Two patients treated with simeprevir plus pegylated-interferon and ribavirin triple therapy for recurrent hepatitis C after living donor liver transplantation: case report.

We previously reported our data on telaprevir (TVR) used in combination with pegylated-interferon and ribavirin (PEG-IFN/RBV) for the treatment of recurrent hepatitis C virus (HCV) genotype 1 infection after liver transplantation (LT). TVR substantially increases the blood levels of immunosuppressive agents such as cyclosporine and tacrolimus for drug-drug interactions. On the other hand, the effect of simeprevir (SMV) on the blood levels of these immunosuppressive agents is unclear. We report 2 patients who achieved viral responses with little effect on the blood levels of cyclosporine and tacrolimus using SMV plus PEG-IFN/RBV treatment. The first was a 71-year-old woman with HCV-related liver cirrhosis and hepatocellular carcinoma who failed to respond to PEG-IFN/RBV after living donor LT. She was treated with 40 mg/d of cyclosporine, and received SMV plus PEG-IFN/RBV treatment. The second was a 65-year-old man with HCV-related liver cirrhosis who failed to respond to PEG-IFN/RBV after living donor LT. He was treated with 3 mg/d of tacrolimus, and received SMV plus PEG-IFN/RBV treatment. Serum HCV RNA became undetectable using TaqMan polymerase chain reaction (PCR) test after 4 weeks of treatment in both patients, and no remarkable fluctuation in blood concentration was observed either in cyclosporine or tacrolimus during the 12 weeks of SMV treatment. Completion of 12-week SMV triple therapy was followed by PEG-IFNα2b plus RBV, and both patients achieved sustained virological response 12 weeks after the end of treatment. SMV plus PEG-IFNRBV treatment showed a remarkable viral response with little effect on blood levels of immunosuppressive agents for recurrent HCV genotype 1 infection after LT.

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.

The effect of ascorbate on minor recurrent aphthous stomatitis.

AIM: Minor recurrent aphthous stomatitis (MRAS) is a common, painful and inflammatory ailment of the oral cavity with juvenile onset and unknown aetiology. The purpose of this study was to evaluate the potential of ascorbate (vitamin C) to reduce the frequency of MRAS and severity of pain. PATIENTS AND METHODS: Sixteen MRAS patients (9 boys and 7 girls: mean age, 12.0 +/- 2.4 years old) were assigned to take an oral dosage of 2000 mg/m(2)/day ascorbate. SUBJECTS: Their baseline frequency of outbreaks and the level of pains were compared during the treatment; in addition, a crossover clinical trial was performed. Polymorphonuclear leucocytes play a role in the pathogenesis, and then superoxide anion production was evaluated in prior to ascorbate treatment. RESULTS: The data indicated a statistically significant 50% reduction in oral ulcer outbreaks and a decline of pain level. Neutrophils were primed for superoxide anion production in the patients with MRAS. CONCLUSION: Ascorbate may modulate the generation of reactive oxygen species and augment neutrophil apoptosis, which could prevent neutrophil-mediated inflammation. Ascorbate seems to be effective, but the findings of our study were preliminary and it should be re-evaluated with a larger randomized controlled clinical trials.

Thalidomide prevents formation of multinucleated giant cells (Langhans-type cells) from cultured monocytes: possible pharmaceutical applications for granulomatous disorders.

Thalidomide is an effective drug for chronic inflammatory diseases, but the mechanism underlying its immunomodulatory action remains uncertain. Thalidomide has been reported to clinically improve chronic inflammatory granulomatous disorders. In such disorders, the granulomas consist of epithelioid cells, scattered lymphocytes and multinucleated giant cells (MNGC; Langhans-type cells). The present experimental approach permitted the reproduction of MNGC formation from peripheral blood monocytes and examination of thalidomides effect on it. MNGC can be effectively generated from monocytes cultured in the presence of interleukin-4 (IL-4) and macrophage colony-stimulating factor(M-CSF) for 14 days. Thalidomide can inhibit the formation of MNGC in a dose-dependent manner. MNGC formation was partly inhibited by the presence of neutralizing TNF-alpha antibody in the responses induced by IL-4 and M-CSF. Autocrinal TNF-alpha production and modulation of cadhelin expression to regulate cell adhesion might be involved in this inhibitory action of thalidomide. Our results support thalidomides clinical efficacy in the treatment of chronic granulomatous disorders (granulomatosis).

