LaRusso, Nicholas F.

ABSTRACTInternalization 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.

In this review, we have emphasized the importance of cholangiocytes in biology and pathology and highlighted recent advances in experimental approaches to study these increasingly important cells. It is anticipated that major advances will continue to be made in our understanding of how these cells funciton and the processes which result in their dysfunction. The techniques are now available to allow major inroads in our understanding of the pathobiology of cholangiocytes.

There are opportunities to improve quality and safety of care provided to adult patients. The Plummer Project of the Department of Medicine at the Mayo Clinic (Rochester, MN, USA) is an initiative to redesign outpatient practice. We used multidisciplinary teams to standardize the tasks essential to improve patient care. With the initiative to standardize the rooming process, patient care and safety improved with greater accuracy of the medication list. The standardization also improved physician efficiency because trained clinical assistants helped address the needs of the patient. Physicians were satisfied by the new process and the technology enhancements. Clinical assistants were also highly satisfied by the training process. The quality and safety of patient care can be significantly improved by practice redesign. This practice redesign was satisfying for all, especially the patients, physicians and support team in our practice.

Cholangiocytes, the epithelial cells lining bile ducts, provide the first line of defense against lumenal microbes in the biliary system. Recent advances in biliary immunity indicate that cholangiocytes express a variety of pathogen‐recognition receptors and can activate a set of intracellular signaling cascades to initiate a profound antimicrobial defense, including release of proinflammatory cytokines and chemokines, production of antimicrobial peptides and maintenance of biliary epithelial integrity. Cholangiocytes also interact with other cell types in the liver (for example, lymphocytes and Kupffer cells) through expression and release of adhesion molecules and immune mediators. Subsequently, through an intricate feedback mechanism involving both epithelial and other liver cells, a set of intracellular signaling pathways are activated to regulate the functional state of cholangiocyte responses during microbial infection. Thus, cholangiocytes are actively involved in mucosal immunity of the biliary system and represent a fine‐tuned, integral component of liver immunity.

Cholangiocytes, epithelial cells lining the biliary tree, have primary cilia extending from their apical membrane into the ductal lumen. Although important in disease, cilia also play a vital role in normal cellular functions. We reported that cholangiocyte cilia are sensory organelles responding to mechanical stimuli (i.e., luminal fluid flow) by alterations in intracellular Ca 2+ and cAMP. Because cholangiocyte cilia are also ideally positioned to detect changes in composition and tonicity of bile, we hypothesized that cilia also function as osmosensors. TRPV4, a Ca 2+ -permeable ion channel, has been implicated in signal transduction of osmotic stimuli. Using purified rat cholangiocytes and perfused intrahepatic bile duct units (IBDUs), we found that TRPV4 is expressed on cholangiocyte cilia, and that hypotonicity induces an increase in intracellular Ca 2+ in a TRPV4-, ciliary-, and extracellular calcium-dependent manner. The osmosensation of luminal tonicity by ciliary TRPV4 induces bicarbonate secretion, the main determinant of ductal bile formation, by a mechanism involving apical ATP release. Furthermore, the activation of TRPV4 in vivo , by its specific agonist, 4αPDD, induces an increase in bile flow as well as ATP release and bicarbonate secretion. Our results suggest that cholangiocyte primary cilia play an important role in ductal bile formation by acting as osmosensors.

ABSTRACTWhileCryptosporidium parvuminfection of the intestine has been reported in both immunocompetent and immunocompromised individuals, biliary infection is seen primarily in adult AIDS patients and is associated with development of AIDS cholangiopathy. However, the mechanisms of pathogen-induced AIDS cholangiopathy remain unclear. Since we previously demonstrated that the Fas/Fas ligand (FasL) system is involved in paracrine-mediatedC. parvumcytopathicity in cholangiocytes, we also tested the potential synergistic effects of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat)-mediated FasL regulation onC. parvum-induced apoptosis in cholangiocytes by semiquantitative reverse transcription-PCR, immunoblotting, immunofluorescence analysis, and immunogold electron microscopy. H69 cells do not express CXCR4 and CCR5, which are receptors required for direct HIV-1 viral infection. However, recombinant biologically active HIV-1-associated Tat protein increased FasL expression in the cytoplasm of cholangiocytes without a significant increase in apoptosis. We found thatC. parvum-induced apoptosis was associated with translocation of intracellular FasL to the cell membrane surface and release of full-length FasL from infected H69 cells. Tat significantly (P< 0.05) increasedC. parvum-induced apoptosis in bystander cells in a dose-dependent manner. Moreover, Tat enhanced bothC. parvum-induced FasL membrane translocation and release of full-length FasL. In addition, the FasL neutralizing antibody NOK-1 and the caspase-8 inhibitor Z-IETD-fmk both blockedC. parvum-induced apoptosis in cholangiocytes. The data demonstrated that HIV-1 Tat enhancesC. parvum-induced cholangiocyte apoptosis via a paracrine-mediated, FasL-dependent mechanism. Our results suggest that concurrent active HIV replication, with associated production of Tat protein, andC. parvuminfection synergistically increase cholangiocyte apoptosis and thus jointly contribute to AIDS-related cholangiopathies.

