Prominent Intrapulmonary Shunt Vessels and Altered Lung Development in Infants With Sudden Unexplained Infant Death.

The purpose of this study was to evaluate intrapulmonary arteriovenous shunts in patients with and without sudden unexplained infant death. We identified open intrapulmonary bronchopulmonary anastomoses as potential pathways for right-to-left shunt in a subset of infants with sudden unexplained infant death.

Association of sickle cell trait with adverse pregnancy outcomes in a population-based cohort.

INTRODUCTION: Sickle cell trait (SCT) is common in African descendants. Its association with several adverse pregnancy outcomes (APOs) has been reported but remains inconsistent. The objectives of this study are to test associations of SCT with APOs in non-Hispanic Black women, including (1) validate the associations of SCT with previously reported APOs, (2) test novel associations of SCT with broad spectrum of APOs, and (3) estimate the attributable risk of SCT for implicated APOs. MATERIAL AND METHODS: This is a retrospective analysis of a prospectively designed population-based cohort. Women/participants were self-reported non-Hispanic Black women from the UK Biobank (UKB). SCT status was determined based on heterozygous Glu6Val in the HBB gene. Several APOs were studied, including four previously reported SCT-associated APOs (preeclampsia, bacteriuria, pregnancy loss, and preterm delivery), and broad conditions related to pregnancy, childbirth, and the puerperium. APOs were curated by experts' peer review and consensus processes. Associations of SCT with APOs were tested by estimating its relative risk and 95% confidence interval (95% CI), adjusting for number of live births and age at first birth. Attributable risk proportion (ARP) and population attributable risk proportion (PARP) of SCT to APOs were estimated. RESULTS: Among the 4057 self-reported non-Hispanic Black women with pregnancy records in the UKB, 581 (14.32%) were SCT carriers. For four previously reported SCT-associated APOs, two were confirmed at a nominal P < 0.05; relative risk (RR) was 2.39 (95% CI 1.09-5.23) for preeclampsia, and 4.85 (95% CI 1.77-13.27) for bacteriuria. SCT contributed substantially to these two APOs among SCT carriers, with attributable risk proportion estimated at 61.00% and 68.96% for preeclampsia and bacteriuria, respectively. SCT also contributed substantially to these two APOs in the population (self-reported Black UK women), with population attributable risk proportion estimated at 18.30% and 24.14% for preeclampsia and bacteriuria, respectively. In addition, novel associations were found for seven other APOs (nominal P < 0.05). CONCLUSIONS: SCT is significantly associated with APOs in this study and contributes substantially to APOs among self-reported Black women in the UK. Confirmation of these findings in independent study populations is required.

Electrocution Due to Fractal Wood Burning: Two Case Reports and a Review of the Medical Literature.

ABSTRACT: Fractal wood burning is a new technique of pyrography that passes an electrical current through a piece of wood resulting in decorative electrical burns. This practice has become increasingly popular with many walk-through tutorials of the process found online. This includes videos of how to build homemade devices fashioned from disassembled microwave oven transformers. There have been 31 reported deaths and many serious injuries due to fractal wood burning resulting in news headlines, warning statements, and an outright ban of the practice at certain woodworking events. The medical community has begun to recognize the danger of fractal wood burning with a few cases of severe burn injuries reported. We report 2 cases of electrocution from fractal wood burning accidents. The scene investigations were examined, including the similarities in the homemade microwave oven transformers that were used, as well as the autopsy findings. The pathophysiology of fractal wood burning and the creation of Lichtenberg figures is discussed as well as the high-voltage injury patterns seen in cases of fractal wood burning accidents. Other cases of electrical injury from fractal wood burning accidents reported in the news and medical literature were then examined in terms of demographics, burn pattern, cardiac findings, and whether a homemade wood burning device was involved.

