, Rokitansky-Aschoff sinuses were found close to small intraparietal vessels and sometimes they contained black pigment microstones. After the fourth to fifth decades of life, black gallstones can be found in the Rokitansky-Aschoff sinuses and in the main gallbladder lumen. Black pigment gallstones can form in Rokitansky-Aschoff sinuses of the gallbladder in absence of the typical risk factors for bilirubin suprasaturation of bile.

Structural analysis of gallstones with thin-section petrographic microscopy: a study of 100 gallstones from Taiwanese patients.

Cholecystolithiasis is a common disease, making cholecystectomy a commonly performed surgical procedure. The gross appearance of gallstones differs from case to case. To classify gallstones on the basis of their structure, we randomly collected gallstones from 100 (64 of them women) of 3,289 patients who underwent cholecystectomy at our hospital. The stones were grossly classified into five major types: pure-cholesterol stones, combination stones, mixed stones, black stones, and calcium bilirubinate stones. We then used thin-section petrographic microscopic study (TSPMS) to inspect each stone under a polarizing microscope. Final classification of the stone depended on TSPMS findings. Of the 100 patients, 35 had pure-cholesterol stones, 12 had combination stones, 17 had mixed stones, 25 had black stones, and 8 had calcium bilirubinate stones; the stones from 3 patients could not be classified on TSPMS. Accurate structural classification of gallstones could be made by gross inspection with confirmation by TSPMS, a useful method of classifying gallstones.

Biofilm formation and interaction with the surfaces of gallstones by Salmonella spp.

Salmonellae can exist in an asymptomatic carrier state in the human gallbladder. Individuals with gallstones are more likely to become typhoid carriers, and antibiotic treatments are often ineffectual against Salmonella enterica serovar Typhi in carriers with gallstones. Therefore, we hypothesized that Salmonella spp. form biofilms on the surfaces of gallstones, where the bacteria are protected from high concentrations of bile and antibiotics. A number of methods were utilized to examine biofilm formation on human gallstones and glass coverslips in vitro, including confocal, light, and scanning electron microscopy. In our assays, salmonellae formed full biofilms on the surfaces of gallstones within 14 days and appeared to excrete an exopolysaccharide layer that bound them to the surfaces and to other bacteria. Efficient biofilm formation on gallstones was dependent upon the presence of bile, as a biofilm did not form on gallstones within 14 days in Luria-Bertani broth alone. The biofilms formed by a Salmonella enterica serovar Typhi Vi antigen mutant, as well as strains with mutations in genes that eliminate production of four different fimbriae, were indistinguishable from the biofilms formed by the parents. Mutants with an incomplete O-antigen, mutants that were nonmotile, and mutants deficient in quorum sensing were unable to develop complete biofilms. In addition, there appeared to be selectivity in salmonella binding to the gallstone surface that did not depend on the topology or surface architecture. These studies should aid in the understanding of the Salmonella carrier state, an important but underresearched area of typhoid fever pathogenesis. If the basis of carrier development can be understood, it may be possible to identify effective strategies to prevent or treat this chronic infection.

Milk of calcium cholelithiasis in children.

Milk of calcium bile is uncommon and occurs mainly in the adult population. The authors report on 2 children, each having a distinct clinical history and presentation, and each with milk of calcium bile/calculi possessing differing chemical composition and highly notable gross morphology. J Pediatr Surg 36:644-647.

Ultrasonic parameters and relationship between compressive strength, microstructure of gall bladder stones.

Patients with symptomatic stones are at a great risk for complications and these complications are a major cause of morbidity. The gall bladder stones may have a complex structure and variable composition. In the present investigation stones have been grouped into three categories namely cholesterol, bilirubinate and mixed, and a correlation between the surface structure, ultrasonic parameters and compressive strength is estimated. A double-probe through-transmission technique was used for the ultrasonic parameters study, a universal testing instrument for hardness and a scanning electron microscope (SEM) for microstructure study. Gall bladder stones of mixed type with higher ultrasonic velocity, less attenuation and higher crushing strength were found to be more difficult to break in comparison to other types of stones. SEM of mixed type stones showed rough surface as compared to bilirubinate and cholesterol stones. The results obtained as well as the relationship might be useful in the design of a focussed ultrasonic 0lithotripter.

