Multiplex nucleic acid suspension bead arrays for detection and subtyping of filoviruses.

Here we describe multiplex suspension bead array systems that allow fast and reliable detection of reverse transcriptase (RT) PCR amplified filovirus genomes and also enable subtyping of Ebola virus species and Marburg virus strains. These systems have an analytical sensitivity equivalent to that of RT-PCR.

Iron-regulatory gene expression during liver regeneration.

BACKGROUND: In rat, the first 18-24 h after partial hepatectomy (PH) are characterized by an acute-phase reaction, after which liver regeneration predominates. Interleukin-6 (IL-6) induces the iron hormone hepcidin, which blocks iron uptake and may compromise iron uptake in the growing liver. The expressions of hepcidin and the iron-regulatory pathway of hepcidin gene expression during the late phase of liver regeneration are unknown. AIM: To characterize the expression pattern of hepcidin and the iron-sensing pathway of hepcidin regulation during liver regeneration. METHODS: Rats were subjected to PH or sham operation. Liver weights, number of S-phase nuclei, and serum levels of iron and IL-6 were determined. Messenger-RNA levels of hepcidin, ferritin, hemojuvelin, transferrin receptor 1 and 2, HFE, divalent metal transporter 1, ferroportin, and ceruloplasmin were determined with qPCR at different time points. Protein levels of STAT3 and SMAD4 were determined with western blot. RESULTS: During the acute-phase response, IL-6 release induced STAT3 protein and hepcidin mRNA, whereas mRNA levels of proteins in the iron-sensing pathway (HFE, hemojuvelin, and transferrin receptor 2) decreased. The mRNA levels of proteins involved in cellular iron uptake were increased and cellular iron export unchanged. During liver regeneration >24 h after PH, gene expressions in the iron-sensing pathway were continuously suppressed and hepcidin mRNA levels declined 3-7 days after surgery. CONCLUSIONS: Hepcidin gene expression peaks during the acute-phase response, but a sustained down-regulation of the iron-sensing pathway of hepcidin regulation gradually reduces hepcidin gene expression until regeneration is complete, thereby promoting iron mobilization to the regenerating liver.

A novel mutation in the biliverdin reductase-A gene combined with liver cirrhosis results in hyperbiliverdinaemia (green jaundice).

BACKGROUND: Hyperbiliverdinaemia is a poorly defined clinical sign that has been infrequently reported in cases of liver cirrhosis or liver carcinoma, usually indicating a poor long-term prognosis. AIMS: To clarify the pathogenesis of hyperbiliverdinaemia in an extended case report. METHODS: A 64-year-old man with alcoholic cirrhosis was admitted to hospital with severe bleeding from oesophageal varices. Ultrasonography showed ascites, but no dilatation of the biliary tree. The skin, sclerae, plasma, urine and ascites of the patient showed a greenish appearance. Bilirubin levels were normal, and there were no signs of haemolysis. Biliverdin was analysed in plasma and urine with liquid chromatography coupled to mass spectrometry. The seven exonic regions of the biliverdin reductase-A (BVR-A) gene was amplified by polymerase chain reaction and sequenced. RESULTS: Biliverdin was present in plasma and urine. In nucleotide 52 of exon I of the DNA isolated from the hyperbiliverdinaemic patient, we discovered a novel heterozygous C-->T nonsense mutation converting an arginine (CGA) in position 18 into a stop codon (TGA) (R18Stop) predicted to truncate the protein N-terminally to the active site Tyr97. Two children of the proband were heterozygous for the identical mutation in the BVR-A gene, but had no clinical signs of liver disease and had normal levels of biliverdin. The BVR-A gene mutation was not found in 200 healthy volunteers or nine patients with end-stage liver cirrhosis. CONCLUSION: Hyperbiliverdinaemia (green jaundice) with green plasma and urine may be caused by a genetic defect in the BVR-A gene in conjunction with decompensated liver cirrhosis.

Expression of iron regulatory genes in a rat model of hepatocellular carcinoma.

