Emergence of resistant variants detected by ultra-deep sequencing after asunaprevir and daclatasvir combination therapy in patients infected with hepatitis C virus genotype 1.

Daclatasvir (DCV) and asunaprevir (ASV) are NS5A and NS3 protease-targeted antivirals respectively, currently under development for the treatment of chronic hepatitis C virus (HCV) infection. We analysed the relationship between pre-existing drug-resistant variants and clinical outcome of the combination treatment with DCV and ASV. Ten patients with HCV genotype 1b were orally treated with a combination of ASV and DCV for 24 weeks. The frequencies of amino acid (aa) variants at NS3 aa positions 155, 156 and 168 and at NS5A aa31 and 93 before and after treatment were analysed by ultra-deep sequencing. We established a minimum variant frequency threshold of 0.3% based on plasmid sequencing. Sustained virological response (SVR) was achieved in 8 out of 10 patients (80%), and relapse of HCV RNA after cessation of the treatment and viral breakthrough occurred in the other two patients. Pre-existing DCV-resistant variants (L31V/M and/or Y93H; 0.9-99.4%) were detected in three out of eight patients who achieved SVR. Pre-existing DCV-resistant variants were detected in a relapsed patient (L31M, Y93H) and in a patient with viral breakthrough (Y93H); however, no ASV-resistant variants were detected. In these patients, HCV RNA rebounded with ASV- and DCV- double resistant variants (NS3 D168A/V plus NS5A L31M and Y93H). While pre-existing DCV-resistant variants might contribute to viral breakthrough in DCV and ASV combination therapy, the effectiveness of prediction of the outcome of therapy based on ultra-deep sequence analysis of pre-existing resistant variants appears limited.

Ribavirin dose reduction during telaprevir/ribavirin/peg-interferon therapy overcomes the effect of the ITPA gene polymorphism.

Treatment success of chronic hepatitis C virus genotype 1 infection has improved with the advent of telaprevir plus peg-interferon/ribavirin triple combination therapy. However, the effect of inosine triphosphatase (ITPA) polymorphism on dose reduction during triple therapy, especially during the postmarketing phase, has not been sufficiently evaluated. We analysed 273 patients with genotype 1 infection who were treated with triple therapy and assessed the effect of the ITPA polymorphism on dose reduction. ITPA and IFNL4 SNP genotypes were determined by the Invader assay. A stepwise multivariate regression analysis was performed to identify factors associated with outcome of the therapy. The overall sustained viral response (SVR) rate 12 weeks after the end of therapy was 80.2% (219/273). Decline of haemoglobin was significantly faster, and ribavirin was more extensively reduced in patients with ITPA SNP rs1127354 genotype CC than CA/AA. Extensive reduction of ribavirin resulted in mild reduction of telaprevir and peg-interferon, but no significant increase in viral breakthrough. Although the amount of telaprevir given was slightly higher in CA/AA patients, the total dose of peg-interferon and the SVR rate did not differ between the two groups. Multivariate analysis showed that IFNL4 but not ITPA SNP genotype, platelet count and peg-interferon adherence were significantly associated with outcome of therapy. Postmarketing-phase triple therapy resulted in a high SVR rate in spite of extensive ribavirin dose reduction in a diverse patient population, indicating the importance of treatment continuation and appropriate management of adverse events.

Human microRNA hsa-miR-1231 suppresses hepatitis B virus replication by targeting core mRNA.

Pathogen-specific miRNA profiles might reveal potential new avenues for therapy. To identify miRNAs directly associated with hepatitis B virus (HBV) in hepatocytes, we performed a miRNA array analysis using urokinase-type plasminogen activator (uPA)-severe combined immunodeficiency (SCID) mice where the livers were highly repopulated with human hepatocytes and human immune cells are absent. Mice were inoculated with HBV-infected patient serum samples. Eight weeks after HBV infection, human hepatocytes were collected from liver tissues, and miRNAs were analysed using the Toray 3D array system. The effect of miRNAs on HBV replication was analysed using HBV-transfected HepG2 cells. Four miRNAs, hsa-miR-486-3p, hsa-miR-1908, hsa-miR-675 and hsa-miR-1231 were upregulated in mouse and human livers with HBV infection. These miRNAs were associated with immune response pathways such as inflammation mediated by chemokine and cytokine signalling. Of these miRNAs, hsa-miR-1231, which showed high homology with HBV core and HBx sequences, was most highly upregulated. In HBV-transfected HepG2 cells, overexpression of hsa-miR-1231 resulted in suppression of HBV replication with HBV core reduction. In conclusion, a novel interaction between hsa-miR-1231 and HBV replication was identified. This interaction might be useful in developing new therapeutic strategies against HBV.