[Gallstones and pancreatic necrosis in acute pancreatitis]. / Galdesten og pancreasnekroser ved akut pancreatitis.

The purpose of this study was to investigate the occurrence of gallstones and the severity of acute pancreatitis in a Danish population, and in severe cases, the relation between pancreatic necrosis and gallstones. We used Ranson's prognostic scoring system to measure the severity of acute pancreatitis, ultrasonography for detecting biliary stones, and computed tomography or laparotomy for detecting pancreatic necrosis. During a two year period, we registered 101 patients. Approximately 70% were mild cases, and we found 18 patients with and 53 patients without gallstones. Among patients with severe cases, we observed 14 with and 16 without gallstones. There was a significantly higher proportion of gallstones in severe cases. Eight of 30 patients with severe pancreatitis had pancreatic necrosis, but we found no relation between the occurrence of necrosis and gallstones. We conclude that patients with gallstone related pancreatitis have more severe disease than patients without stones, but the complication of pancreatic necrosis is not related to gallstones.

Cryoelectron microscopy of a nucleating model bile in vitreous ice: formation of primordial vesicles.

Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23-31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10-20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation.

Investigation of cholesterol, bilirubin, and protein distribution in human gallstones by color cathodoluminescence scanning electron microscopy and transmission electron microscopy.

The application of color cathodoluminescent scanning electron microscopy (CCL-SEM) for qualitative luminescence analysis of cholesterol, bilirubin, and protein in human gallastones was demonstrated. Images of these deposits (cholesterol, bilirubin, and protein) were formed in real colors (blue-cholesterol, red, orange-bilirubin, yellow, green-protein) in accordance with the cathodoluminescent spectrum for each control material. The other method described for transmission electron microscopy (TEM) of ultrathin sections provides more detailed characterization of the ultrastructure of cholesterol-containing regions and their spatial interrelations with bilirubin-containing regions. Using CCL-SEM combined with TEM permits the receipt of more complete information about the chemical composition and ultrastructure of gallstones and may lead to more effective understanding of the pathogenesis of cholesterol cholelithiasis.

Fine structure of cholesterolosis in the human gallbladder and the mechanism of lipid accumulation.

Gallbladders with cholesterolosis removed surgically for cholelithiasis were studied by light and electron microscopy as well as by cytochemical methods to demonstrate the presence of free cholesterol in the epithelial cells. Lipid droplets were found not only in the submucosa, but also in the infranuclear cytoplasm of epithelial cells. These contained well developed mitochondria and an agranular endoplasmic reticulum. Macrophages were often present between the epithelial cells and the submucosa, and protruded numerous processes, which also contained well developed cell organelles, abundant lysosomes and lipid droplets. With the excessive lipid deposition, macrophages were filled with lipid droplets and became foam cells. In the epithelial cells, many reaction precipitates occurred after digitonin treatment and some of them were observed in the endoplasmic reticulum. It is suggested, therefore, that free cholesterol is absorbed by epithelial cells and thereafter becomes esterified in the endoplasmic reticulum and thus appears as lipid droplets. Lipid droplets synthesized in the epithelial cells may then be released into the intercellular space, and phagocytosed there by macrophages. It is thus suggested that macrophages filled with lipid droplets may become too large and rigid to pass through the endothelium of lymph vessels, and those large "foam cells" may cause the destruction of lymph vessels. Those sequential events should eventually advance the accumulation of foam cells in the submucosa.

Ultrastructural aspects of human cystic duct epithelium as a result of cholelithiasis and cholesterolosis.

