Growth activation of influenza virus by trypsin and effect of T-705 (favipiravir) on trypsin-optimized growth condition.

Influenza virus is activated by proteolytic cleavage of hemagglutinin by trypsin. After determining the optimal trypsin concentration, intracellular and extracellular influenza A/PR/8/34 (H1N1) and A/Victoria/361/2011 (H3N2) virus productions were compared in cultures treated with T-705 (favipiravir) and GS 4071 (an active form of oseltamivir). Although both drugs efficiently inhibited extracellular viral RNA release in a dose-dependent manner, T-705 inhibited it to the level of the inoculum without trypsin treatment, while GS 4071 inhibited it to a final level 10 times higher than that without trypsin. T-705 inhibited intracellular viral RNA production to the level of input virus in both trypsin-treated and untreated cells. In contrast, GS 4071 dose-dependently inhibited intracellular viral RNA production in cells treated with trypsin but allowed viral RNA synthesis. The level of maximum inhibition by GS 4071was 10 times higher than that of cells without trypsin and 1,000 times greater than the inoculum titer in cells without trypsin. T-705 inhibited both intracellular and extracellular virus production 1,000 and 10 times more strongly, respectively, than GS 4071. T-705 has powerful anti-influenza activity in the absence of trypsin and even in the trypsin-optimized growth condition, suggesting the therapeutic advantage in treatment of influenza complicated with bacterial pneumonia. Keywords: influenza; T-705; Tamiflu; trypsin; bacterial trypsin-like protease.

Persistent Primary Cytomegalovirus Infection After Deceased Donor Kidney Transplant: Ganciclovir Susceptibility of Human Cytomegalovirus With UL97 D605E Mutation: A Case Report.

BACKGROUND: Cytomegalovirus (CMV) could cause rejection in immunocompromised patients during early post-renal transplant stage. The American Transplant Society guidelines recommend prophylactic therapy with ganciclovir (GCV) for 3 to 6 months to prevent CMV infections in adult renal transplant patients. However, there is no recommended CMV treatment regimen for pediatric patients. MAIN FINDINGS: We performed deceased donor kidney transplant from an anti-CMV antibody-positive donor to an anti-CMV antibody-negative 15-year-old female recipient with end-stage renal disease caused by bilateral renal hypoplasia. One month after transplant, increase in positive cells in the CMV antigenemia assay indicated a primary CMV infection in the patient, who immediately received GCV. She was switched to foscarnet after 4 months of anti-CMV therapy because of clinical GCV resistance. CMV was isolated from the peripheral blood mononuclear cells but neutralizing antibody was not detected. Isolated CMV was susceptible to GCV and foscarnet, although it carried the UL97 D605E mutation, assumed to be associated with GCV resistance. CONCLUSIONS: The primary CMV infection presented a phenotypic clinical drug resistance, but all recovered CMV isolates were drug-susceptible even if isolated after prolonged anti-CMV therapy, indicating that immune status was more important for recovery from primary CMV infection than anti-CMV therapy.

T-705 (Favipiravir) suppresses tumor necrosis factor α production in response to influenza virus infection: A beneficial feature of T-705 as an anti-influenza drug.

Influenza virus infection induces the production of various cytokines, which play important roles in the pathogenesis of infection. Among the cytokines induced by influenza, tumor necrosis factor α (TNF-α) production has been correlated with the severity of lung lesions. We investigated the effects of T-705 (Favipiravir, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) on cytokine production due to influenza virus infection in vitro and in vivo, compared with oseltamivir or GS 4071, an active form of oseltamivir. TNF-α production in mouse macrophage-derived P388D1 cells infected with the influenza virus was lower following treatment with T-705 at concentrations of 0.3 to 100 µg/ml than treatment with GS 4071 at the same concentrations. The effect of treatment with T-705 on the cytokine production induced by the influenza virus infection was investigated in mouse influenza virus infection model. At 48 h post-infection (p.i.) T-705 significantly suppressed the viral load in the lungs and TNF-α production in the airways of infected mice even when viral loads were high. Furthermore, T-705 suppressed only TNF-α production from the early phase of infection. In this study, T-705 showed the antiviral activity of reducing pulmonary viral load compared with oseltamivir, thereby suppressing the TNF-α production. This feature of T-705 is benefit against severe influenza infection.