BACKGROUND/AIMS: The altered iron metabolism in hepatocellular carcinomas (HCCs), characterized by the iron-deficient phenotype, is suggested to be of importance for tumour growth. However, the underlying molecular mechanisms remain poorly understood. We asked whether these iron perturbations would involve altered expression of genes controlling iron homeostasis. METHODS: HCCs were induced in rats by the Solt and Farber protocol of chemical hepatocarcinogenesis, and to evaluate the effects of iron loading, one group of animals were supplemented with dietary iron during tumour progression. Tissue iron contents were determined, labelling indices of S-phase nuclei were calculated, and mRNA levels of iron-regulatory genes were quantitated. Protein levels of ferroportin1 were determined with Western blot. RESULTS: HCCs displayed reduced amount of tissue iron and lack of histologically stainable iron. HCCs expressed significantly higher mRNA levels of genes involved in iron uptake (transferrin receptor-1, divalent metal ion transporter-1), ferroxidase activity (Ferritin-H), and iron extrusion (ferroportin1). The protein levels of ferroportin1 in iron-deficient HCCs were similar as in control livers, and did not increase in HCCs exposed to iron. Hepcidin mRNA levels were decreased in iron-deficient HCCs, rose in response to iron loading and correlated to the tissue iron content. CONCLUSIONS: Taken together, the altered expressions of iron-regulatory genes in HCCs possibly reflect an increased demand for bioavailable iron and a high iron turnover in neoplastic cells.

Regulatory effects of tumor necrosis factor-alpha and interleukin-6 on HAMP expression in iron loaded rat hepatocytes.

BACKGROUND/AIMS: To study the effect of iron and proinflammatory cytokines on the expression of HAMP and other iron regulatory genes in primary rat hepatocytes. METHODS: Primary hepatocytes from rats fed a control or iron-enriched diet were plated on extracellular matrix and incubated with inflammatory stimuli in the presence or absence of serum. Cells were also incubated with desferrioxamine or ferric ammonium citrate. mRNA levels were determined by Real-Time PCR. RESULTS: Hepatocytes from control rats increased their HAMP expression during culturing, whereas the opposite was seen in hepatocytes from carbonyl-iron loaded animals. In the presence of serum, tumor necrosis factor-alpha, lipopolysaccharide and interleukin-6 increased HAMP expression in hepatocytes from both control and iron-loaded rats. Under serum-free conditions only tumor necrosis factor-alpha increased HAMP mRNA levels. Desferrioxamine and ferric ammonium citrate decreased HAMP gene expression. Tumor necrosis factor-alpha significantly increased mRNA levels of TfR2 and decreased those of DMT1 and IREG1. CONCLUSIONS: HAMP expression differs in cultured as compared with freshly isolated hepatocytes, and decreases in iron-loaded hepatocytes in serum free-media, suggesting that additional serum factors influence HAMP expression. Tumor necrosis factor-alpha regulates the mRNA levels of HAMP, IREG1, DMT1 and TfR2 in cultured hepatocytes from both iron-loaded and control animals.

Structure and liver cell expression pattern of the HFE gene in the rat.

BACKGROUND/AIMS: Very little is known about the HFE gene in the rat. The aim of the present study was to determine: (1) the structure of the rat HFE gene; and (2) the tissue expression of the HFE mRNA in the rat, with special emphasis on the liver. METHODS: Cloning of the rat HFE gene was performed using library screening and PCR. Exon-intron borders were assigned by DNA sequencing. Parenchymal and non-parenchymal liver cells were isolated by fractionation of normal rat liver. HFE mRNA levels were determined by Northern blot (tissues) and real-time PCR (isolated liver cells). RESULTS: The rat HFE gene contained six exons and five introns. The HFE gene is expressed in multiple tissues in the rat, including bone marrow, with the highest expression in the liver. We observed HFE transcripts in several categories of isolated rat liver cells. Unexpectedly, expression also occurred in rat hepatocytes. CONCLUSIONS: The exon-intron pattern of the HFE gene is strongly conserved between rat and mouse. The pattern of tissue expression of the HFE gene is rather similar in humans and rodents. The finding of HFE gene expression in rat hepatocytes raises interesting questions regarding its role in the hepatocyte iron metabolism.