The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC 50 of ϳ0.5 M, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.Hepatitis C virus (HCV), 1 a positive strand RNA virus of the Flaviviridae family, is the major etiological agent of post-transfusion and sporadic non-A, non-B hepatitis (1). An estimated 2-3% of the world population is chronically infected with HCV, which causes significant liver disease, cirrhosis, and can eventually lead to the development of hepatocellular carcinoma. In infected cells, translation of the viral RNA yields a 3011-residue polyprotein chain (2-4), which is subsequently cleaved to generate envelope and core proteins, for assembly of new virus particles and nonstructural enzymes essential for viral replication (5-7). Studies using recombinant NS5B polymerase have provided direct evidence for RNA-dependent RNA polymerase activity (8, 9), and this catalytic activity has been confirmed to be required for infectivity in chimpanzees (10).NS5B polymerase contains a hydrophobic C-terminal domain thought to be responsible for anchoring the protein to mammalian cell membranes. Removal of the C-terminal 21 residues has been reported to facilitate protein isolation from Escherichia coli without compromising RdRp activity (11). The HCV RdRp initiates RNA synthesis preferentially from the 3Ј terminus of the template RNA (12, 13-15) but lacks specificity for HCV RNA in vitro, because it readily utilizes heterologous nonviral templates (8). Based on crystallographic studies of the enzyme containing C-terminal truncations (16, 17), the hydrophobic tail present in the full-length ...

We report 5 cases of Aggressive Natural Killer-Cell Leukemia (ANKL) diagnosed and treated in our institution within a period of 5 years. Presented are the clinical, hematological, immunophenotypic, immunogenotypic and cytogenetic data. ANKL is a very rare disorder. On review of the English-language literature only 68 published cases were identified. Analysis was performed on a total number of 73 cases (68 from the literature and the series of 5 presented hereby). Presented and discussed are the epidemiology, clinical picture, morphology, cell marker, immunogenotype, cytogenetics, and survival data of the analysis, as well as the associations with the Epstein-Barr virus (EBV). To our knowledge this is the largest series of cases of ANKL analyzed and therefore it is hoped to contribute towards a better characterization of the disorder.

The ICT is simple to perform, with higher sensitivity than HbH-i, and gives the result in a short time and at a lower cost. This can be used by clinical laboratories to replace HbH-i for α-thalassaemia detection.

The dilute tissue thromboplastin inhibition (DTTI) test (Schleider et al, 1976) is a sensitive but non-specific test for lupus anticoagulant (LA). False positive results are seen in patients with clotting factor deficiency involving the extrinsic pathway and also in some patients with specific factor inhibitors (Triplett et al, 1983; Rosove et al, 1986). Since the effect of LA is phospholipid dependent but those of factor deficiency and specific inhibitors are not, we analyse the test results by comparing the degree of inhibition using different dilutions of tissue thromboplastin and express it as the DTTI index. This is defined as the clotting time ratio with 0.2% tissue thromboplastin divided by the clotting time ratio with 2% tissue thromboplastin. We also perform a dilute tissue thromboplastin time with platelet substitution to see if this could neutralize the inhibition caused by LA. Both of these modifications can reliably distinguish LA from other conditions associated with prolonged APTT better than the original DTTI test.