Neutrophil extracellular trap inhibition increases inflammation, bacteraemia and mortality in murine necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease affecting primarily premature infants. The disease is characterized by intestinal inflammation and leucocyte infiltration, often progressing to necrosis, perforation, systemic inflammatory response and death. Neutrophil extracellular traps (NETs), denoting nuclear DNA, histone and antimicrobial protein release, have been suggested to play a role in NEC. This study aimed to determine the role of NETs in NEC and explore the effect of chloramidine, a NET inhibitor, on a murine NEC-like intestinal injury model. Blood and intestinal tissues were collected from infants diagnosed with ≥ Stage II NEC, and levels of nucleosomes and NETs, respectively, were compared with those of case-matched controls. In mice, NEC was induced with dithizone/Klebsiella, and mice in the treatment group received 40 mg/kg chloramidine. Bacterial load, intestinal histology, plasma myeloperoxidase and cytokine levels, and immunofluorescent staining were compared with controls. Nucleosomes were significantly elevated in both human and mouse NEC plasma, whereas NET staining was only present in NEC tissue in both species. Chloramidine treatment increased systemic inflammation, bacterial load, organ injury and mortality in murine NEC. Taken together, our findings suggest that NETs are critical in the innate immune defence during NEC in preventing systemic bacteraemia.

Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ.

Polycystin-1 (PC1), encoded by the PKD1 gene that is mutated in the autosomal dominant polycystic kidney disease, regulates a number of processes including bone development. Activity of the transcription factor RunX2, which controls osteoblast differentiation, is reduced in Pkd1 mutant mice but the mechanism governing PC1 activation of RunX2 is unclear. PC1 undergoes regulated cleavage that releases its C-terminal tail (CTT), which translocates to the nucleus to modulate transcriptional pathways involved in proliferation and apoptosis. We find that the cleaved CTT of PC1 (PC1-CTT) stimulates the transcriptional coactivator TAZ (Wwtr1), an essential coactivator of RunX2. PC1-CTT physically interacts with TAZ, stimulating RunX2 transcriptional activity in pre-osteoblast cells in a TAZ-dependent manner. The PC1-CTT increases the interaction between TAZ and RunX2 and enhances the recruitment of the p300 transcriptional co-regulatory protein to the TAZ/RunX2/PC1-CTT complex. Zebrafish injected with morpholinos directed against pkd1 manifest severe bone calcification defects and a curly tail phenotype. Injection of messenger RNA (mRNA) encoding the PC1-CTT into pkd1-morphant fish restores bone mineralization and reduces the severity of the curly tail phenotype. These effects are abolished by co-injection of morpholinos directed against TAZ. Injection of mRNA encoding a dominant-active TAZ construct is sufficient to rescue both the curly tail phenotype and the skeletal defects observed in pkd1-morpholino treated fish. Thus, TAZ constitutes a key mechanistic link through which PC1 mediates its physiological functions.

Randomized Controlled Trial of Early Docosahexaenoic Acid and Arachidonic Acid Enteral Supplementation in Very Low Birth Weight Infants.

OBJECTIVE: To determine feasibility of providing a concentrated emulsified long-chain polyunsaturated fatty acids (LCPUFA) supplement to very low birth weight infants, and to evaluate blood LCPUFA concentrations at 2 and 8 weeks of study supplementation. STUDY DESIGN: This prospective, randomized, double-blind, placebo-controlled trial randomized infants to receive (1) LCPUFA-120 (a supplement of 40 mg/kg/day docosahexaenoic acid [DHA] and 80 mg/kg/day arachidonic acid [ARA]; DHA:ARA at 1:2 ratio), (2) LCPUFA-360 (a supplement of 120 mg/kg/day DHA and 240 mg/kg/day ARA), or (3) sunflower oil (placebo control). Infants received supplement daily for 8 weeks or until discharge, whichever came first. Whole blood LCPUFA levels (wt%; g/100 g) were measured at baseline, 2 weeks, and 8 weeks. RESULTS: Infants were 28 weeks of gestation (IQR, 27-30 weeks of gestation) and weighed 1040 g (IQR, 910-1245 g). At 2 weeks, the change in blood DHA (wt%) from baseline differed significantly among groups (sunflower oil, n = 6; -0.63 [IQR, -0.96 to -0.55]; LCPUFA-120: n = 12; -0.14 [IQR, -0.72 to -0.26]; LCPUFA-360, n = 12; 0.46 [IQR, 0.17-0.81]; P = .002 across groups). Change in blood ARA (wt%) also differed by group (sunflower oil: -2.2 [IQR, -3.9 to -1.7]; LCPUFA-120: 0.1 [IQR, -2.1 to 1.1] vs LCPUFA-360: 2.9 IQR, 1.5 to 4.5]; P = .0002). Change from baseline to 8 weeks significantly differed between groups for DHA (P = .02) and ARA (P = .003). CONCLUSIONS: Enteral LCPUFA supplementation supported higher blood DHA by 2 weeks. LCPUFA supplementation at 360 mg of combined DHA and ARA is likely necessary to reduce declines as well as allow increases in whole blood concentrations in the first 8 weeks of life. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03192839.

