Novel surface markers directed against adult human gallbladder.

Novel cell surface-reactive monoclonal antibodies generated against extrahepatic biliary cells were developed for the isolation and characterization of different cell subsets from normal adult human gallbladder. Eleven antigenically distinct gallbladder subpopulations were isolated by fluorescence-activated cell sorting. They were classified into epithelial, mesenchymal, and pancreatobiliary (PDX1(+)SOX9(+)) subsets based on gene expression profiling. These antigenically distinct human gallbladder cell subsets could potentially also reflect different functional properties in regards to bile physiology, cell renewal and plasticity. Three of the novel monoclonal antibodies differentially labeled archival sections of primary carcinoma of human gallbladder relative to normal tissue. The novel monoclonal antibodies described herein enable the identification and characterization of antigenically diverse cell subsets within adult human gallbladder and are putative tumor biomarkers.

Hnf1b controls pancreas morphogenesis and the generation of Ngn3+ endocrine progenitors.

Heterozygous mutations in the human HNF1B gene are associated with maturity-onset diabetes of the young type 5 (MODY5) and pancreas hypoplasia. In mouse, Hnf1b heterozygous mutants do not exhibit any phenotype, whereas the homozygous deletion in the entire epiblast leads to pancreas agenesis associated with abnormal gut regionalization. Here, we examine the specific role of Hnf1b during pancreas development, using constitutive and inducible conditional inactivation approaches at key developmental stages. Hnf1b early deletion leads to a reduced pool of pancreatic multipotent progenitor cells (MPCs) due to decreased proliferation and increased apoptosis. Lack of Hnf1b either during the first or the secondary transitions is associated with cystic ducts. Ductal cells exhibit aberrant polarity and decreased expression of several cystic disease genes, some of which we identified as novel Hnf1b targets. Notably, we show that Glis3, a transcription factor involved in duct morphogenesis and endocrine cell development, is downstream Hnf1b. In addition, a loss and abnormal differentiation of acinar cells are observed. Strikingly, inactivation of Hnf1b at different time points results in the absence of Ngn3(+) endocrine precursors throughout embryogenesis. We further show that Hnf1b occupies novel Ngn3 putative regulatory sequences in vivo. Thus, Hnf1b plays a crucial role in the regulatory networks that control pancreatic MPC expansion, acinar cell identity, duct morphogenesis and generation of endocrine precursors. Our results uncover an unappreciated requirement of Hnf1b in endocrine cell specification and suggest a mechanistic explanation of diabetes onset in individuals with MODY5.

Roles of cholesterol and bile salts in the pathogenesis of gallbladder hypomotility and inflammation: cholecystitis is not caused by cystic duct obstruction.

BACKGROUND AND PURPOSE: A large number of human and animal studies have challenged the hypothesis that cystic duct obstruction by gallstones causes cholecystitis. These studies suggest that lithogenic bile that can deliver high cholesterol concentrations to the gallbladder wall causes hypomotility and creates a permissive environment that allows normal concentrations of hydrophobic bile salts to inflame the mucosa and impair muscle function inhibiting gallbladder emptying. High concentrations of cholesterol increase its diffusion rates through the gallbladder wall where they are incorporated into the sarcolemmae of muscle cells by caveolin proteins. High caveolar cholesterol levels inhibit tyrosine-induced phosphorylation of caveolin proteins required to transfer receptor-G protein complexes into recycling endosomes. The sequestration of these receptor-G protein complexes in the caveolae results in fewer receptors recycling to the sarcolemmae to be available for agonist binding. Lower internalization and recycling of CCK-1 and other receptors involved in muscle contraction explain gallbladder hypomotility. PGE2 receptors involved in cytoprotection are similarly affected. Cells with a defective cytoprotection failed to inactivate free radicals induced by normal concentrations of hydrophobic bile salts resulting in chronic inflammation that may lead to acute inflammation. Ursodeoxycholic acid salts (URSO) block these bile salts effects thereby preventing the generation of free radicals in muscle cells in vitro and development of cholecystitis in the ligated common bile duct in guinea pigs in vivo. Treatment with URSO improves muscle contraction and reduces the oxidative stress in patients with symptomatic cholesterol gallstones by lowering cholesterol concentrations and blocking the effects of hydrophobic bile salts on gallbladder tissues.

Computational analysis of the flow of bile in human cystic duct.

