Serum microRNAs as novel biomarkers for primary sclerosing cholangitis and cholangiocarcinoma.

The diagnosis of primary sclerosing cholangitis (PSC) is difficult due to the lack of sensitive and specific biomarkers, as is the early diagnosis of cholangiocarcinoma (CC), a complication of PSC. The aim of this study was to identify specific serum miRNAs as diagnostic biomarkers for PSC and CC. The levels of 667 miRNAs were evaluated in 90 human serum samples (30 PSC, 30 CC and 30 control subjects) to identify disease-associated candidate miRNAs (discovery phase). The deregulated miRNAs were validated in an independent cohort of 140 samples [40 PSC, 40 CC, 20 primary biliary cirrhosis (PBC) and 40 controls]. Receiver operating characteristic (ROC) curves were established and only miRNAs with an area under the curve (AUC) > 0·70 were considered useful as biomarkers. In the discovery phase we identified the following: 21 miRNAs expressed differentially in PSC, 33 in CC and 26 in both in comparison to control subjects as well as 24 miRNAs expressed differentially between PSC and CC. After the validation phase, miR-200c was found to be expressed differentially in PSC versus controls, whereas miR-483-5p and miR-194 showed deregulated expression in CC compared with controls. We also demonstrate a difference in the expression of miR-222 and miR-483-5p in CC versus PSC. Combination of these specific miRNAs further improved the specificity and accuracy of diagnosis. This study provides a basis for the use of miRNAs as biomarkers for the diagnosis of PSC and CC.

Anti-CD40 ligand monoclonal antibody delays the progression of murine autoimmune cholangitis.

While there have been significant advances in our understanding of the autoimmune responses and the molecular nature of the target autoantigens in primary biliary cirrhosis (PBC), unfortunately these data have yet to be translated into new therapeutic agents. We have taken advantage of a unique murine model of autoimmune cholangitis in which mice expressing a dominant negative form of transforming growth factor ß receptor II (dnTGFßRII), under the control of the CD4 promoter, develop an intense autoimmune cholangitis associated with serological features similar to human PBC. CD40-CD40 ligand (CD40L) is a major receptor-ligand pair that provides key signals between cells of the adaptive immune system, prompting us to determine the therapeutic potential of treating autoimmune cholangitis with anti-CD40L antibody (anti-CD40L; MR-1). Four-week-old dnTGFßRII mice were injected intraperitoneally with either anti-CD40L or control immunoglobulin (Ig)G at days 0, 2, 4 and 7 and then weekly until 12 or 24 weeks of age and monitored for the progress of serological and histological features of PBC, including rigorous definition of liver cellular infiltrates and cytokine production. Administration of anti-CD40L reduced liver inflammation significantly to 12 weeks of age. In addition, anti-CD40L initially lowered the levels of anti-mitochondrial autoantibodies (AMA), but these reductions were not sustained. These data indicate that anti-CD40L delays autoimmune cholangitis, but the effect wanes over time. Further dissection of the mechanisms involved, and defining the events that lead to the reduction in therapeutic effectiveness will be critical to determining whether such efforts can be applied to PBC.

An adult case of acute lymphoblastic leukaemia presenting as hepatic dysfunction.

Acute hepatic dysfunction is a rare and often fatal presentation of haematological malignancies. We describe an adult case of acute lymphoblastic leukaemia presenting as an acute hepatitis. Due to the elevation in the patient's transaminases and bilirubin, standard acute lymphoblastic leukaemia induction therapy could not be used. Instead the combination of prednisone and asaparaginase were used to successfully induce remission.

Costs of hepatitis C.

