Liver X receptor ß regulates bile volume and the expression of aquaporins and cystic fibrosis transmembrane conductance regulator in the gallbladder.

The gallbladder is considered an important organ in maintaining digestive and metabolic homeostasis. Given that therapeutic options for gallbladder diseases are often limited to cholecystectomy, understanding gallbladder pathophysiology is essential in developing novel therapeutic strategies. Since liver X receptor ß (LXRß), an oxysterol-activated transcription factor, is strongly expressed in gallbladder cholangiocytes, the aim was to investigate LXRß physiological function in the gallbladder. Thus, we studied the gallbladders of WT and LXRß-/- male mice using immunohistochemistry, electron microscopy, qRT-PCR, bile duct cannulation, bile and blood biochemistry, and duodenal pH measurements. LXRß-/- mice presented a large gallbladder bile volume with high duodenal mRNA levels of the vasoactive intestinal polypeptide (VIP), a strong mediator of gallbladder relaxation. LXRß-/- gallbladders showed low mRNA and protein expression of Aquaporin-1, Aquaporin-8, and cystic fibrosis transmembrane conductance regulator (CFTR). A cystic fibrosis-resembling phenotype was evident in the liver showing high serum cholestatic markers and the presence of reactive cholangiocytes. For LXRß being a transcription factor, we identified eight putative binding sites of LXR on the promoter and enhancer of the Cftr gene, suggesting Cftr as a novel LXRß regulated gene. In conclusion, LXRß was recognized as a regulator of gallbladder bile volume through multiple mechanisms involving CFTR and aquaporins.NEW & NOTEWORTHY This report reveals a novel and specific role of the nuclear receptor liver X receptor ß (LXRß) in controlling biliary tree pathophysiology. LXRß-/- mice have high gallbladder bile volume and are affected by a cholangiopathy that resembles cystic fibrosis. We found LXRß to regulate the expression of both aquaporins water channels and the cystic fibrosis transmembrane conductance regulator. This opens a new field in biliary tree pathophysiology, enlightening a possible transcription factor controlling CFTR expression.

A cholecystic extracellular matrix-based hybrid hydrogel for skeletal muscle tissue engineering.

Tailoring the properties of extracellular matrix (ECM) based hydrogels by conjugating with synthetic polymers is an emerging method for designing hybridhydrogels for a wide range of tissue engineering applications. In this study, poly(ethylene glycol) diacrylate (PEGDA), a synthetic polymer at variable concentrations (ranging from 0.2 to 2% wt/vol) was conjugated with porcine cholecyst derived ECM (C-ECM) (1% wt/vol) and prepared a biosynthetic hydrogel having enhanced physico-mechanical properties, as required for skeletal muscle tissue engineering. The C-ECM was functionalized with acrylate groups using activated N-hydroxysuccinimide ester-based chemistry and then conjugated with PEGDA via free-radical polymerization in presence of ammonium persulfate and ascorbic acid. The physicochemical characteristics of the hydrogels were evaluated by Fourier transform infrared spectroscopy and environmental scanning electron microscopy. Further, the hydrogel properties were studied by evaluating rheology, swelling, gelation time, percentage gel fraction, in vitro degradation, and mechanical strength. Biocompatibility of the gel formulations were assessed using the C2C12 skeletal myoblast cells. The hydrogel formulations containing 0.2 and 0.5% wt/vol of PEGDA were non-cytotoxic and found suitable for growth and proliferation of skeletal myoblasts. The study demonstrated a method for modulating the properties of ECM hydrogels through conjugation with bio-inert polymers for skeletal muscle tissue engineering applications.

Endogenous Developmental Cycle of the Human Coccidian Cyclospora cayetanensis.

