Chronic Proliferative Cholangitis Research Articles

Gross and histological lesions in the livers of sika deer with particular emphasis on fascioliasis.

We performed gross and histological examinations of the livers of sika deer (Cervus nippon yesoensis) in Hokkaido, Japan. Out of 1,381 deer slaughtered for venison production, thickening and dilation of the large intrahepatic bile ducts and Fasciola flukes in the duct lumens were detected in 621 deer (45.0%). Furthermore, 107 non-bile lesions (75 intrahepatic and 32 capsular lesions) were detected during gross examinations. Histologically, the bile duct lesions included chronic proliferative cholangitis, papillary hyperplasia, goblet cell and pyloric gland metaplasia, and periductal fibrosis. Many of the intrahepatic non-bile duct lesions (53/75, 71%) were considered to be Fasciola fluke migration-associated lesions, including two lesion types: necrosis, hemorrhage, and eosinophilic granuloma formation (29 lesions), and lymphoid tissue formation (24 lesions). Lymphoid tissue formation was considered to result from the persistent immune responses against dead Fasciola flukes. An epidermoid liver cyst was found incidentally, which has not been reported in the veterinary literature. In summary, this study demonstrated the predominance of fascioliasis-associated lesions in sika deer livers. The gross and histological lesions caused by Fasciola flukes in sika deer were similar to fascioliasis in other animals. Moreover, we described lymphoid tissue formation as a fascioliasis-associated lesion for the first time. The fact that bile duct lesions (45.0%) had a markedly higher prevalence than fascioliasis-associated parenchymal lesions (53/1,381, 3.8%) indicated that sika deer are a permissive host for fascioliasis. Our results provide information that will aid pathological examinations of sika deer.

Biliary antibiotics irrigation for E. coli-induced chronic proliferative cholangitis and hepatolithiasis: A pathophysiological study in rabbits

The gram-negative bacteria secreted endotoxin, Lipopolysaccharide (LPS), plays important roles in the formation and recurrence of hepatolithiasis and chronic biliary inflammation in patients of Southeast Asia. We aimed to elucidate the anti-inflammatory effect and mechanism of local antibiotics irrigation on chronic proliferative cholangitis (CPC) and hepatolithiasis. Escherichia coli was injected into rabbit bile ducts to induce CPC. Rabbits were divided into sham operation (SO), povidone-iodine, Metronidazole plus chlorhexidine, ofloxacin, furacillin, Neosporin® G.U., and CPC groups. Local irrigation was performed for 28 days after CPC was established. Residual E.coli and LPS, and the expression of MCP-1, CD14, COX-2, VEGF, IL-6, NF-κB, TNF-α, Fas, TGF-β1, α-SMA, Collagen-I, β-glucuronidase, PKC, C-myc, and Mucin 5AC were assessed in bile duct tissues. The residual E.coli and LPS, and expression of MCP-1, CD14, COX-2, IL-6, NF-κB, TNF-α, Fas, TGF-β1, α-SMA, β-glucuronidase, PKC, C-myc, and Mucin 5AC in the SO, povidone-iodine, Metronidazole plus chlorhexidine, ofloxacin, and Neosporin® G.U. groups were significantly lower than those in the furacillin and CPC groups (P<0.05). VEGF and Collagen-I levels in the SO, povidone-iodine, metronidazole plus chlorhexidine, and ofloxacin groups were significantly lower than those in the furacillin, Neosporin® G.U., and CPC groups (P<0.05). LPS affects the pathophysiology of E.coli caused chronic proliferative cholangitis and hepatolithiasis recurrence. Local antibiotics irrigation could prevent chronic proliferative cholangitis and stones formation by decreasing LPS-induced proinflammatory and profibrotic cytokines release. Povidone iodine, metronidazole plus chlorhexidine, and ofloxacin were more effective than Neosporin® G.U. and furacillin.