Visual and Patient-Reported Outcomes of a Diffractive Trifocal Intraocular Lens Compared with Those of a Monofocal Intraocular Lens.

PURPOSE: To evaluate the effectiveness and safety of a trifocal intraocular lens (IOL), the TFNT00 (Alcon, Fort Worth, TX), versus a monofocal IOL, the SN60AT (Alcon). DESIGN: Food and Drug Administration-approved, prospective, multicenter, nonrandomized, parallel-group, assessor-masked, confirmatory trial. PARTICIPANTS: Patients enrolled were 22 years of age or older with a diagnosis of bilateral cataract with planned removal by phacoemulsification with a clear corneal incision. METHODS: Consented participants selected their preferred IOL, which was implanted sequentially into each eye of patients meeting eligibility criteria. MAIN OUTCOME MEASURES: The coprimary effectiveness outcomes were mean photopic monocular best-corrected distance visual acuity (BCDVA; 4 m) and distance-corrected near visual acuity (DCNVA; 40 cm) at 6 months after surgery. Secondary effectiveness outcomes included mean monocular distance-corrected intermediate visual acuity (DCIVA; 66 cm) and proportion of participants responding "never" to question 1 of the Intraocular Lens Satisfaction questionnaire (regarding frequency of spectacle use in the past 7 days). Safety outcomes included frequency of "severe" and "most bothersome" visual disturbances. RESULTS: Two hundred forty-three patients underwent cataract surgery with bilateral implantation of the TFNT00 (n = 129) or SN60AT (n = 114) and were followed up for 6 months. Noninferiority of TFNT00 to SN60AT in mean photopic monocular BCDVA (95% upper confidence limit of the difference was <0.1 logarithm of the minimum angle of resolution [logMAR] margin), and superiority in mean photopic monocular DCNVA (difference of 0.42 logMAR; P < 0.001) and DCIVA (difference of 0.26 logMAR; P < 0.001) were demonstrated. The proportion of patients never requiring glasses overall was superior for TFNT00 versus SN60AT (80.5% and 8.2%, respectively). Starbursts, halos, and glare were the most frequently rated severe symptoms with TFNT00; however, less than 5% of patients were very bothered at month 6. CONCLUSIONS: The TFNT00 exhibited superior monocular DCNVA and DCIVA to a spherical monofocal IOL, with comparable monocular BCDVA. Binocular visual acuity was 20/25 or better for distance to near (+0.5 D to -2.5 D), resulting in high levels of spectacle independence. Less than 5% of patients were very bothered by the photic visual disturbances associated with the TFNT00 at 6 months after surgery.

Holding open the door reveals a new view of polycystin channel function.