Cyclospora cayetanensis is a coccidian parasite of humans of known and growing importance. However, we are surprisingly naïve as to our understanding of how to diagnose it and how it develops inside the human body. Here we provide details of the developmental stages of C. cayetanensis in the gallbladder of a 33-yr-old male with human immunodeficiency virus. The gallbladder was removed surgically in 2001 because of severe abdominal pain. For the present study, the archived paraffin block of gallbladder was processed for light microscopy and transmission electron microscopy (TEM). Histological sections were examined after staining with hematoxylin and eosin (HE) or using the periodic acid Schiff (PAS) reaction. Immature and mature asexual stages, gamonts, and oocysts were seen in epithelial cells, both in the superficial epithelium and in glands. The merozoites were present singly, in pairs, and 3 or more in a single parasitophorous vacuole in the host cytoplasm. Up to 6 nuclei were seen in immature schizonts without evidence of merozoite formation. Mature schizonts were 7.6 × 5.1 µm and contained up to 10, 3-4 µm long merozoites. Merozoites were 0.6 to 2.0 µm wide, and their shape varied from pear-shaped to slender. Merozoites were generally PAS-positive; however, some were intensely positive, some had only minute granules, while others were PAS-negative. The microgamonts (male) were 6.6 × 5.2 µm and contained fewer than 20 microgametes around a residual body. The microgametes were up to 2 µm long and were flagellated. Macrogamonts (female) contained distinctive eosinophilic wall-forming bodies that varied in size and were less than 1 µm in HE-stained sections. Macrogamonts were 5.8-6.5 × 5.3-6.5 µm. Oocysts in sections were unsporulated and had a diameter of 5.7-7.5 µm. The TEM examination confirmed the histologic findings. The DNA extracted from paraffin sections was confirmed as C. cayetanensis with real-time PCR. The detailed description of the life cycle stages of C. cayetanensis reported here in an immunosuppressed patient could facilitate histopathologic diagnosis of this parasite. We have shown that the parasite's development more closely resembles that of Cystoisospora than Eimeria and that the parasite has multiple nuclei per immature meront indicating schizogony, and we have undermined evidence for a Type II meront.

Ultrastructural Characteristics of Gallbladder Epithelial Inclusions Mimicking Cystoisospora.

OBJECTIVES: There is recently reported increased prevalence of Isospora organisms in cholecystectomy specimens from immunocompetent patients, especially in acalculous cholecystectomies. We performed an ultrastructural and molecular evaluation of these specimens. METHODS: From 28 gallbladders with intraepithelial inclusions, two specimens with diffuse involvement of the gallbladder epithelium were analyzed by electron microscopy. Polymerase chain reaction was performed on five samples for the ITS2 region of C belli and eukaryotic 18S region. The 18S products were sequenced by next-generation sequencing. RESULTS: Electron microscopic analysis showed cytoplasmic condensations leading to vacuole formation. In contrast with true C belli, there were no identifiable organelles or organization. None of these cases showed amplified products other than human on molecular analysis. CONCLUSIONS: Electron microscopic analysis demonstrates that the inclusions are condensed cytoplasmic material and not true organisms.

Immunohistochemical study of aquaporin 1 and 2 in the hepatobiliary system of Qinghai lizard (Pphrynocephalus vlangalii ) / Estudio inmunohistoquímico de aquaporin 1 y 2 en la sistema hepatobiliar del lagarto de Qinghai ( Phrynocephalus vlangalii )

A serous membrane covering the liver and the hepatic parenchyma, consists of hepatocytes, arteries, veins, hepatic sinusoids and biliary ductuli. There are erythrocytes, thrombocytes, melanin particles and Kupffer cell in the hepatic sinusoids and the blood vessels. The gall bladder wall consists of a mucous layer a muscle layer and a serous layer. The bottom of the epithelium abounds with round or oval secretory. In liver, immunohistochemistry results show that AQP1 have intense reaction in hepatic lobule, Kupffer cells (Macrophagocytus stellatus), hepatocytes, portal tract, blood islands, vein and artery, but almost no reaction of AQP2 was detected. In gallbladder, mucous epithelium, endothelial cells from vein and artery all have strong AQP1 expression, AQP2 showed minor diffused positive reaction in gallbladder, which suggesting that AQP1 may have the main role in the absorption and transportation of fluid in hepatobiliary system of Qinghai Lizard.

Una membrana serosa cubre el hígado y el parénquima hepático el cual está formado por hepatocitos, arterias, venas, sinusoides hepáticos y conductos biliares. Se encuentran eritrocitos, trombocitos, partículas de melanina y células de Kupffer en los sinusoides hepáticos y en los vasos sanguíneos. La pared de la vesícula biliar presenta tres capas: mucosa, muscular y serosa. En el hígado, la inmunohistoquímica mostró que AQP1 tiene una reacción intensa en el lóbulo hepático, células de Kupffer, hepatocitos, tracto portal e islotes sanguíneos. En venas y arterias, no se detectó reacción alguna de AQP2. En la vesícula biliar, el epitelio mucoso, las células endoteliales venosas y arteriales tuvieron una importante expresión de AQP1, sin embargo, AQP2 mostró una reacción positiva difusa menor, lo que sugiere que la AQP1 podría tener una función principal en la absorción y transporte de líquido en el sistema hepatobiliar del Lagarto Qinghai.

