GSK2878175, a pan-genotypic non-nucleoside NS5B polymerase inhibitor, in healthy and treatment-naïve chronic hepatitis C subjects.

GSK2878175 is a potent, pan-genotypic, non-nucleoside, nonstructural protein 5B palm polymerase inhibitor being developed for the treatment of chronic hepatitis C (CHC). A first-in-human, randomized, placebo-controlled, dose escalation study, evaluated the safety and pharmacokinetics of GSK2878175 administered as single and repeat oral doses (once daily for 14 days) to healthy volunteers. A separate proof-of-concept, placebo-controlled, repeat dose (once daily for 2 days) study evaluated the safety, pharmacokinetics and antiviral activity of GSK2878175 monotherapy in treatment-naïve, noncirrhotic, subjects with hepatitis C virus (HCV) genotype 1 [1a and 1b], 2, or 3. No deaths or SAEs were reported in either study, and treatment was well-tolerated. Across all the HCV genotypes, GSK2878175 monotherapy at doses of 10, 30 or 60 mg once daily for 2 days produced a statistically significant multilog reduction (P<.001) in plasma HCV RNA log10 IU/mL from Baseline to 24, 48 and 72 hours after the first dose of GSK2878175 compared to placebo. The reduction in HCV RNA was sustained for a prolonged period across all of the active treatment groups, consistent with the long apparent half-life of GSK2878175 that was observed (mean t1/2 range: 60-63 hours in the CHC subjects). In summary, GSK2878175, when administered to healthy subjects and subjects with CHC, did not reveal any safety concerns that would limit or preclude further clinical development. GSK2878175 monotherapy across a wide dose range produced substantial reduction in HCV RNA, irrespective of HCV genotype. The results from these studies support further evaluation of GSK2878175-based regimens.

Longitudinal assessment of hepatitis C fibrosis progression by collagen and smooth muscle actin morphometry in comparison to serum markers.

BACKGROUND: Assessment of fibrosis progression in chronic liver disease relies upon non-invasive tools and changes in semi-quantitative histopathology scores that may not be reliable. AIM: To assess the diagnostic performance of the FibroSURE (FS) index and collagen/alpha smooth muscle actin (α-SMA) morphometry in relation to longitudinal changes in fibrosis on paired biopsies. METHODS: The study cohort included 201 chronic hepatitis C (CHC) nonresponders enrolled in a prior phase II anti-fibrotic study. Serum FS and paired biopsies, with both collagen and α-SMA morphometry, were evaluated at baseline and week 52. RESULTS: Study patients were mostly male (67%) and Caucasian (77%), with Ishak stages 2 (n = 79), 3 (n = 88) and 4 (n = 30), excluded (n = 4 stage 1 or 5). Mean biopsy length was 22.9 mm. For baseline Ishak 2/3 vs. 4, there were no significant differences in AUROCs for collagen (0.71), SMA (0.66) or FS (0.70). At week 52, 62% of patients had no change in Ishak stage, but collagen/α-SMA increased by 34-51% (P < 0.0001), and FS decreased by 5% (P = 0.008). Among the 33% of patients with +/-1 Ishak stage change, FS changes were not significant, but α-SMA increased 29-72%, and collagen increased by 12-38% (P = 0.01 for +1 only). CONCLUSIONS: Longitudinal changes in collagen and α-SMA morphometry are apparent prior to change in histological stage or FibroSURE in CHC nonresponders with intermediate fibrosis. This likely reflects quantitative morphological differences that are not detected by routine histological staging or serum markers such as FibroSURE.

Long-term lamivudine treatment of children with chronic hepatitis B: durability of therapeutic responses and safety.

