Search

Enterovirus infections of the central nervous system (CNS) are common and important causes of morbidity in immunocompromised children and adults. In this study we identify and characterize coxsackievirus B4-induced neuronal death. To investigate the CNS pathophysiology resulting from this viral infection, cultured rat neurons were infected with coxsackievirus B4 (CVB4) and nuclear morphology, phosphatidylserine (PS) externalization, and the effects of Actinomycin D or cycloheximide (CHX) were examined. CVB4 induced neuronal cell death within 24 h while PS externalization was apparent in cell bodies 16 h after CVB4 infection. Actinomycin D or CHX significantly reduced CVB4 induced-neuronal cell death in a dose-dependent manner. Pretreatment with CHX or actinomycin D also inhibited nuclear condensation, which occurred after CVB4 infection. However, the changes were relatively unresponsive to zVAD-fmk. These results suggest that CVB4 induces CHX- and actinomycin D-sensitive, but zVAD-fmk-insensitive neuronal apoptosis. [Copyright &y& Elsevier]

An abstract of the article "Epstein-Barr Virus LF2: an Antagonist to Type I Interferon," by Liguo Wu, Even Fossum, Chul Hyun Joo and colleagues is presented.

Retinoic-acid-inducible gene-I (RIG-I; also called DDX58) is a cytosolic viral RNA receptor that interacts with MAVS (also called VISA, IPS-1 or Cardif) to induce type I interferon-mediated host protective innate immunity against viral infection. Furthermore, members of the tripartite motif (TRIM) protein family, which contain a cluster of a RING-finger domain, a B box/coiled-coil domain and a SPRY domain, are involved in various cellular processes, including cell proliferation and antiviral activity. Here we report that the amino-terminal caspase recruitment domains (CARDs) of RIG-I undergo robust ubiquitination induced by TRIM25 in mammalian cells. The carboxy-terminal SPRY domain of TRIM25 interacts with the N-terminal CARDs of RIG-I; this interaction effectively delivers the Lys 63-linked ubiquitin moiety to the N-terminal CARDs of RIG-I, resulting in a marked increase in RIG-I downstream signalling activity. The Lys 172 residue of RIG-I is critical for efficient TRIM25-mediated ubiquitination and for MAVS binding, as well as the ability of RIG-I to induce antiviral signal transduction. Furthermore, gene targeting demonstrates that TRIM25 is essential not only for RIG-I ubiquitination but also for RIG-I-mediated interferon-β production and antiviral activity in response to RNA virus infection. Thus, we demonstrate that TRIM25 E3 ubiquitin ligase induces the Lys 63-linked ubiquitination of RIG-I, which is crucial for the cytosolic RIG-I signalling pathway to elicit host antiviral innate immunity. [ABSTRACT FROM AUTHOR]

Abstract: RNA interference (RNAi) is a ‘knock-down’ reaction to reduce expression of a specific gene through highly regulated, enzyme-mediated processes. Small interfering RNAs (siRNAs) are RNA molecules that play an effector role in RNAi and can bind the PAZ domains present in Dicer and RISC. We investigated the interaction between the PAZ domain and the siRNA-like duplexes through dissociation molecular dynamics (DMD) simulations. Specifically, we focused on the response of the PAZ domain to various 3′-overhang structures of the siRNA-like duplexes. We found that the siRNA-like duplex with the 3′ UU-overhang made relatively more stable complex with the PAZ domain compared to those with 3′ CC-, AA-, and GG-overhangs. The siRNA-like duplex with UU-overhang was easily dissociated from the PAZ domain once the structural stability of the complex is impaired. Interestingly, the 3′ UU-overhang spent the least time at the periphery region of the binding pocket during the dissociation process, which can be mainly attributable to UU-overhang''s smallest number of hydrogen bonds. [Copyright &y& Elsevier]

Abstract: Gelsolin consists of six related domains (G1–G6) and the C-terminal half (G4–G6) acts as a calcium sensor during the activation of the whole molecule, a process that involves large domain movements. In this study, we used targeted molecular dynamics simulations to elucidate the conformational transitions of G4–G6 at an atomic level. Domains G4 and G6 are initially ruptured, followed by a rotation of G6 by ∼90°, which is the dominant conformational change. During this period, local conformational changes occur at the G4 and G5 calcium-binding sites, facilitating large changes in interdomain distances. Alterations in the binding affinities of the calcium ions in these three domains appear to be related to local conformational changes at their binding sites. Analysis of the relative stabilities of the G4–G6-bound calcium ions suggests that they bind first to G6, then to G4, and finally to G5. [Copyright &y& Elsevier]

