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Nicholas F LaRusso

Publications and source records attributed to Nicholas F LaRusso.

At least 19 recordsLinked to original sources
PubMed2007

T cell targeting and phagocytosis of apoptotic biliary epithelial cells in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterized by loss of tolerance against ubiquitously expressed mitochondrial autoantigens followed by biliary and salivary gland epithelial cell (BEC and SGEC) destruction by autoreactive T cells. It is unclear why BECs and SGECs are targeted. Previous work demonstrated that the reduced form of the major PBC autoantigen predominated in apoptotic BECs and SGECs as opposed to an oxidized form in other apoptotic cells. This led to the hypothesis that presentation of novel self-peptides from phagocytosed apoptotic BECs might contribute to BEC targeting by autoreactive T cells. The effect of autoantigen redox status on self-peptide formation was examined along with the phagocytic ability of BECs. Oxidation of PBC autoantigens first was shown to be due to protein S-glutathionylation of lipoyllysine residues. Absence of protein S-glutathionylation generated novel self-peptides and affected T cell recognition of a lipoyllysine containing peptide. Liver biopsy staining revealed BEC phagocytosis of apoptotic BECs (3.74+/-2.90% of BEC) was present in PBC (7 of 7 cases) but not in normal livers (0 of 3). BECs have the ability to present novel mitochondrial self-peptides derived from phagocytosed apoptotic BECs. Apoptotic cell phagocytosis by non-professional phagocytes may influence the tissue specificity of autoimmune diseases.

Animals↗
PubMed2006

Cryptosporidium parvum infects human cholangiocytes via sphingolipid-enriched membrane microdomains.

Cryptosporidium parvum attaches to intestinal and biliary epithelial cells via specific molecules on host-cell surface membranes including Gal/GalNAc-associated glycoproteins. Subsequent cellular entry of this parasite depends on host-cell membrane alterations to form a parasitophorous vacuole via activation of phosphatidylinositol 3-kinase (PI-3K)/Cdc42-associated actin remodelling. How C. parvum hijacks these host-cell processes to facilitate its infection of target epithelia is unclear. Using specific probes to known components of sphingolipid-enriched membrane microdomains (SEMs), we detected aggregation of host-cell SEM components at infection sites during C. parvum infection of cultured human biliary epithelial cells (i.e. cholangiocytes). Activation and membrane translocation of acid-sphingomyelinase (ASM), an enzyme involved in SEM membrane aggregation, were also observed in infected cells. Pharmacological disruption of SEMs and knockdown of ASM via a specific small interfering RNA (siRNA) significantly decreased C. parvum attachment (by approximately 84%) and cellular invasion (by approximately 88%). Importantly, knockdown of ASM and disruption of SEMs significantly blocked C. parvum-induced accumulation of Gal/GalNAc-associated glycoproteins at infection sites by approximately 90%. Disruption of SEMs and knockdown of ASM also significantly blocked C. parvum-induced activation of host-cell PI-3K and subsequent accumulation of Cdc42 and actin by up to 75%. Our results suggest an important role of SEMs for C. parvum attachment to and entry of host cells, likely via clustering of membrane-binding molecules and facilitating of C. parvum-induced actin remodelling at infection sites through activation of the PI-3K/Cdc42 signalling pathway.

Actins↗
PubMed2006

Aquaporins in the hepatobiliary system.

The review focuses on the potential physiological and pathophysiological roles of aquaporins (AQPs), a family of water channel proteins, in the hepatobiliary system. Among 13 aquaporins (AQP0-AQP12) cloned in mammals, seven AQPs have been identified in the liver and biliary tree. Accumulating evidence suggests that AQPs are likely involved in canalicular and ductal bile secretion, gluconeogenesis and microbial infection and may have other novel roles that affect liver function.

Animals↗
PubMed2006

Isolation and characterization of lipid microdomains from apical and basolateral plasma membranes of rat hepatocytes.