EGFR monoclonal antibody panitumumab inhibits chronic proliferative cholangitis by downregulating EGFR.

In hepatolithiasis, chronic proliferative cholangitis (CPC), an active and longstanding inflammation of stone-containing bile ducts with enhanced mucin-producing activity, not only affects the progression of the disease, it can also induce biliary carcinogenesis. The present study aimed to examine the effect of the epidermal growth factor receptor (EGFR) monoclonal antibody panitumumab (Pani) on CPC. Following the establishment of CPC rat models, periodic acid Schiff staining was used to observe the positive rate of EGFR expression. The expression levels of EGFR, mucin 5AC (MUC5AC), Ki-67, type I collagen and mammalian target of rapamycin (mTOR), and the activity of β-glucuronidase (β-G), were measured. The rats treated with Pani demonstrated a significantly lower degree of hyperproliferation of the epithelium and submucosal glands of the bile duct and collagen fibers of the bile duct wall, a significantly decreased positive rate of EGFR, reduced phosphorylation of mTOR, decreased expression of EGFR, MUC5AC, Ki-67 and type I collagen, and reduced β-G activity. The therapeutic effects in rats treated with 4 and 6 mg/kg of Pani were more marked than those in rats treated with 2 mg/kg of Pani. Collectively, the data obtained in the present study suggest that the EGFR monoclonal antibody Pani can effectively inhibit the excessive proliferation and stone-forming potential of bile duct mucosa in CPC with a receptor saturation effect. Therefore, Pani offers promise as a treatment for the prevention and control of intra-hepatic choledocholithiasis caused by CPC.

Corrigendum to "Effective Treatment of Chronic Proliferative Cholangitis by Local Gentamicin Infusion in Rabbits".

[This corrects the article DOI: 10.1155/2018/6751952.].

Effective Treatment of Chronic Proliferative Cholangitis by Local Gentamicin Infusion in Rabbits.

Background Hepatolithiasis is highly prevalent in East Asia characterized by the presence of gallstones in the biliary ducts of the liver. Surgical resection is the potentially curative treatment but bears a high risk of stone recurrence and biliary restenosis. This is closely related to the universal presence of chronic proliferative cholangitis (CPC) in the majority of patients. Recent evidence has indicated the association of bacterial infection with the development of CPC in hepatolithiasis. Thus, this study aims to investigate the feasibility and efficacy of local infusion of gentamicin (an antibiotic) for the treatment of CPC in a rabbit model. Methods The rabbit CPC model was established based on previously published protocols. Bile duct samples were collected from gentamicin-treated or control animals for pathological and molecular characterization. Results Histologically, the hyperplasia of biliary epithelium and submucosal glands were inhibited and the thickness of the bile duct wall was significantly decreased after gentamicin therapy. Consistently, the percentage of proliferating cells marked by ki67 was significantly reduced by the treatment. More importantly, this treatment inhibited interleukin 2 production, an essential inflammatory cytokine, and the enzyme activity of endogenous β-Glucuronidase, a key factor in the formation of bile pigment. Conclusions Local gentamicin infusion effectively inhibits the inflammation, cell proliferation, and lithogenesis in a rabbit model of CPC. This approach represents a potential treatment for CPC and thus prevents recurrent hepatolithiasis.

Immunohistochemically detected expression of Skp2, p27 kip1, and p-p27 (Thr187) in patients with cholangiocarcinoma.

