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During bile formation by the liver, large volumes of water are transported across two epithelial barriers consisting of hepatocytes and cholangiocytes (i.e. intrahepatic bile duct epithelial cells). We recently reported that a water channel, aquaporin-channel-forming integral protein of 28 kDa, is present in cholangiocytes and suggested that it plays a major role in water transport by these cells. Since the mechanisms of water transport across hepatocytes remain obscure, we performed physiological, molecular, and biochemical studies on hepatocytes to determine if they also contain water channels. Water permeability was studied by exposing isolated rat hepatocytes to buffers of different osmolarity and measuring cell volume by quantitative phase contrast, fluorescence and laser scanning confocal microscopy. Using this method, hepatocytes exposed to hypotonic buffers at 23 degrees C increased their cell volume in a time and osmolarity-dependent manner with an osmotic water permeability coefficient of 66.4 x 10(-4) cm/s. In studies done at 10 degrees C, the osmotic water permeability coefficient decreased by 55% (p0.001, at 23 degrees C; t test). The derived activation energy from these studies was 12.8 kcal/mol. After incubation of hepatocytes with amphotericin B at 10 degrees C, the osmotic water permeability coefficient increased by 198% (p0.001) and the activation energy value decreased to 3.6 kcal/mol, consistent with the insertion of artificial water channels into the hepatocyte plasma membrane. Reverse transcriptase polymerase chain reaction with hepatocyte RNA as template did not produce cDNAs for three of the known water channels. Both the cholesterol content and the cholesterol/phospholipid ratio of hepatocyte plasma membranes were significantly (p0.005) less than those of cholangiocytes; membrane fluidity of hepatocytes estimated by measuring steady-state anisotropy was higher than that of cholangiocytes. Our data suggests that the osmotic flow of water across hepatocyte membranes occurs mainly by diffusion via the lipid bilayer (not by permeation through water channels as in cholangiocytes).

The cholangiopathies are a group of hepatobiliary diseases in which intrahepatic bile duct epithelial cells, or cholangiocytes, are the target for a variety of destructive processes, including immune-mediated damage. We tested the hypothesis that cholangitis could be induced in rodents by immunization with highly purified cholangiocytes. Inbred Wistar rats were immunized with purified hyperplastic cholangiocytes isolated after bile duct ligation from either syngeneic Wistar or allogeneic Fischer 344 rats; control rats were immunized with bovine serum albumin (BSA) or hepatocytes. After immunization with cholangiocytes, recipient animals developed histologic evidence of nonsuppurative cholangitis without inflammation in other organs; groups immunized with BSA or hepatocytes showed no cholangitis. Immunohistochemical studies revealed that portal tract infiltrates around bile ducts consisted of CD3-positive lymphocytes, some of which expressed major histocompatibility complex class II antigen; B cells and exogenous monocytes/macrophages were essentially absent. Transfer of unfractionated ConA-stimulated spleen cells from cholangiocyte-immunized (but not BSA-immunized) rats into recipients also caused nonsuppurative cholangitis. Moreover, these splenocytes from cholangiocyte-immunized (but not BSA-immunized) rats were cytotoxic in vitro for cultured rodent cholangiocytes; no cytotoxicity was observed against a rat hepatocyte cell line. Also, a specific antibody response in sera of cholangiocyte-immunized rats was demonstrated by immunoblots against cholangiocyte proteins. Finally, cholangiograms in cholangiocyte-immunized rats showed distortion and tortuosity of the entire intrahepatic biliary ductal system. This unique rodent model of experimental cholangitis demonstrates the importance of immune mechanisms in the pathogenesis of cholangitis and will prove useful in exploring the mechanisms by which the immune system targets and damages cholangiocytes.

Cholangiocytes possess specific membrane transport systems for a large number of substrates. As a result of the secretion or absorption of solutes, water moves passively across plasma membranes, very likely through water channels; as a result, the volume and composition of primary bile are modified. Cholangiocytes express receptors for secretin and somatostatin, hormones that alter intracellular levels of cAMP in opposite directions; these alterations very likely alter the activation (i.e., phosphorylation state) or number (i.e., by coupled exocytic-endocytic membrane modification) of transporters in the apical or basolateral cholangiocyte membranes. Although our understanding of these mechanisms is sill embryonic, it is anticipated that accelerated research efforts by laboratories utilizing recently available experimental models will provide important advances in the knowledge of the molecular mechanisms involved in the transport of water and solutes by cholangiocytes. The challenge will be to integrate the emerging, often isolated, findings in a physiologically relevant manner to understand how cholangiocytes function normally and in pathological conditions.

Primary cilia are multisensory organelles recently found to be absent in some tumor cells, but the mechanisms of deciliation and the role of cilia in tumor biology remain unclear. Cholangiocytes, the epithelial cells lining the biliary tree, normally express primary cilia and their interaction with bile components regulates multiple processes, including proliferation and transport. Using cholangiocarcinoma as a model, we found that primary cilia are reduced in cholangiocarcinoma by a mechanism involving histone deacetylase 6 (HDAC6). The experimental deciliation of normal cholangiocyte cells increased the proliferation rate and induced anchorage-independent growth. Furthermore, deciliation induced the activation of mitogen-activated protein kinase and Hedgehog signaling, two important pathways involved in cholangiocarcinoma development. We found that HDAC6 is overexpressed in cholangiocarcinoma and overexpression of HDAC6 in normal cholangiocytes induced deciliation and increased both proliferation and anchorage-independent growth. To evaluate the effect of cilia restoration on tumor cells, we targeted HDAC6 by short hairpin RNA (shRNA) or by the pharmacologic inhibitor, tubastatin-A. Both approaches restored the expression of primary cilia in cholangiocarcinoma cell lines and decreased cell proliferation and anchorage-independent growth. The effects of tubastatin-A were abolished when cholangiocarcinoma cells were rendered unable to regenerate cilia by stable transfection of IFT88-shRNA. Finally, inhibition of HDAC6 by tubastatin-A also induced a significant decrease in tumor growth in a cholangiocarcinoma animal model. Our data support a key role for primary cilia in malignant transformation, provide a plausible mechanism for their involvement, and suggest that restoration of primary cilia in tumor cells by HDAC6 targeting may be a potential therapeutic approach for cholangiocarcinoma. Cancer Res; 73(7); 2259–70. ©2013 AACR.