Canalicular bile is formed by the osmotic filtration of water in response to osmotic gradients generated by active transport at the apical and basolateral plasma membrane domains of hepatocytes. We recently demonstrated that mixed plasma membrane fractions isolated from rat hepatocyte couplets contain lipid microdomains ("rafts") enriched in cholesterol and sphingolipids and AQP8 and 9. We isolated lipid microdomains from hepatocyte apical and basolateral plasma membrane domains using Triton X-100 as detergent, and characterized their lipid and protein composition. A Triton-insoluble band ("raft fraction") at the 5%/30% sucrose interface in both apical and basolateral fractions was enriched for alkaline phosphatase (apical) and Na/K ATPase (basolateral) and was negative for amino peptidase-N. This detergent-insoluble band was also positive for caveolin-1 (a "raft" associated protein) and negative for clathrin (a "raft" negative protein). Lipid analysis showed that, the Triton-insoluble fraction was highly enriched in cholesterol and sphingolipids. Immunofluorescence staining on hepatocyte couplets for both caveolin-1 and cholera toxin B showed a punctate distribution on both the apical and basolateral plasma membranes, consistent with localized membrane microdomains. Dot blot analysis showed that the "raft" associated ganglioside GM1 was enriched in the detergent-insoluble fraction both domains. Furthermore, exposure of isolated hepatocytes to glucagon, a choleretic agonist, significantly increased the expression of AQP8 associated with the apical microdomain fractions but had no effect on AQP9 expression in the basolateral microdomain fractions. In conclusion, "rafts" represent target microdomains for exocytic insertion and retrieval of "flux proteins", including AQPs, involved in canalicular bile secretion.

Animals↗
PubMed2006

Primary sclerosing cholangitis: summary of a workshop.

Primary sclerosing cholangitis (PSC) is a rare but important liver disease that leads to cirrhosis and need for liver transplantation in a high proportion of cases. The disease occurs in approximately 1 per 100,000 population per year, usually presents in adulthood, and affects men more often than women. Typical serum biochemical results, autoantibodies and liver biopsy are suggestive but not diagnostic of PSC, the diagnosis requiring cholangiographic demonstration of stricturing and dilatation of the intra- and/or extra-hepatic bile ducts. The natural history of PSC is variable, the average survival being 12 to 17 years. The cause of PSC is still unknown. Although considered an autoimmune disease, PSC has several atypical features and a strong genetic component. The therapy of PSC is unsatisfactory. Standard doses of ursodeoxycholic acid (UDCA) lead to improvements in biochemical abnormalities but not in histology, cholangiographic appearance or survival. Several innovative therapies have been tried in PSC, but with scant evidence of benefit. For patients with high grade strictures, endoscopic dilatation is beneficial. Liver transplantation is successful for end-stage liver disease due to PSC and improves survival. PSC may recur after transplantation but is rarely progressive. The most dreaded complication of PSC is cholangiocarcinoma. Diagnosis of this highly malignant tumor is difficult, and there are no biomarkers for its early detection. Liver transplantation for cholangiocarcinoma has an exceedingly poor outcome, although transplantation with neoadjuvant chemoirradiation holds promise in selected patients. Thus, significant opportunities remain for basic and clinical research into the cause, natural history, and therapy of PSC.

Biopsy↗
PubMed2006

Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma.

The oncolytic measles virus Edmonston strain (MV-Edm), a nonpathogenic virus targeting cells expressing abundant CD46, selectively destroys neoplastic tissue. Clinical development of MV-Edm would benefit from noninvasive monitoring strategies to determine the speed and extent of the spread of the virus in treated patients and the location of virus-infected cells. We evaluated recombinant MV-Edm expressing carcinoembryonic antigen (CEA) or the human sodium iodide symporter (hNIS) for oncolytic potential in hepatocellular carcinoma (HCC) and efficiency in tracking viruses in vivo by noninvasive monitoring. CD46 expression in human HCC and primary hepatocytes was assessed by flow cytometry and immunohistochemistry. Infectivity, syncytium formation, and cytotoxicity of recombinant MV-Edm in HCC cell lines were evaluated by fluorescence microscopy, crystal violet staining, and the MTS assay. Transgene expression in HCC cell lines after infection with recombinant MV-Edm in vitro and in vivo was assessed by CEA concentration, 125I-uptake, and 123I-imaging studies. Toxicology studies were performed in Ifnar(KO)xCD46 transgenic mice. The CD46 receptor was highly expressed in HCC compared to nonmalignant hepatic tissue. Recombinant MV-Edm efficiently infected HCC cell lines, resulting in extensive syncytium formation followed by cell death. Transduction of HCC cell lines and subcutaneous HCC xenografts with recombinant MV-Edm resulted in high-level expression of transgenes in vitro and in vivo. MV-Edm was nontoxic in susceptible mice. Intratumoral and intravenous therapy with recombinant MV-Edm resulted in inhibition of tumor growth and prolongation of survival with complete tumor regression in up to one third of animals. In conclusion, engineered MV-Edm may be a potent and novel cancer gene therapy system for HCC. MV-Edm expressing CEA or hNIS elicited oncolytic effects in human HCC cell lines in vitro and in vivo, enabling the spread of the virus to be monitored in a noninvasive manner.