Lamivudine has been demonstrated safe and efficacious in the short term in a large cohort of children with chronic hepatitis B (CHB), but optimal duration of treatment has not been elucidated and limited data on the safety of long-term lamivudine administration have been reported. In addition, the durability of favourable therapeutic outcomes after lamivudine therapy in children has not been well characterized. The aim of this study was to examine the safety of lamivudine and the durability of clinical responses in a group of children who received up to 3 years of treatment for CHB. One hundred and fifty-one children from centres in nine countries who had previously received lamivudine in a large prospective trial were enrolled. During the first year, children had been randomized to either lamivudine or placebo treatment. Subsequently, in a separate extension study, those who remained hepatitis B e antigen (HBeAg) positive were given lamivudine for up to 2 years and those who were HBeAg negative were observed for additional 2 years. Results of these studies have been previously reported. In this study, these children were followed for 2 additional years. Data gathered from medical record review included weight, height, signs and symptoms of hepatitis, alanine aminotransferase (ALT) levels, serologic markers, hepatitis B virus (HBV) DNA levels and serious adverse events (SAEs). Other pharmacological treatments for CHB were allowed according to the practices of individual investigators and were documented. Subjects were divided into two groups for analysis, those who had achieved virological response (VR), defined as HBeAg negative and undetectable HBV DNA by the bDNA assay by the end of the extension study at 3 years, and those who had not. In those who had achieved VR by the end of the extension study, long-term durability of HBeAg seroconversion was 82% and >90% in those who had received lamivudine for 52 weeks and at least 2 years respectively. This compares to 75% for those who had achieved seroconversion after placebo. In those who had not achieved VR by the end of the extension study, an additional 11% did so by the end of the study; they had all received lamivudine in the previous trial, and none had received further treatment during the study. Eight children lost hepatitis B surface antigen during the study and all had received lamivudine at some point during the previous trials. Evaluation of safety data revealed no SAEs related to lamivudine. There was no effect of treatment on weight or height z scores. Clinically benign ALT flares (>10 times normal) were seen in 2% of children. Favourable outcomes from lamivudine treatment of CHB in children are maintained for at least several years after completion of treatment. Up to 3 years of lamivudine treatment is safe in children.

Increased leaf area expansion of hybrid poplar in elevated CO2. From controlled environments to open-top chambers and to FACE.

We examined the response of hybrid poplar to elevated CO2 in contrasting growth environments: controlled environment chamber (CE). open-top chamber (OTC) and poplar free air CO2 enrichment (POPFACE) in order to compare short versus long-term effects and to determine whether generalisations in response are possible for this fast growing tree. Leaf growth, which for poplar is an important determinant of stemwood productivity was followed in all environments, as were the determinants of leaf growth-cell expansion and cell production. Elevated CO2 (550-700 micromol mol(-1), depending on environment) resulted in an increase in final leaf size for Populus trichocarpa x Populus deltoides (Populus x interamericana) and P. deltoides x Populus nigra (Populus x euramericana), irrespective of whether plants were exposed during a short-term CE glasshouse study (90 days), a long-term OTC experiment (3 years) or during the first year of a POPFACE experiment. An exception was observed in the closed canopy POPFACE experiment, where final leaf size remained unaltered by CO2. Increased leaf extension rate was observed in elevated CO2 in all experiments, at some point during leaf development, as determined by leaf length. Again the exception were the POPFACE experiment, where effects were not statistically significant. Leaf production and specific leaf area (SLA) were increased and decreased, respectively, on five out of six occasions, although both were only statistically significant on two occasions and interestingly for SLA never in the FACE experiment. Although both cell expansion and cell production were sensitive to CO2 concentration, effects appeared highly dependent on growth environment and genotype. However, increased leaf cell expansion in elevated CO2 was often associated with changes in the biophysical properties of the cell wall, usually increased cell wall plasticity. This research has shown that enhanced leaf area development was a consistent response to elevated CO2 but that the magnitude of this response is likely to decline, in long-term exposure to elevated CO2. Effects on SLA and leaf production suggest that CE and OTC experiments may not always provide good predictors of the 'qualitative' effects of elevated CO2 in long-term ecosystem experiments.

Purification and characterization of sn-1-stearoyl-2-arachidonoylglycerol kinase from pig testes.

1-Stearoyl-2-arachidonoylglycerol (SAG) kinase was identified in the particulate fraction of pig testes. This activity was enriched by hydroxyapatite and blue dye chromatography. The enzyme was selective for polyunsaturated diradylglycerol species and activity was not modulated by other diradylglycerol species or sphingomyelin metabolites. Further purification resulted in the isolation of 55 and 50 kDa proteins that corresponded with SAG kinase activity. These results support the view that the phosphorylation of polyunsaturated diradylglycerol is regulated by structural determinants in the molecule.

The antigen receptors on mature and immature T lymphocytes are coupled to phosphatidylcholine-specific phospholipase D activation.

