Gallbladder Inflammation is Associated with Increase in Mucin Expression and Pigmented Stone Formation

Mucin is a high molecular weight glycoprotein that plays an important role in protecting the gallbladder epithelium from the detergent effect of bile. However, it also participates in gallstone formation. There is little information about a possible relationship between gallbladder inflammation and mucin expression or gallbladder stones’ characteristics. The aims of this study were to investigate stone characteristics and patterns of mucin expression in the gallbladder epithelium and bile of gallstone patients, in relation to inflammation. Gallbladder bile and tissue samples from 21 patients were obtained at surgery. Mucin content was evaluated by gel filtration on a Sepharose CL-4B column. Dot blot for bile mucin apoproteins and immunohistochemistry staining for gallbladder mucosal mucin apoproteins were performed with antibodies to MUC2, MUC3, MUC5AC, MUC5B and MUC6. Staining intensity score (0–3) was used for assessment of antigen expression and the level of inflammation. Gallstone cholesterol content was determined in 16 patients. MUC 5AC and MUC 5B were demonstrated in 95.4 and 100% of gallbladder bile samples, respectively. Immunohistochemistry staining with antibodies to MUC 2, MUC 3, MUC 5AC, MUC 5B and MUC 6 were positive in 0, 100, 85.7, 100 and 95.4% of the gallbladder mucosal samples, respectively. Pigmented brown stones were associated with a higher level of gallbladder inflammation. Mucin species expressed in gallbladder epithelium are MUC3, MUC5AC, MUC5B and MUC6. MUC5AC and MUC5B are secreted into bile. Inflammation of the gallbladder is accompanied by a higher level of MUC5AC expression and is associated with pigmented brown stones.     

Altered Mucin Core Peptide Expression in Acute and Chronic Cholecystitis

Human mucin genes include membrane-bound mucins (MUC1, MUC3, MUC4) and secretory mucins (MUC2, MUC5AC, MUC5B, MUC6). Our aim was to determine mucin gene expression in human gallbladder cell lines, normal gallbladder from liver donors (N = 7) and surgical specimens with mild chronic cholecystitis (N = 29), chronic cholecystitis (N = 48), and acute and chronic cholecystitis (N = 27). MUC1 mRNA was ubiquitous; however, only rare MUC1 immunoreactivity was detected. MUC3, MUC5AC, MUC5B, and MUC6 mRNA were present in all gallbladder specimens and cell lines examined. Prominent MUC3, MUC5AC, MUC5B, and MUC6 immunoreactivity was present in 86–100% of normal gallbladders. The frequency of MUC5AC reactivity was decreased in specimens with acute cholecystitis (P < 0.05). In contrast, MUC2-reactivity was absent in normal gallbladder and present in 53.8% of acute cholecystitis specimens (P < 0.05). Surface epithelium is characterized by MUC3, MUC5AC, and MUC5B, whereas deeper mucosal folds display MUC5B and MUC6 immunoreactivity. Gallbladder epithelium demonstrates a unique and diverse pattern of mucin core proteins that becomes altered with increasing degrees of inflammation.     

Altered Mucin Gene Expression in Stone-Containing Intrahepatic Bile Ducts and Cholangiocarcinomas

Neoplastic transformation of epithelial cells is commonly associated with alterations in the synthesis and structures of mucin. Mucin protein epitopes and mRNA levels were frequently altered in adenocarcinomas compared to corresponding normal tissues. Clinically, hepatolithiasis has been regarded as a risk factor for cholangiocarcinoma. The aims of this study were to determine the possible alteration of mucin gene expression in stone-containing intrahepatic bile ducts and cholangiocarcinomas and to try to predict whether or not hepatolithiasis has a predisposition to development of cholangiocarcinoma. In situ hybridization with DIG-tailed oligonucleotides was performed on sections of paraffin-embedded tissues of stone-containing intrahepatic bile ducts, cholangiocarcinomas, and normal controls to identify the expression of MUC2, MUC3, MUC4, MUC5B, and MUC5AC in nonneoplastic and neoplastic biliary epithelium. The findings showed that (1) while multiple diverse mucin genes were expressed in the biliary epithelium, MUC3 and MUC5B mRNA were the main mucin genes expressed in the biliary epithelium of stone-containing intrahepatic bile ducts and normal controls; (2) absent or decreased expression of MUC2, MUC3, and MUC5B of mRNA was found in cholangiocarcinomas in contrast to nonneoplastic biliary epithelium; and (3) increased expression of MUC4 and MU5AC of mRNA was found in cholangiocarcinomas and the biliary epithelium, especially for dysplastic cells of stone-containing intrahepatic bile ducts compared with normal controls. In this study, using in situ hybridization we demonstrated that neoplastic transformation of the biliary epithelium is accompained by alterations in mucin gene expression, the altered mucin gene expression in dysplastic cells of stone-containing intrahepatic bile ducts may reflect a higher potential for malignant transformation in these cells, and it could be a precursor of cholangiocarcinoma in the presence of hepatolithiasis.     