Although there is a large body of data on the gallbladder and the importance of the cystic duct in surgical procedures, there is insufficient data regarding the morphology of the human cystic duct. In the present study, transmission electron microscopic (TEM) and scanning electron microscopic (SEM) survey of several surgical and autopsy cystic ducts in cholelithiasis and cholesterolosis is reported. In cholelithiasis, similar to gallbladder epithelium, the cystic duct epithelial cells display minor-to-severe alterations of the epithelial surface accompanied by variable erosion of the epithelium. Areas of intact surface epithelium demonstrate microvilli-covered cells coated by a rich glycocalyx and mucous production. In other areas, apical excrescences are associated with mucus hyperproduction and secretory events. Lipoid bodies are also present in many cells and especially in many of the cells' subliminal apical areas. In cholesterolosis, mucous secretory granules appear dilated, fatty deposits are infrequent, and peculiar intracellular cholesterol deposits can be detected in the apical and subapical region of cells and around condensed mitochondria. Following elective cholecystectomies, predominantly in association with cholelithiasis, eroded areas were detected; therefore, it appears that the action of intraluminal calculi may be a principal causative factor in discrete epithelial erosions of the cystic duct. Intraluminal calculi/ debris, along with the alteration of mucus, cell sloughing, and a decreased pool of bile acids and motility may participate in the gallstone nucleation process. The peculiar cholesterol inclusions may also play a role in that nucleating process.

Ground squirrel model for cholelithiasis: role of epithelial glycoproteins.

The cholesterol-fed Richardson's ground squirrel (Spermophilus richardsonii) has proven to be an effective animal model in which to study factors that influence cholesterol gallstone formation and associated alterations in the gallbladder epithelium. Ground squirrels of either sex, fed a 2% cholesterol-enriched diet, exhibit cholesterol monohydrate crystal precipitation within 24 hours and macroscopically visible cholesterol stones by 3 weeks. Data on bile chemistry, biliary cholesterol precipitation, and various mucosal alterations occurring prior to, during, and after stone formation were collected using sampling intervals from 6 hours to 20 weeks on the diet. The results indicate that mucin hypersecretion appears to be more closely related to the initiation of nucleation than does either bile calcium of pH. Mucus hypersecretion begins within 18 hours of diet initiation and continues throughout the 20 week experimental period. Apical excrescences became more common and were larger in size during the early stages of cholelithiasis. Administration of aspirin during the experimental period demonstrated an inhibition of mucin synthesis and release. Gallstones were not formed in these aspirin-treated animals. A lectin-binding panel for 10 epithelial glycoprotein-related sugars indicated the mucin secreted by the gallbladder epithelium of 7 day experimental animals differed from that of controls. The most obvious difference was the abolition of WGA binding in the experimental animals, suggesting an absence of sialic acid expression in the mucin during the lithogenic process. Ultrastructural histochemistry indicated that both sulphomucin and sialomucin were present in the secretory granules and within the surface mucus layer of both experimental and control animals. Experimental animals, however, exhibited a significant predominance for sulphomucin. This pattern varies from that typically seen in other regions of the gastrointestinal tract where sialomucins predominate during pathologic processes.

Cholelithiasis induced in the Syrian hamster: evidence for an intramucinous nucleating process and down regulation of cholesterol 7 alpha-hydroxylase (CYP7) gene by medroxyprogesterone.