Phosphatidylcholine-phospholipase D has been proposed to play a key role in the transduction of the proliferative responses of a wide range of mitogens and growth factors. We now report that the antigen receptors on T lymphocytes derived from human tonsillar or murine splenic preparations are coupled to phosphatidylcholine (PtdCho)-phospholipase D (PLD) activation following stimulation of these T cells with anti-CD3 antibodies. However, since we also demonstrate that the antigen receptors on murine thymocytes are coupled to PtdCho-PLD activation, we propose that it is unlikely that this PLD pathway plays a central role in the transduction of T-cell proliferative responses, but rather, may be involved in either driving cells into cycle or maintaining cell cycle progression, processes required both for proliferation and activation-induced cell death. Whilst the molecular mechanisms underlying T-cell receptor (TCR)-coupling to PtdCho-PLD activation in these cells have not been fully defined, kinetics studies and experiments using pharmacological inhibitors of protein tyrosine phosphatases (PTPases) and reconstituting CD3-coupled PtdCho-PLD activity in streptolysin-O permeabilized cells, suggest that the TCR/CD3 complex, under optimal conditions of activation, may be predominantly coupled to PtdCho-PLD activation downstream of tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma), phosphatidylinositol (PtdIns)P2 hydrolysis, calcium mobilization and protein kinase C (PKC) activation.

The JC virus antibody response in serum and cerebrospinal fluid in progressive multifocal leucoencephalopathy.

BACKGROUND: A clinical diagnosis of progressive multifocal leucoencephalopathy (PML) can be confirmed by histological or virological examination of brain material. Whilst a less invasive method is provided by the detection of JC DNA in cerebrospinal fluid (CSF), very few studies have been done to assess the value of JC virus (JCV) serology in PML diagnosis. OBJECTIVES: To study the JCV antibody response in the serum and CSF of PML patients. STUDY DESIGN: A retrospective study was done using haemagglutination inhibition (HI), M-antibody capture radioimmunoassay (MACRIA) and JC-specific oligoclonal IgG banding on one or more sera and/or CSFs from 28 confirmed PML patients. Seventy-one serum and CSF samples were tested from patients with memory loss or dementia as a control group. RESULTS: Twenty-seven PML patients (96%) had detectable JCV HI antibody in the serum, with titres ranging from 1 : 10 to > 1 : 20480, compared to 48 (68%) of the controls (P = <0.005). JCV IgM antibody was detected in the serum of 12/22 (55%) PML patients. JCV HI antibody was detected in the CSF in 12 of 18 (67%) PML patients, antibody index measurements being used to control for a possible breakdown of the blood-brain barrier. Intrathecal JCV antibody was not found in any control patient. Locally produced JCV-specific IgG bands were detected in the CSF of 7 PML patients tested, confirming the intrathecal origin and specificity of the HI antibody. CONCLUSIONS: The presence of intrathecal JCV antibody indicates active central nervous system infection with JC virus, and provides a useful diagnostic test for PML, with a sensitivity of 67% and a specificity of 100%. The absence of serum JCV antibody nearly always excludes a diagnosis of PML, but the titre of antibody, IgG or IgM, correlates with the underlying condition rather than the development of neurological symptoms.

Preparation of monoclonal antibodies to JC virus and their use in the diagnosis of progressive multifocal leucoencephalopathy.

Seven monoclonal antibodies (MAbs) to polyomavirus JC were produced, and one was selected for use in immunofluorescence (IF) tests on brain material from patients with suspected progressive multifocal leucoencephalopathy (PML). The selected MAb (5.12.2) reacted by IF with JC-infected primary human foetal glial (PHFG) cell cultures (titre 1/200,000) but not with BK-infected human embryo lung (HEL) fibroblasts (titre less than 1/20). Its haemagglutination-inhibition (HI) titre was high (greater than 1/5 x 10(6)) against JC virus but low (less than 1/5) against BK virus. A diagnosis of PML was confirmed on brain biopsy or at postmortem in four patients, two of whom were also infected with human immunodeficiency virus (HIV). In one of the patients, specific detection of JC virus antigen had not been possible using our routine high titred JC and BK human sera due to interference by JC antibody present in the cerebrospinal fluid (CSF). Viral antigen was, however, detected with the MAb 5.12.2.