Krüppel-like factor 11 (KLF11) and the highly homologous KLF10 proteins are transcription factors originating from duplication of the Drosophila melanogaster ancestor cabut. The function of these proteins in epithelial cells has been previously characterized. In the current study, we report a functional role for KLF11 in mesenchymal cells and in mesenchymal cell dysfunction, namely, fibrosis, and subsequently perform a detailed cellular, molecular, and in vivo characterization of this phenomenon. We find that, in cultured mesenchymal cells, enhanced expression of KLF11 results in activated extracellular matrix pathways, including collagen gene silencing and matrix metalloproteinases activation without changes in tissue inhibitors of metalloproteinases. Combined, reporter and chromatin immunoprecipitation assays demonstrate that KLF11 interacts directly with the collagen 1a2 (COL1A2) promoter in mesenchymal cells to repress its activity. Mechanistically, KLF11 regulates collagen gene expression through the heterochromatin protein 1 gene-silencing pathway as mutants defective for coupling to this epigenetic modifier lose the ability to repress COL1A2. Expression studies reveal decreased levels of KLF11 during liver fibrogenesis after chemically induced injury in vivo. Congruently, KLF11(-/-) mice, which should be deficient in the hypothesized anti-fibrogenic brake imposed by this transcription factor, display an enhanced response to liver injury with increased collagen fibril deposition. Thus, KLFs expands the repertoire of transcription factors involved in the regulation of extracellular matrix proteins in mesenchymal cells and define a novel pathway that modulates the fibrogenic response during liver injury.

Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense., Author Summary Exosomes are secreted membranous nanovesicles produced by a variety of cells. Exosomes shuttle various molecules to transfer them to neighboring or distant cells, and have been implicated as mediators in cell-cell communications to modulate physiological and pathological procedures. Here, we report that luminal release of exosomal vesicles is an important component of Toll-like receptor 4 (TLR4)-associated gastrointestinal epithelial defense against infection by Cryptosporidium parvum, an obligate intracellular protozoan that infects gastrointestinal epithelial cells. Activation of TLR4 signaling in host epithelial cells following C. parvum infection promotes luminal release of epithelial exosomes and exosomal shuttling of antimicrobial peptides from the epithelium. By direct binding to the C. parvum surface, exosomal vesicles reveal anti-C. parvum activity. Activation of TLR4 signaling in epithelial cells after LPS stimulation also increases exosomal release and exosome-associated anti-C. parvum activity. Therefore, we speculate that TLR4-mediated exosome release may be relevant to innate mucosal immunity in general, representing a new target for therapeutic intervention for infectious diseases at the mucosal surface.

Previous investigators measuring the pH of lysosomes have used digitized video microscopy (DVM) in freshly isolated or cultured cells. Although useful, this technique is time consuming, requires the use of an image analysis system, and is limited by the fact that measurements can be made in only a relatively small number of cells. The aim of this study was to develop and initially apply a technique using flow cytometry to make dynamic measurements of lysosomal pH in a large number of living hepatocytes. Rats were injected intraperitoneally with fluorescein isothiocyanate-dextran (FITC-Dex), a pH-sensitive fluorescent probe that is sequestered into lysosomes. Hepatocytes were isolated 16 hours after injection by collagenase perfusion. Lysosomal pH was measured in 20,000 hepatocytes per animal using flow cytometry with excitation at 488 nm and emission at 530 nm (pH sensitive) and 585 nm (pH insensitive). A standard curve of pH versus the 530/585 nm ratio was generated with FITC-Dex-loaded hepatocytes by equilibrating intralysosomal pH with extracellular pH using ionophores and metabolic inhibitors. The acute effects of chloroquine and methylamine were determined by exposing isolated hepatocytes to these lysosomotropic agents. The effect of chronic administration of chloroquine and Triton WR-1339 (Rutger Chemical, Inc., Irvington, NJ) on lysosomal pH was also measured. Intralysosomal pH was 4.67 + 0.02, nearly identical to the value 4.70 + 0.05 previously measured by us using DVM. Both chloroquine and methylamine caused both rapid (

Cholangiocytes, the epithelial cells that line intrahepatic bile ducts, are composed of plasma membranes with discrete apical (lumenal) and basolateral domains. While these domains are thought to contain different transporters, exchangers, channels, and receptors, no methodology currently exists for the isolation of these different membrane compartments. Thus, our aim was to develop a technique to isolated plasma membranes from cholangiocytes enriched in apical or basolateral domains. We isolate a cholangiocyte-enriched population of cells from rats 3 weeks after bile duct ligation (BDL), a maneuver which stimulates selective cholangiocyte proliferation. Using isopycnic centrifugation on linear sucrose gradients, we prepared a mixed cholangiocyte plasma membrane (MCPM) fraction from which we further generated separate apical and basolateral cholangiocyte plasma membrane (ACPM and BCPM, respectively). We characterized these fractions by specific marker enzyme assays, transmission electron microscopy (TEM), lipid analysis, anisotropy measurements, one- and two-dimensional gel electrophoresis, and quantitative immunoblots of the cystic fibrosis transmembrane conductance regulator (CFTR). Marker enzyme assays and TEM revealed that the MCPM fraction was essentially devoid of other organelles but was enriched approximately 70-fold in phosphodiesterase I, a general plasma membrane marker; the ACPM and BCPM were appropriately enriched in the respective apical and basolateral markers. TEM of ACPM and BCPM revealed homogeneous preparations of vesiculated membranes without contamination by other organelles. Lipid analysis, one- and two-dimensional gel electrophoresis, CFTR immunoblots, and anisotropy measurements showed unequivocal differences in lipid and protein composition and in fluidity between the ACPM and BCPM domains.(ABSTRACT TRUNCATED AT 250 WORDS)

PSC is the most common and most important hepatobiliary disease seen in association with IBD. Approximately 5% of all patients with CUC have PSC, and most patients with PSC ultimately develop IBD, usually CUC. PSC and CUC appear to be associated diseases-one does not cause the other, but common pathogenic mechanisms are likely involved. PSC alone does not differ from PSC with IBD with regard to clinical, biochemical, cholangiographic, and hepatic histologic features. There is an overlap syndrome of CAH and PSC in patients with CUC suggesting that patients with CAH and CUC should have a cholangiogram. Colectomy in patients with PSC and CUC does not influence the PSC and, if done for colitic indications, should be accompanied by an ileal pouch-anal anastomosis. Serologic markers are being identified, which are frequently found in PSC with or without CUC, including markers for the dreaded complication of cholangiocarcinoma. Unfortunately, patients with PSC and CUC are doubly at risk for malignancies of the colon and biliary system. Medical therapies are being assessed that may beneficially affect both PSC and CUC, and liver transplantation is life-saving for patients with advanced PSC. Although CAH and gallstones are also found in association with IBD, they are much less common and of considerably less clinical importance than PSC associated with IBD.