The functions of polycystin 1 and polycystin 2 (PC1 and PC2) have been surprisingly difficult to establish. PC1 and PC2 are encoded by the Pkd1 and Pkd2 genes that are implicated in autosomal dominant polycystic kidney disease (ADPKD). ADPKD is the most common potentially lethal genetic disorder, affecting ~1 in 1,000 people. Over the course of decades, ADPKD patients' kidneys acquire numerous fluid-filled cysts whose expansion compresses the surrounding parenchyma, leading to end-stage renal disease in ~50% of afflicted individuals [1]. Identification of the genes encoding the PC proteins 20 years ago led to the hypothesis that they form an ion channel, since the sequence of PC2 marks it as a member of the TRP family of cation channels. In the ensuing 2 decades, tremendous effort has been devoted to determining whether this is indeed true and, if so, what characteristics that channel might manifest. A recent paper by Wang et al in this issue of EMBO Reports [2] demonstrates that assembly with PC1 changes the properties of the polycystin channel in ways that may help explain the complex behaviors that have been attributed to it.

Newly synthesized polycystin-1 takes different trafficking pathways to the apical and ciliary membranes.

Mutations in the genes encoding polycystin-1 (PC1) and polycystin 2 (PC2) cause autosomal dominant polycystic kidney disease. These transmembrane proteins colocalize in the primary cilia of renal epithelial cells, where they may participate in sensory processes. PC1 is also found in the apical membrane when expressed in cultured epithelial cells. PC1 undergoes autocatalytic cleavage, producing an extracellular N-terminal fragment that remains noncovalently attached to the transmembrane C-terminus. Exposing cells to alkaline solutions elutes the N-terminal fragment while the C-terminal fragment is retained in the cell membrane. Utilizing this observation, we developed a "strip-recovery" synchronization protocol to study PC1 trafficking in polarized LLC-PK1 renal epithelial cells. Following alkaline strip, a new cohort of PC1 repopulates the cilia within 30 minutes, while apical delivery of PC1 was not detectable until 3 hours. Brefeldin A (BFA) blocked apical PC1 delivery, while ciliary delivery of PC1 was BFA insensitive. Incubating cells at 20°C to block trafficking out of the trans-Golgi network also inhibits apical but not ciliary delivery. These results suggest that newly synthesized PC1 takes distinct pathways to the ciliary and apical membranes. Ciliary PC1 appears to by-pass BFA sensitive Golgi compartments, while apical delivery of PC1 traverses these compartments.

Mechanisms involved in AMPK-mediated deposition of tight junction components to the plasma membrane.

AMP-activated protein kinase (AMPK) activation promotes early stages of epithelial junction assembly. AMPK activation in MDCK renal epithelial cells facilitates localization of the junction-associated proteins aPKCζ and Par3 to the plasma membrane and promotes conversion of Cdc42, a key regulator of epithelial polarization and junction assembly, to its active GTP bound state. Furthermore, Par3 is an important regulator of AMPK-mediated aPKCζ localization. Both aPKCζ and Par3 serve as intermediates in AMPK-mediated junction assembly, with inhibition of aPKCζ activity or Par3 knockdown disrupting AMPK's ability to facilitate zonula occludens (ZO-1) localization. AMPK phosphorylates the adherens junction protein afadin and regulates its interaction with the tight-junction protein zonula occludens-1. Afadin is phosphorylated at two critical sites, S228 (residing within an aPKCζ consensus site) and S1102 (residing within an AMPK consensus site), that are differentially regulated during junction assembly and that exert different effects on the process. Expression of phospho-defective mutants (S228A and S1102A) perturbed ZO-1 localization to the plasma membrane during AMPK-induced junction assembly. Expression of S228A increased the ZO-1/afadin interaction, while S1102A reduced this interaction during extracellular calcium-induced junction assembly. Inhibition of aPKCζ activity also increased the ZO-1/afadin interaction. Taken together, these data suggest that aPKCζ phosphorylation of afadin terminates the ZO-1/afadin interaction and thus permits the later stages of junction assembly.