Multi-nucleated giant cell formation from human cord blood monocytes in vitro, in comparison with adult peripheral blood monocytes.

Multi-nucleated giant cells (MGCs; Langhans-type cell), formed from macrophage fusion, are recognized as a hallmark histological feature in chronic inflammation. However, their precise pathological role is still poorly understood, especially for microorganism pathogens in the neonatal immune system, which are capable of surviving intracellularly in phagocytes. To conduct a partial evaluation of the monocyte function of neonates, we investigated the ability of human cord blood monocytes to form MGCs in vitro by stimulating various cytokines and comparing them with adult peripheral blood monocytes. Monocytes from cord blood and adult peripheral blood were isolated and cultured for 14 days with cytokines known to induce MGC in vitro. The fusion index in experiments with a combination of interleukin (IL)-4 and macrophage colony-stimulating factor (M-CSF) and a combination of IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly lower in cord blood than in adult blood monocytes (P = 0.0018 and P = 0.0141, respectively). The number of nuclei per MGC was significantly lower in cord blood than in adult blood monocytes in experiments with IL-4 alone, the combination of IL-4 and M-CSF, and the combination of IL-4 and GM-CSF (P < 0.0001). These results suggest the possibility that the susceptibility of newborns to mycobacterium infection is due partly to impaired MGC formation.

Theophylline inhibits the differentiation of human monocyte into dendritic cell potentially via adenosine receptor antagonism.

BACKGROUND: Theophylline has an anti-inflammatory action that may account for its clinical effectiveness in the reduction of inflammatory cells in the airways. Dendritic cells (DCs) are professional antigen-presenting cells, capable of priming naïve T cells, and play key roles in the activation of immune responses in asthma. OBJECTIVE: The purpose of this study was to investigate the effects of theophylline on human monocyte differentiation into DCs and whether this involved antagonism of adenosine receptors. METHODS: Peripheral human blood monocytes were cultured in the presence of granulocyte/macrophage-colony stimulating factor and IL-4 to induce DC differentiation. The cells were incubated with theophylline, KF17837 (a selective A2a receptor antagonist) and enprofylline (A2b receptor antagonist) and co-incubated with selective adenosine A1 and A2a receptor agonists, a phosphodiesterase inhibitor (rolipram) and adenosine deaminase (ADA) to determine their effects on DC differentiation. In addition, depletion of adenosine receptors by small interfering RNA (siRNA) was also examined. RESULTS: Monocytes differentiated into myeloid DCs in the culture system. The number of DCs was remarkably reduced by 60-70% when theophylline was administered at a therapeutic concentration. This effect was concentration-dependently exacerbated, was partly mediated by cellular apoptosis and was effectively reversed by the addition of the A1 agonists [2-chloro-N(6)-cyclopentyladenosin, N(6)-cyclohexyladenosine, and N-ethylcarboxamidoadenosine (NECA)] or the A2a agonist (CGS-21680, NECA). The depletion of the adenosine A1 receptor by siRNA and addition of ADA remarkably reduced DC differentiation. Meanwhile, both enprofylline and rolipram had little effect. CONCLUSION: Our findings suggest that the adenosine A1 (and possibly coordinated with A2a) receptors contribute to DC differentiation and survival. These findings provide further evidence that theophylline has an anti-inflammatory action in bronchial asthma.

Enhanced methyltransferase activity of SMYD3 by the cleavage of its N-terminal region in human cancer cells.

Histone methylation is involved in the regulation of gene expression and DNA replication through alteration of chromatin structure. We earlier showed that SMYD3, a histone H3-lysine 4-specific methyltransferase, is frequently upregulated in human colorectal, liver and breast cancer compared to their matched non-cancerous cells, and that its activity is associated with the growth of these tumors. In the present study, we found that human cancer cells express both the full-length and a cleaved form of SMYD3 protein. Amino acid sequence analysis uncovered that the cleaved form lacks the 34 amino acids in the N-terminal region of the full-length protein. Interestingly, the cleaved protein and mutant protein containing substitutions at glycines 15 and 17, two highly conserved amino acids in the N-terminal region, revealed a higher histone methyltransferase (HMTase) activity compared to the full-length protein. Furthermore, the N-terminal region is responsible for the association with heat shock protein 90alpha (HSP90alpha). These data indicate that the N-terminal region plays an important role for the regulation of its methyltransferase activity and suggest that a structural change of the protein through the cleavage of the region or interaction with HSP90alpha may be involved in the modulation. These findings may help for a better understanding of the mechanisms that modulate the HMTase activity of SMYD3, and contribute to the development of novel anticancer drugs targeting SMYD3 methyltransferase activity.