Serum PAI-1 is a novel predictor for response to pegylated interferon-α-2b plus ribavirin therapy in chronic hepatitis C virus infection.

Obesity and insulin resistance have been reported as negative predictors for sustained virological response (SVR) in hepatitis C virus (HCV) genotype 1 infected patients treated with pegylated interferon-α plus ribavirin. They are also known to affect serum levels of several cytokines including adipocytokines. But the association between these cytokines and treatment outcome has not been fully elucidated. We examined pretreatment serum levels of 14 cytokines among 190 patients who were treated with pegylated interferon-α-2b plus ribavirin for chronic HCV-1b infection with high viral load (≥ 5 log IU/mL) and analyzed their contribution to treatment response. Plasminogen activator inhibitor-1 (PAI-1), vascular endothelial growth factor, and 11 clinical factors showed significant association with SVR in univariate logistic regression analysis. Four significant factors in multivariate analysis; serum PAI-1 (odds ratio [OR] = 15.42), body mass index (OR = 4.56), rs8099917 (OR = 4.95) and fibrosis stage (OR = 5.18) were identified as independent predictors. We constructed a simple and minimally invasive prediction score for SVR based on the presence of these factors except for fibrosis stage. The accuracy of this score was 73%, and was confirmed using an independent validation cohort consisting of 31 patients (68%). The strongest correlation was between PAI-1 level and platelet count (r = 0.38, P = 1.8 × 10(-7)), and PAI-1 level was inversely correlated with fibrosis stage. Serum PAI-1 is a novel predictor for the response to combination therapy against chronic HCV-1b infection and may be associated with liver fibrosis.

IL28B polymorphism may guide pegylated interferon plus ribavirin therapy even after curative treatment for hepatitis C virus-related hepatocellular carcinoma.

The present study was designed to determine the predictive factors for the viral response to pegylated interferon-alpha plus ribavirin combination therapy (PEGIFN/RBV) administered after curative treatment for hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). The study group was 78 patients treated between January 2005 and January 2009. The sustained viral response (SVR) rate was 25.8% (15/58) in patients infected with HCV-genotype 1 and 55.0% (11/20) in those with genotype 2. Among the 78 patients, 32 (41.0%) could not complete the treatment protocol, and this was because of HCC recurrence in 17 (53%) of them. Multivariate analysis identified partial early viral response (pEVR) as the only independent determinant of SVR [odds ratio (OR) 14.73, P = 0.013] for patients with genotype 1. Multivariate analysis identified male gender (OR 8.72, P = 0.001) and interleukin-28B (IL-28B) genotype (rs8099917) TT (OR 7.93, P = 0.007) as independent predictors of pEVR. Multivariate analysis also identified IL-28B genotype GG+TG (OR 14.1, P = 0.021) and α-fetoprotein >30 (OR 5.4, P = 0.031) as independent predictors of null response. Patients with SVR showed a better survival rate than those without SVR (P = 0.034). The second HCC recurrence rate tended to be lower in patients with SVR than in those without SVR (P = 0.054). With regard to the prognosis of patients with SVR, it is desirable to achieve SVR with interferon therapy even when administered after HCC treatment. IL-28B genotype is a potentially useful marker for the response to PEGIFN/RBV therapy administered after curative treatment of HCV-related HCC.

Perceptions of positive relationship traits in gay and lesbian couples.