Helicase-primase inhibitor amenamevir for herpesvirus infection: Towards practical application for treating herpes zoster.

Valacyclovir and famciclovir enabled successful systemic therapy for treating herpes simplex virus (HSV) and varicella zoster virus (VZV) infection by their phosphorylation with viral thymidine kinase. Helicase-primase inhibitors (HPIs) inhibit the progression of the replication fork, an initial step in DNA synthesis to separate the double strand into two single strands. The HPIs amenamevir and pritelivir have a novel mechanism of action, once-daily administration with nonrenal excretory characteristics, and clinical efficacy for genital herpes. Amenamevir exhibits anti-VZV and anti-HSV activity while pritelivir only has anti-HSV activity. A clinical trial of amenamevir for herpes zoster has been completed, and amenamevir has been licensed and successfully used in 20,000 patients with herpes zoster so far in Japan. We have characterized the features of the antiviral action of amenamevir and, unlike acyclovir, the drug's antiviral activity is not influenced by the viral replication cycle. Amenamevir is opening a new era of antiherpes therapy.

Negative prognostic impact of renal replacement therapy in adult living-donor liver transplant recipients: preoperative recipient condition and donor factors.

BACKGROUND: In deceased-donor liver transplantation settings, post-transplantation acute renal failure with the induction of renal replacement therapy (RRT) is known to have negative effects on graft and patient survivals. However, the impact of RRT in living-donor liver transplantation (LDLT) has not been well investigated. The aim of this study was to elucidate risk factors requiring RRT and prognostic factors after its induction. METHODS: Clinical data on the consecutive 113 adult patients who underwent LDLT from March 2002 to May 2013 were retrospectively reviewed. They were divided into 2 groups: RRT (n = 33) and Non-RRT (n = 80). The primary reasons for receiving RRT were hepatorenal syndrome (n = 17), sepsis (n = 12), and renal hypoperfusion (n = 4). RESULTS: Although there were no significant differences in age or sex, the Model for End-Stage Liver Disease (MELD) score was significantly higher in the RRT group than in the Non-RRT group (23 ± 13 vs 16 ± 7; P = .002). The graft-recipient weight ratio (GRWR) was significantly lower in the RRT group (0.86 ± 0.3 vs 0.99 ± 0.2; P = .025). The 1- and 5-year patient survival rates were significantly higher in the Non-RRT group than in the RRT group (respectively, 91.3% and 84.3% vs 42.9% and 25.5%; P < .001). In multivariate analysis, independent risk factors for receiving RRT were MELD score >20 (P = .044) and GRWR <0.7 (P = .039). In the RRT group, donor age >50 years (P = .042) and preoperative serum creatinine level >1.5 mg/dL (P = .049) were significant prognostic risk factors. CONCLUSIONS: In adult LDLT patients, the induction of RRT after LDLT was a negative predictor of survival. In addition to the preoperative recipient's condition, donor factors including graft size and donor age influenced prognosis after the induction of RRT.

Biliary complications in 108 consecutive recipients with duct-to-duct biliary reconstruction in living-donor liver transplantation.

BACKGROUND: Biliary complications remain the leading cause of postoperative complications after living-donor liver transplantation (LDLT) in patients undergoing duct-to-duct choledochocholedochostomy. The aim of this study was to analyze the causes of these complications. METHODS: One hundred eight patients who underwent LDLT with duct-to-duct biliary reconstruction at Mie University Hospital were enrolled in this study. The mean follow-up time was 58.4 months (range, 3-132). The most recent 18 donors underwent indocyanine green (ICG) fluorescence cholangiography for donor hepatectomy. The development of biliary complications was retrospectively analyzed. Biliary complications were defined as needing endoscopic or radiologic treatment. RESULTS: Biliary leakages and strictures occurred in 6 (5.6%) and 15 (13.9%) of the recipients, respectively, and 3 donors (2.7%) experienced biliary leakage. However, since the introduction of ICG fluorescence cholangiography, we have not encountered any biliary complications in either donors or recipients. Biliary leakage was an independent risk factor for the development of biliary stricture (P = .013). Twelve (80%) of the 15 recipients with biliary stricture had successful nonoperative endoscopic or radiologic management, and 3 patients underwent surgical repair with hepaticojejunosotomy. CONCLUSIONS: Biliary leakage was an independent factor for biliary stricture. ICG fluorescence cholangiography might be helpful to reduce biliary complications after LDLT in both donors and recipients.