Defining necrotizing enterocolitis: current difficulties and future opportunities.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in hospitalized infants. First classified through Bell staging in 1978, a number of additional definitions of NEC have been proposed in the subsequent decades. In this review, we summarize eight current definitions of NEC, and explore similarities and differences in clinical signs and radiographic features included within these definitions, as well as their limitations. We highlight the importance of a global consensus on defining NEC to improve NEC research and outcomes, incorporating input from participants at an international NEC conference. We also highlight the important role of patient-families in helping to redefine NEC.

Grading the evidence to identify strategies to modify risk for necrotizing enterocolitis.

Although risk for necrotizing enterocolitis (NEC) is often presented from the perspective of a premature infant's vulnerability to nonmodifiable risk factors, in this paper we describe the evidence and present recommendations to manage modifiable risks that are amenable to clinical actions. Using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria, we present recommendations in the context of their supporting evidence in a way that balances risks (e.g. potential harm, cost) and benefits. Across the prenatal, intrapartum, early and late clinical course, strategies to limit NEC risk in premature infants are presented. Our goal is to summarize modifiable NEC risk factors, grade the evidence to offer quality improvement (QI) targets for healthcare teams and offer a patient-family advocate's perspective on how to engage parents to recognize and reduce NEC risk.

Newly synthesized and recycling pools of the apical protein gp135 do not occupy the same compartments.

Polarized epithelial cells sort newly synthesized and recycling plasma membrane proteins into distinct trafficking pathways directed to either the apical or basolateral membrane domains. While the trans-Golgi network is a well-established site of protein sorting, increasing evidence indicates a key role for endosomes in the initial trafficking of newly synthesized proteins. Both basolateral and apical proteins have been shown to traverse endosomes en route to the plasma membrane. In particular, apical proteins traffic through either subapical early or recycling endosomes. Here we use the SNAP tag system to analyze the trafficking of the apical protein gp135, also known as podocalyxin. We show that newly synthesized gp135 traverses the apical recycling endosome, but not the apical early endosomes (AEEs). In contrast, post-endocytic gp135 is delivered to the AEE before recycling back to the apical membrane. The pathways pursued by the newly synthesized and recycling gp135 populations do not detectably intersect, demonstrating that the biosynthetic and post-endocytic pools of this protein are subjected to distinct sorting processes.

Implications of AMPK in the Formation of Epithelial Tight Junctions.

Tight junctions (TJ) play an essential role in the epithelial barrier. By definition, TJ are located at the demarcation between the apical and baso-lateral domains of the plasma membrane in epithelial cells. TJ fulfill two major roles: (i) TJ prevent the mixing of membrane components; and (ii) TJ regulate the selective paracellular permeability. Disruption of TJ is regarded as one of the earliest hallmarks of epithelial injury, leading to the loss of cell polarity and tissue disorganization. Many factors have been identified as modulators of TJ assembly/disassembly. More specifically, in addition to its role as an energy sensor, adenosine monophosphate-activated protein kinase (AMPK) participates in TJ regulation. AMPK is a ubiquitous serine/threonine kinase composed of a catalytic α-subunit complexed with regulatory ß-and γ-subunits. AMPK activation promotes the early stages of epithelial TJ assembly. AMPK phosphorylates the adherens junction protein afadin and regulates its interaction with the TJ-associated protein zonula occludens (ZO)-1, thereby facilitating ZO-1 distribution to the plasma membrane. In the present review, we detail the signaling pathways up-and down-stream of AMPK activation at the time of Ca2+-induced TJ assembly.

Sequence-specific detection of different strains of LCMV in a single sample using tentacle probes.

BACKGROUND: Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems. METHODS: We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe. RESULTS: Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA. CONCLUSIONS: Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.

Investigation of peanut oral immunotherapy with CpG/peanut nanoparticles in a murine model of peanut allergy.