OBJECTIVE: To estimate the direct and indirect costs of the hepatitis C virus (HCV) in the United States in 1997. DESIGN: Aggregation and analysis of national data sets collected by the National Center for Health Statistics, the Health Care Financing Administration, and other government bureaus and private firms. To estimate costs, we used the human capital method, which decomposes costs into direct categories, such as medical expenses, and indirect categories, such as lost earnings and lost home production. We consider HCV that results in chronic liver disease separate from HCV that results in primary liver cancer. RESULTS: We estimate $5.46 billion as the cost of HCV in 1997. Costs are split as follows: 33% for direct and 67% for indirect costs. Hepatitis C virus that results in chronic liver disease contributes roughly 92% of the costs, and HCV that results in primary liver cancer contributes the remaining 8%. The total estimate of $5.46 billion is conservative, because we ignore costs associated with pain and suffering and the value of care rendered by family members. CONCLUSIONS: To our knowledge, only one estimate of the annual costs of HCV in the 1990s has appeared in the literature, $0.6 billion. However, that estimate was not supported by an explanation of the methods. Our estimate, which relies on detailed methods, is nearly 10 times the original estimate. Our estimate of $5.46 billion is on a par with the cost of asthma ($5.8 billion [1994]).

DMT1 gene expression and cadmium absorption in human absorptive enterocytes.

Divalent metal transporter 1 (DMT1) is a transmembrane, proton-coupled metal ion transporter that is upregulated in the duodenum of iron-deficient rodents and in hereditary hemochromatosis patients, suggesting that it may constitute a key factor in the uptake of dietary iron. Functional expression studies in Xenopus oocytes have shown that DMT1 not only mediates transport of iron but also other divalent metal ions, including the toxic metal cadmium. In the present study, the correlation between the cadmium absorption process and gene expression of DMT1 was investigated in an experimental model of human absorptive enterocytes. Fully differentiated Caco-2 cells were grown in monolayers and treated with iron supplemented medium or control medium for 1, 3 or 7 days. At each time point, cadmium transport experiments across the Caco-2 cell monolayers were performed and gene expression of DMT1 measured. Iron treatment for 3 and 7 days resulted in a 50% reduction in the cadmium uptake and a 75% reduction in the transport of the metal across the basolateral membrane. No effects were observed at 24 h. Gene expression of DMT1 in the iron-treated Caco-2 cells was reduced by about 50% at 3 and 7 days and thus, correlated well with the uptake of cadmium. In summary, our results indicate that the uptake of cadmium into human absorptive enterocytes may be mediated by DMT1.

Granular cells as a marker of early amiodarone hepatotoxicity.

Hepatotoxicity due to chronic amiodarone (AD) use is well described. However, hepatitis occurring after acute administration of AD has only occasionally been reported and the pathologic findings in the liver in this condition have not been well characterized. We describe an idiosyncratic reaction, in a 40-year-old man after 6 weeks of oral AD therapy, consisting of acute hepatitis, which resolved after withdrawal of the drug. The liver biopsy showed clusters of cells with granular cytoplasm. These cells were characterized as macrophages, and phospholipid membranous inclusions were demonstrated ultrastructurally in the granular cells and in the hepatocytes. Pathologists and clinicians should be aware of this subtle histologic finding when looking for evidence to support AD hepatotoxicity.

Functional and molecular responses of human intestinal Caco-2 cells to iron treatment.

BACKGROUND: Divalent metal transporter 1 (DMT1), HFE, and stimulator of iron transport (SFT) are transmembrane proteins that have been implicated in the regulation of iron homeostasis. OBJECTIVE: The objective of this study was to investigate whether absorption and transepithelial movement of iron correlated with gene expression of DMT1, HFE, and SFT in an experimental model of human absorptive enterocytes. DESIGN: Caco-2 cells were exposed to iron-supplemented media in either the presence or the absence of serum for 24, 72, and 168 h. At each time point, the uptake and transepithelial movement of iron were examined and gene expression of DMT1, HFE, and SFT was measured. Manganese and zinc absorption was also examined at 168 h. RESULTS: Iron treatment in the presence or absence of serum reduced the uptake and transepithelial movement of iron by approximately 50% after 72 and 168 h. No effect was observed at 24 h. The uptake and transepithelial movement of manganese were similar to those of iron at 168 h, whereas the effects on zinc were less pronounced. In the absence of serum, iron treatment was associated with a reduction of DMT1 expression by 50% at 72 and 168 h. HFE expression was dependent on serum, but iron treatment did not alter HFE expression. SFT expression was not affected by iron. CONCLUSIONS: Iron treatment decreased cellular uptake of iron, manganese, and zinc, suggesting that these metals may utilize the same apical transporter. The transepithelial movement of iron and manganese, but not of zinc, was reduced across iron-treated Caco-2 cells, suggesting that iron and manganese are regulated by the same mechanism at the basolateral membrane. The gene expression of DMT1, HFE, and SFT did not fully correlate with the functional responses of Caco-2 cells. This may have been a result of posttranscriptional regulation of these genes or regulation of other genes involved in the uptake and transepithelial movement of iron in Caco-2 cells.