Excess Secretion of Gel-Forming Mucins and Associated Innate Defense Proteins with Defective Mucin Un-Packaging Underpin Gallbladder Mucocele Formation in Dogs.

Mucosal protection of the gallbladder is vital yet we know very little about the mechanisms involved. In domestic dogs, an emergent syndrome referred to as gallbladder mucocele formation is characterized by excessive secretion of abnormal mucus that results in obstruction and rupture of the gallbladder. The cause of gallbladder mucocele formation is unknown. In these first mechanistic studies of this disease, we investigated normal and mucocele-forming dog gallbladders to determine the source, identity, biophysical properties, and protein associates of the culprit mucins with aim to identify causes for abnormal mucus behavior. We established that mucocele formation involves an adoptive excess secretion of gel forming mucins with abnormal properties by the gallbladder epithelium. The mucus is characterized by a disproportionally significant increase in Muc5ac relative to Muc5b, defective mucin un-packaging, and mucin-interacting innate defense proteins that are capable of dramatically altering the physical and functional properties of mucus. These findings provide an explanation for abnormal mucus behavior and based on similarity to mucus observed in the airways of people with cystic fibrosis, suggest that abnormal mechanisms for maintenance of gallbladder epithelial hydration may be an instigating factor for mucocele formation in dogs.

The role of interstitial Cajal-like cells in the formation of cholesterol stones in guinea pig gallbladder.

OBJECTIVES: To investigate the effect of interstitial Cajal-like cells (ICLCs) on contraction of gallbladder muscle strips; and to analyze the changes of ICLCs during cholesterol gallstone formation in guinea pig. METHODS: The cholesterol gallstone animal model was made by feeding guinea pig with high cholesterol diet (HCD). In vitro isolated gallbladder muscle strips were prepared. Gallbladder motility was assessed by the contraction frequency and amplitude of slow wave in response to CCK-8. The alteration in ICLC density was estimated by using immunohistochemistry. The expression of c-kit and stem cell factor (SCF) were determined. RESULTS AND CONCLUSIONS: The amplitude and frequency of slow wave was significantly lower in gallbladder muscle strips with the impaired ICLCs. And it is correlated with the decreased contractile response to CCK-8. In HCD guinea pig, the ICLC density and bile flow in response to CCK-8 were remarkably decreased. The results indicated that gallbladder ICLCs can create slow wave potential, and also get involved in the regulation of CCK-8 induced gallbladder smooth muscle motility. In the process of cholesterol gallstone formation, ICLC density clearly decreased. This further impaired gallbladder motility. The decrease in ICLC density may result from decreased expression of c-kit and SCF during cholesterol gallstone formation.

Liver, biliary and pancreatic injuries in pancreaticobiliary maljunction model in cats.

BACKGROUND: Pancreaticobiliary maljunction is a high risk factor of pancreatitis and biliary tract cancer. How this maljunction affects the liver remains obscure. This study aimed to examine the effects of pancreaticobiliary maljunction on the liver, pancreas and gallbladder in a cat model. METHODS: A model of choledocho-pancreatic side-to-side ductal anastomosis was created in ten cats. Before the procedure, a small piece of tissue from the liver, pancreas and gallbladder was collected as a control. The common channel formation was checked by cholecystography. The livers, pancreases and gallbladders of these cats were harvested for histological examination. The expression of proliferating cell nuclear antigen in the gallbladder was examined with immunohistochemistry. RESULTS: Seven of the 10 cats survived for 6 months after surgery. The color of the liver was darker in the PBM model than the control specimen, with nodules on the surface. Histological examination showed ballooning changes and inflammatory infiltrations and the histopathological score increased significantly (P<0.05). Also, mitochondria swelling and lipid droplet in cytoplasm were observed under an electron microscope. The pancreas also appeared darker in the PBM model than the control specimen and dilated pancreatic ducts were found in three cats. Histopathological examination revealed vascular proliferation and inflammatory infiltration with numerous neutrophils. Gallbladder epithelial cells were featured by expanded endoplasmic reticulum, increased intercellular space and cellular nucleus deformation. The positive cells of proliferating cell nuclear antigen were increased significantly (P<0.05). CONCLUSION: The present study demonstrated that pancreaticobiliary maljunction can lead to the injuries of the liver, pancreas and gallbladder.