BACKGROUND: Treatments to reverse peanut allergy remain elusive. Current clinical approaches using peanut oral/sublingual immunotherapy are promising, but concerns about safety and long-term benefit remain a barrier to wide use. Improved methods of delivering peanut-specific immunotherapy are needed. OBJECTIVE: We sought to investigate the efficacy and safety of peanut oral immunotherapy using CpG-coated poly(lactic-co-glycolic acid) nanoparticles containing peanut extract (CpG/PN-NPs) in a murine model of peanut allergy. METHODS: C3H/HeJ mice were rendered peanut allergic by means of oral sensitization with peanut and cholera toxin. Mice were then subjected to 4 weekly gavages with CpG/PN-NPs, vehicle (PBS), nanoparticles alone, peanut alone, CpG nanoparticles, or peanut nanoparticles. Untreated mice served as naive controls. After completing therapy, mice underwent 5 monthly oral peanut challenges. Anaphylaxis was evaluated by means of visual assessment of symptom scores and measurement of body temperature and plasma histamine levels. Peanut-specific serum IgE, IgG1, and IgG2a levels were measured by using ELISA, as were cytokine recall responses in splenocyte cultures. RESULTS: Mice with peanut allergy treated with CpG/PN-NPs but not vehicle or other treatment components were significantly protected from anaphylaxis to all 5 oral peanut challenges, as indicated by lower symptom scores, less change in body temperature, and a lower increase of plasma histamine levels. Importantly, CpG/PN-NP treatment did not cause anaphylactic reactions. Treatment was associated with a sustained and significant decrease in peanut-specific IgE/IgG1 levels and an increase in peanut-specific IgG2a levels. Compared with vehicle control animals, peanut recall responses in splenocyte cultures from nanoparticle-treated mice showed significantly decreased levels of TH2 cytokines (IL-4, IL-5, and IL-13) but increased IFN-γ levels in cell supernatants. CONCLUSIONS: Preclinical findings indicate that peanut oral immunotherapy with CpG/PN-NPs might be a valuable strategy for peanut-specific immunotherapy in human subjects.

Polycystin-1 Is a Cardiomyocyte Mechanosensor That Governs L-Type Ca2+ Channel Protein Stability.

BACKGROUND: L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress-induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. METHODS AND RESULTS: We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction-induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals CONCLUSION: PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein.

Knockdown of ezrin causes intrahepatic cholestasis by the dysregulation of bile fluidity in the bile duct epithelium in mice.

UNLABELLED: Cholangiopathies share common features, including bile duct proliferation, periportal fibrosis, and intrahepatic cholestasis. Damage of biliary epithelium by autoimunne disorder, virus infection, toxic compounds, and developmental abnormalities causes severe progressive hepatic disorders responsible for high mortality. However, the etiologies of these cholestatic diseases remain unclear because useful models to study the pathogenic mechanisms are not available. In the present study, we have found that ezrin knockdown (Vil2(kd/kd) ) mice develop severe intrahepatic cholestasis characterized by extensive bile duct proliferation, periductular fibrosis, and intrahepatic bile acid accumulation without developmental defects of bile duct morphology and infiltration of inflammatory cells. Ezrin is a membrane cytoskeletal cross-linker protein, which is known to interact with transporters, scaffold proteins, and actin cytoskeleton at the plasma membrane. We found that the normal apical membrane localizations of several transport proteins including cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE-2), aquaporin 1 (AQP1), and Na(+) /H(+) exchanger regulatory factor were disturbed in bile ducts of Vil2(kd/kd) mice. Stable expression of a dominant negative form of ezrin in immortalized mouse cholangiocytes also led to the reduction of the surface expression of CFTR, AE-2, and AQP1. Reduced surface expression of these transport proteins was accompanied by reduced functional expression, as evidenced by the fact these cells exhibited decreased CFTR-mediated Cl(-) efflux activity. Furthermore, bile flow and biliary HCO3 (-) concentration were also significantly reduced in Vil2(kd/kd) mice. CONCLUSION: Dysfunction of ezrin mimics important aspects of the pathological mechanisms responsible for cholangiopathies. The Vil2(kd/kd) mouse may be a useful model to exploit in the development and testing of potential therapies for cholangiopathies.