This report reviews previously published studies from our laboratory and shows some recent morphological data obtained with scanning and transmission electron microscopy regarding gallstone formation and alteration of the gallbladder epithelium in the Syrian hamster model. Both male and female hamsters were treated with female sex steroids (estradiol alone, estradiol and medroxyprogesterone, medroxyprogesterone alone) during one month. The results show that the Syrian hamster is a good model to study bile changes, gallbladder structure changes, including gallstone formation, and the regulation of cholesterol metabolism at the molecular level. Arguments in favor of this animal model are presented and, during gallstone formation, epithelial cell changes, anionic mucus secretion, and formation of gallbladder luminal deposits can be demonstrated. Recent molecular biology observations related to the effect of female sex steroids on liver cholesterol 7 alpha-hydroxylase (CYP7) gene suggest that progestin alone or primed by estrogen down regulates CYP7 transcription and activity. In addition, progesterone in cell culture systems has been shown to enhance intracellular accumulation of free cholesterol by increasing its uptake and synthesis and by decreasing its esterification by inhibiting the activity of acylcoenzyme A: cholesterol acyltransferase. Non-esterified cholesterol is free to migrate to the extracellular spaces and may contribute to nucleation within the bile. It is suggested that these effects of progesterone on cholesterol metabolism combined with the CYP7 gene down regulation, physical changes in the mucus and the hypomotility of the gallbladder and biliary ducts result in hypersaturation of cholesterol in the bile which favors gallstone formation.

Ultrastructure of cholesterol gallstones as observed by electron microscopy after freeze-fracturing.

The ultrastructure of cholesterol gallstones (mixed type) was studied in detail for the first time under the transmission electron microscope after freeze-fracturing. Gallstones consisted essentially of cholesterol crystals, some impurities, and fluid. In accord with the theoretical 3.4 nm bilayered structure of cholesterol crystals, 3-4 nm periodicity of crystal layering was observed. However, gallstone cholesterol crystals were not perfect and often showed structural defects. Between crystals, complete edge-to-surface, edge-to-edge and surface-to-surface adhesions, and overall block-like aggregations were found. These may represent the structural basis for the stability of cholesterol crystal aggregation. The easy breakdown of cholesterol gallstones by extracorporeal shock wave lithotripsy is discussed in relation to their ultrastructure.

Chemolysis of gallbladder debris left over after contact litholysis with methyl tert-butyl ether.

Rapid and safe gallbladder clearance from residual, post-MTBE stone debris is believed to be absolutely necessary to reduce stone recurrence after contact litholysis. Because the clearing effect of prokinetic agents is considered an uncertain postdissolution trial, we investigated by in vitro experiments whether and to what extent debris from various cholesterol and "mixed stones" could be removed by direct (topical) chemolysis. Debris from radiolucent cholesterol stones could be dissolved very easily, using the aqueous solvent S-01, composed of EDTA-2Na (1-2%), lauryl sulfobetaine-12 (0.1 M), and 0.1 M sodium carbonate/boric acid buffer, pH 9,5. Its dissolution capacity (DC) was 8.06 +/- 2.3 mg debris/ml and its dissolution efficacy (DE) was 16.2 +/- 4.6 mg debris/ml/hr. Debris from mixed, slightly to moderately calcified stones needed another treatment with S-05, composed of sodium citrate (0.25 M), lauryl sulfobetaine-12 (0.01 M), and citric acid. The initial pH was 5.2. The DC of S-05 ranged from 1.61 +/- 1.1 (debris enriched with Ca-phosphate) to 3.94 +/- 1.3 mg/ml (debris enriched with Ca-carbonate). Stones which did not respond immediately to MTBE because of a thin rim of inorganic or/and organic Ca salts could be made ready for MTBE litholysis by pretreatment with S-01 or S-05 or with a combination of both solvents. Debris containing large portions of biliary mucus could be dissolved most effectively by successive application of S-01 and S-06 (2 M urea).

Deconjugation of bilirubin accelerates coprecipitation of cholesterol, fatty acids, and mucin in human bile--in vitro study.