The paper was aimed to detect the expression of Skp2, p27(kip1) (p27), and p-p27 (Thr187) in patients with cholangiocarcinoma (CCA). Western blot and immunohistochemistry were used to analyze the expression and subcellular localization of p27, Skp2, and p-p27 (Thr187) in tissue specimens of 53 patients with CCA and 10 with chronic proliferative cholangitis (CPC). Besides, the relationships of p27, Skp2, and p-p27 (Thr187) with clinicopathologic findings were examined, followed by analysis of the relationships of p27 expression with p-p27 (Thr187) and Skp2 in CCA tissue. The expression of p27 was lower in CCA than CPC tissue, and the expression of Skp2 and p-p27 (Thr187) was higher in CCA than CPC tissue (P < 0.05). The positive p27 and Skp2 proteins were mainly expressed in nucleus and cytoplasm of CCA, and p-p27 (Thr187) was only observed in nucleus. The expression of p27, Skp2, and p-p27 (Thr187) was closely associated with tumor grade and TNM stage (P < 0.05). A significantly negative correlation between the expression of p27 and Skp2 (r = -0.480, P < 0.05) and between the expression of p27 and p-p27 (Thr187) (r = -0.387, P < 0.05) was observed. Skp2-dependent proteasomal degradation of p27 plays role in the malignant transformation of CCA. Besides, the expression of p27, Skp2, and p-p27 (Thr187) may serve as markers for the progression of CCA.

Therapeutic effects of antisense gene targeting at PCNA on proliferative cholangitis

Objective In recent years, high stone recurrence and biliary restenosis rates in hepatolithiasis patients have been confirmed to be closely related to chronic proliferative cholangitis（PC）.Since PC is a kind of chronic proliferative disease, we designed this study to investigate the feasibility and effectiveness of applying PCNA shRNA to inhibit the hyperplasia and lithogenic potentiality of PC and to explore a new treatment approach for the prevention of stone recurrence and biliary restenosis.Methods The common bile duct of a PC animal model was given an intralumenal administration of 0.5 ml of PCNA shRNA to investigate its influence on PC.Results The PCNA shRNA could efficiently inhibit the hyperplasia of the biliary epithelium, submucosal gland, and collagen fiber by inhibiting mRNA or protein expressions of PCNA and Procollagen Ⅲ, thus showing promise to control or reverse PC and its secondary biliary stricture.In addition, it could inhibit the lithogenic potentiality of PC by inhibiting the expression of mucin 5AC.Conclusion PCNA shRNA treatment might achieve the effect of controlling or reversing the PC and its subsequent biliary stricture and concurrently assist in preventing the recurrence of intrahepatic stone. Key words: Cholelithiasis; Proliferative cholangitis; Recurrence; Biliary duct restenosis; PCNA

Significance of Controlling Chronic Proliferative Cholangitis in the Treatment of Hepatolithiasis

Recently, high stone recurrence and biliary restenosis rates in hepatolithiasis patients have been confirmed to be closely related to chronic proliferative cholangitis (CPC). However, the effective management of CPC has not yet been established. A vicious cycle exists between the presence of intrahepatic calculi and CPC: both the stone itself and secondary biliary infection can stimulate persistent hyperplasia in the biliary duct wall, leading to the occurrence of CPC and biliary stricture. The recurrent attacks of CPC will, in turn, facilitate new stone formation via mucoglycoprotein production, or induced biliary stricture and cholestasis. Thus, even when the stone is completely removed and the biliary tract stenosis is corrected, residual CPC will persist and progress, with an underlying risk for postoperative stone recurrence and biliary tract restenosis. Therefore, the perfect hepatolithiasis treatment would target stone removal and correction of the biliary tract stricture, as well as control of postoperative residual CPC. In fact, CPC, the management of which has been traditionally ignored, is the key to breaking this vicious cycle. Overall, the subsequent treatment of residual CPC after operation or choledochoscopic lithotomy would be helpful to decrease postoperative stone recurrence and the rate of biliary restenosis. Adding such treatment would reduce the incidence of surgical reintervention and choledochoscopic lithotomy, and it would also improve the postoperative hepatolithiasis outlook.