Previous work from our laboratory has implicated hormone-induced plasma membrane movement (i.e., endo- and exocytosis) in water and electrolyte transport by the epithelial cells that line the ducts in the liver (i.e., cholangiocytes). To further explore the cellular mechanisms regulating ductal bile secretion, we infused somatostatin and/or secretin intravenously into rats 2 wk after either bile duct ligation (BDL), a procedure that induces selective proliferation of cholangiocytes, or sham surgery and measured bile flow and biliary constituents. We also determined the effect of somatostatin on basal and secretin-induced exocytosis by purified cholangiocytes isolated from rat liver after BDL. Finally, we studied the expression of the somatostatin receptor gene by both ribonuclease (RNase) protection and nuclear run-on assays using cDNA encoding for two subtypes of the somatostatin receptor gene (i.e., SSTR1 and SSTR2). In vivo, somatostatin infusion caused a dose-dependent bicarbonate-poor decrease (57% maximal decrease below baseline; P < 0.05) in bile flow in BDL but not in sham-operated rats; in contrast, secretin caused a dose-dependent bicarbonate-rich choleresis (228% maximal increase above baseline; P < 0.05) in BDL but not in sham-operated rats. Simultaneous or prior infusion of somatostatin inhibited the secretin-induced hypercholeresis in BDL rats. In vitro, somatostatin had no effect on basal exocytosis by cholangiocytes isolated from BDL rats; however, somatostatin inhitibed (88% maximal inhibition; P < 0.05) secretin-induced exocytosis by cholangiocytes in a dose-dependent fashion. In addition, somatostatin inhibited secretin-induced increases in levels of adenosine 3',5'-cyclic monophosphate (cAMP) in cholangiocytes isolated from BDL rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The turnover rate of an individual protein is a function of the rates of synthesis and loss of that protein. For most intracellular proteins, loss occurs through digestion by lysosomal or cytosolic proteases. Although a significant proportion of hepatic lysosomal enzymes is released from the hepatocyte by excretion into bile, the contribution of biliary excretion to the turnover of hepatic lysosomal enzymes has never been measured. Thus, we used in vivo pulse-labeling to determine the half-lives of two hepatic hydrolases, beta-galactosidase (beta-gal) and beta-glucuronidase (beta-glu). Each enzyme was purified by immunoisolation from hepatic lysosomes that were isolated at various times after injection of rats with 3H-labeled leucine. The decay curves for the specific radioactivities of beta-gal and beta-glu were used to calculate the half-lives of the proteins, which were 3.8 and 5.1 days, respectively. To determine the percent of total hepatic contents of each enzyme that was lost per day by biliary excretion, we collected bile from bile fistula rats for 24 hours and then used radioimmunoassays to quantitate the amounts of beta-gal and beta-glu in bile and liver samples of the same rats. We found that approximately 4% of the total hepatic contents of both beta-gal and beta-glu was excreted into bile per day. Finally, we used these data to calculate that 31% and 41% of hepatic losses of beta-gal and beta-glu, respectively, were due to biliary excretion. These results suggest that extracellular release through biliary excretion is a major mechanism contributing to the turnover of lysosomal hydrolases.

We reviewed the initial lipid and fat-soluble vitamin levels in 56 patients with primary sclerosing cholangitis (PSC) enrolled in a randomized, placebo-controlled trial evaluating ursodeoxycholic acid. We also evaluated lipid and vitamin levels in a group of 87 patients with advanced PSC being evaluated for liver transplantation. Of the patients entering the therapeutic trial, 41% had total serum cholesterol levels greater than the 95th percentile, whereas only 20% had high-density lipoprotein cholesterol levels above normal, and only one (2%) had an elevated triglyceride level. Total cholesterol levels were correlated with serum bilirubin levels and were lower in early versus later histologic stages (206 +/- 61 vs. 248 +/- 79, p = 0.04). Of the 87 pretransplant patients, 29% had elevated serum cholesterol levels and 17% had elevated serum triglyceride levels. Total serum cholesterol levels correlated inversely with total serum bilirubin levels in this group. In patients in the therapeutic trial, vitamin A deficiency was seen in 40%, vitamin D deficiency in 14%, and vitamin E deficiency in 2% of those tested. More prominent deficiencies of fat-soluble vitamins occurred in the pretransplant group of patients, with 82% deficient in vitamin A, 57% deficient in vitamin D, and 43% deficient in vitamin E. We conclude that hypercholesterolemia and fat-soluble vitamin deficiencies are frequent in patients with PSC and are more common with more severe disease. Patients with PSC, especially with advanced liver disease, should be screened for fat-soluble vitamin deficiencies and supplemented accordingly.

Intermediate-stage hepatocellular carcinoma (HCC) is usually treated with locoregional therapy using transarterial chemoembolization (TACE). Transarterial radioembolization (TARE) using β-emitting yttrium-90 integral to the glass matrix of the microspheres is an alternative to TACE. This retrospective case-control study compared the outcomes and safety of TARE versus TACE in patients with unresectable HCC.Patients with unresectable HCC without portal vein thrombosis treated with TARE between 2005 and 2008 (n = 61) were retrospectively frequency-matched by age, sex, and liver dysfunction with TACE-treated patients (n = 55) in the Mayo Clinic Hepatobiliary Neoplasia Registry. Imaging studies were reviewed, and clinical and safety outcomes were abstracted from the medical records.Complete tumor response was more common after TARE (12 %) than after TACE (4 %) (p = 0.17). When complete response was combined with partial response and stable disease, there was no difference between TARE and TACE. Median survival did not differ between the two groups (15.0 months for TARE and 14.4 months for TACE; p = 0.47). Two-year survival rates were 30 % for TARE and 24 % for TACE. TARE patients received fewer treatments (p0.001). Fifty-nine (97 %) TARE patients received outpatient treatment. In contrast, 53 (98 %) TACE patients were hospitalized for ≥1 day (p0.001). Compared with TACE, TARE was more likely to induce fatigue (p = 0.003) but less likely to cause fever (p = 0.02).There was no significant difference in efficacy between TARE and TACE. TARE patients reported more fatigue but had less fever than TACE patients. Treatment with TARE required less hospitalization than treatment with TACE. These findings require confirmation in randomized trials.