The current research examined perceptions of positive traits in homosexual relationships. Students (n = 216) and professional counselors (n = 96) read one of three variations of a transcript of a couple's counseling session that were identical in all aspects except for the names of the couple members, which implied sexual orientation (either John and Amy, Amy and Jennifer, or John and David). Participants then rated the couple's level of commitment, satisfaction, investment, and closeness. Surprisingly, the student group perceived no differences between the couples, but the counselor group perceived the gay and lesbian couples as having higher levels of the positive relationship traits. Implications regarding counselor bias are discussed.

Comparison of scleral buckling and vitrectomy for retinal detachment resulting from flap tears in superior quadrants.

PURPOSE: To compare the surgical results of vitrectomy and scleral buckling for uncomplicated superior retinal detachment caused by flap tears. METHODS: Included in the study were 225 patients (225 phakic eyes) undergoing primary surgery by three surgeons between January 1990 and December 1996 for superior retinal detachment caused by flap tears (138 eyes by scleral buckling, 87 eyes by vitrectomy); all patients had been followed up for longer than 6 months after surgery. The choice of one of the two procedures was based on each surgeon's preference. The surgical outcome and the rate of complications were retrospectively compared between the two groups of eyes. RESULTS: Initial and final anatomical success rate were 92% and 100% after each procedure. Retinal redetachment after the initial procedure was due to new retinal breaks in 5 eyes, reopening of original breaks in 2 eyes of vitrectomy cases, and due to malpositioned buckle in 11 eyes of scleral buckling cases. Proliferative vitreoretinopathy occurred in 3 eyes of vitrectomy cases. CONCLUSION: Primary vitrectomy was as successful as scleral buckling for treating superior rhegmatogenous retinal detachment. Even though the high incidence of postoperative cataract formation was the major drawback, vitrectomy had some advantages over scleral buckling.

Photocrosslinkable polysaccharides for in situ hydrogel formation.

In situ photopolymerization is an exciting new technique for tissue engineering. Two photocrosslinkable polysaccharides composed of alginate and hyaluronan are described that upon photolysis form soft, flexible, and viscoelastic hydrogels. The degree of methacrylate modification and thus covalent affects mechanical properties such as swelling, compression, and creep compliance. Significant swelling is observed in aqueous solution; these hydrogels can swell up to 14 times their dry weight. Both hydrogels exhibit low phase angles and (G*) values indicative of viscoelastic materials. The hyaluronan based hydrogel is stronger and more resilient than the corresponding alginate gel. SEM and AFM studies on both hydrogels show smooth and uniform surfaces at the macroscopic level with salient features observed only on the nanometer scale. Rapid polymerization by an optical trigger allows for controlled in situ photopolymerization in a minimally invasive manner, indicating that these hydrogels are relevant for biomedical applications such as sealing wounds and reconstructing soft tissues.

[Comparison of scleral buckling and vitrectomy for superior retinal detachment caused by flap tears].

PURPOSE: We compared the surgical results of vitrectomy and scleral buckling for uncomplicated superior retinal detachment caused by flap tears. SUBJECTS AND METHODS: Included in the study were 225 phakic eyes of 225 patients undergoing primary surgery from January 1990 to December of 1996 for superior retinal detachment caused by flap tears; all eyes had been followed for longer than six months after surgery. The choice of procedures was based on each surgeon's preference. The cases were evaluated retrospectively and the surgical outcome and the rate of complications compared between the two groups of eyes. RESULTS: Initial and final anatomical success rate were 92% and 100% after each procedure. Redetachment after the first procedure was due to new retinal breaks in 5 eyes, reopening of original breaks in 2 eyes of vitrectomy cases, and malpositioned buckle in 11 eyes of scleral buckling cases. Proliferative vitreoretinopathy occurred in 3 eyes of vitrectomy cases. CONCLUSION: Primary vitrectomy was as successful as scleral buckling for superior rhegmatogenous retinal detachment. Although a high incidence of postoperative cataract formation was a major drawback, vitrectomy had some advantages over to scleral buckling.

[Characteristics of retinal detachment in eyes with choroidal colobomas].