Mucin gene expression in bile of patients with and without gallstone disease, collected by endoscopic retrograde cholangiography

AIM： To investigate the pattern of mucin expression and concentration in bile obtained during endoscopic retrograde cholangiography （ERC） in relation to gallstone disease.METHODS： Bile samples obtained at ERC from 29 consecutive patients, 17 with and 12 without gallstone disease were evaluated for mucin content by gel filtration on a Sepharose CL-4B column. Dot blot analysis for bile mucin apoproteins was performed with antibodies to Mucin 1 （MUC1）, MUC2, MUC3, MUC5AC, MUC5B and MUC6. Staining intensity score （0-3） was used as a measure of antigen expression.RESULTS： MUCl, MUC2, MUC3, MUCSAC, MUC5B and MUC6 were demonstrated in 34.4%, 34.4%, 51.7%, 51.7%, 55.1% and 27.5% of bile samples, respectively.The staining intensity scores were 0.62 ± 0.94, 0.58 ± 0.90, 0.79 ± 0.97, 1.06 ± 1.22, 1.20 ± 2.26 and 0.41 ± 0.73, respectively. Mean mucin concentration measured in bile by the Sepharose CL-4B method was 22.8 ± 24.0 mg/mL （range 3.4-89.0 mg/mL）. Mean protein concentration was 8.1 ± 4.8 mg/mL （range 1.7-23.2 mg/mL）.CONCLUSION： High levels of MUC3, MUC5AC and MUC5B are expressed in bile aspirated during ERC examination. A specific pattern of mucin gene expression or change in mucin concentration was not found in gallstone disease.     

Gallbladder glycoprotein secretion in mice with hemolytic anemia and pigment gallstones

The nb/nb mouse with hereditary hemolytic anemia provides an animal model for the study of pigment gallstone disease. We measured glycoprotein synthesis and secretion in gallbladder neck and fundus of 6-month-old mice without stones, and in 12-month old mice with and without stones in order to determine the effect of age and presence or absence of stones on mucin release. We observed that the gallbladder necks of 12-month-old nb/nb mice with pigment stones secreted more 3H-glucosamine-labeled glycoprotein (68.2 dpm per microgram protein) than did the gallbladder necks of mice without pigment stones (31.1 dpm per microgram protein). When expressed as a percentage of total glycoprotein synthesis in the gallbladder neck, secretion of glycoprotein was 23.4% in 6-month-old hemolytic mice and 28.7% in 12-month-old hemolytic mice without pigment stones. However, in the presence of pigment stones, the percentage of secreted glycoprotein rose to 4.08%, which differed significantly from both 6-month-old (p < 0.02) and 12-month-old mice without stones (p < 0.05). Our results suggest that the presence of gallstones in nb/nb mice was associated with a localized increase in glycoprotein release from gallbladder neck. The mechanism for this increase may be precipitation of pigment-mucin concentrations in the gallbladder neck glands which has been previously described in this animal model.     