Proliferating cell nuclear antigen shRNA treatment attenuates chronic proliferative cholangitis in rats

Chronic proliferative cholangitis (CPC) is currently considered as a pathological basis and major cause for the high recurrence rate of intrahepatic stones. Since CPC is a form of chronic proliferative disease, this study was designed to preliminarily investigate the inhibitory effect of proliferating cell nuclear antigen (PCNA) shRNA on the hyperplastic behavior and lithogenic potentiality of CPC. The rat model of CPC was given an intralumenal administration of 0.5 mL PCNA shRNA through a 20-gauge venous retained needle. PCNA shRNA-mediated effects on CPC-associated hyperplastic behavior and lithogenic potential were assessed by investigating histological changes, immunohistochemistry for Ki-67, biochemistry for beta-glucuronidase, real-time polymerase chain reaction, and western blot analysis of PCNA, procollagen I, and mucin-3. PCNA shRNA treatment could efficiently inhibit the mRNA and protein expressions of the proliferation-related gene, PCNA, and Ki-67, which efficiently inhibited the hyperplastic behavior of the biliary epithelium, submucosal gland, and collagen fibers in the diseased bile duct wall. This novel treatment could efficiently inhibit the formation of acidic mucus glands, the expression of mucin-3 mRNA, and the secretion of endogenous beta-glucuronidase, thus effectively inhibiting the lithogenic potentiality of CPC. A further analysis revealed that PCNA shRNA-1 might display a more robust inhibitory effect on CPC-associated hyperplastic behavior and lithogenic potential than other gene sequences targeted in this study. PCNA shRNA-1 treatment could effectively inhibit the hyperplastic behavior and lithogenic potentiality of CPC, which might facilitate the prevention of stone recurrence and biliary restenosis.

Treatment of chronic proliferative cholangitis with c-myc shRNA

To investigate the feasibility and effectiveness of c-myc shRNA in inhibiting the hyperplastic behavior and lithogenic potentiality of chronic proliferative cholangitis (CPC), in order to prevent stone recurrence and biliary restenosis. An animal model of CPC was established by giving intralumenally 0.5 mL of c-myc shRNA. Then, the effects of c-myc shRNA on hyperplastic behavior and lithogenic potentiality of CPC were evaluated by histological observation, immunohistochemistry, real-time PCR and Western blotting for c-myc, proliferating cell nuclear antigen (PCNA), procollagen III, mucin 5AC, enzymatic histochemistry for beta-glucuronidase, and biochemistry for hydroxyproline in the diseased bile duct. Treatment with c-myc shRNA efficiently suppressed the hyperplasia of biliary epithelium, submucosal gland, and collagen fiber by inhibiting mRNA and protein expression of c-myc. More importantly, it decreased the lithogenic potentiality of CPC by inhibiting the expression of mucin 5AC and the secretion of endogenous beta-glucuronidase. Further investigation indicated that c-myc shRNA-3 had a better inhibitory effect on CPC. Treatment with c-myc shRNA-3 can control CPC and reduce the lithogenic potentiality of CPC.

Preliminary study of chemical bile duct embolization to treat hepatolithiasis in rabbits

The high recurrence rate of hepatolithiasis is still a problem to be solved. The purpose of the present study was therefore to carry out a preliminary study of the practical value of chemical bile duct embolization (CBDE) to treat hepatolithiasis in rabbits. Chemical bile duct embolization was performed with phenol or absolute ethanol along with N-butyl-cyanoacrylate. The feasibility and effectiveness of CBDE for chemical hepatectomy was assessed by investigating histological changes, biochemistry for hydroxyproline and in situ hybridization for collagen I. Histologically, the mucosal epithelia of the embolized bile ducts were entirely replaced by collagen fibers, thus effectively eradicating chronic proliferative cholangitis. Also of note, the diseased biliary duct lumens were completely filled with N-butyl-cyanoacrylate, thus effectively preventing calculus formation. The hepatocytes also disappeared completely in the periphery of the embolized lobe, demonstrating that the desired effects of chemical hepatectomy were achieved through CBDE. In a further comparison of embolizing agents, the phenol-cyanoacrylate embolized livers and bile ducts had a higher level of hydroxyproline and collagen I than those embolized with ethanol plus cyanoacrylate. Chemical bile duct embolization is a promising approach to prevent the recurrence of hepatolithiasis and to achieve the effect of chemical hepatectomy.

