Short interfering RNA-directed inhibition of hepatitis B virus replication

AbstractRNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of mRNA. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of short interfering RNA (siRNA). We examined effects of siRNA on hepatitis B virus (HBV) replication. Human hepatoma cells were transfected with HBV DNA and siRNA against HBV-pregenome RNA. Transfection experiments demonstrated that the siRNA reduced the amount of HBV-pregenome RNA and resulted in reduction of the levels of replicative intermediates and viral protein. Our results indicate that siRNA-mediated gene silencing inhibits HBV replication through suppression of viral RNA, which may be useful as a potential therapeutic modality

Shape analysis of rectus extraocular muscles with age and axial length using anterior segment optical coherence tomography

Purpose This study aimed to evaluate the shape of the extraocular muscles (EOMs) in normal subjects using the en-face images of anterior segment optical coherence tomography (AS-OCT). The EOM insertion and the direction of the muscle fibers were investigated. Subjects and methods A total of 97 healthy normal subjects (194 eyes) at Okayama University Hospital (age, 47.1±21.5 years; range, 8–79 years) participated in the study. A series of 256 tomographic images of the rectus EOMs were captured using the C-scan function of the AS-OCT (CASIA2, TOMEY Co., Japan), and the images were converted to en-face images in multi-TIFF format. The anterior chamber angle to EOM insertion distance (AID) and the angle of the muscle fibers from the insertion site (angle of muscles) were measured from the images. The correlations of AID and angle of muscles with age and axial length were investigated and evaluated. Results AID and angle of muscles were significantly correlated with age or axial length in some EOMs. The AIDs of medial rectus (MR) (P = 0.000) and superior rectus (SR) (P = 0.005) shortened with age. The AIDs of MR (P = 0.001) and inferior rectus (IR) (P = 0.035) elongated with axial length, whereas lateral rectus (LR) (P = 0.013) shortened. The angles of MR (P = 0.001) and LR (P = 0.000) were found to have a more downward direction toward the posterior in older subjects. Conclusion En-face images can be created by AS-OCT, and the shape of the EOMs in normal subjects using these image measurements was available. With the ability to assess the EOMs, AID and angle of muscles are expected give useful information for treating and diagnosing strabismus-related diseases

A comparative study of dynamic CT and ultrasonic pulsed Doppler method for estimation of the portal blood flow.

A new dynamic CT method for evaluating the portal blood flow is described. Thirty healthy volunteers were injected with non-ionic hypo-osmotic iodine contrast medium to estimate the portal blood flow. Time density curves (TD-curves) for the abdominal aorta and the main trunk of the portal vein were determined on the basis of data obtained by dynamic CT. From the TD-curves, portal blood flow coefficient and circulation time to flow into the portal vein (P-P time) were calculated. More detailed data of the TD-curves could be obtained by the new dynamic CT than by the previous methods. Subjects were simultaneously studied by an ultrasonic pulsed Doppler method which has been clinically accepted. There was a significant correlation between our dynamic CT method (portal blood flow coefficient) and the ultrasonic pulsed Doppler method concerning the measurement of portal blood flow. Therefore, it may be concluded that this CT method is reliable and clinically acceptable.</p

Attenuation of Responsiveness to Interferon-α Treatment by Preceded Overactivation of Interferon-mediated Pathway in Patients Chronically Infected by Hepatitis C Virus

The Interferon (IFN) receptor-mediated signal transduction pathways involve the two novel DNA-binding factors, interferon regulatory factor-1 (IRF-1) and IRF-2. Both recognize the same DNA sequence, in which the expression ratio of IRF-1 to IRF-2 is relevant to induction of IFN-inducible genes, because IRF-1 acts as a transcriptional activator and IRF-2 as a counterpart. In the present study, 54 patients with chronic hepatitis C and the age-and sex-matched 7 subjects with fatty liver as a control were subjected to analysis of the expression ratio of IRF-1 to IRF-2 mRNA in the liver tissue obtained at the time of livert biopsy by reverse transcription-polymerase chain reaction in combination with a restriction fragment length polymorphism assay. The expression ratio of IRF-1 to IRF-2 mRNA in the liver tissue in patients chronically infected by hepatitis C virus (HCV) was significantly higher than that in control, although the values did not correlate with the serum levels of HCV-RNA. Of 54 patients, 28 received IFN treatment, resulting in complete response in 8 patients. With respect to responsiveness to IFN treatment, patients who had complete response had the relatively lower ratios of IRF-1 to IRF-2 mRNA in the liver tissue, compared with those who did not. These results indicate that the IFN-mediated pathway is spontaneously activated in patients with chronic hepatitis C, and that its preceded overactivation counteracts on the efficacy of IFN treatment in these patients