Cloning of a novel MHC-encoded serine peptidase highly expressed by cortical epithelial cells of the thymus.

Antigen presentation by cortical thymic epithelial cells (cTEC) during the positive selection of T cells has been shown to differ from that of other antigen-presenting cells. In the case of MHC class II presentation, cathepsin L as opposed to cathepsin S is responsible at least in part for the degradation of invariant chain. Other proteases, however, must be involved. We have identified a putative serine protease that is specifically expressed in the thymus. Encoded within the class I major histocompatibility complex (MHC) region, this gene has sequence homology with lysosomal prolylcarboxypeptidase, suggesting that it is a serine protease. We have, therefore, designated this gene thymus-specific serine protease (TSSP). In situ hybridization and immunofluorescence staining reveal that TSSP is expressed exclusively by cortical thymic epithelial cells, with the strongest staining noted around vessels and the thymic capsule. The identification of TSSP further supports the theory that MHC class II antigen processing and presentation in the thymic cortex involves a proteolytic milieu that differs from that of other antigen-presenting cells.

Characterization of three different elements in the 5'-flanking region of the fibronectin gene which mediate a transcriptional response to cAMP.

A cAMP regulatory element (CRE) at nucleotide position -170 of the fibronectin gene was characterized previously (Dean, D. C., Blakeley, M. S., Newby, R. F., Ghazal, P., Hennighausen, L., and Bourgeois, S. (1989) Mol. Cell. Biol. 9, 1498-1506). Here we identify two additional low affinity CREs at nucleotide positions -260 and -415 which differ in sequence by 1 base pair. Interestingly, these CREs did not compete for binding of nuclear proteins in gel retardation assays and partial tryptic digestion of protein-DNA complexes produced a different pattern with each CRE, indicating that they bind different proteins. CRE (-170) competed for binding of proteins to both CREs, suggesting that it may represent a composite of the two elements. CRE (-415) competed effectively for binding of nuclear proteins to the somatostatin gene CRE, suggesting that, like the somatostatin CRE, it binds the nuclear protein CREB. On the other hand, CRE (-260) appears to bind the nuclear protein PEA-2, which also binds a site in the polyoma virus enhancer. In summary, disruption of dyad symmetry in the 3' region of the CRE, as occurs with CRE (-260) and CRE (-415), results in a lower affinity site and may also change the specificity for different nuclear proteins.

Cloning and analysis of the promotor region of the human fibronectin gene.

Human fibronectin (FN) genomic clones were isolated by screening a human genomic library with a 75-base oligonucleotide. The sequence of the oligonucleotide corresponds to a region near the 5' end of the human FN cDNA clone pFH6 that contains the amino-terminal coding sequences but does not extend to the 5' end of the mRNA [Kornblihtt, A. R., Umezawa, K., Vibe-Pedersen, K. & Baralle, F. E. (1985) EMBO J. 4, 1755-1759]. The 5' end of the FN gene is found on a 3.7-kilobase-pair EcoRI fragment that contains about 2.7 kilobase pairs of flanking sequence. The first exon is 414 base pairs long, with a 5' untranslated region of 267 base pairs. As deduced on the basis of the position of the initiation codon, FN is synthesized with a 31-residue amino acid extension on the amino terminus that is not present in the mature polypeptide. This amino-terminal extension appears to contain both a signal peptide and a propeptide. The first 200 base pairs of 5'-flanking sequence is very G + C rich. Upstream of this the sequence becomes relatively A + T rich. The sequence ATATAA is found at -25 and the sequence CAAT is present at -150. The sequence GGGGCGGGGC at -102 exhibits homology to the binding site for the transcription factor SP1, and the sequence TGACGTCA at -173 exhibits homology to 5'-flanking sequences important for induction by cAMP.