The Practical Value of Applying Chemical Biliary Duct Embolization to Chemical Hepatectomy for Treatment of Hepatolithiasis

The high recurrence rate of hepatolithiasis, together with the high operative risk of hepatectomy for specifically located stones, has not been effectively settled until now. Thus, the aim of this study was to investigate the feasibility and effectiveness of using chemical biliary duct embolization (CBDE) to achieve chemical hepatectomy in a rabbit model of hepatolithiasis. The animal model of hepatolithiasis was established using the methods of obstruction plus infection. Seven days later, the left hepatic ducts were embolized using phenol plus cyanoacrylate or absolute ethanol plus cyanoacrylate. Subsequently, the influence of CBDE on bile duct, liver, and stone formation was analyzed by histology, RT-PCR for procollagen, biochemistry, and enzymatic histochemistry for beta-glucuronidase (beta-G). CBDE resulted in the entire ablation of the diseased biliary duct mucosa and the complete occlusion of the diseased biliary duct lumen, thus effectively eradicating chronic proliferative cholangitis and preventing stone formation. More importantly, CBDE also resulted in the complete fibrosis and "self-cut" in the periphery of the embolized lobe, thus achieving chemical hepatectomy. Also of note, the embolized lobe exhibited a much lower level of endogenous beta-G than the nonembolized lobe, indicating an inhibitory effect of CBDE on beta-G. Besides, the mRNA level of procollagen I in the embolized bile duct wall of phenol embolization group was significantly higher than the ethanol embolization group. Chemical biliary duct embolization, especially using phenol plus cyanoacrylate, may prevent the recurrence of intrahepatic stone and concurrently achieve the effect of chemical hepatectomy.

Mutations identified in the human multidrug resistance P-glycoprotein 3 (ABCB4) gene in patients with primary hepatolithiasis

Primary hepatolithiasis (HL), highly prevalent in the Far East, including Japan, is characterized clinically by chronic proliferative cholangitis with frequent recurrences. In HL patients, hepatic hyposecretion of phospholipid due to decreased multidrug resistance P-glycoprotein 3 (MDR3; now referred to as ABCB4) expression levels (Hepatology 2001;33:1194-1205) may contribute to the formation of aggressive ductular lesions through a decreased formation of mixed micelles. However, specified factors underlying the decreased expression levels of MDR3 have not been well defined. To determine whether the decreased MDR3 expression level is associated with the gene mutations, mutation analysis of cDNA of the MDR3 gene with focus on the coding region was performed using liver specimens. Heterozygous mutations were detected in only two of 16 HL patients. By sequence analysis of the gene, a 77-bp deletion at nucleotides 537-613 in exon 7 in transmembrane domain (TM) 3, which results in a frameshift at codon 179 and an early stop codon predicting a truncated protein, was found as a heterozygous mutation in two of the 16 patients. A 1-bp deletion at nucleotide 1015 in exon 10 in TM 6 was found as a heterozygous mutation in one of those two patients, and a 242-bp deletion at nucleotides 2683-2924 in exons 22-23 in TM 11 was found as a heterozygous mutation in the same patient. No other mutations were found in the other 14 patients. In real-time polymerase chain reaction (PCR), no significant difference was found between the mRNA levels of MDR3 in the two HL patients with mutations nor in the other 14 patients without mutations. Immunostaining of MDR3 protein was found in the bile canaliculi of liver sections from the two patients with mutations. The results suggest that in primary HL the decreased transcription levels of MDR3 in the liver are not due to the mutations detected in the coding region of the gene.