Polycystic kidney (PKD) and liver (PLD) diseases cause significant morbidity and mortality. A large body of evidence indicates that cyclic AMP plays an important role in their pathogenesis. Clinical trials of drugs that reduce cyclic AMP levels in target tissues are now in progress. Secretin may contribute to adenylyl cyclase-dependent urinary concentration and is a major agonist of adenylyl cyclase in cholangiocytes. To investigate the role of secretin in PKD and PLD, we have studied the expression of secretin and the secretin receptor in rodent models orthologous to autosomal recessive ( PCK rat) and dominant ( Pkd2−/WS25mouse) PKD; the effects of exogenous secretin administration to PCK rats, PCK rats lacking circulating vasopressin ( PCKdi/di), and Pkd2−/WS25mice; and the impact of a nonfunctional secretin receptor on disease development in Pkd2−/WS25:SCTR−/−double mutants. Renal and hepatic secretin and secretin receptor mRNA and plasma secretin were increased in both models, and secretin receptor protein was increased in the kidneys and liver of Pkd2−/WS25mice. However, exogenous secretin administered subcutaneously via osmotic pumps had minimal or negligible effects and the absence of a functional secretin receptor had no influence on the severity of PKD or PLD. Therefore, it is unlikely that by itself secretin plays a significant role in the pathogenesis of PKD and/or PLD.

Polycystic liver (PLD) and kidney (PKD) diseases are incurable genetic disorders linked to disturbances in many intracellular signaling pathways and cell functions.(1–3) One of the well-defined mechanisms involved in hepato-renal cystogenesis is increased accumulation of intracellular cAMP that triggers cell hyper-proliferation, cell cycle deregulation and fluid secretion. Basal cAMP levels in cholangiocytes are maintained by the coordinated functioning of: 1) secretin receptors (activation of which by secretin increases cAMP); 2) somatostatin receptors ([SSTRs], activation of which by somatostatin inhibits cAMP); 3) adenylyl cyclases (crucial for cAMP production); and 4) phosphodiesterases (critical for cAMP degradation).(1, 2) Activation of SSTRs induces multiple transduction pathways and mediates several cellular functions; however inhibition of cell proliferation is one of the major effects.(4, 5) Cholangiocytes express all five SSTRs (i.e., SSTR1 through 5).(6, 7) Importantly, targeting of SSTRs with synthetic somatostatin analogues in patients with PLD and PKD has limited but unequivocal benefit by reducing cyst growth.(3, 7–10) The natural SSTR ligand, somatostatin, is susceptible to proteolytic degradation and has a short half-life (~3 minutes) limiting its clinical utility.(5) Over the years, multiple stable somatostatin analogues have been developed. Octreotide [(OCT), Sandostatin®, SMS 201-95], a synthetic octapeptide with a half-life of 2 hours, was the first clinically introduced analogue. We and others have tested the effects of OCT in preclinical and clinical trials in PLD and PKD.(7–11) However, despite beneficial results (i.e., symptom relief and cyst reduction), changes in liver and kidney volumes in patients are moderate; thus, additional pharmacologic approaches are needed. OCT binds with high affinity to SSTR2 and SSTR3, with moderate affinity to SSTR5, and has no affinity to SSTR1 and SSTR4.(5, 12, 13) Since all five SSTRs coexist in cholangiocytes and renal epithelia(6, 7, 14), somatostatin analogues with higher binding affinity to a broader range of SSTRs might be more effective. Recently, cyclohexapeptide Pasireotide (PAS, SOM-230) has been evaluated experimentally and clinically for the treatment of different pathological conditions.(12, 13) PAS has a high affinity to SSTR1, SSTR2, SSTR3 and SSTR5 and a half-life of 12 hours.(12, 13, 15) Thus, we hypothesized that PAS should have more potent suppressive effects than OCT on cyst growth. We used in vitro and in vivo experimental systems and models to compare the effects of OCT and PAS on cAMP levels, cell proliferation, cell cycle distribution and hepatic cyst growth in vitro; and hepato-renal cystogenesis in two animal models of PLD and PKD, the PCK rat and Pkd2WS25/− mice. Expression of SSTRs in control and cystic cholangiocytes was assessed under basal conditions and after treatment. In addition, we examined concentrations of IGF1 and VEGF in response to OCT and PAS since these growth factors are linked to indirect inhibitory action of somatostatin analogues and known to trigger hepato-renal cystogenesis via autocrine and paracrine mechanisms.(15–19) We show that PAS has stronger suppressive effects on hepato-renal cystogenesis compared to OCT and thus might be more beneficial than OCT in patients with PLD and PKD.

The development of new and refined separation techniques--including FACS, FFE, CFE and isopyknic gradients--has had a profound impact on the ability of investigators to isolate specific cell types from the liver. Although some of these techniques, such as FFE, may be of limited preparative value, they are nonetheless important analytical tools that detect subtle differences among cell subpopulations. The isolation of highly purified preparations of liver cells in large yields requires the use of more conventional purification methods such as CFE and isopyknic centrifugation. Immunological approaches represent a key development for the isolation of specific liver cell types, especially when they are used in combination with other techniques. Excellent, reliable and relatively simple techniques now exist to isolate highly purified preparations of hepatocytes, cholangiocytes, KCs, SCs, FSC, myofibroblasts and pit cells. Additional work is necessary to refine techniques for the isolation of dendritic cells and lymphocytes.

Secretion stimulates ductular bile secretion by binding to receptors on intrahepatic bile duct epithelial cells (i.e., cholangiocytes). In the rat, this choleretic effect increases after bile duct ligation (BDL). Although cholangiocyte proliferation induced by BDL contributes to secretin-induced hypercholeresis, the mechanisms modulating these alterations in secretin-induced ductular bile secretion are obscure. Thus we studied the expression of secretin receptor mRNA (SR-mRNA) in purified liver cells from normal and BDL rats. Northern blot analysis and RNase protection assays with mRNA from purified liver cells demonstrated SR-mRNA only in cholangiocytes; moreover, SR gene expression showed a seven- to ninefold increase in individual cholangiocytes from BDL rats compared with controls. This increase in SR-mRNA expression was related to a similar increase in the rate of transcription of SR-mRNA in cholangiocytes from BDL rats. Thus our studies indicate that 1) SR-mRNA is detected in liver only in cholangiocytes; 2) BDL causes an increase in SR-mRNA in individual cholangiocytes; and 3) the increase in SR-mRNA after BDL is partly related to an increase in the rate of transcription of SR-mRNA by cholangiocytes after BDL. Our data suggest that upregulation of the SR gene may contribute to secretin-induced hypercholeresis.

A cholecystokinin octapeptide (CCK-8)-degrading peptidase was purified from rat liver cytosol by heat precipitation of other proteins followed by gel filtration, ion exchange chromatography and preparative gel electrophoresis, using a silicate binding assay to quantitate the degradation of radiolabeled CCK-8. The purified peptidase (M(r) approximately 60,000) had a pH optimum of 6.0; its activity was inhibited by EDTA and 1, 10-phenanthroline but not by phosphoramidon, calpain inhibitor I, bestatin or bacitracin. CCK-8 peptidase rapidly degraded radiolabeled Met-enkephalin as well as 125I-CCK-8, but not a series of other unrelated peptides. Unlabeled Leu-enkephalin, beta-casomorphin and neurotensin competitively inhibited the degradation of 125I-CCK-8, suggesting that these opioids are also substrates for the enzyme. These data suggest that this protein is a novel hepatic enzyme which may play a role in the degradation of neuropeptides.

Meaningful health reform in the United States must improve the health of the population while lowering costs. In an effort to provide a framework for doing so, the Institute of Health Care Improvement created the triple aim, which encompasses the goals of (1) improving individual health and experience with the health care system, (2) improving population health, and (3) decreasing the rate of per capita health care costs. Current reform efforts have focused on the development of Patient-Centered Medical Homes (an innovative team-based model of care that facilitates a partnership between the patient's personal physician coordinating care throughout a patient's lifetime to maximize health outcomes), but these relatively narrow efforts are focused on office practice and payment methods and are not generally oriented toward community needs. We sought to apply design research in assessing a community opportunity to apply the triple aim as a strategy to transform health care delivery. Mixed methodology provides greater insight into the unexpressed health needs of individuals and into the creation of delivery systems more likely to achieve the triple aim. In a small, midwestern town, a mixed methods approach was used to assess community health needs to facilitate design and implementation of care delivery systems. The research findings suggest that health system design concepts should focus on the creation of health, not health care; foster simplicity; create nurturing relationships; eliminate user fear; and contain costs. These observations can be helpful to health care professionals who are developing new methods of care delivery and policymakers and payers contemplating new payment systems to achieve the goals of the triple aim.

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, are ciliated cells. Each cholangiocyte has a primary cilium consisting of (i) a microtubule-based axoneme and (ii) the basal body, centriole-derived, microtubule-organizing center from which the axoneme emerges. Primary cilia in cholangiocytes were described decades ago, but their physiological and pathophysiological significance remained unclear until recently. We now recognize that cholangiocyte cilia extend from the apical plasma membrane into the bile duct lumen and, as such, are ideally positioned to detect changes in bile flow, bile composition and bile osmolality. These sensory organelles act as cellular antennae that can detect and transmit signals that influence cholangiocyte function. Indeed, recent data show that cholangiocyte primary cilia can activate intracellular signaling pathways when they sense modifications in the flow, molecular constituents and osmolarity of bile. Their ability to sense and transmit signals depends on the participation of a growing number of specific ciliary-associated proteins that act as receptors, channels and transporters. Cholangiocyte cilia, in addition to being important in normal biliary physiology, likely contribute to the cholangiopathies when their normal structure or function is disturbed. Indeed, the polycystic liver diseases that occur in combination with autosomal dominant and recessive polycystic kidney disease (i.e. ADPKD and ARPKD) are two important examples of such conditions. Recent insights into the role of cholangiocyte cilia in cystic liver disease using in vitro and animal models have already resulted in clinical trials that have influenced the management of cystic liver disease.

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, express multiple toll-like receptors (TLRs) and thus have the capacity to recognize and respond to microbial pathogens. In previous work we demonstrated that TLR4, which is activated by gram-negative lipopolysaccharide (LPS), is upregulated in cholangiocytes in response to infection with Cryptosporidium parvum in vitro and contributes to NFkB activation. Here, using an in vivo model of biliary cryptosporidiosis we addressed the functional role of TLR4 in C. parvum infection dynamics and hepatobiliary pathophysiology. We observed that C57BL mice clear the infection by three weeks post-infection. In contrast, parasites were detected in bile and stool in TLR4 deficient mice at four weeks post-infection. The liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST), and the proinflammatory cytokines Tumor Necrosis Factor (TNF)-α, Interferon (IFN)-γ, and Interleukin (IL)-6 peaked at one to two weeks postinfection and normalized by four weeks in infected C57BL mice. C57BL mice also demonstrated increased cholangiocyte proliferation (PCNA staining) at one-week post-infection, which was resolved by two weeks post-infection. In contrast, TLR4 deficient mice showed persistently elevated serum ALT and AST, elevated hepatic IL-6 levels, and histological evidence of hepatocyte necrosis, increased inflammatory cell infiltration, and cholangiocyte proliferation through four weeks post-infection. These data suggest that a TLR4-mediated response is required for efficient eradication of biliary C. parvum infection in vivo, and lack of this pattern recognition receptor contributes to an altered inflammatory response and an increase in hepatobiliary pathology.