We reviewed 6 eyes of 6 patients (1 male and 5 females) with retinal detachment associated with choroidal coloboma. Three of these 6 eyes had visible retinal breaks within or at the margin of the coloboma. In one eye, no retinal breaks were noted before or during vitreous surgery, and shallow retinal detachment was localized in the posterior fundus. One eye with bullous retinal detachment with retinal breaks within the coloboma had been having serous retinal detachment localized in the nasal area close to the optic nerve head. In all of these five eyes; vitrectomy, relaxing retinotomy inside the coloboma close to its edges, and cyanoacrylate retinopexy resulted in successful reattachment of the retina. Schisis-like separation similar to that in pit-macular syndrome was noted in the posterior retinal in one eye which had no visible retinal break. Non-rhegmatogenous retinal detachment may exist in association with choroidal colobomas.

[Posterior segment complications after macular hole surgery].

Fifty-three patients with stage III or stage IV idiopathic macular holes who underwent vitrectomy were reviewed. Posterior segment complications were noted in 20 (38%) of patients. These included peripheral retinal breaks (21%), peripheral visual field loss of unknown etiology (8%), rhegmatogenous retinal detachment caused by peripheral retinal break (6%), late reopening of the hole (4%), and retinal pigment epithelium loss under the hole (4%). The final visual acuity was two lines or more worse than preoperative visual acuity in 10% of these complicated eyes and in 9% of eyes without posterior segment complications. Patients with peripheral retinal breaks or retinal detachment had significantly shorter duration of macular hole symptoms (p < 0.01) than those without these complications. Posterior segment complications after vitrectomy for macular hole appear to be more common than expected, but the effect of such complications on final visual outcome was not significant.

[Proliferative vitreoretinopathy treated with daunorubicin].

Nine eyes with severe proliferative vitreoretinopathy underwent vitrectomy and infusion of Daunorubicin to prevent reproliferation. In 5 eyes (56%) the retina was reattached. In 4 eyes redetachment occurred because of reproliferation or incomplete sealing of the break. After a second vitrectomy, complete reattachment of the retina was obtained eventually in all eyes including one eye with silicone oil tamponade. As postoperative complications, conjunctival dehiscience occurred in 3 eyes. In two of the 3 eyes scleral buckling occurred, and orbital cellulitis occurred in one eye. Daunorubicin seemed to be effective to suppress reproliferation, but care should be taken to avoid postoperative complications.

Cloning, sequencing and sites of expression of genes for the hydroxyarginine-containing adhesive-plaque protein of the mussel Mytilus galloprovincialis.

A segment of Mytilus galloprovincialis foot protein 3 (Mgfp-3) cDNA was amplified by means of reverse-transcription (RT)/PCR with degenerate primers. The 5' and 3' regions of the cloned segment were amplified by means of rapid amplification of cDNA ends. The 5'-region clones had almost identical nucleotide sequences, but two sequences were found among 3'-region clones. The Mgfp-3 coding region was amplified between a 5' untranslated sequence and one of two 3' untranslated sequences. Two cDNA clones which encoded variants Mgfp-3A and Mgfp-3B, were isolated. These two clones encoded proteins with 70 and 77 amino acid residues, of which the first 24 residues are predicted to be signal peptides. The existence of additional variants was suggested by the sequences of other clones. Thus, it was suggested that Mgfp-3 genes constitute a gene family. RT/PCR of RNA from developing larvae indicates that Mgfp-3 genes are transcribed after settlement. RT/PCR of RNA from major organs and in situ hybridization of the foot indicate that Mgfp-3 genes are transcribed in a limited part of the foot, i.e., the phenol gland and a distal part of the accessory gland.

[Vitrectomy in Coats' disease].

Four eyes of four patients with Coats' disease underwent vitrectomy because of exudative or tractional detachments involving the macula or premacular fibrosis. All cases had gelatinous vitreous and had no complete posterior vitreous detachment. The exudates decreased and the retina reattached after removing vitreous traction and coagulating abnormal vessels with endodiathermy and not removing subretinal fluid in 3 eyes with retinal detachment. In one eye with tractional detachment, retinal breaks were found beneath the proliferative membrane during the initial vitrectomy procedure. This eye needed multiple operations because of recurrent traction by the remaining peripheral vitreous. Exudation into the vitreous and vitreous traction may cause mutual progression in these eyes, and vitrectomy is an effective treatment, although there are difficulties in removing vitreous traction completely.