Anti-hepatitis C virus activity of geranylgeranylacetone treatment in hepatitis C-infected patients

Background. Geranylgeranylacetone (GGA), which is an isoprenoid compound, has been used orally as an antiulcer drug inJapan. GGA induces antiviral gene expression by stimulating the formation of interferon-stimulated gene factor 3 in humanhepatoma cells. This study verified the anti-hepatitis C virus (HCV) activity of GGA in chronic hepatitis C-infected patients.Methods. The present prospective study included 20 consecutive anti-HCV antibody-positive, HCV-genotype 1b, and chronicgastritis patients who visited Nagasaki University Hospital between January 1999 and December 1999. GGA (150 mg per day,which is the dose generally used for chronic gastritis) was taken orally for four weeks. We evaluated HCV-RNA titers and otherclinical parameters at pretreatment, posttreatment, and at the endpoint of the study. Pretreatment was the beginning point ofGGA treatment. Posttreatment was the termination point of GGA treatment. The endpoint was the point four weeks after theposttreatment point.Results. All patients completed four weeks of GGA treatment and four weeks of observation. HCV-RNA titers at postpointwere not significantly diminished compared to those at pretreatment. However, HCV-RNA titers were significantly diminishedat endtreatment compared to pretreatment. Unfortunately, we did not observe a case with no titer of HCV-RNA. Alanineaminotransferase values and other parameters were not affected by GGA treatment.Conclusion. GGA has anti-HCV activities in chronic hepatitis C-infected patients. In the future, it will be necessary to examinethe clinical effectiveness of the combination of treatment with both GGA and interferon in HCV patients

Development of Kupffer cell targeting type-I interferon for the treatment of hepatitis via inducing anti-inflammatory and immunomodulatory actions

Because of its multifaceted anti-inflammatory and immunomodulatory effects, delivering type-I interferon to Kupffer cells has the potential to function as a novel type of therapy for the treatment of various types of hepatitis. We report herein on the preparation of a Kupffer cell targeting type-I interferon, an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, Man-HSA(D494N)-IFNα2b, attached by combining albumin fusion technology and site-directed mutagenesis. The presence of this unique oligosaccharide permits the protein to be efficiently, rapidly and preferentially distributed to Kupffer cells. Likewise IFNα2b, Man-HSA(D494N)-IFNα2b caused a significant induction in the mRNA levels of IL-10, IL-1Ra, PD-L1 in RAW264.7 cells and mouse isolated Kupffer cells, and these inductions were largely inhibited by blocking the interferon receptor. These data indicate that Man-HSA(D494N)-IFNα2b retained the biological activities of type-I interferon. Man-HSA(D494N)-IFNα2b significantly inhibited liver injury in Concanavalin A (Con-A)-induced hepatitis model mice, and consequently improved their survival rate. Moreover, the post-administration of Man-HSA(D494N)-IFNα2b at 2 h after the Con-A challenge also exerted hepato-protective effects. In conclusion, this proof-of-concept study demonstrates the therapeutic effectiveness and utility of Kupffer cell targeting type-I interferon against hepatitis via its anti-inflammatory and immunomodulatory actions

Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager

Summary: Autophagy is an intracellular self-degradation process in which part of the cytoplasm, aggregates, or damaged organelles are degraded in lysosomes. Lysophagy is a specific form of selective autophagy responsible for clearing damaged lysosomes. Here, we present a protocol for inducing lysosomal damage in cultured cells and assessing lysosomal damage using a high-content imager and software program. We describe steps for induction of lysosomal damage, image acquisition with spinning disk confocal microscopy, and image analysis using Pathfinder. We then detail data analysis of the clearance of damaged lysosomes.For complete details on the use and execution of this protocol, please refer to Teranishi et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics