LaRusso, Nicholas F.

Extracellular vesicles (EVs) are nanometer‐sized, membrane‐bound vesicles released by cells into the extracellular milieu. EVs are now recognized to play a critical role in cell‐to‐cell communication. EVs contain important cargo in the form of proteins, lipids, and nucleic acids and serve as vectors for delivering this cargo from donor to acceptor or target cell. EVs are released under both physiologic and pathologic conditions, including liver diseases, and exert a wide range of effects on target cells. This review provides an overview on EV biogenesis, secretion, cargo, and target cell interactions in the context of select liver diseases. Specifically, the diverse roles of EVs in nonalcoholic steatohepatitis, alcoholic liver disease, viral hepatitis, cholangiopathies, and hepatobiliary malignancies are emphasized. Liver diseases often result in an increased release of EVs and/or in different cargo sorting into these EVs. Either of these alterations can drive disease pathogenesis. Given this fact, EVs represent a potential target for therapeutic intervention in liver disorders. Because altered EV composition may reflect the underlying disease condition, circulating EVs can be exploited for diagnostic and prognostic purposes as a liquid biopsy. Furthermore, ex vivo modified or synthesized EVs can be engineered as therapeutic nano‐shuttles. Finally, we highlight areas that merit further investigation relevant to understanding how EVs regulate liver disease pathogenesis. (H epatology 2016;64:2219‐2233).

AbstractBackground and Aim:Liver stiffness (LS) measured by magnetic resonance elastography (MRE) is emerging as an important biomarker in chronic liver diseases. We examined the diagnostic performance of MRE, factors associated with an increased LS and the prognostic value of LS as measured by MRE among patients with primary sclerosing cholangitis (PSC).Methods:We performed a retrospective review of 266 patients with PSC to examine whether LS was associated with the primary endpoint of hepatic decompensation (ascites, variceal hemorrhage and hepatic encephalopathy). The ability of MRE to differentiate stages of fibrosis was examined in a subset of patients who underwent a liver biopsy (n = 20).Results:An LS of 4.93 kPa was the optimal point to detected F4 fibrosis (sensitivity, 1.00; 95% confidence interval (CI), 0.40–1.00; specificity, 0.94; 95%CI, 0.68–1.00). While a serum alkaline phosphatase <1.5 times the upper limit of normal excluded the presence of advanced LS, it was not associated with the primary endpoint (hazard ratio, 0.26; 95%CI, 0.01–1.33). However, LS was associated with the development of decompensated liver disease (hazard ratio, 1.55; 95%CI, 1.41–1.70). The optimal LS thresholds that stratified patients at a low, medium and high risk for hepatic decompensation were <4.5, 4.5–6.0 and >6.0 kPa (respectively).Conclusion:Magnetic resonance elastography is able to detect cirrhosis with high specificity and an alkaline phosphatase <1.5 times the upper limit of normal makes the presence of advanced LS unlikely. Moreover, LS obtained by MRE is predictive of hepatic decompensation in PSC.

Abstract Increasing evidence points to the contribution of the intestinal microbiome as a potentially key determinant in the initiation and/or progression of hepatobiliary disease. While current understanding of this dynamic is incomplete, exciting insights are continually being made and more are expected given the developments in molecular and high‐throughput omics techniques. In this brief review, we provide a practical and updated synopsis of the interaction of the intestinal microbiome with the liver and its downstream impact on the initiation, progression and complications of hepatobiliary disease.

The cholangiopathies are a group of liver diseases resulting from different etiologies but with the cholangiocyte as the primary target. As a group, the cholangiopathies result in significant morbidity and mortality and represent one of the main indications for liver transplant in both children and adults. Contributing to this situation is the absence of a thorough understanding of their pathogenesis and a lack of adequate diagnostic and prognostic biomarkers. MicroRNAs are small non-coding RNAs that modify gene expression post-transcriptionally. They have been implicated in the pathogenesis of many diseases, including the cholangiopathies. Thus, in this review we provide an overview of the literature on miRNAs in the cholangiopathies and discuss future research directions.

Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.

Primary sclerosing cholangitis (PSC) is a chronic, idiopathic, fibroinflammatory cholangiopathy. The role of the microbiota in PSC etiopathogenesis may be fundamentally important, yet remains obscure. We tested the hypothesis that germ‐free (GF) mutltidrug resistance 2 knockout (mdr2 −/− ) mice develop a distinct PSC phenotype, compared to conventionally housed (CV) mdr2 −/− mice. Mdr2 −/− mice (n = 12) were rederived as GF by embryo transfer, maintained in isolators, and sacrificed at 60 days in parallel with age‐matched CV mdr2 −/− mice. Serum biochemistries, gallbladder bile acids, and liver sections were examined. Histological findings were validated morphometrically, biochemically, and by immunofluorescence microscopy (IFM). Cholangiocyte senescence was assessed by p16 INK4a in situ hybridization in liver tissue and by senescence‐associated β‐galactosidase staining in a culture‐based model of insult‐induced senescence. Serum biochemistries, including alkaline phosphatase, aspartate aminotransferase, and bilirubin, were significantly higher in GF mdr2 −/− ( P < 0.01). Primary bile acids were similar, whereas secondary bile acids were absent, in GF mdr2 −/− mice. Fibrosis, ductular reaction, and ductopenia were significantly more severe histopathologically in GF mdr2 −/− mice ( P < 0.01) and were confirmed by hepatic morphometry, hydroxyproline assay, and IFM. Cholangiocyte senescence was significantly increased in GF mdr2 −/− mice and abrogated in vitro by ursodeoxycholic acid (UDCA) treatment. Conclusions : GF mdr2 −/− mice exhibit exacerbated biochemical and histological features of PSC and increased cholangiocyte senescence, a characteristic and potential mediator of progressive biliary disease. UDCA, a commensal microbial metabolite, abrogates senescence in vitro . These findings demonstrate the importance of the commensal microbiota and its metabolites in protecting against biliary injury and suggest avenues for future studies of biomarkers and therapeutic interventions in PSC. (H epatology 2016;63:185–196)

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Abstract Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C. parvum infection of cultured human biliary epithelia (i.e., cholangiocytes). We found that normal human cholangiocytes express all known TLRs. C. parvum infection of cultured cholangiocytes induces the selective recruitment of TLR2 and TLR4 to the infection sites. Activation of several downstream effectors of TLRs including IL-1R-associated kinase, p-38, and NF-κB was detected in infected cells. Transfection of cholangiocytes with dominant-negative mutants of TLR2 and TLR4, as well as the adaptor molecule myeloid differentiation protein 88 (MyD88), inhibited C. parvum-induced activation of IL-1R-associated kinase, p-38, and NF-κB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-κB activation. Moreover, C. parvum selectively up-regulated human β-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-κB activation were each associated with a reduction of C. parvum-induced human β-defensin-2 expression. A significantly higher number of parasites were detected in cells transfected with a MyD88 dominant-negative mutant than in the control cells at 48–96 h after initial exposure to parasites, suggesting MyD88-deficient cells were more susceptible to infection. These findings demonstrate that cholangiocytes express a variety of TLRs, and suggest that TLR2 and TLR4 mediate cholangiocyte defense responses to C. parvum via activation of NF-κB.