Advances in characterization of neuroprotective peptide, humanin.

Humanin (HN), a short amino acid peptide, protects neurons as well as other cells from amyloid ß-induced toxicities and other stresses. A number of HN binding proteins have been identified and their involvements in HN-mediated neuroprotection have been suggested in some cases. However, the way HN binds to the target molecules has never been clarified. Here we will review the structures of HN and HN analogs in solution as a function of solvent conditions and attempt to relate their structural characteristics to the functional properties.

High incidence of tumors in diabetic thrombin activatable fibrinolysis inhibitor and apolipoprotein E double-deficient mice.

BACKGROUND: Activation of the complement system has been implicated in tumor growth. The antifibrinolytic protein, activated thrombin-activatable fibrinolysis inhibitor (TAFIa), can modulate the activation of the complement system by inactivating the anaphylatoxins C3a and C5a. The apolipoprotein-E (ApoE) genotype has been associated with carcinogenesis. OBJECTIVE: The aim of this study was to evaluate whether TAFIa can affect the development of cancer in the ApoE-deficient mouse model. METHODS: TAFI and ApoE double-knockout mice were generated. A group of mice was treated with the diabetogenic and carcinogenic compound streptozotocin (stz). Mice treated with saline, single knockout mice and wild-type (wt) mice served as controls. RESULTS: Six months after treatment with stz, mice were sacrificed. Hepatic tumors were found in male double-knockout mice treated with stz but none was found in control animals that were not treated with stz or in single knockout of ApoE or wt animals. There was no significant difference in coagulation system activation between the groups of mice. The plasma concentrations of C5a, factor D and transforming growth factor-ß1 were increased in TAFI/ApoE double-deficient mice treated with stz compared with the mice of the same genotype treated with saline. CONCLUSION: Apo-E deficiency alone was not associated with tumors but the lack of TAFI appears to make the mice permissive for tumor formation in ApoE mice.

Potential application of arginine in interaction analysis.

Aqueous solution of 0.1-2 M arginine at mildly acidic to neutral pH is widely used in biotechnology and protein research, including protein refolding, purification, and formulation. This is largely because of its ability to suppress non-specific protein-protein and protein-surface interactions. Here we propose potential applications of arginine in interaction analysis for proteins. One of the important goals of such analysis is discovery of small molecule antagonistic or agonistic ligands that bind to target proteins and thereby modulate their function. Such research is often hampered by the low solubility of the small molecules, the instability of target proteins and the non-specific protein-ligand interactions. Aqueous arginine solution increases the solubility of small molecules, which should give an alternative to conventional dissolution method of small molecules by organic solvents. Arginine may also directly impact on the analysis of protein-protein or protein-ligand interactions by suppressing weak non-specific interactions.

Interleukin 12 is a primary cytokine responding to influenza virus infection in the respiratory tract of mice.

We have reported previously that an increase in interleukin 12 (IL-12) production in the lungs of mice infected with Influenza A virus or an intranasal (i.n.) administration of IL-12 to the infected mice alleviated pneumonia (Tsurita et al., J. Pharmacol. Exp. Therapeut. 298, 362-368, 2001). In this study, we found that in the bronchoalveolar lavage fluids (BALF) obtained from mice infected i.n. with Influenza A virus IL-12 was elevated on day 1 post infection (p.i.) and was followed by tumor necrosis factor alfa (TNF-alfa), IL-18, and interferons alfa, beta, and gama (IFN-alfa, -beta, and -gama) on day 2 p.i. Histochemical analyses of the infected lungs on day 1 p.i. showed the presence of IL-12 and IL-12 mRNA in mononuclear and macrophage-like cells and co-localization of macrophages with viral antigen, while other cytokines were absent. Thus, IL-12 was produced by macrophages infiltrating the infected epithelium as the first response cytokine and its production at the site of infection may direct an early immune defense to alleviate the severity of infection.

Predictive factors for distant recurrence of HCV-related hepatocellular carcinoma after radiofrequency ablation combined with chemoembolization.