To examine the initial step of brown pigment gallstone formation, sterile human gallbladder bile samples were incubated with or without beta-glucuronidase in vitro. Enhanced bilirubin deconjugation achieved by adding beta-glucuronidase significantly accelerated the formation of a precipitate that contained bilirubin (28.2 +/- 3.8% of dry weight), cholesterol (14.3 +/- 5.2%), free fatty acids (12.0 +/- 1.3%), and glycoprotein (10.0 +/- 6.7%). Both the composition and scanning electron microscopic appearance of the precipitate were similar to these features in brown pigment gallstones. The cholesterol saturation index and nucleation time in the supernatant did not change with various incubation periods. The weight ratios of bilirubin to cholesterol in the precipitates correlated with those in bile (r = 0.76; P = 0.017). Gel chromatography of the precipitate showed high molecular weight glycoprotein to be the major constituent. Bilirubin, cholesterol, fatty acids, and mucin were found to coprecipitate in accordance with bilirubin deconjugation, which process may play an important role in an early stage of the formation of brown pigment gallstones.

Role of bacterial biofilms in the chemotherapy of cholangitis with brown pigment stones.

The purpose of the present study was to clarify the role of biofilms in the chemotherapy of cholangitis. In 1 case of recurrent cholangitis with intrahepatic stones, the patient underwent right hepatectomy after chemotherapy was performed for 42 weeks. In the other case, chemotherapy including ciprofloxacin was performed during an attack of recurrent cholangitis. Repeated culture of bile specimens yielded negative results, whereas culture of the choledochal stone yielded Pseudomonas aeruginosa. We observed an acute transient IgM response to a component of the biofilm of P. aeruginosa (alginate) in this case. In both cases, electron microscopy revealed viable bacteria covered with biofilm in the component of brown pigment stones. It was concluded that biofilm is a factor of drug resistance.

Backscattered electron imaging and energy-dispersive X-ray microanalysis studies of evidence for calcium salt heterogeneity in fifteen gallstones from an elderly human.

We examined 15 variably-sized gallstones, taken from an elderly male, by backscattered electron imaging and energy-dispersive X-ray microanalysis to learn the structural and distribution patterns of gallstone calcium (Ca-) salts. Of the 13 cholesterol-rich stones, nine stones had peripheral concentric layers of Ca-carbonate, whereas 2 stones had peripheral layers of Ca-phosphate. No Ca-salts were detected from 2 cholesterol-rich stones. The 2 stones containing Ca-phosphate had no Ca-salt cores, whereas the stones containing Ca-carbonate were separated into 3 different types: two stones with a Ca-carbonate core, four stones with several Ca-bilirubinate cores of glass-like structure, and 3 stones lacking Ca-salt cores. A closer view of the Ca-salt layers, which may be occasionally coexistent with Ca-bilirubinate, mainly showed either laminate deposits or numerous globules with a few laminae. Of the 2 cholesterol-poor stones, one had dispersed particles mainly of Ca-phosphate, and the other had loosely dispersed particles with small amounts of Ca-phosphate, bilirubinate, and/or palmitate. Some relationship between the size and Ca-salt species of these gallstones was suggested. Gallstones collected from the same individual showed a considerable heterogeneity of Ca-salts.

Interaction of human gallbladder mucin with calcium hydroxyapatite: binding studies and the effect on hydroxyapatite formation.

Calcium hydroxyapatite (HAP) crystals formed in vitro in the presence of polymeric human gallbladder mucin (1.0 mg/mL) were smaller (0.75 +/- 0.39 microns) than control crystals (7.86 +/- 2.76 microns), but the mucin did not affect the kinetics of crystal formation or alter the amount of mineral phase present at equilibrium. In contrast, glycopeptide subunits produced by proteolysis of the native mucin had no effect on HAP crystal size. Both native mucin and glycopeptides bound to mature HAP crystals, but the glycopeptides were much more readily displaced by phosphate ions. Therefore, in experiments where HAP was being formed, the phosphate ions inhibited the interaction of glycopeptides with the nascent HAP. These results indicate that gallbladder mucin may modulate HAP formation in vivo, and that this ability may be altered during pathological states, such as neutrophil infiltration or bacterial colonization, that may cause the release of proteinases capable of digesting mucin.