Regulation of bombesin-stimulated inositol 1,4,5-trisphosphate generation in Swiss 3T3 fibroblasts by a guanine-nucleotide-binding protein.

The stimulation of inositol phosphate generation by bombesin and GTP analogues was studied in Swiss 3T3 cells permeabilized by electroporation. Bombesin-stimulated inositol phosphate generation is potentiated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and inhibited by guanosine 5'-[beta-thio]diphosphate at all peptide concentrations tested, with no change in the EC50 value (concn. giving half-maximal response) for the agonist. Kinetic analysis showed that, although bombesin-stimulated [3H]InsP3 generation in [3H]inositol-labelled cells was rapid (maximal by 5-10 s), the response to GTP[S] alone displayed a distinct lag time of 20-30 s. This lag time was significantly decreased by the addition of bombesin, suggesting that in this system agonist-stimulated GTP/GDP exchange occurs. In addition, bombesin-stimulated generation of Ins(1,4,5)P3 mass at 10 s was enhanced by GTP[S] in the absence of a nucleotide response alone, a result consistent with this proposal. Pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent inhibition of bombesin-, but not GTP[S]-, stimulated inositol phosphate generation. Furthermore, although PMA pretreatment did not affect the lag time for InsP3 formation in response to GTP[S] alone, the degree of synergy between bombesin and the nucleotide was severely decreased at early time points. The results therefore demonstrate that the high-affinity bombesin receptor is coupled via a G-protein to phospholipase C in a manner consistent with a general model for receptor-G-protein interactions and that this coupling is sensitive to phosphorylation by protein kinase C.

Human papillomavirus types in anogenital warts of children.

Tissue from anogenital warts of 25 children, 10 of whom were suspected of being victims of sexual abuse, was investigated by dot blot and Southern blot techniques for human papillomavirus (HPV) types. HPV DNA was detected in 22 children, two of whom had double infections. The genital HPV types 6 and/or 11 were detected in 20 children, and in three children other HPV types were found. One had HPV 18 (as well as 11); in a second child a possible skin type, HPV 2, was detected; and the third child was infected with an unidentified type. In three cases genital wart material was available from one of the parents, and in all three the HPV type was the same as that of the child. For nine other children one or both parents were reported to have genital warts. The source of infection appeared to be the adult genital tract, but sexual contact might not be the only means of transmission.

Progressive multifocal leukoencephalopathy-remission with cytarabine.

A 51-year-old woman who was in complete remission from non-Hodgkin's lymphoma, developed a rapidly progressive dementia. Progressive multifocal leukoencephalopathy (PML) was diagnosed on the basis of a rising antibody titre to JC polyomavirus in cerebro-spinal fluid and serum and the presence of diffuse white matter changes on magnetic resonance imaging. She was treated initially with intravenous cytarabine and showed minimal improvement. Rapid improvement occurred when intrathecal cytarabine was added and the patient is in complete remission from both lymphoma and PML 20 months later.

An indirect immunofluorescence method for detection of infectious BK virus in urine.

An indirect immunofluorescence (IF) method is described for the detection of infectious BK virus in urine within seven days in contrast to up to three months or longer using routine tissue culture. Virus is pelleted from the urine, inoculated onto pre-formed monolayers of human embryo lung (HEL) fibroblasts, and infected cells are detected in an indirect fluorescent antibody test using a human serum. The sensitivity of the IF method is 91%. Positive isolates may be confirmed as BK virus using specific rabbit antisera on the original inoculated cultures. Furthermore, a virus stock may be grown up by passage from the original culture fluids for further studies such as DNA analysis. As a broadly-reactive human serum is used for screening the cultures, other viruses which grow in HEL cells, such as CMV, may also be detected.

Serological typing scheme for BK-like isolates of human polyomavirus.

Human polyomavirus BK-like isolates were subjected to restriction endonuclease analysis with the enzymes EcoRI and Hind III. End-point dilution was used to obtain homogeneous virus pools for DNA analysis and to remove JC virus from a mixed stock. The results of Hind III digestion suggested that two subgroups could be distinguished. Several BK-like isolates were purified and rabbit antisera raised. The isolates were compared with each other and with BK and JC viruses by haemagglutination-inhibition (HI) and by neutralisation. JC virus was serologically distinct, but all the other isolates showed some cross-reactivity. Two subgroups were again evident: GS and PG were with prototype BK in subgroup 1, and MG and IV were in subgroup 2. Two isolates, AS and SB, reacted with isolates of both subgroups 1 and 2 but were distinct from one another: their genome was similar to subgroup 1 isolates. Typing by HI or by neutralisation may form a basis for grouping BK-like polyomavirus isolates.