Animals↗
PubMed2006

Cholangiocyte cilia detect changes in luminal fluid flow and transmit them into intracellular Ca2+ and cAMP signaling.

BACKGROUND & AIMS: Cholangiocytes have primary cilia extending from the apical plasma membrane into the ductal lumen. While the physiologic significance of cholangiocyte cilia is unknown, studies in renal epithelia suggest that primary cilia possess sensory functions. Here, we tested the hypothesis that cholangiocyte cilia are sensory organelles that detect and transmit luminal bile flow stimuli into intracellular Ca2+ ([Ca2+]i) and adenosine 3',5'-cyclic monophosphate (cAMP) signaling. METHODS: Scanning electron microscopy, transmission electron microscopy, and immunofluorescent confocal microscopy of rat isolated intrahepatic bile duct units (IBDUs) were used to detect and characterize cholangiocyte cilia. The fluid flow-induced changes in Ca2+ and cAMP levels in cholangiocytes of microperfused IBDUs were detected by epifluorescence microscopy and a fluorescence assay, respectively. RESULTS: In microperfused IBDUs, luminal fluid flow induced an increase in [Ca2+]i and caused suppression of the forskolin-stimulated cAMP increase. The fluid flow-induced changes in [Ca2+]i and cAMP levels were significantly reduced or abolished when cilia were removed by chloral hydrate or when ciliary-associated proteins polycystin-1 (a mechanoreceptor), polycystin-2 (a Ca2+ channel), and the Ca2+-inhibitable adenylyl cyclase isoform 6 were individually down-regulated by small interfering RNAs. CONCLUSIONS: Cholangiocyte cilia are sensory organelles containing polycystin-1, polycystin-2, and adenylyl cyclase isoform 6 through which luminal fluid flow affects both [Ca2+]i and cAMP signaling in the cell. The data suggest a new model for regulation of ductal bile secretion involving cholangiocyte cilia.

Animals↗
PubMed2005

Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-kappaB.

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-kappaB 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-kappaB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-kappaB activation. Moreover, C. parvum selectively up-regulated human beta-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-kappaB activation were each associated with a reduction of C. parvum-induced human beta-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-kappaB.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Expression and subcellular localization of aquaporin water channels in the polarized hepatocyte cell line, WIF-B.

BACKGROUND: Recent data suggest that canalicular bile secretion involves selective expression and coordinated regulation of aquaporins (AQPs), a family of water channels proteins. In order to further characterize the role of AQPs in this process, an in vitro cell system with retained polarity and expression of AQPs and relevant solute transporters involved in bile formation is highly desirable. The WIF-B cell line is a highly differentiated and polarized rat hepatoma/human fibroblast hybrid, which forms abundant bile canalicular structures. This cell line has been reported to be a good in vitro model for studying hepatocyte polarity. RESULTS: Using RT-PCR, immunoblotting and confocal immunofluorescence, we showed that WIF-B cells express the aquaporin water channels that facilitate the osmotically driven water movements in the liver, i.e. AQP8, AQP9, and AQP0; as well as the key solute transporters involved in the generation of canalicular osmotic gradients, i.e., the bile salt export pump Bsep, the organic anion transporter Mrp2 and the chloride bicarbonate exchanger AE2. The subcellular localization of the AQPs and the solute transporters in WIF-B cells was similar to that in freshly isolated rat hepatocytes and in intact liver. Immunofluorescent costaining studies showed intracellular colocalization of AQP8 and AE2, suggesting the possibility that these transporters are expressed in the same population of pericanalicular vesicles. CONCLUSION: The hepatocyte cell line WIF-B retains the expression and subcellular localization of aquaporin water channels as well as key solute transporters for canalicular bile secretion. Thus, these cells can work as a valuable tool for regulatory and mechanistic studies of the biology of bile formation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Localized glucose and water influx facilitates Cryptosporidium parvum cellular invasion by means of modulation of host-cell membrane protrusion.

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.

Actins↗
PubMed2005

Aquaporin-8 is involved in water transport in isolated superficial colonocytes from rat proximal colon.