Background & Aims In polycystic kidney disease and polycystic liver disease (PLD), the normally nonproliferative hepato-renal epithelia acquire a proliferative, cystic phenotype that is linked to overexpression of cell division cycle 25 (Cdc25)A phosphatase and cell-cycle deregulation. We investigated the effects of Cdc25A inhibition in mice and rats via genetic and pharmacologic approaches. Methods Cdc25A +/− mice (which have reduced levels of Cdc25A) were cross-bred with polycystic kidney and hepatic disease 1 ( Pkhd1 del2/del2 ) mice (which have increased levels of Cdc25A and develop hepatic cysts). Cdc25A expression was analyzed in livers of control and polycystic kidney (PCK) rats, control and polycystic kidney 2 ( Pkd2 ws25/− ) mice, healthy individuals, and patients with PLD. We examined effects of pharmacologic inhibition of Cdc25A with vitamin K3 (VK3) on the cell cycle, proliferation, and cyst expansion in vitro; hepato-renal cystogenesis in PCK rats and Pkd2 ws25/− mice ; and expression of Cdc25A and the cell-cycle proteins regulated by Cdc25A. We also examined the effects of the Cdc25A inhibitor PM-20 on hepato-renal cystogenesis in Pkd2 ws25/− mice. Results Liver weights and hepatic and fibrotic areas were decreased by 32%–52% in Cdc25A +/− :Pkhd1 del2/del2 mice, compared with Pkhd1 del2/del2 mice. VK3 altered the cell cycle and reduced proliferation of cultured cholangiocytes by 32%–83% and decreased growth of cultured cysts by 23%–67%. In PCK rats and Pkd2 ws25/− mice, VK3 reduced liver and kidney weights and hepato-renal cystic and fibrotic areas by 18%–34%. PM-20 decreased hepato-renal cystogenesis in Pkd2 ws25/− mice by 15%. Conclusions Cdc25A inhibitors block cell-cycle progression and proliferation, reduce liver and kidney weights and cyst growth in animal models of polycystic kidney disease and PLD, and might be developed as therapeutics for these diseases.

Background: Although Wilson's disease is characterized by an accumulation of copper within hepatocyte lysosomes, the effects of excess copper on hepatic lysosomes are unknown. We studied the effects of excess copper on the structure, physicochemical properties, and pH of hepatocyte lysosomes using a rodent model. Methods: Rats were copper loaded with 0.125% copper acetate in water for 6 weeks. Copper was measured by atomic absorption spectrophotometry. Morphology was studied by electron microscopy. Lysosomal membrane fluidity was studied by fluorescence polarization, and lipid composition was determined by gas chromatography. Hepatocyte lysosomal pH was determined by flow cytometry. Results: Copper overload resulted in a 10-fold increase in hepatic copper. Hepatocyte lysosomes were enlarged and abnormally shaped with a 27-fold increase in copper, increased in vitro fragility, and decreased lysosomal membrane fluidity. Thiobarbituric acid reactive substances, a measure of lipid peroxidation, doubled in isolated lysosomal membranes. Polyunsaturated fatty acids increased, saturated fatty acids decreased, and membrane content of selected fatty acids was modified after copper overload. Lysosomal pH increased from 4.67 ± 0.02 to 4.87 ± 0.02. Conclusions: Copper overload causes alterations in lysosomal morphology, increases lysosomal fragility, decreases membrane fluidity, alters membrane fatty acid composition, and increases lysosomal pH. Copper catalyzed lipid peroxidation represents the likely mechanism for these alterations.

While intrahepatic bile duct epithelial cells secrete bile through transport of ions and water, the physiological mechanisms regulating ductular bile secretion are obscure, in part because of the lack of suitable experimental models. We report here the successful micropuncture of the lumen of isolated intrahepatic bile ducts and direct measurements of ductular ion secretion. Intact, polarized bile duct units (BDUs) were isolated from livers of normal rats by enzymatic digestion and microdissection. BDUs were cultured and mounted on a microscope in bicarbonate-containing buffer, and the lumens were microinjected with 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF)-dextran. Lumenal pH was measured by ratio imaging of BCECF fluorescence using digitized video fluorescent microscopy. After 36 hr in culture, the ends of BDUs sealed, forming closed compartments. After lumenal microinjection of BCECF-dextran, fluorescence was stable at the pH-insensitive wavelength, indicating no dye leakage. Serial changes in pH of extralumenal buffers containing pH-gradient collapsing ionophores allowed us to establish reliable standard curves relating fluorescence ratio to lumenal pH (r = 0.99; P < 0.001). By this approach, the basal pH inside the lumen of BDUs was 7.87 +/- 0.08 units (n = 9), 0.47 unit higher (P < 0.001) than the bathing buffer pH. Addition of 100 microM forskolin increased (P = 0.02) the lumenal pH from 7.78 +/- 0.06 to 7.97 +/- 0.06 units (n = 5); the forskolin effect was completely abolished by incubation of BDUs in HCO3-/CO2-free buffer. Moreover, forskolin caused a 50-fold increase in cAMP levels in BDUs. The observations are consistent with cAMP-dependent, active lumenal HCO3- secretion by BDUs. Furthermore, they demonstrate the suitability of the BDU model for studying regulatory and mechanistic aspects of ductular bile secretion.

The study determined predictive factors for postoperative complications and outcome after ileal pouch-anal anastomosis in patients with ulcerative colitis and primary sclerosing cholangitis. Patients with ulcerative colitis and primary sclerosing cholangitis treated by colectomy and ileostomy are at high risk of troublesome bleeding from peristomal varices. Postoperative complications and outcome were assessed in 40 patients with ulcerative colitis and sclerosing cholangitis who received in ileal pouch-anal anastomosis between January 1981 and February 1990. Immediate postoperative and remote ileoanal anastomosis-related complications were high but related directly to the severity of liver disease. No patient had perianastomotic anal bleeding. In patients with both ulcerative colitis and primary sclerosing cholangitis, ileal pouch-anal anastomosis is safe and is not associated with perianastomotic bleeding.