Prostaglandin E receptors in bile ducts of hepatolithiasis patients and the pathobiological significance for cholangitis

Background & Aims: In hepatolithiasis, chronic proliferative cholangitis may influence the progression of the disease. Prostaglandin (PG) E 2 experimentally causes morphologic changes to intrahepatic bile ducts, analogous to the changes found in cholangitis. This study was designed to gain an understanding of the involvement of PGE 2 and PGE receptor (EP) subtypes in the development of cholangitis. Methods: The expression levels of secretory-type group IIA phospholipase A 2 (sPLA 2-IIA) and cyclooxygenase (COX)-2 as well as EP subtypes were determined in the bile ducts with change of cholangitis. In in vitro experiments, growth promotion and mucin secretagogue properties of biliary epithelial cells in response to EP-selective agonists or antagonists were studied. Results: The messenger RNA (mRNA) level of sPLA 2-IIA and the protein and mRNA levels of COX-2 were significantly increased in the bile ducts of patients with hepatolithiasis compared with the levels of the bile ducts of control subjects. These changes were associated with a concomitant increase in PGE 2 and total mucin concentrations in the bile. The mRNAs of EP subtypes EP 2, EP 3, and EP 4 but not EP 1 were amplified in the bile ducts. Treatment with an EP 4-selective agonist (ONO-AE1-329) caused a dose-dependent increase in DNA synthesis, colony number, and mucin secretion in the cells. Conversely, treatment with an EP 4-selective antagonist (ONO-AE3-208) abolished the biological effects of PGE 2 on the cells. Conclusions: In hepatolithiasis, an enhanced synthesis of sPLA 2-/COX-2-derived PGE 2 and its actions mediated via the EP 4 receptor in the bile ducts may be of pathobiological significance for chronic proliferative cholangitis.

Involvement of prostaglandin E2 and its specific receptor subtype EP4 in chronic proliferative cholangitis in the bile ducts of patients with hepatolithiasis

Involvement of prostaglandin E2 and its specific receptor subtype EP4 in chronic proliferative cholangitis in the bile ducts of patients with hepatolithiasis

Cystic fibrosis transmembrane conductance regulators (CFTR) in biliary epithelium of patients with hepatolithiasis.

Cystic fibrosis transmembrane conductance regulator (CFTR) has been considered to be involved in the regulatory pathway of biliary mucin secretion. We investigated expression of CFTR protein and mRNA in 24 livers with hepatolithiasis, in 6 with cholangiocarcinoma, and in 12 histologically normal livers. According to the histologic features of chronic proliferative cholangitis, hepatolithiasis was subdivided into inflammatory cell infiltration predominant (N = 14) and fibrosis predominant (N = 10). The mean signal density of CFTR in overall hepatolithiasis and in histologically normal livers was 1.23 +/- 0.15 and 1.01 +/- 0.13, respectively (P > 0.05). The CFTR protein (1.60 +/- 0.18) and mRNA (1.09 +/- 0.15) in inflammatory cell infiltration predominant patients were significantly higher (CFTR protein, 1.01 +/- 0.13; mRNA, 0.75 +/- 0.11) than in control subjects (P < 0.05), whereas those in fibrosis-predominant patients (CFTR protein, 0.72 +/- 0.15; mRNA, 0.55 +/- 0.13) were less than in control subjects (P < 0.05). CFTR protein (1.27 +/- 0.17) in patients with cholangiocarcinoma was not different from that of control subjects (1.01 +/- 0.13). CFTR expression in hepatolithiasis patients was rather heterogeneous but was closely related to the histology of bile ducts.

Etiologic significance of defects in cholesterol, phospholipid, and bile acid metabolism in the liver of patients with intrahepatic calculi.