Bone mineral density and osteo sono assessment index in adolescents.

The standard value for bone mineral density in the distal radius (R-BMD) and the osteo sono assessment index (OSI) in the os calcaneus for each sex and age in teenagers have not yet been fully reported. The R-BMD and OSI of junior and senior high school students were measured by dual energy X-ray absorptiometry (DEXA) or by a quantitative ultrasound technique. Subjects measured by DEXA included 635 junior and senior high school students (274 males and 361 females, aged 12-17 years). Ultrasound measurements were made for 2878 subjects (1733 males, 1145 females, aged 12-18 years). All subjects filled out questionnaires about their past history, family history, past and present eating habits, sports activities, and for females, the presence of menses, regularity of menses, and so on. The R-BMD in 15- to 17-year-old males was significantly higher than that in females. The R-BMD rate of increase in males was almost linear; the rate of increase in females was significantly highest from ages 12 to 13, after which R-BMD increased gradually. The OSI in 15- to 18-year-old males was significantly higher than that in females. The OSI rate of increase in males was almost linear from ages 12 to 17. The OSI in females, except in 14-year-olds, was roughly equal at each age. The OSI was significantly higher in those who regularly participated in sports, either currently or in the past. It was significantly higher in those who previously or currently consumed milk on a daily basis compared with those who had consumed little or no milk. To prevent osteoporosis, increasing peak bone mass is very important. Adequate calcium intake from dairy products which are rich in calcium and absorbed easily, and exercise in adolescence, are expected to result in increased bone formation and increased OSI.

[A case of Churg-Strauss syndrome presented as right thalamic hemorrhage].

Intracerebral hemorrhage is an uncommon sequel of Churg-Strauss syndrome (CSS). We report the first case of CSS accompanied by right thalamic hemorrhage. The patient was 59-year-old male who experienced a sudden onset of left hemiplegia. Magnetic resonance imaging of the brain revealed right thalamic hemorrhage. Although several reports had pointed out a close relationship between hypertension and cerebral hemorrhage in patients with CSS, this patient had no apparent prior history of hypertension. The cause of thalamic hemorrhage in this patient might be probably due to cerebral vasculitis and an effect of abnormal coagulation and fibrinolytic agents.

[Phase II study of cisplatin in advanced esophageal cancer and gastric cancer].

Twenty-four patients with advanced gastric cancer and 4 patients with advanced esophageal cancer were treated with cisplatin at a dose of 80-100 mg/m2 for one day or 10-20 mg/m2 for 5 consecutive days every 3-4 weeks. As for gastric cancer, 21 of 24 were evaluable for this study according to the criteria of the Japan Society for Cancer Therapy. Four of 21 patients (19%) showed partial response (PR), 7 displayed no change (NC), and 10 evidenced progressive disease (PD). Among 4 PR cases, only one had effective primary lesion. As for esophageal cancer, all 4 patients were evaluable, while 2 showed no change (NC) and another 2 exhibited progressive disease (PD). Gastrointestinal toxicity occurred in 20 patients despite the use of anti-emetic drugs. Nephrotoxicity and hematological toxicity were observed in 14 patients and 22 patients, respectively. These did not impede the continuous treatment except one case of hematological toxicity. It was concluded that cisplatin is more effective for the metastatic lesion of gastric cancer than primary lesion.

[Clinical evaluation of cefmenoxime in internal medicine, with special reference to infection associated with hematological disorders].

Clinical evaluation of cefmenoxime (CMX, Bestcall) was performed against infections associated with hematological, respiratory tract and other disorders. Clinical effectiveness of CMX against severe infections with hematological disorders including sepsis, pneumonia, pyelitis and so on was 74.4% for good responses and against the respiratory tract infections, 96.2% for good responses was obtained. Neither objective or subjective side effects nor extreme abnormalities in laboratory tests were observed in these patients. It can be concluded, therefore, that CMX is one of the most useful drugs against infectious diseases associated with hematological disorders, respiratory tract and other disorders.