Exosomes are small extracellular vesicles that are thought to participate in intercellular communication. Recent work from our laboratory suggests that, in normal and cystic liver, exosome-like vesicles accumulate in the lumen of intrahepatic bile ducts, presumably interacting with cholangiocyte cilia. However, direct evidence for exosome-ciliary interaction is limited and the physiological relevance of such interaction remains unknown. Thus, in this study, we tested the hypothesis that biliary exosomes are involved in intercellular communication by interacting with cholangiocyte cilia and inducing intracellular signaling and functional responses. Exosomes were isolated from rat bile by differential ultracentrifugation and characterized by scanning, transmission, and immunoelectron microscopy. The exosome-ciliary interaction and its effects on ERK1/2 signaling, expression of the microRNA, miR-15A, and cholangiocyte proliferation were studied on ciliated and deciliated cultured normal rat cholangiocytes. Our results show that bile contains vesicles identified as exosomes by their size, characteristic “saucer-shaped” morphology, and specific markers, CD63 and Tsg101. When NRCs were exposed to isolated biliary exosomes, the exosomes attached to cilia, inducing a decrease of the phosphorylated-to-total ERK1/2 ratio, an increase of miR-15A expression, and a decrease of cholangiocyte proliferation. All these effects of biliary exosomes were abolished by the pharmacological removal of cholangiocyte cilia. Our findings suggest that bile contains exosomes functioning as signaling nanovesicles and influencing intracellular regulatory mechanisms and cholangiocyte proliferation through interaction with primary cilia.

There are no proven, effective therapies for polycystic kidney disease (PKD) or polycystic liver disease (PLD). We enrolled 42 patients with severe PLD resulting from autosomal dominant PKD (ADPKD) or autosomal dominant PLD (ADPLD) in a randomized, double-blind, placebo-controlled trial of octreotide, a long-acting somatostatin analogue. We randomly assigned 42 patients in a 2:1 ratio to octreotide LAR depot (up to 40 mg every 28 ± 5 days) or placebo for 1 year. The primary end point was percent change in liver volume from baseline to 1 year, measured by MRI. Secondary end points were changes in total kidney volume, GFR, quality of life, safety, vital signs, and clinical laboratory tests. Thirty-four patients had ADPKD, and eight had ADPLD. Liver volume decreased by 4.95% ± 6.77% in the octreotide group but remained practically unchanged (+0.92% ± 8.33%) in the placebo group (P = 0.048). Among patients with ADPKD, total kidney volume remained practically unchanged (+0.25% ± 7.53%) in the octreotide group but increased by 8.61% ± 10.07% in the placebo group (P = 0.045). Changes in GFR were similar in both groups. Octreotide was well tolerated; treated individuals reported an improved perception of bodily pain and physical activity. In summary, octreotide slowed the progressive increase in liver volume and total kidney volume, improved health perception among patients with PLD, and had an acceptable side effect profile.

Internalization of the obligate intracellular apicomplexan parasite,Cryptosporidium parvum, results in the formation of a unique intramembranous yet extracytoplasmic niche on the apical surfaces of host epithelial cells, a process that depends on host cell membrane extension. We previously demonstrated that efficientC. parvuminvasion of biliary epithelial cells (cholangiocytes) requires host cell actin polymerization and localized membrane translocation/insertion of Na+/glucose cotransporter 1 (SGLT1) and of aquaporin 1 (Aqp1), a water channel, at the attachment site. The resultant localized water influx facilitates parasite cellular invasion by promoting host-cell membrane protrusion. However, the molecular mechanisms by whichC. parvuminduces membrane translocation/insertion of SGLT1/Aqp1 are obscure. We report here that cultured human cholangiocytes express several nonmuscle myosins, including myosins IIA and IIB. Moreover,C. parvuminfection of cultured cholangiocytes results in the localized selective aggregation of myosin IIB but not myosin IIA at the region of parasite attachment, as assessed by dual-label immunofluorescence confocal microscopy. Concordantly, treatment of cells with the myosin light chain kinase inhibitor ML-7 or the myosin II-specific inhibitor blebbistatin or selective RNA-mediated repression of myosin IIB significantly inhibits (P< 0.05)C. parvumcellular invasion (by 60 to 80%). Furthermore ML-7 and blebbistatin significantly decrease (P< 0.02)C. parvum-induced accumulation of SGLT1 at infection sites (by approximately 80%). Thus, localized actomyosin-dependent membrane translocation of transporters/channels initiated byC. parvumis essential for membrane extension and parasite internalization, a phenomenon that may also be relevant to the mechanisms of cell membrane protrusion in general.

Primary cilia are nonmotile, tubular organelles extending from the cell plasma membrane. They are important in maintaining normal cell physiology and in many clinical disorders (i.e., cilia-related diseases or ciliopathies) associated with abnormalities in ciliary structure and/or function.

In summary, we answer the three questions we have previously posed: (a) Can liver transplantation prolong survival? Evolving data from several centers indicate that liver transplantation indeed prolongs survival in patients with PBC and PSC as compared with estimated survival using disease-specific risk scores based on the natural history of PBC and PSC. (b) Can we optimize timing of liver transplantation? Although many factors enter into the timing of liver transplantation, including when the patient is actually referred for liver transplantation and the individual desires of the patient to pursue liver transplantation, evidence is growing that having patients with chronic liver diseases like PBC and PSC undergo transplantation a little earlier in the course of the disease rather than waiting until the patients have experienced life-threatening complications or are on life-support measures can indeed improve early postliver transplant survival. In patients with PBC and PSC, the survival risk score, which reflects disease severity, can serve as an objective measurement to assess and evaluate the effect of liver disease severity on transplant outcome. Indeed, a number of studies have strongly suggested that optimal timing of liver transplantation may indeed be important to improve outcome, decrease morbidity and decrease cost. (c) Does the present allocation system in the United States allow for optimal use of our scarce donor organ resource?(ABSTRACT TRUNCATED AT 250 WORDS)