Excess membrane cholesterol alters human gallbladder muscle contractility and membrane fluidity

BACKGROUND & AIMS: The relationship between muscle contractility, plasma membrane cholesterol, and fluidity was investigated in human gallbladders with gallstones. METHODS: Isolated gallbladder muscle cells were used to measure contraction. Plasma membranes of gallbladder muscle were purified in a sucrose gradient and measured for cholesterol content and cholesterol/phospholipid mole ratio. Membrane fluidity was determined by using fluorescence polarization and was expressed as the reciprocal of anisotropy. RESULTS: The maximal contraction induced by cholecystokinin octapeptide was significantly less in gallbladders with cholesterol stones than in those with pigment stones. The membrane cholesterol content and cholesterol/phospholipid mole ratio were significantly higher in gallbladders with cholesterol stones than in those with pigment stones. Membrane anisotropy was also higher than in gallbladders with pigment stones, reflecting lower membrane fluidity in gallbladders with cholesterol stones. After muscle cells from cholesterol stone gallbladders were incubated with cholesterol-free liposomes for 4 hours, cholecystokinin octapeptide-induced contraction, membrane cholesterol content and cholesterol/phospholipid ratio, and membrane fluidity returned to normal levels. CONCLUSIONS: Gallbladder muscle from patients with cholesterol stones has increased membrane cholesterol/phospholipid mole ratio and decreased membrane fluidity resulting in impaired muscle contractility. These abnormalities are corrected by removing the excess cholesterol from the plasma membranes.     

Frequent expression of mucin core protein MUC1 in non-neoplastic gallbladder mucosa from patients with pancreaticobiliary maljunction.

BACKGROUND: Gallbladder carcinoma is known to develop frequently in patients with pancreaticobiliary maljunction, though the causal relationship remains speculative. METHODS: Histopathologic changes, expression of mucin core protein MUC1 and MUC2, and cell proliferative activities in the gallbladder mucosa from 27 patients with panceaticobiliary maljunction and 21 control gallbladders were examined. Three cases of pancreaticobiliary maljunction were associated with gallbladder carcinoma. RESULTS: The lining epithelia of the non-neoplastic gallbladder mucosa of pancreaticobiliary maljunction showed frequently papillary hyperplasia and higher proliferative activities, when compared to the control. In 3 cases with carcinoma, MUC1 was expressed on the luminal border and in the cytoplasm of carcinoma cells, particularly in de-differentiated and invasive areas. MUC1 was variably expressed on the luminal surface of the lining epithelia of non-neoplastic gallbladder mucosa in babies, children, youths and adults with pancreaticobiliary maljunction. However, such expression was focally seen in 2 of the 21 control cases (p<0.01). MUC2 was scattered in the hyperplastic and carcinomatous epithelial cells appearing as goblet cells in pancreaticobiliary maljunction and control groups. CONCLUSIONS: This study suggests that persistent MUC1 expression and increased cell proliferative activities of non-neoplastic gallbladder epithelium of the patients with pancreaticobiliary maljunction after birth reflect an altered phenotype of epithelial cells and these abnormalities may be related to carcinogenesis in such patients.     

Human gallbladder mucin accelerates nucleation of cholesterol in artificial bile

The gallbladder bile of patients with cholesterol gallstones is characterized by two abnormalities: (a) supersaturation with cholesterol and (b) accelerated nucleation of cholesterol monohydrate crystals. We studied the ability of purified human gallbladder mucin to nucleate artificial bile in vitro. Human gallbladder mucin at concentrations of 2 and 4 mg/ml accelerated the nucleation time of cholesterol crystals in model bile. The mean number of cholesterol crystals in artificial bile incubated for 10 days with 4 mg/ml of human gallbladder mucin was 2327/mm3 (p less than 0.01) vs. control of 51/mm3. The number of crystals found in model bile was dependent on the concentration of human gallbladder mucin (2-16 mg/ml) and the time of incubation (4-14 days). Human gallbladder mucin was associated with an increase in the number of liquid crystals after 4 days of incubation, which then decreased in number as solid cholesterol monohydrate crystals formed. Nucleation by human gallbladder mucin was significantly increased only with cholesterol saturation indices greater than 1.0, and in biles containing 10% but not 3% total lipid by weight. Pooled human gallbladder mucin from gallbladders with and without stones both increased nucleation significantly when compared with controls. Increased nucleation of saturated model bile was also observed with purified monkey cervical and bovine gallbladder mucin, but not with porcine gastric mucin. These observations provide further evidence that human gallbladder mucin may contribute to cholesterol gallstone formation in humans by accelerating nucleation of cholesterol monohydrate crystals from supersaturated gallbladder bile.     

