Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1–4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1–4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1–4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1–4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

Fgf15 is necessary to maintain BA homeostasis and prevent liver injury during liver regeneration. Moreover, Fgf15 is an essential mediator of the liver growth-promoting effects of BA. Preoperative administration of this enterokine to patients undergoing liver resection might be useful to reduce damage and foster regeneration.

Cl−/HCO3−anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here we tested the potential role of hsa-microRNA 506 (miR-506) – predicted as candidate to target AE2 mRNA – for the decreased expression of AE2 in PBC. Real-time qPCR showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is upregulated in the intrahepatic bile ducts of PBC livers compared with normal and primary-sclerosing-cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in 3D-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the three-dimensional conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3’UTR region of AE2 mRNA and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity together with miR-506 overexpression compared to normal human cholangiocytes, and, transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. Conclusion miR-506 is upregulated in cholangiocytes from PBC patients, binds the 3’UTR region of AE2 mRNA and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease.

No abstract

PCK rats, an animal model of autosomal recessive polycystic kidney disease (ARPKD), develop cholangiocyte-derived liver cysts associated with increased intracellular adenosine 3 ,5 -cyclic adenosine monophosphate (cAMP), the inhibition of which suppresses cyst growth. We hypothesized that elevated cAMP stimulates cholangiocyte proliferation via two downstream effectors, exchange proteins activated by cAMP (Epac1 and Epac2 isoforms) and protein kinase A (PKA), and that intracellular calcium is also involved in this process. Assessment of Epac isoforms and PKA regulatory subunits at the messenger RNA and protein level showed that cultured normal rat cholangiocytes express Epac1, Epac2, and all regulatory PKA subunits. Epac isoforms and the PKA RI␤ subunit were overexpressed in cultured PCK cholangiocytes. Proliferation analysis in response to Epac and PKA activation indicated that both normal and PCK cholangiocytes increase their growth upon Epac-specific stimulation, while PKA-specific stimulation results in differential effects, suppressing proliferation in normal cholangiocytes but accelerating this process in PCK cholangiocytes. On the other hand, both PKA and Epac activation of cystic structures generated by normal and PCK cholangiocytes when cultured under three-dimensional conditions resulted in increased cyst growth, particularly in PCK-cholangiocyte derived cysts. Pharmacological inhibitors and small interfering RNA-mediated gene silencing demonstrated the specificity of each effector activation, as well as the involvement of MEK-ERK1/2 signaling in all the observed effector-associated proliferation changes. Hyperproliferation of PCK cholangiocytes in response to PKA stimulation, but not to Epac stimulation, was found to be associated with decreased intracellular calcium, and restoration of calcium levels blocked the PKA-dependent proliferation via the PI3K/AKT pathway. Conclusion: Our data provide strong evidence that both cAMP effectors, Epac and PKA, and the levels of intracellular calcium are involved in the hepatic cystogenesis of ARPKD. (HEPATOLOGY 2009;49:160-174.)

One of the mechanisms by which epithelial cells regulate intracellular pH is exchanging bicarbonate for Cl − . We tested the hypothesis that in ameloblasts the anion exchanger-2 (Ae2) is involved in pH regulation during maturation stage amelogenesis. Quantitative X-ray microprobe mineral content analysis, scanning electron microscopy, histology, micro-computed tomography and Ae2 immunolocalisation analyses were applied to Ae2-deficient and wild-type mouse mandibles. Immunolocalisation of Ae2 in wild-type mouse incisors showed a very strong expression of Ae2 in the basolateral membranes of the maturation stage ameloblasts. Strikingly, zones of contiguous ameloblasts were found within the maturation stage in which Ae2 expression was extremely low as opposed to neighbouring cells. Maturation stage ameloblasts of the Ae2 a,b −/− mice failed to stain for Ae2 and showed progressive disorganisation as enamel development advanced. Maturation stage enamel of the Ae2 a,b −/− mice contained substantially less mineral and more protein than wild-type enamel as determined by quantitative X-ray microanalysis. Incisor enamel was more severely affected than molar enamel. Scanning electron microscopy revealed that the rod-inter-rod structures of the Ae2 a,b −/− mice incisor enamel were absent. Mineral content of dentine and bone of Ae2 a,b −/− mice was not significantly different from wild-type mice. The enamel from knockout mouse teeth wore down much faster than that from wild-type litter mates. Basolateral bicarbonate secretion via the anionic exchanger Ae2 is essential for mineral growth in the maturation stage enamel. The observed zonal expression of Ae2 in the maturation stage ameloblasts is in line with a model for cyclic proton secretion during maturation stage amelogenesis.

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Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl ؊ efflux and subsequent biliary HCO 3 ؊ secretion, possibly via Cl ؊ /HCO 3 ؊ anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl ؊ /HCO 3 ؊ exchange in secretin-stimulated HCO 3 ؊ secretion all remain unclear. Here, secretin was administered to normal rats with maintained bile acid pool via continuous taurocholate infusion. Bile flow and biliary HCO 3 ؊ and Cl ؊ excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl ؊ channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) or the Cl ؊ /HCO 3 ؊ exchange inhibitor 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion of HCO 3 ؊ and Cl ؊ . Interestingly, secretin effects were not observed in the absence of taurocholate. Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin-induced increases in bile flow and HCO 3 ؊ excretion but not the increased Cl ؊ excretion, revealing a role of biliary Cl ؊ /HCO 3 ؊ exchange in secretininduced, bicarbonate-rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na ؉ -independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused a marked inhibition of unstimulated and secretin-stimulated Cl ؊ /HCO 3 ؊ exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate-rich choleresis in the normal rat and that this occurs via a chloride-bicarbonate exchange process consistent with AE2 function. (HEPATOLOGY 2006;43:266-275.) S ecretin is known to induce bicarbonate-rich hydrocholeresis in many animal species. 1-7 Its interaction with a G-protein-coupled receptor selectively localized to the epithelial bile duct cells 8 results in increased intracellular levels of cyclic adenosine monophosphate (cAMP) [cAMP] i 7,9,10 and protein kinase A activation. 11,12 Phosphorylation and opening of a cAMP-dependent Cl Ϫ channel, the cystic fibrosis transmembrane conductance regulator (CFTR), 13 causes Cl Ϫ efflux to the ductular lumen. This appears to stimulate an apical Na ϩ -independent Cl Ϫ /HCO 3 Ϫ anion exchange (AE), 14 with HCO 3 Ϫ efflux and Cl Ϫ influx, that is facilitated by the outside to inside transmembrane gradient of Cl Ϫ at relatively high intracellular HCO 3 Ϫ concentration. [10][11][12]15,16 Several bicarbonate transporters, most of them encoded by the SLC4 and SLC26 gene families, 17 have been described to exert AE activity. A decade ago, we localized one of those polypeptides, the SLC4A2 or AE2, 18 to the apical membrane in...