Intrahepatic bile duct epithelial cells, or cholangiocytes, contribute to bile secretion in response to hormones, including secretin. However, the mechanism by which secretin stimulates ductular bile flow is unknown. Since recent data in nonhepatic epithelia have suggested a role for exocytosis in fluid secretion, we tested the hypothesis that secretin stimulates exocytosis by isolated cholangiocytes. Cholangiocytes were isolated from normal rat liver by a newly described method employing enzymatic digestion and mechanical disruption followed by immunomagnetic separation using specific monoclonal antibodies, and exocytosis was measured using a fluorescence unquenching assay employing acridine orange. Secretin caused a dose-dependent (10(-12)-10(-7) M) increase in acridine orange fluorescence by acridine orange-loaded cholangiocytes with a peak response at 10 min; the half-maximal concentration of secretin was 7 x 10(-9) M. The secretin effect was inhibited by preincubation of cholangiocytes with colchicine (30% inhibition, p less than 0.05) or trypsin (90% inhibition, p less than 0.001); no inhibition was seen with lumicolchicine and heat-inactivated trypsin. Cholecystokinin, insulin, and somatostatin had no effect on fluorescence of acridine orange-loaded cholangiocytes; secretin had no effect on fluorescence of acridine orange-loaded hepatocytes or hepatic endothelial cells. Exposure of isolated cholangiocytes to secretin at doses that stimulated exocytosis caused a dose-dependent increase in cyclic AMP levels (218% maximal increase, p less than 0.05); moreover, an analogue of cyclic AMP stimulated exocytosis by cholangiocytes. Secretin had no effect on intracellular calcium concentration using Fura-2-loaded cholangiocytes assessed by digitized video microscopy. Our results demonstrate, for the first time, that secretin stimulates exocytosis by rat cholangiocytes. The effect is cell- and hormone-specific, dependent on intact microtubules, on a protein(s) on the external surface of cholangiocytes, and on changes in cellular levels of cyclic AMP. The results are consistent with the hypothesis that secretin-induced changes in bile flow may involve an exocytic process.

MicroRNAs, central players of numerous cellular processes, regulate mRNA stability or translational efficiency. Although these molecular events are established, the mechanisms regulating microRNA function and expression remain largely unknown. The microRNA let-7i regulates Toll-like receptor 4 expression. Here, we identify a novel transcriptional mechanism induced by the protozoan parasite Cryptosporidium parvum and Gram(-) bacteria-derived lipopolysaccharide (LPS) mediating let-7i promoter silencing in human biliary epithelial cells (cholangiocytes). Using cultured cholangiocytes, we show that microbial stimulus decreased let-7i expression, and promoter activity. Analysis of the mechanism revealed that microbial infection promotes the formation of a NFkappaB p50-C/EBPbeta silencer complex in the regulatory sequence. Chromatin immunoprecipitation assays (ChIP) demonstrated that the repressor complex binds to the let-7i promoter following microbial stimulus and promotes histone-H3 deacetylation. Our results provide a novel mechanism of transcriptional regulation of cholangiocyte let-7i expression following microbial insult, a process with potential implications for epithelial innate immune responses in general.

Clinical research centers that support hypothesis-driven investigation have long been a feature of academic medical centers but facilities in which clinical care delivery can be systematically assessed and evaluated have heretofore been nonexistent. The Institute of Medicine report “Crossing the Quality Chasm” identified six core attributes of an ideal care delivery system that in turn relied heavily on system redesign. Although manufacturing and service industries have leveraged modern design principles in new product development, healthcare has lagged behind. In this article, we describe a methodology utilized by our facility to study the clinical care delivery system that incorporates modern design principles.

Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1(del2/del2) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.

Primary cilia are present in most mammalian cells and have lately been recognized as important cellular sensors that integrate and transduce extracellular signals into functional responses. Development of approaches to isolate primary cilia of sufficient quantity and quality for biochemical and molecular studies are crucial to understand their roles and functions under normal and pathological conditions. Two separate but complementary techniques (i.e., peel-off and slide pulling) to isolate enriched ciliary fractions from cultured epithelial cells are described. The purity and quantity of isolated cilia is verified by immunofluorescent confocal microscopy, light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM), and western blot analysis. Examples of detection of ciliary-associated proteins using isolated cilia are shown. These techniques will allow the isolation of primary cilia from cultured epithelial cells and permit further examination of the expression and localization of proteins of interest, helping to elucidate the role of primary cilia in health and disease.

The incidence and severity of osteopenic bone disease in primary sclerosing cholangitis is poorly defined. Clinical, biochemical and radiographic assessment and bone mineral density measurements of the lumbar spine were carried out in two groups of patients. Group 1 consisted of 30 patients with advanced primary sclerosing cholangitis; group 2 consisted of 18 patients with newly diagnosed primary sclerosing cholangitis. Only one patient had bone pain. All patients were normocalcemic; two had elevated serum parathormone levels. Fourteen patients (47%) from group 1 but no patients from group 2 had low serum 25-hydroxyvitamin D levels. Mean bone mineral density was significantly reduced in group 1 patients (0.97 ± 0.04 gm/cm2) compared with age-matched and sex-matched controls (1.25 ± 0.01 gm/cm2, p < 0.0001), and in 15 patients (50%) bone mineral density was below the fracture threshold (0.98 gm/cm2). The bone mineral density in group 2 was not significantly different from controls, and no patient was below the fracture threshold. In neither group did bone mineral density correlate with serum bilirubin, 25-hydroxyvitamin D, fecal fat excretion, previous drug therapy or the presence of chronic ulcerative colitis. Histomorphometrical examination of bone from four group 1 patients showed increased bone resorption, reduced bone formation, moderate-to-severe osteopenia and no osteomalacia. In conclusion, severe osteopenic bone disease is common in advanced primary sclerosing cholangitis and, like that seen in other cholestatis diseases, is consistent with osteoporosis. (HEPATOLOGY 1991;14:257–261.)

Whether serum autoantibodies to neutrophil cytoplasmic components, previously found in ulcerative colitis, are also associated with primary sclerosing cholangitis was determined. In an enzyme-linked immunosorbent assay for immunoglobulin G neutrophil antibodies, neutrophil binding by primary sclerosing cholangitis sera was significantly greater than that for primary biliary cirrhosis, chronic hepatitis B, and chronic non-A, non-B hepatitis. Similar differences were seen when sera from patients with primary sclerosing cholangitis without evidence for ulcerative colitis were compared with sera from liver disease controls. Perinuclear immunofluorescence staining of neutrophils was exhibited by the majority of ulcerative colitis, primary sclerosing cholangitis, and primary sclerosing cholangitis without ulcerative colitis sera. The combination of elevated immunoglobulin G neutrophil antibodies and a perinuclear pattern was 65% sensitive and 100% specific for primary sclerosing cholangitis compared with the liver disease control sera. It is concluded that neutrophil cytoplasmic antibodies in ulcerative colitis and primary sclerosing cholangitis may be markers of shared underlying immunopafho-genic mechanisms. Identification of the target antigene) may facilitate understanding of the underlying immune response and development of an improved disease marker assay.