Black pigment gallstones with cholesterol gallstones in the same gallbladder

We studied 1312 consecutive patients who underwent surgery for gallstones in the biliary tract at one university hospital in Siena, Italy, with a systematic classification of gallstones found within the gallbladder. Of these patients, 1226 were found to have gallbladder stones; 94 of these had black pigment gallstones. Of these, 13 patients were found to have black pigment gallstones and cholesterol gallstones within their gallbladder. They all had multiple black pigment gallstones, usually very small (all <6 mm diameter), in association with larger cholesterol stones in the gallbladder lumen. The cholesterol gallstones were single in seven cases, double in two cases, and multiple in four cases. All 13 of these patients with black pigment stones in association with cholesterol stones had histologic evidence of either adenomyomatosis or Rokitansky-Aschoff sinuses in the gallbladder wall. In nine of the 13 patients, the black pigment stones were located both in the gallbladder lumen and in close association with the gallbladder wall (in areas of adenomyomatosis or in Rokitanski-Aschoff sinuses). In the other four patients, the stones were found in close association with the gallbladder wall alone and not freely mobile within the gallbladder lumen. It is concluded that cholesterol stones and black pigment stones may be found in the same gallbladder. This association is infrequent with an incidence of 13 of 1226 (1.06%) in our series. There appears to be some relationship between the formation of the black pigment stones and the presence of adenomyomatosis or Rokitanski-Aschoff sinuses. However, the pathogenesis of these two compositionally distinctly different types of stones within the same gallbladder is not understood and deserves further study.     

人体胆囊结石时胆囊组织PGE、PGI2、LTC4的含量

本文测定非结石组（ｎ＝１６）与结石组（ｎ＝４７）人体胆囊粘膜内前列腺素Ｅ（ＰＧＥ）、前列环素（ＰＧＩ２）白三烯０４（ＬＴＣ４）的含量及磷脂酶Ａ２（ＰＬＡ２）的活性，发现结石组四者的水平均非常显著地高于非结石组。提示可能由于脂类代谢紊乱，胆囊组织的ＰＬＡ２活性增加．致使ＰＧｓ、ＬＴｓ水平增高，引起胆囊上皮细胞活化，合成分泌粘蛋白功能亢进，从而参与促进结石的形成。     

Mucin gene expression in intestinal epithelial cells in Crohn's disease

BACKGROUND: Crohn's disease (CD) is a chronic relapsing inflammatory bowel disease of unknown origin. It is characterised by chronic mucosal ulcerations which affect any part of the intestine but most commonly are found in the ileum and proximal colon. AIMS: Studies were undertaken to provide information regarding cell specific expression of mucin genes in the ileum of patients with CD. PATIENTS AND METHODS: Expression of mucin genes was analysed in the ileal mucosa of patients with CD and controls by in situ hybridisation and immunohistochemistry. RESULTS: In healthy ileal mucosa, patients with CD showed a pattern identical to normal controls with main expression of MUC2 and MUC3, lesser expression of MUC1 and MUC4, and no expression of MUC5AC, MUC5B, MUC6, or MUC7. In the involved mucosa, the pattern was somewhat comparable although heterogeneous to that observed in healthy ileal mucosa. Importantly, a particular mucin gene expression pattern was observed in ileal mucosa close to the ulcer margins in ulcer associated cell lineage, with the appearance of MUC5AC and MUC6 mRNAs and peptides, which are normally restricted to the stomach (MUC5AC and MUC6) and duodenum (MUC6), and disappearance of MUC2. CONCLUSIONS: Our results suggest that gel forming mucins (more particularly MUC5AC and MUC6) may have a role in epithelial wound healing after mucosal injury in inflammatory bowel diseases in addition to mucosal protection.     