Computational fluid dynamic (CFD) simulations of the three-dimensional flow structures in realistic cystic ducts have been performed to obtain quantitative readings of the flow parameters to compare with clinical measurements. Resin casts of real patients' cystic ducts lumen that possess representative anatomical features were scanned to obtain three-dimensional flow domains that were used in the numerical analysis. The convoluting nature of the studied cystic ducts resulted in strong secondary flow that contributed towards a dimensionless pressure drop that is four times higher than those of a straight circular tube of an equivalent length and average diameter. The numerical pressure drop results across the cystic duct compared very well with those obtained from clinical observations which indicate that CFD is an appropriate tool to investigate the flow and functions of the biliary system. From the hydrodynamic point of view, the cystic duct lumen seems to serve as a passive resistor that strives to provide a constant amount of resistance to control the flow of bile out of the gallbladder. This is mainly achieved by the coupling of the secondary flow effects and bile rheology to provide flow resistance.

Papillary carcinoma with diffuse papillomatosis of gallbladder and cystic duct.

Biliary papillomatosis and papillary carcinoma are rare tumors of biliary tract; and because of their morphologic similarities, papillomatosis-papillary carcinoma sequel has been proposed. We report an unusual case of polypoid minimally invasive papillary carcinoma located at the junction between cystic and common bile ducts, complicated with biliary papillomatosis of gallbladder and cystic duct, showing focal areas of malignant change. Intrahepatic ducts, hepatic ducts, and distal common bile duct were spared. Both papillomatosis and papillary carcinoma showed areas of high p53 and p21 expression with high proliferative index. Patient is still alive for 4 years without evidence of disease after modified Whipple operation. Possible pathogenetic mechanisms are further discussed.

Muscularis mucosae versus muscularis propria in gallbladder, cystic duct, and common bile duct: smoothelin and desmin immunohistochemical study.

The muscle layer in the cystic duct and common bile duct is not well defined, and it is unresolved whether it represents muscularis mucosae or muscularis propria. Smoothelin is a novel smooth muscle-specific contractile protein expressed only in fully differentiated smooth muscle cells of the muscularis propria and not in proliferative or noncontractile smooth muscle cells of the muscularis mucosae. In this study, we characterize the histologic aspects of the muscle layer in gallbladder, cystic duct, and common bile duct by evaluation of routine histologic sections and the utilization of immunohistochemistry using desmin and smoothelin. Formalin-fixed, paraffin-embedded sections of the gallbladder (15 cases), cystic duct (11 cases), and common bile duct (10 cases) were stained for smoothelin and desmin. Staining intensity was evaluated as weak or strong. The staining pattern score was evaluated as follows: 0 or negative = less than or equal to 5% positivity, +1 or focal = 6% to 10% positivity, +2 or moderate = 11% to 50% positivity, and +3 = greater than 50% muscle cells positivity. With desmin, strong and diffuse (+3) staining was observed in all gallbladder cases (15/15, 100%), highlighting one continuous muscle layer. The muscle layer was discontinuous and interrupted in all cystic duct cases and in most common bile ducts, highlighted by the desmin stain. Smoothelin intensely stained (at least +2) muscle fibers in the gallbladder in 11 (73%) of 15 cases similar to that observed with desmin staining. In contrast, common bile ducts predominantly had absent or weak and focal immunostaining (0 or +1 staining) with smoothelin (7/10, 70%), with only a few cases (3/10, 30%) having +2 staining (no cases with +3). Cystic ducts also showed absent or weak and focal immunostaining with smoothelin, with 5 (44%) of 11 cases showing 2+ immunostaining with smoothelin (no cases with 3+). Based on our findings, we conclude that, in the gallbladder wall, the muscle layer is muscularis propria and there is no muscularis mucosae present. In the cystic duct and common bile duct, only an attenuated and incomplete muscle layer of muscularis mucosae is present; because there is no muscularis propria, there probably is limited contractile function. Differentiating these anatomical muscle structures may be important for the pathologic staging of carcinoma in these organs.

Effects of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid on T2-weighted MRCP.