The effect of cholera toxin on the inhibition of vasopressin-stimulated inositol phospholipid hydrolysis is a cyclic AMP-mediated event at the level of receptor binding.

Incubation of L6 skeletal myoblasts for 16 h with cholera toxin but not with pertussis toxin, led to the inhibition of inositol phosphate generation induced by subsequent exposure to vasopressin. The effects of the toxin on inositol lipid metabolism were accompanied by the total ADP-ribosylation of the available cholera-toxin substrates within the cells. Immunological analysis demonstrated that the two polypeptides modified in vivo by cholera toxin were different forms of Gs alpha (alpha subunit of Gs). No novel cholera-toxin substrate(s) were detected. The cholera-toxin-mediated inhibition of vasopressin-stimulated inositol phosphate generation could be mimicked by both forskolin and dibutyryl cyclic AMP, but not by the separated subunits of the toxin. Receptor-binding studies demonstrated that the inhibition of agonist-stimulated inositol phosphate generation was accompanied by a decrease in cell-surface vasopressin-binding sites, with no effect on the affinity of these for the hormone. We suggest that the effect of cholera toxin and agents which increase intracellular cyclic AMP on vasopressin-stimulated inositol lipid hydrolysis is an effect on receptor number, and that there is no requirement to postulate a role for a novel G-protein, which is a substrate for cholera toxin, in the regulation of inositol phospholipid metabolism.

Nucleotide sequence of the human polyomavirus AS virus, an antigenic variant of BK virus.

The complete DNA sequence of the human polyomavirus AS virus (ASV) is presented. Although ASV can be differentiated antigenically from the other human polyomaviruses (BK and JC viruses), it shares 94.9% homology at the nucleotide level with the Dunlop strain of BK virus. Differences found in ASV relative to BK virus include the absence of tandem repeats in its regulatory region, the deletion of 32 nucleotides in the late mRNA leader region (altering the initiation codon for the agnoprotein), the presence of a cluster of base pair substitutions within the coding region of the major capsid protein, VP1, and the absence of 4 amino acids in the carboxy-terminal region of the early protein, T antigen. The 43 nucleotides deleted in the Dunlop strain of BK virus relative to the Gardner prototype strain of BK virus are present in ASV. Possible reasons for the distinct antigenicity of the ASV capsid, given the high degree of nucleotide homology with BK virus, are discussed. To reflect the high degree of sequence homology between ASV and BK virus, we suggest ASV be renamed BKV(AS).

Characterization of a new polyomavirus (Polyomavirus papionis-2) isolated from baboon kidney cell cultures.

Viruses with papovavirus morphology were seen in fluids from baboon kidney cell cultures on three separate occasions (isolates A, B, and C). The size of the virions, 47.9 nm, placed the virus in the polyomavirus genus. It grew well in baboon kidney and Vero cells and less well in human embryo lung (HEL) fibroblasts. The virus could not be identified as the previously described baboon polyomavirus, SA 12, or as any of the other known primate polyomaviruses BK, JC or SV 40, the non-primate viruses mouse polyoma, K, rabbit kidney vacuolating virus (RKV) or bovine polyomavirus (FRKV) by immunofluorescence, immune electron microscopy or hemagglutination inhibition (HI) tests. A rabbit antiserum to the new virus (isolate A) reacted only with the three isolates and not with the other primate polyomaviruses studied. Thirteen percent of 118 wild-caught baboons (Papio anubis) had HI antibody to the new polyomavirus and 21 percent were seropositive for SA 12; only two baboons had antibody to both viruses. These results suggest that in baboons there are two antigenically distinct polyomaviruses which circulate independently. The two viruses may also be distinguished by their hemagglutinating properties: SA 12 agglutinated erythrocytes from a wider range of species but only the newly recognized polyomavirus agglutinated baboon erythrocytes. We propose that the two baboon viruses, SA 12 and the new virus, should be named Polyomavirus papionis-1 and Polyomavirus papionis-2 respectively.