Intrahepatic calculi, highly prevalent in the Far East, including Japan, are characterized clinically by chronic proliferative cholangitis with frequent stone recurrences. Intrahepatic calculi consist of 2 groups, i.e., brown pigment stones, including a high cholesterol content, and cholesterol stones, with the former predominating. To gain insights into the pathogenesis of intrahepatic calculi, cholesterol and bile acid biosynthesis, as well as alterations in intracellular transport and/or canalicular secretion of phospholipid and bile acid were investigated in liver of patients with intrahepatic calculi. Enzyme activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase were increased (12.8 +/- 1.9 pmol/min/mg protein, mean +/- SEM vs. 5.5 +/- 0.4 in controls; P < .01) and cholesterol 7 alpha-hydroxylase activities were decreased (1.3 +/- 0.4 vs. 4.9 +/- 0.6; P < .01) in liver specimens of patients with brown pigment stones. In addition, messenger RNA (mRNA) levels of multidrug resistance P-glycoprotein 3 (MDR3 Pgp) and phosphatidylcholine transfer protein (PCTP) were markedly low in the liver specimens compared with the levels in specimens of control subjects, gallbladder stone patients, and patients with obstructive cholestasis. The protein levels and the immunohistochemical staining were decreased for MDR3 Pgp and PCTP in the liver. Consistently, the concentrations of phospholipid were markedly reduced in the hepatic bile from both affected and unaffected hepatic segments. In patients with intrahepatic calculi, biliary cholesterol supersaturation and the formation of cholesterol-rich brown pigment as well as cholesterol stones may be attributed to decreased hepatic transport and biliary secretion of phospholipids, in the setting of increased cholesterogenesis and decreased bile acid synthesis.

Expression of nerve growth factor in hepatolithiasis

Nerve growth factor (NGF) has recently been shown to influence the survival and function of non-neuronal inflammatory cells, possibly through its activity as a colony-stimulating factor. It may also play an important role in acute inflammation and tissue repair. However, no prior report has focused on NGF in chronic inflammatory diseases of the gastrointestinal and biliary tracts. The aim of this study was to examine the expression of NGF in hepatolithiasis. Twenty-six liver specimens resected from 22 patients with intrahepatic calcium bilirubinate stones and from 4 patients with intrahepatic cholesterol stones were examined immunohistochemically. The 22 patients with calcium bilirubinate stones demonstrated NGF immunoreactivity associated with surrounding inflammatory cells that was localized to the epithelia of proliferative peribiliary glands in the ductal wall. However, neither the surface lining of the bile duct nor hepatocytes expressed detectable NGF immunoreactivity. In the cholesterol stones cases in contrast, peribiliary glandular elements and inflammatory cell infiltration were less extensive than those observed in cases of calcium bilirubinate stones, and NGF immunoreactivity was not noted. These observations suggest that proliferative peribiliary glands express NGF protein in chronic proliferative cholangitis. This is characteristic of intrahepatic calcium bilirubinate stones.

Secretory low-molecular-weight phospholipases A2 and their specific receptor in bile ducts of patients with intrahepatic calculi: factors of chronic proliferative cholangitis.