Cellular and subcellular localization of cholecystokinin (CCK)-1 receptors in the pancreas, gallbladder, and stomach of mice.

Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.

A new exploration for gallbladder polyps: gallbladder polypectomy by endolap technique.

Abstract Gallbladder polyps are most commonly treated with cholecystectomy, which is associated with various complications. For benign disease, preserving the gallbladder is preferable. Since 1994, we have been exploring percutaneous polypectomy and have recently developed an improved new technique. This study reports a new endoscopic-laparoscopic (Endolap) technique for the removal of polyps and the preservation of the gallbladder. Nine Chinese mini-pigs were used to observe mucosal regeneration. Microwaves of 50-70 mA for 9 seconds were safe, and the gallbladder mucosa of pigs recovered to nearly normal 2 weeks later. In the clinical cases, 60 patients with gallbladder polyps were studied. With the patient under general anesthesia, each polyp stem was coagulated, and then the polyp was removed. All procedures were successful at between 60 and 135 minutes. The success rate was 93.33% (56/60). A retrospective analysis was conducted to assess the recovery of gallbladder function. All patients were followed up and symptom-free, without recurrence of the polyps; 3 months after the operation, the volume and contraction of the gallbladder recovered to preoperative levels. Thus the Endolap technique is reliable for removing benign gallbladder polyps and is applicable to a wider range of clinical situations than percutaneous polypectomy.

Defining the human gallbladder proteome by transcriptomics and affinity proteomics.

Global protein analysis of human gallbladder tissue is vital for identification of molecular regulators and effectors of its physiological activity. Here, we employed a genome-wide deep RNA sequencing analysis in 28 human tissues to identify the genes overrepresented in the gallbladder and complemented it with antibody-based immunohistochemistry in 48 human tissues. We characterized human gallbladder proteins and identified 140 gallbladder-specific proteins with an elevated expression in the gallbladder as compared to the other analyzed tissues. Five genes were categorized as enriched, with at least fivefold higher levels in gallbladder, 60 genes were categorized as group enriched with elevated transcript levels in gallbladder shared with at least one other tissue and 75 genes were categorized as enhanced with higher expression than the average expression in other tissues. We explored the localization of the genes within the gallbladder through cell-type specific antibody-based protein profiling and the subcellular localization of the genes through immunofluorescent-based profiling. Finally, we revealed the biological processes and metabolic functions carried out by these genes through the use of GO, KEGG Pathway, and HMR2.0 that is compilation of the human metabolic reactions. We demonstrated the results of the combined analysis of the transcriptomics and affinity proteomics.

Angioarchitecture of gallbladder in pig: LM and SEM study on vascular microcorrosion casts.

The study focused on the description of pig gallbladder angioarchitecture, with particular emphasis on the specifics of the course of blood vessels in individual layers of the gallbladder wall. Furthermore, the vascular systems of the pig gallbladder were analyzed in terms of the adaptation of this organ to changes in its volume during cyclical bile storage and discharge. The gallbladder is supplied by the cystic artery, which in the pig represents a mixed pinnate and bipinnate pattern of branching. The light microscopic and scanning electron microscopic observations of three-dimensional vascular corrosion casts showed the presence of two main complex vascular networks in the wall of the gallbladder, one located in the subserosal and the other in the mucosa. The unique features in the pig, connected with the size of the gallbladder, is the well-developed horizontal venous plexus under folds of the mucosa, which is a voluminous reservoir of fluids absorbed from bile and vascular networks around mucous glands. Superficial blood vessels of the gallbladder run in vascular pairs or triads, where a single artery runs between two veins. The structures of blood flow control, that is, venous valves, were observed only in venules of the subserosal plexus. Spatial arrangement of the vascular network in the pig gallbladder shows functional plasticity during changes in gallbladder volume. The course of superficial blood vessels in the well-filled gallbladder is arcuate, while in the empty gallbladder it is undulated or spiral. In the mucosal and intramural vessels the direction of blood vessels may change from perpendicular to oblique.

Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice.

Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute 'embryonic hepatitis', leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17(+/-) B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17(+/-) gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.

The gallbladder of the electric ray Torpedo marmorata Risso displays excrescent cholecystocytes with merocrine and apocrine-like secretions.