Water is an essential nutrient because it must be introduced from exogenous sources to satisfy metabolic demand. Under physiologic conditions, the colon can absorb and secrete considerable amounts of water even against osmotic gradients, thus helping to maintain the body fluid balance. Here we describe studies on both aquaporin (AQP) expression and function using cells isolated from the superficial and lower crypt regions of the rat proximal colon. The expression of AQP-3, -4, and -8 in isolated colonocytes was determined by semiquantitative RT-PCR and by immunoblotting. The localization of AQP-8 in the colon was evaluated by immunohistochemistry. A stopped-flow light scattering method was used to examine osmotic water movement in isolated colonocytes. Moreover, the contribution of AQP-8 to overall water movement through isolated colonocytes was studied using RNA interference technology. Colonocytes from the proximal colon express AQP-3, -4, and -8 with increasing concentrations from the lower crypt cells toward those on the surface. Osmotic water permeability was higher in surface than in crypt colonocytes (P < 0.05); it was significantly inhibited by the water channel blocker dimethyl sulfoxide, and reversed by beta-mercaptoethanol (P < 0.05). Immunohistochemistry revealed a strong AQP-8 labeling in the apical membrane of the superficial colonocytes. Inhibition of aquaporin-8 expression by small interfering RNA significantly decreased osmotic water permeability (approximately 38%; P < 0.05). Current results indicate that aquaporin-8 may play a major role in water movement through the colon by acting on the apical side of the superficial cells.

Animals↗
PubMed2005

Cholangiocyte biology.

PURPOSE OF REVIEW: Cholangiocytes are increasingly recognized as biologically important because of the diverse array of cellular processes in which they participate. Collectively, these processes define normal function and, when disturbed, account for abnormalities that cause disease. Advances in animal models and sophisticated technology in imaging and gene silencing have allowed progress in defining the roles that cholangiocytes play in signaling, transport of water, ions and solutes, and alterations that result in cholestasis. The pace of advances in technology justifies a yearly summary to identify the most important developments in cholangiocyte biology. RECENT FINDINGS: The main areas of recent progress include insights into the molecular mechanisms of bile secretion and the development of new experimental models and technologies. SUMMARY: Understanding the critical components and key biologic processes in cholangiocytes responsible for regulation of ductal bile secretion is an initial and required step in generating hypotheses relevant to disease. With regard to the pathologic relevance of this work, cholestatic liver diseases represent a broad group of hepatobiliary disorders with which hepatologists must deal. In addition to genetic defects, the study of the normal and altered trafficking of cholangiocyte transport systems involved in bile secretion may provide a molecular correlate for the functional changes that occur in disease. Critical to this understanding is the ongoing development of experimental models and techniques to interpret data to answer key hypothesis-driven questions. Second, the collegial sharing and exchange of novel concepts, ideas, reagents, and probes promotes positive advances in the field.

Bile Acids and Salts↗
PubMed2005

Distribution of Cryptosporidium parvum sporozoite apical organelles during attachment to and internalization by cultured biliary epithelial cells.

Although accumulating evidence supports an active role for host cells during Cryptosporidium parvum invasion of epithelia, our knowledge of the underlying parasite-specific processes triggering such events is limited. In an effort to better understand the invasion strategy of C. parvum, we characterized the presence and distribution of the apical organelles (micronemes, dense granules, and rhoptry) through the stages of attachment to, and internalization by, human biliary epithelia, using serial-section electron microscopy. Novel findings include an apparent organized rearrangement of micronemes upon host cell attachment. The apically segregated micronemes were apposed to a central microtubule-like filamentous structure, and the more distal micronemes localized to the periphery and apical region of the parasite during internalization, coinciding with the formation of the anterior vacuole. The morphological observations presented here extend our understanding of parasite-specific processes that occur during attachment to, and internalization by, host epithelial cells.

Animals↗
PubMed2004

Phosphatidylinositol 3-kinase and frabin mediate Cryptosporidium parvum cellular invasion via activation of Cdc42.