Intrahepatic calculi is characterized by an intractable course and frequent recurrences, requiring multiple operative interventions. Chronic proliferative cholangitis, an active and long-standing inflammation of the stone-containing bile ducts with the hyperplasia of epithelia and the proliferation of the duct-associated mucus glands, may underlie the complex nature of the disease. In terms of the pathophysiology, interest has been focused on the role of secretory low-molecular-weight phospholipases A2 (sPLA2s) as inflammatory mediators or factors modulating cell functions via their specific sPLA2-receptor, and also on the production and secretion of altered mucin molecules from the inflamed bile ducts. In search of factors involving chronic proliferative cholangitis, the sPLA2 isoforms in the bile such as the pancreatic-type sPLA2 (group IB sPLA2) and the arthritic-type sPLA2 (group IIA sPLA2), were assayed to correlate protein masses of the sPLA2s with alterations in biliary composition. Furthermore, the steady-state messenger RNA (mRNA) levels of the sPLA2s, the membrane-bound sPLA2-receptor, cystic fibrosis transmembrane conductance regulator (CFTR), and mucin core polypeptide (MUC) genes in the bile ducts were assayed by reverse- transcriptase polymerase chain reaction (RT-PCR). Immunoreactive sPLA2-IB and sPLA2-IIA levels were significantly higher in the bile from the stone-containing hepatic ducts (2315 +/- 677 for sPLA2-IB; 281 +/- 42 for sPLA2-IIA ng/dL, mean +/- SEM; n = 20) than in the ductal bile from gallbladder stone patients (609 +/- 92, P <.01; 22 +/- 2, P <.01; n = 24). The increased sPLA2 levels were associated with a concomitant increase in lysophosphatidylcholine, prostaglandin E2 (PGE2), and total mucin concentrations. The affected bile ducts showed an increased mRNA level of sPLA2-IB and sPLA2-IIA compared with the ducts from control subjects, in whom the mRNAs of the sPLA2-receptor and other sPLA2 isoforms, such as groups V and X sPLA2s, were coincidently expressed. Reflecting the increased amounts of total biliary mucins, the affected ducts showed an increase in mRNA levels of CFTR as well as MUC2, MUC3, MUC5AC, MUC5B, and MUC6 compared with the ducts from control subjects. In intrahepatic calculi, an enhanced expression of the sPLA2s and their possible cross-talk via sPLA2-receptor may be of pathophysiological significance for the chronic proliferative cholangitis, in association with the enhanced CFTR expression and the alterations in mucin gene expression in the bile ducts, probably through potentiating arachidonate metabolism with associated biliary alterations favoring growth of preexisting stones and even further progressions.

Proliferating cell nuclear antigen expression in peribiliary glands of stone-containing intrahepatic bile ducts.

All cases of hepatolithiasis showed features of chronic proliferative cholangitis, and it has been speculated that the atypical glandular proliferation might be a precursor to overt cholangiocarcinoma. Proliferative cell nuclear antigen (PCNA) is a nuclear protein synthesized in the G1/S phase of the cell cycle and therefore is related to cell proliferative activity. In an attempt to assess the activity of cell proliferation of stone-containing intrahepatic bile ducts, we conducted a study using immunohistochemical staining with monoclonal antibody to score PCNA in intrahepatic bile ducts. Thirty patients (10 men, 20 women; mean age 52.4 years) having hepatolithiasis surgically resected were studied. Ten stone-free patients served as controls. All 40 specimens were immunostained for PCNA using PC 10 monoclonal antibody. PCNA of both stone-containing and stone-free intrahepatic bile ducts were assessed by counting positive staining nuclei per 500 cells and expressed as labeling index (LI), ie, percentage of positive nuclei to the total number of nuclei. The PCNA LI in stone-free intrahepatic bile ducts was generally low: 10.0+/-13.2%, 10.4+/-10.7% and 7.9+/-9.6% for extramural glands, intramural glands, and epithelial lining, respectively. In contrast, the PCNA LI for stone-containing intrahepatic bile ducts were significantly higher than those of controls (P < 0.001): 49.4+/-8.3%, 40.6+/-7.0% and 34.1+/-6.8% for extramural glands, intramural glands, and epithelial lining, respectively. The extramural glands had a significantly higher PCNA LI (P < 0.001) than the intramural glands and controls. Hyperplasia was found in all specimens, while dysplasia was found in six of 30 cases with hepatolithiasis. The dysplastic cells also had a higher PCNA LI (P < 0.001) than the hyperplastic cells and normal epithelium. Our findings showed that there is marked increase of activity of cell proliferation in stone-containing intrahepatic bile ducts. It is well known that genetic mutations are facilitated in proliferating cells. Therefore, our results suggest that the high epithelial turnover in dysplastic cells and extramural glands had higher potential for proliferation and neoplastic transformation in long-standing untreated hepatolithiasis.