The gallbladder of Torpedo marmorata exhibits a mucosal surface layer of simple columnar epithelium with very tall cholecystocytes. The apical domain of each cell has few microvilli, but many mucous vesicles that are secreted by exocytosis at the cell apices. The apical regions may also elongate and undergo self-excision while shedding mucus and cell debris into the gallbladder lumen in a manner similar to that described in mammals as a result of sex steroid treatment to induce gallstones and to that found in the cholecystitis associated with cholelithiasis. Numerous small mitochondria, spherical to elongated, are distributed throughout the cells, while the nuclei are often located in the lower third of each cell. In the lower part of the cholecystocytes, large and very densely contrasted lysosomes can be found. All cells are tightly joined by junctional complexes, including long, highly contrasted desmosomes. The fibromuscular layer is made of a loose stroma with a limited muscular component and a poor blood supply. Large diameter blood vessels can only be found in the subserosal layer. It is hypothesized that the obligatorily carnivorous diet of this ureotelic fish has resulted in the evolution of a gallbladder ultrastructure resembling that found in cholecystitis but without the associated cholelithiasis.

Are short-term focused training courses on a phantom model using porcine gall bladder useful for trainees in acquiring basic laparoscopic skills?

The best training method in laparoscopic surgery has not been defined. We evaluated the efficacy of laparoscopic skills acquisition in a short-term focused program. Two hundred fifty-six participants undergoing training on a phantom model were divided into 2 groups. Group 1 had no exposure and group 2 had performed a few laparoscopic surgeries. Acquisition of laparoscopic skills was assessed by operation time and the modified Global Operative Assessment of Laparoscopic Skills (GOALS) scale. A questionnaire was sent to the participants after 3 to 6 months for assessment of impact of training. There was a statistically significant improvement in the assessed parameters and in the mean score of all 5 domains of GOALS. The participants in group 2 performed better than those in group 1 in the first case. The difference between both the groups disappeared after the training. Participants who responded to the questionnaire felt that training helped them in improving their performance in the operation theater.

Metachromatic leukodystrophy and its effects on the gallbladder: a case report.

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder caused by a deficiency of arylsulfatase A enzyme. This deficiency leads to accumulation of sulfatides in the central nervous system and other organs, such as the gallbladder. Here the authors discuss a 9-year-old Middle Eastern patient with late-infantile-type MLD who presented with symptoms of cholecystitis. Radiographic studies revealed an enlarged gallbladder with a thickened wall and a pericholecystic fluid collection with peripheral calcifications. Gross examination of the gallbladder showed multiple small to medium-sized papillary projections involving the entire mucosal surface. Sections through the gallbladder wall revealed multilocular dilated mucin-producing cystic spaces. Microscopically, the mucosa showed numerous papillary projections with complex folds lined by mucin-producing cuboidal to tall columnar cells. The cystic spaces were composed of numerous markedly distended Rokitansky-Aschoff sinuses filled with mucin. Ultrastructurally, the epithelial cells and macrophages showed frequent secondary lysosomes containing closely packed lamellar amorphous to prismatic material with alternating leaflets and tubules, imparting a "herringbone" or "tuffstone" pattern. This case illustrates the features of gallbladder involvement in MLD and the potential role of ultrastructural examination in diagnosis of MLD.

The gallbladder of Uranoscopus scaber L. (teleost perciform fish) is lined by specialized cholecystocytes.

The gallbladder of Uranoscopus exhibits a mucosal surface layer of simple columnar epithelium composed of specialized cholecystocytes. The apices show storage and mucous secretions, typical microvilli, and very apical projections extending deep into the luminal contents. Many organelles and heterogeneous vesicles of diverse size fill the cytoplasm, including neutral mucins, mitochondria, peroxisomes, lysosomal bodies, and lipid-rich deposits with cholesterol inclusions. The fibromuscular layer shows little blood supply and contains scattered lymph-like walls with minute cholesterol inclusions. The remaining muscular, subserosal, and serosal or adventitial layers of this species do not show any histologic differences to those of other vertebrates. It was unexpected to find cholesterol inclusions in the fatty deposits of the cholecystocytes, similar to those noted in human cholesterolosis and in some forms of hypercholesterolemia, in this teleostean. In addition, aggregations of mitochondria and anomalous mitochondrial morphologies were found that resemble oncocytoma-like changes.

Interstitial cells of Cajal are present in human extrahepatic bile ducts.