Expression sites of two byssal protein genes of Mytilus galloprovincialis.

Mussels form byssal threads that can attach tenaciously to wet and irregular surfaces. The byssus consists of a fibrous collagenous core, and at least two types of polyphenolic proteins surround it. One of these proteins, designated Mgfp-1, coats the collagenous core; the other, designated Mgfp-2, is the major component of the terminal adhesive plaque of byssal threads. Both proteins contain 3,4-dihydroxyphenylalanine (DOPA) in their primary sequences. In this study, the sites of expression of the genes encoding the polyphenolic proteins were investigated in Mytilus galloprovincialis. By northern blot analysis, we found that the expression of both genes is foot-specific. Northern blot analysis of RNA isolated from the distal end and the remaining proximal portion of the foot indicated that the Mgfp-2 gene is expressed primarily in the distal part, whereas Mgfp-1 expression occurs in both parts. In situ hybridization indicated that the Mgfp-1 gene transcript is localized in the accessory gland along the ventral groove of the foot, and the Mgfp-2 gene transcript is localized in the phenol gland near the foot apex. Thus, it was shown that tissues expressing Mgfp-1 and Mgfp-2 are located around the ventral groove in an arrangement appropriate for byssus formation.

Mussel adhesive plaque protein gene is a novel member of epidermal growth factor-like gene family.

A mussel (Mytilus galloprovincialis) cDNA encoding Mgfp2, a major component of the adhesive plaque that anchors mussels tightly to underwater surfaces was isolated. It encoded a protein mainly consisted of epidermal growth factor-like repeats, containing tyrosine residues that will be converted to 3,4-dihydroxyphenylalanine near C and N termini. Amino acid residues important for cell-cell interaction in other epidermal growth factor-like proteins were, however, not conserved in the structure of Mgfp2. RNA blot analysis on adult tissues showed foot-specific expression of this gene, while the analysis on developing larvae showed that the expression starts with formation of the foot. These results suggest that the function of Mgfp2 has been specialized to form the adhesive plaque.

Preparation of monoclonal antibodies against the IL-6 signal transducer, gp130, that can inhibit IL-6-mediated functions.

mAbs specific to human gp130, a signal transducing component of the IL-6 receptor complex, were prepared by immunizing mice with a previously described recombinant human soluble gp130. Some of the mAbs inhibited the IL-6-induced association of soluble gp130 and soluble IL-6 receptor. Three mAbs (GPX7, GPX22 and GPZ35) were shown to inhibit IL-6-mediated biological responses such as Ig production in a human B cell line and proliferative responses of a human Lennert's lymphoma-derived T cell line, a human myeloma cell line, and a mouse pro-B cell line-derived transfectant expressing human gp130.

Functional inhibition of hematopoietic and neurotrophic cytokines by blocking the interleukin 6 signal transducer gp130.

Functional pleiotropy and redundancy are characteristic features of cytokines. To understand the signaling mechanisms of such cytokines, we have proposed a two-chain interleukin (IL) 6 receptor model: IL-6 triggers the association of a ligand-binding chain (IL-6 receptor) and a nonbinding signal transducer (gp130) to form a high-affinity receptor complex, resulting in transmission of the signal by the cytoplasmic portion of gp130. This model would explain the functional redundancy of cytokines if we were to assume that gp130 interacts with several different receptor chains. Here we present data indicating that gp130 functions as a common signal transducer for IL-6, oncostatin M, leukemia inhibitory factor, and ciliary neurotrophic factor. We show that anti-gp130 monoclonal antibodies completely block the biological responses induced by all of these factors. Since leukemia inhibitory factor functions as a cholinergic differentiation factor in nerve cells, as does ciliary neurotrophic factor, these results suggest that gp130 may also play a role in the nervous system.