Apolipoproteins A-1 and A-2 were purified from human plasma. At concentrations present in human bile these proteins prolonged the nucleation time of cholesterol monohydrate crystals when added to model systems of supersaturated bile. In contrast, apolipoprotein C-3 and other serum proteins did not have this effect. Also, when human gallbladder bile was fractionated by gel filtration chromatography, apolipoproteins A-1 and A-2 were among the proteins present in a fraction of bile enriched in potent inhibitors of cholesterol crystal nucleation. These findings suggest that apolipoproteins A-1 and A-2 in supersaturated human gallbladder bile could inhibit the rate of formation of solid cholesterol crystals and thus help to prevent spontaneous cholesterol gallstone formation in humans.

Cholangiocytes line the intrahepatic bile ducts and regulate salt and water secretion during bile formation, but the mechanism(s) regulating ductal water movement remains obscure. A water-selective channel, the aquaporin CHIP, was recently described in several epithelia, so we tested the hypothesis that osmotic water movement by cholangiocytes is mediated by CHIP. Isolated rodent cholangiocytes showed a rapid increase in volume in the presence of hypotonic extracellular buffers; the ratio of osmotic to diffusional permeability coefficients was > 10. The osmotically induced increase in cholangiocyte volume was inversely proportional to buffer osmolality, independent of temperature, and reversibly blocked by HgCl2. Also, the luminal area of isolated, enclosed bile duct units increased after exposure to hypotonic buffer and was reversibly inhibited by HgCl2. RNase protection assays, anti-CHIP immunoblots, and immunocytochemistry confirmed that CHIP transcript and protein were present in isolated cholangiocytes but not in hepatocytes. These results demonstrate that (i) isolated cholangiocytes and intact, polarized bile duct units manifest rapid, mercury-sensitive increases in cell size and luminal area, respectively, in response to osmotic gradients and (ii) isolated cholangiocytes express aquaporin CHIP at both the mRNA and the protein level. The data implicate aquaporin water channels in the transcellular movement of water across cholangiocytes lining intrahepatic bile ducts and provide a plausible molecular explanation for ductal water secretion.

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.

Dynamic membrane protrusions such as lamellipodia and filopodia are driven by actin polymerization and often hijacked by intracellular microbes to enter host cells. The overall rate of membrane protrusion depends on the actin polymerization rate and the increase of localized cell volume. Although the signaling pathways involving actin polymerization are well characterized, the molecular mechanisms regulating local cell volume associated with membrane protrusion are unclear. Cryptosporidium parvum , an intracellular parasite, depends on host-cell membrane protrusion to accomplish cell entry and form the parasitophorous vacuole. Here, we report that C. parvum infection of cholangiocytes recruits host-cell SGLT1, a Na + /glucose cotransporter, and aquaporin 1 (AQP1), a water channel, to the attachment site. SGLT1-dependent glucose uptake occurs at the attachment site. Concordantly, the region of attachment displays localized water influx that is inhibited by either suppression of AQP1 by means of AQP1-small interfering RNA (siRNA) or inhibition of SGLT1 by a specific pharmacologic inhibitor, phlorizin. Inhibition of SGLT1 does not affect actin accumulation but decreases the membrane protrusion at the attachment site. Moreover, functional inhibition of host-cell AQP1 and SGLT1 hampers C. parvum invasion of cholangiocytes. Thus, glucose-driven, AQP-mediated localized water influx is involved in the membrane protrusion during C. parvum cellular invasion, phenomena that may also be relevant to the mechanisms of cell membrane protrusion in general.