BACKGROUND AND AIMS: Interstitial cells of Cajal (ICC) are distributed with smooth muscle throughout the gastrointestinal tract and are involved in regulating motility. ICC were recently discovered in the wall of the human gallbladder. This study sought to determine whether ICC are present in human bile ducts. METHODS: Biliary tract samples were obtained from several sources: surgical specimens (n = 16, 11 women, mean age 61 years); archival post-mortem specimen (n = 1, 86 years, man); and cadavers (n = 2, 68 and 80 years, men). Paraffin-embedded sections (3 microm) from the gallbladder (fundus, body and neck) and both extrahepatic and intrahepatic bile ducts were investigated. A double immunofluorescence protocol using polyclonal and monoclonal c-kit antibodies and mast cell tryptase was used to distinguish c-kit-positive cells with typical ICC morphology from c-kit-positive mast cells. Small bowel samples were used as positive controls. ICC in the gallbladder were confirmed by ultrastructural study. RESULTS: c-kit-positive cells with characteristic ICC morphology were identified in the subepithelial and muscular layers of the gallbladder and extrahepatic bile ducts. They were most prominent within the muscle layer of the extrahepatic bile ducts where they were organized into loosely arranged laminae running parallel to circular smooth muscle fibers. ICC were not found in intrahepatic bile ducts. CONCLUSION: This study demonstrates for the first time that ICC are present in human extrahepatic bile ducts where they are more densely aggregated than in the gallbladder. This cellular network is likely to be involved in biliary tract motility and its related disorders.

Non-adhesive organ culture of human biliary epithelium with stroma.

OBJECTIVE: Explanted tissue has been shown to keep adult human cells in organ culture with a preserved morphology for at least one month as spheres in a non-adhesive organ culture. In the present study, we explored whether also human biliary epithelium can be grown in this manner, because the result may be of interest in studies of hepato-biliary-pancreatic carcinogenesis. MATERIAL AND METHODS. Small tissue samples were obtained from the gallbladder wall of patients who had been operated upon with cholecystectomy. Fragments of about 300 microm in diameter from each patient were cultured and investigated with light microscopy at the time of explantation and after 5, 10, 20, 30 and 40 days of culture. Scanning and transmission electron microscopy were performed to demonstrate the ultrastructure. Incubation of cultured fragments with the vital dyes revealed a viable epithelium. RESULTS: At the time of explantation, all the tissue fragments had a rough appearance with an uneven, torn periphery, while during the first few days of culture they became rounder with a smooth-looking surface covering the entire circumference. This spheroid morphology persisted for the remainder of the culture period. The core of the fragments harboured connective tissue with vascular elements, fibroblasts and leucocytes. Immunostaining for cytokeratin 7, 19 and 20 revealed a strong positive staining of the epithelium. CONCLUSIONS: These results show that biliary epithelium can be grown in vitro in a non-adhesive organ culture with their stroma.

Interstitial Cajal-like cells in human gallbladder.

We describe here an interstitial Cajal-like cell type (ICLC) in human gallbladder, resembling the archetypal enteric interstitial cells of Cajal. Gallbladder ICLC were demonstrated in fresh preparations (tissue cryosections) using methylene-blue, and fixed specimens in Epon semi-thin sections stained with toluidine blue or transmission electron microscopy (TEM). The positive diagnosis of gallbladder ICLC was further verified by immunohistochemistry: CD117/c-kit, CD34, and another 16 antigens: vimentin, desmin, nestin, alpha-smooth muscle actin, NK-1, S-100, PGP-9.5, tau protein, chromogranin A, NSE, GFAP, CD1a, CD62-P, CD68, estrogen and progesterone receptors. Double immunostaining was performed for CD117, CD34 and CD117 and nestin, respectively. In fresh specimens, the spatial density of gallbladder ICLC was 100-110 cells/mm(2). ICLC mainly appeared beneath the epithelium and in muscularis (about 7%, and approximately 5%, respectively). In toto, ICLC represent in gallbladder approximately 5.5% of subepithelial cells. TEM showed that diagnostic criteria were fulfilled by ICLC. Moreover, TEM indicated that the main ultrastructural distinctive feature for ICLC, the cell processes, develop into the characteristic shape at a relatively early stage of development. It remains to be established if, in humans, ICLC are involved in gallbladder (dis)functions (e.g. pace-making, secretion (auto-, juxta- and/or paracrine), intercellular signaling, or stone formation).