PURPOSE: Gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) is a recently developed liver-specific contrast agent for magnetic resonance (MR) imaging that is excreted equally via the kidneys and the biliary system. To our knowledge, its effects on T(2)-weighted MR cholangiopancreatography (MRCP) images have not been explored. Acquisition of the hepatobiliary phase is recommended 20 min after administration of Gd-EOB-DTPA. Examination time cannot be extended if the contrast does not take effect on T(2)-weighted MRCP within 20 min after administration. We attempted to assess the change in signal of T(2)-weighted MRCP by excretion of Gd-EOB-DTPA. METHODS: Between March and July 2008, 40 patients (15 women, 25 men; mean age 70.8 years) were examined with abdominal MR imaging. T(2)-weighted MRCP was performed before and 10 and 20 min after administration of Gd-EOB-DTPA. We analyzed signal intensity of the bile duct, gallbladder, cystic duct, and pancreatic duct on MRCP for changes in intensity. RESULTS: T(2)-weighted MRCP 20 min after contrast administration showed loss of signal of the bile duct (intrahepatic bile duct in all cases, upper extrahepatic duct in 36 [90%], middle extrahepatic duct in 33 [85%], and lower extrahepatic duct in 26 [67%]), the gallbladder in 23 cases (72%), and the cystic duct in 25 (64%). This signal change increased with time. We observed no change in signal of the pancreatic duct. CONCLUSION: T(2)-weighted MRCP sequences should not be obtained after administration of Gd-EOB-DTPA because this contrast agent decreases signal intensity of the biliary structure on these images.

Defective development of the gall bladder and cystic duct in Lgr4- hypomorphic mice.

Leucine-rich repeat (LRR) -containing G protein coupled receptor (LGR) family members are characterized by the presence of a seven-transmembrane domain and LRR motifs. We describe a new function for Lgr4 in the development of the gall bladder and cystic duct and in the epithelium-mesenchyme interaction. Lgr4 expression was observed in the gall bladder epithelium when the gall bladder primordium elongated ventrally. Although Lgr4 hypomorphic mutant (Lgr4(Gt/Gt)) embryos developed a normal gall bladder bud at embryonic day (E) 10.25, no further elongation was observed at later stages. At E12.5, the mesenchyme surrounding the gall bladder had completely disappeared in Lgr4(Gt/Gt) embryos, while the gall bladder remained unelongated. Neighboring tissues such as liver and pancreas were unaffected, as revealed by expression of marker genes. This is the first report of a mutant mouse that lacks a gall bladder and cystic duct without affecting the other tissues that derive from the same hepatic diverticulum.

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.

Identification and characterization of specific binding sites for somatostatin in cytosol of bovine cystic duct mucosa.

Specific binding sites for somatostatin have been detected in cytosolic fraction of bovine cystic duct mucosa. At 37 degrees C, the interaction of 125I-Tyr11-somatostatin with cytosolic fraction was rapid, reversible, specific and saturable. At equilibrium, the binding of tracer was competitively inhibited by native peptide in the 1 nM to 2 microM range of concentrations. Scatchard analysis of binding data suggested the presence of two distinct classes of somatostatin binding sites: a class with a high affinity (Kd = 7.8 +/- 0.3 nM) and a low capacity (1.3 +/- 0.3 pmol somatostatin/mg protein) and a class with a low affinity (Kd = 129.1 +/- 2.0 nM) and a high capacity (43.5 +/- 6.7 pmol somatostatin/mg protein). The binding sites were shown to be highly specific for somatostatin since neuropeptides present in cystic duct such as Leu-enkephalin, neurotensin, substance P and vasoactive intestinal peptide did practically not show competition. These findings suggest that somatostatin could contribute to the regulation of the functions of the cystic duct mucosa in physiological and pathological conditions.

Lectin binding sites in the human gallbladder and cystic duct.

A battery of 18 fluorescein isothiocyanate (FITC) labeled lectins was used to histochemically define the features of epithelial cells in the body, neck and the cystic duct of the human gallbladder. Surface epithelium in all three anatomic locations reacted with all lectins, either diffusely or focally, except for lectin type I from Griffonia simplicifolia and type II from Ulex europaeus. No quantitative differences were noted except for the tendency of some lectins to bind more often to the neck and cystic duct epithelium rather than the body and vice versa. In the body the surface epithelium did not differ from cells lining the crypts. On the other hand, glands of the neck and the cystic duct were essentially indistinguishable one from another but differed from the overlying surface epithelium in so far as they reacted with some lectins which were unreactive with surface epithelium and vice versa. Considerable case to case variation in the expression of lectin binding sites in each of three anatomic areas was noted. No consistent differences were noticed between gallbladders removed for cholecystitis--cholelithiasis and those removed for other incidental reasons. We conclude that all epithelial cells in the cholecysto-biliary tract are rich on glyconjugates but the pattern of expression varies depending on the anatomical location and the influence of poorly understood individual determinants.