Prediction of Prognosis in Gallbladder Carcinoma by Mucin and p53 Immunohistochemistry

Mucin core proteins are known to be present in various organs and are specifically expressed with carcinogenesis and closely associated with the prognoses of various malignant tumors in the digestive tract such as colorectal cancer. The present study evaluated correlations between mucin and p53 expression and prognosis of gallbladder cancer using surgically resected tissue specimens from 26 patients with gallbladder carcinoma surgically treated at our hospital. Immunohistochemical staining was performed using MUC1, MUC2, and p53 monoclonal antibody. The level of antigen expression in the lesion was classified into four stages: none(−), slight(+), moderate (++), and severe (+++). According to the UICC classification, histopathological grading, levels of T, N, and M factors, and tumor stages were compared with regard to the correlations with mucin and p53 expression. All cases were classified into two groups according to the results of mucin immunohistochemistry: group A (MUC1, ≥{++}; and MUC2, ≤+) and group B (MUC1, < ++; or MUC2, > +). Postoperative survival periods were compared between the two groups and p53-positive and -negative groups. Neither histological grading nor T factor correlated with mucin or p53 expression, respectively. Moreover, neither N factor nor M factor correlated with mucin or p53 expression. Furthermore, stage grouping did not correlate with mucin or p53 expression. However, when the correlation between the postoperative survival period and mucin expression was evaluated, the mean postoperative surgical period was significantly shorter in Group A than in Group B (1.02 years in Group A vs 2.92 years in Group B; P = 0.016). There was no relationship between postoperative survival period and p53 positivity. Mucin expression was independent of various tumor growth factors and clearly reflected the prognosis of gallbladder cancer. Because the relative malignancy of gallbladder cancer could be evaluated by examining the level of glycoprotein expression in tumor tissue, mucin could be a more important marker than p53 for predicting prognosis in gallbladder carcinoma using surgically resected tissue specimens.     

Mucin gene expression in biliary epithelial cells

Background/Aims: In recent years considerable advances have been made in our knowledge of human mucin genes. Although analysis of their genomic organization is still in progress, the pattern of their expression in different human mucosae is now fairly well established. However, little is known about their expression in the biliary tree. In this study we determined the pattern of expression of the different human mucin genes in gallbladder biliary epithelial cells, intrahepatic bile ducts and liver.Methods: Two complementary methods were used: Northern-blot and in situ hybridization analyses. The experiments were performed with eight probes corresponding to MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6 and MUC7.Results: Our results revealed a strong mRNA expression of MUC3, MUC6 and MUC5B, a week expression of MUC1, MUC5AC and MUC2, and no expression of MUC4 and MUC7. Surprisingly, MUC3, which was the gene which was most expressed in the biliary tree, was also found in hepatocytes, suggesting another function for the MUC3 protein than that of a secreted mucin.Conclusions: We conclude that MUC3, MUC6 and MUC5B were the main mucin genes expressed in biliary epithelial cells.     

Mucin gene expression in Barrett's oesophagus: an in situ hybridisation and immunohistochemical study

BACKGROUND AND AIMS: Mucin genes are expressed in a site specific manner throughout the gastrointestinal tract. Little is known about the expression pattern in the oesophagus. In this study we have investigated MUC gene expression in both the normal oesophagus and specialised intestinal metaplasia (Barrett's oesophagus). PATIENTS: Archived paraffin embedded material from eight specimens of normal oesophagus, 18 Barrett's oesophagus, eight gastric metaplasia, six high grade dysplasia, and six cases of adenocarcinoma were examined for expression of the mucin genes MUC1-6. METHODS: Mucin mRNA was detected by in situ hybridisation using [(35)S] dATP labelled oligonucleotide probes. Mucin core protein was detected by immunohistochemistry. RESULTS: Normal oesophagus expressed MUC5B in the submucosal glands and MUC1 and MUC4 in the stratified squamous epithelium. Barrett's oesophagus strongly expressed MUC5AC and MUC3 in the superficial columnar epithelium, MUC2 in the goblet cells, and MUC6 in the glands. In high grade dysplasia and adenocarcinoma there was downregulation of MUC2, MUC3, MUC5AC, and MUC6, but upregulation of MUC1 and MUC4 in half of the specimens examined. CONCLUSIONS: Normal oesophagus and Barrett's oesophagus have a novel pattern of mucin gene expression. Barrett's oesophagus expressed the mucins associated with normal gastric epithelium and normal intestinal epithelium. While most mucin genes were downregulated in severely dysplastic and neoplastic tissues, there was upregulation of the membrane bound mucins MUC1 and MUC4. This may prove useful in detecting early signs of progression to adenocarcinoma of the oesophagus.     