Cryptosporidium parvum invades target epithelia via a mechanism that involves host cell actin reorganization. We previously demonstrated that C. parvum activates the Cdc42/neural Wiskott-Aldrich syndrome protein network in host cells resulting in actin remodeling at the host cell-parasite interface, thus facilitating C. parvum cellular invasion. Here, we tested the role of phosphatidylinositol 3-kinase (PI3K) and frabin, a guanine nucleotide exchange factor specific for Cdc42 in the activation of Cdc42 during C. parvum infection of biliary epithelial cells. We found that C. parvum infection of cultured human biliary epithelial cells induced the accumulation of PI3K at the host cell-parasite interface and resulted in the activation of PI3K in infected cells. Frabin also was recruited to the host cell-parasite interface, a process inhibited by two PI3K inhibitors, wortmannin and LY294002. The cellular expression of either a dominant negative mutant of PI3K (PI3K-Deltap85) or functionally deficient mutants of frabin inhibited C. parvum-induced Cdc42 accumulation at the host cell-parasite interface. Moreover, LY294002 abolished C. parvum-induced Cdc42 activation in infected cells. Inhibition of PI3K by cellular overexpression of PI3K-Deltap85 or by wortmannin or LY294002, as well as inhibition of frabin by various functionally deficient mutants, decreased C. parvum-induced actin accumulation and inhibited C. parvum cellular invasion. In contrast, the overexpression of the p85 subunit of PI3K promoted C. parvum invasion. Our data suggest that an important component of the complex process of C. parvum invasion of target epithelia results from the ability of the organism to trigger host cell PI3K/frabin signaling to activate the Cdc42 pathway, resulting in host cell actin remodeling at the host cell-parasite interface.

Actins↗
PubMed2004

AQP4 transfected into mouse cholangiocytes promotes water transport in biliary epithelia.

Rodent cholangiocytes express 6 of the 11 known channel proteins called aquaporins (AQPs) that are involved in transcellular water transport in mammals. However, clarifying the role of AQPs in mediating water transport in biliary epithelia has been limited in part because of the absence of physiologically relevant experimental models. In this study, we established a novel AQP4-transfected polarized mouse cholangiocyte cell line suitable for functional studies of transepithelial water transport, and, using this model, we define the importance of this AQP in water transport across biliary epithelia. Polarized normal mouse cholangiocytes (NMCs) lacking endogenous AQP4 were transfected stably with functional AQP4 or cotransfected with functional AQP4 and a transport-deficient AQP4 dominant negative mutant using a retroviral delivery system. In transfected NMCs, AQP4 is expressed on both the mRNA and protein levels and is localized at both the apical and basolateral membranes. In nontransfected NMCs, the transcellular water flow, P(f), value was relatively high (i.e., 16.4 +/- 3.2 microm/sec) and likely was a reflection of endogenous expression of AQP1 and AQP8. In NMCs transfected with AQP4, P(f) increased to 75.7 +/- 1.4 microm/sec, that is, by 4.6-fold, indicating the contribution of AQP4 in channel-mediated water transport across MNCs monolayer. In cotransfected NMCs, AQP4 dominant negative reduced P(f) twofold; no changes in P(f) were observed in NMCs transfected with the empty vector. In conclusion, we developed a novel polarized mouse cholangiocyte monolayer model, allowing direct study of AQP4-mediated water transport by biliary epithelia and generated data providing additional support for the importance of AQP4 in cholangiocyte water transport.

Animals↗
PubMed2004

Cholangiocyte biology.

PURPOSE OF REVIEW: Cholangiocytes are increasingly recognized as biologically important because of the diversity of cellular processes in which they participate. Collectively, these processes define normal function and, when disturbed, account for abnormalities that cause disease. Advances in animal models of disease, sophistication of technology in imaging, and gene silencing have allowed progress in defining the roles that cholangiocytes play in signaling; transport of water, ions, and solutes; and alterations that result in cholestasis. The pace of advances in technology justifies a yearly summary to identify the most important developments in cholangiocyte biology. RECENT FINDINGS: The main areas of recent progress include insights into the molecular mechanisms of hormone-induced bile secretion, development of new experimental models, and a better understanding of the mechanisms of cholestasis. SUMMARY: Understanding the normal components and key biologic processes in cholangiocytes responsible for the regulation of ductal bile secretion is an initial and required step in generating hypotheses relevant to disease. With regard to the pathologic relevance of this work, cholestatic liver diseases represent a broad group of hepatobiliary disorders with which hepatologists must deal. In addition to genetic defects, the study of the normal and altered trafficking of cholangiocyte transport systems involved in bile secretion may provide a molecular correlate for the functional changes that occur in cholestasis. Crucial to this understanding is the ongoing development of experimental models and techniques to answer key hypothesis-driven questions. Additionally, the collegial sharing and exchange of novel concepts, ideas, reagents, and probes promotes positive advances in the field.

Journal Article↗