BACKGROUND: Radiofrequency ablation (RFA) therapy for hepatocellular carcinoma has enabled good local control to be possible. However, after successful local control, distant recurrences frequently occur in the remnant liver. AIM: To identify the predictive factors for distant recurrence after RFA. METHODS: A total of 117 patients with initial non-advanced hepatocellular carcinoma with HCV who underwent RFA in our hospital were selected for this study. After transcatheter chemoembolization, RFA was performed under real-time computed tomography-fluoroscopic guidance. We studied survival rates, local (adjacent to treated tumour) and distant (intrahepatic site distant from the treated tumours) recurrence rates, as well as predictive factors for distant recurrence. RESULTS: After RFA, survival rates were 98.2% and 64.7% at 1 and 5 years, respectively. Child B patients had a significantly worse survival than Child A. Recurrence rates were 2.4% at 5 years for local, and 17.1% and 76.9% at 1 and 5 years, respectively, for distant. The Kaplan-Meier method revealed significantly high recurrence rates in cases with low albumin levels (Alb < 3.5 g/dL), high aspartate aminotransferase levels (AST > 60 IU/L), high alanine aminotransferase levels (ALT > 60 IU/L), low platelet counts (Plt < 10 x 10(4)/microL), and high alpha-fetoprotein levels (AFP > 50 ng/mL). On multivariate analysis, low Alb levels and high AST levels were independent predictive factors for distant recurrence. CONCLUSIONS: Although RFA enables good local control for initial hepatocellular carcinoma, distant recurrence is observed at high rates in HCV patients. Low albumin and high AST levels are predictive factors for distant recurrence.

Different roles of nitric oxide synthase-1 and -2 between herpetic and postherpetic allodynia in mice.

We investigated using the mice role of nitric oxide synthase (NOS) in the spinal dorsal horn in herpetic and postherpetic pain, especially allodynia, which was induced by transdermal inoculation of the hind paw with herpes simplex virus type-1 (HSV-1). The virus inoculation induced NOS2 expression in the lumbar dorsal horn of mice with herpetic allodynia, but not postherpetic allodynia. There were no substantial alternations in the expression level of NOS1 at the herpetic and postherpetic stages. Herpetic allodynia was significantly inhibited by i.p. administration of the selective NOS2 inhibitor S-methylisothiourea, but not the selective NOS1 inhibitor 7-nitroindazole. NOS2 expression was observed around HSV-1 antigen-immunoreactive cells. On the other hand, postherpetic allodynia was significantly inhibited by i.p. administration of 7-nitroindazole, but not S-methylisothiourea. The activity of reduced nicotinamide adenine dinucleotide phosphate diaphorase, an index of NOS1 activity, significantly increased in the laminae I and II of the lumbar dorsal horn of mice with postherpetic allodynia, but not mice without postherpetic allodynia. The expression level of NOS1 mRNA in the dorsal root ganglia was similar between mice with and without postherpetic allodynia. The results suggest that herpetic and postherpetic allodynia is mediated by nitric oxide in the dorsal horn and that NOS2 and NOS1 are responsible for herpetic and postherpetic allodynia, respectively. It may be worth testing the effects of NOS2 and NOS1 inhibitors on herpetic pain and postherpetic neuralgia in human subjects, respectively.

Visible high-speed optical transmission over photonic crystal fiber.

We demonstrated high-speed transmission at visible wavelengths over a 1 km photonic crystal fiber (PCF). We achieved a 1 Gbit/s transmission at 783 nm by using the direct modulation of a cost-effective Fabry-Perot laser diode (FP-LD). By employing the external modulation of the longitudinally single-mode grating-stabilized LD, we obtained the first penalty free 10 Gbit/s transmission at 780 nm.

Energy structure of a finite Haldane chain in Y2BaNi0.96Mg0.04O5 studied by high field electron spin resonance.

This Letter presents the fine structure of energy levels for the edge states of a Haldane chain. In order to investigate the edge states, we have performed high field and multifrequency electron spin resonance (ESR) measurements of finite length S=1 antiferromagnetic chains in Y2BaNi0.96Mg0.04O5. Owing to the high spectral resolution by high fields and high frequencies, observed ESR signals can be separated into the contributions of the finite chains with various chain lengths. Our results clearly show that the edge spins actually interact with each other through the quantum spin chain and the interaction depends on the chain length N. This N dependence has been obtained experimentally for the first time, and shows that the correlation length xi in the real system is somewhat larger than that calculated by a simple Heisenberg model.