Aberrant expression of MUC5AC and MUC6 gastric mucins and sialyl Tn antigen in intraepithelial neoplasms of the pancreas

BACKGROUND & AIMS: It has recently been suggested that infiltrating adenocarcinoma of the pancreas arises from histologically well-defined precursor ductal lesions called pancreatic intraepithelial neoplasia (PanIN-1A, -1B, -2, and -3). This study examined alterations in the pattern and the level of expression of several mucin genes (MUC1, MUC2, MUC5AC, and MUC6) and mucin-associated tumor antigens (Nd2 and sialyl Tn) in these precursor lesions. METHODS: We examined 139 PanINs and 68 infiltrating ductal adenocarcinomas of the pancreas by using immunohistochemistry and in situ hybridization methods. RESULTS: Overexpression of MUC1, a pan-epithelial mucin, and MUC6, a pyloric-gland mucin, and de novo expression of MUC5AC, a gastric foveolar mucin, was observed in all stages of PanINs and invasive ductal adenocarcinoma. In contrast, the expression of mucin-associated carbohydrate antigen, sialyl Tn, was markedly increased only in PanlN-3 and invasive ductal adenocarcinoma. In addition, a decrease in the expression of these mucin-associated peptide and carbohydrate antigens was correlated with the degree of differentiation of the tumor. CONCLUSIONS: Expression of both gastric-foveolar and pyloric-gland mucin in PanINs is an early event, whereas sialyl Tn expression is a late event in the recently defined progression model of pancreatic carcinogenesis. This altered mucin gene expression provides new insight into the role of cell lineage-associated metaplasia in pancreatic carcinogenesis.     

Gallbladder mucin and cholesterol and pigment gallstone formation in hamsters

We measured gallbladder mucin production by hamsters fed diets lithogenic for either cholesterol or pigment gallstones. In hamsters on the cholesterol stone diet, gallbladder production of 3H-glucosamine-labeled mucin was elevated two- and seven-fold after 1 and 3 weeks, respectively. After 1 week cholesterol crystals were seen in a mucus gel on the gallbladder surface. In hamsters on the pigment stone diet, gallbladder mucin production was significantly elevated after 1 and 3 weeks. The first precipitation of pigment crystals was in mucus in bile or on the gallbladder surface. Black pigment stones grew by agglomeration of pigment crystals enmeshed in mucus. In conclusion, gallbladder mucin production is increased before cholesterol or pigment stone formation, and the earliest deposition of crystals is in mucus in bile or on the gallbladder surface.     

Frequent expression of mucin core protein MUC1 in non-neoplastic gallbladder mucosa from patients with pancreaticobiliary maljunction

Abstract: Background: Gallbladder carcinoma is known to develop frequently in patients with pancreaticobiliary maljunction, though the causal relationship remains speculative. Methods: Histopathologic changes, expression of mucin core protein MUC1 and MUC2, and cell proliferative activities in the gallbladder mucosa from 27 patients with panceaticobiliary maljunction and 21 control gallbladders were examined. Three cases of pancreaticobiliary maljunction were associated with gallbladder carcinoma. Results: The lining epithelia of the non-neoplastic gallbladder mucosa of pancreaticobiliary maljunction showed frequently papillary hyperplasia and higher proliferative activities, when compared to the control. In 3 cases with carcinoma, MUC1 was expressed on the luminal border and in the cytoplasm of carcinoma cells, particularly in de-differentiated and invasive areas. MUC1 was variably expressed on the luminal surface of the lining epithelia of non-neoplastic gallbladder mucosa in babies, children, youths and adults with pancreaticobiliary maljunction. However, such expression was focally seen in 2 of the 21 control cases (p<0.01). MUC2 was scattered in the hyperplastic and carcinomatous epithelial cells appearing as goblet cells in pancreaticobiliary maljunction and control groups. Conclusions: This study suggests that persistent MUC1 expression and increased cell proliferative activities of non-neoplastic gallbladder epithelium of the patients with pancreaticobiliary maljunction after birth reflect an altered phenotype of epithelial cells and these abnormalities may be related to carcinogenesis in such patients.