As a new class of cancer therapeutic agents, oncolytic viruses (OVs) have gained much attention not only due to their ability to selectively replicate in and lyse tumor cells, but also for their potential to stimulate antitumor immune responses. As a result, there is an increasing need for in vitro modeling systems capable of recapitulating the 3D physiological tumor microenvironment. Here, we investigated the potential of our recently developed microphysiological system (MPS), featuring a vessel-like channel to reflect the in vivo tumor microenvironment and serving as culture spaces for 3D multicellular tumor spheroids (MCTSs). The MCTSs consist of cancer A549 cells, stromal MRC5 cells, endothelial HUVECs, as well as the extracellular matrix. 3D MCTSs residing in the MPS were infected with oncolytic VSV expressing GFP (oVSV-GFP). Post-infection, GFP signal intensity increased only in A549 cells of the MPS. On the other hand, HUVECs were susceptible to virus infection under 2D culture and IFN-β secretion was quite delayed in HUVECs. These results thus demonstrate that OV antitumoral characteristics can be readily monitored in the MPS and that its behavior therein somewhat differs compared to its activity in 2D system. In conclusion, we present the first application of the MPS, an in vitro model that was developed to better reflect in vivo conditions. Its various advantages suggest the 3D MCTS-integrated MPS can serve as a first line monitoring system to validate oncolytic virus efficacy. [ABSTRACT FROM AUTHOR]

The airway is the major entry route of pathogens due to continuous gas exchange with the environment. In particular, the nasal epithelial layer is the common site of airborne mucotropic virus infections. The inflammatory response to such infections must be tightly controlled due to its non-specific nature. Unrestrained inflammation breaks down the physiological mucosal defense system and leads to secondary bacterial or fungal infections. Chronic rhinosinusitis (CRS) is a prevalent inflammatory disease that compromises quality of life. In spite of its importance in the initiation of inflammation, the role of interferon signaling in nasal airway epithelial cells is largely unknown. We analyzed the expression of interferon signaling genes using clinical lavage specimens and nasal airway epithelial cells collected from CRS patients and controls. Basal expression of IFNAs , IKBKE , STAT1 , and some CXC chemokines was elevated in samples from CRS patients. In subsequent in vitro studies, we found IKKε to be the key molecule and augmented CXCL10 secretion. Based on our findings and review of the literature, we hypothesized that high levels of IKKε might induce intractable inflammation via CXCL10. We tested the hypothesis in an animal model and found not only that IKKε induced severe eosinophilic inflammation with CXCL10 over-production, but also that inhibition of IKKε resolved the inflammation. Image 1 • High basal expression of IFNA in CRS NAEC. • IKKε was the key molecule of augmented secretion of IFNα and CXCL10. • The inhibition of IKKε resolved the inflammation in animal model. [ABSTRACT FROM AUTHOR]

Human interferon alpha-2b (IFNα-2b) has therapeutic applications as an antiviral and antiproliferative drug and has been used for a wide range of indications. Efficient production of IFNα-2b in Escherichia coli has been difficult because the protein tends to form inclusion bodies. This obstacle has garnered interest in efficiently expressing IFNα-2b and overcoming its poor solubility. In this study, seven N-terminal fusion partners - hexahistidine (His6), thioredoxin, glutathione S -transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide bond isomerase (PDI), and b'a' domain of PDI - were tested for soluble overexpression of codon-optimized IFNα-2b in E. coli . Low temperature increased the expression level of all of the tagged proteins except for the GST fusion. All the tags, except for His6 and GST, improved solubility. We purified IFNα-2b from the MBP-tagged fusion using immobilized metal affinity chromatography and anion exchange chromatogra phy, and obtained a final yield of 7.2 mg from an initial 500-ml culture. The endotoxin level was 0.46 EU/μg. Biological activity was demonstrated using a luciferase assay, which showed a dose-dependent response with a calculated EC 50 of 10.3 ± 5.9 p M . Our results demonstrate that using an MBPtagged fusion is an efficient way to produce pure IFNα-2b. [ABSTRACT FROM AUTHOR]

Human rhinovirus (HRV)-A and -B is a common cause of upper respiratory tract infections. Recently, a third species, HRV-C, was categorized based on molecular typing studies. The results showed that the HRV-C genome had diverged from that of HRV-A and -B. Despite its late identification, increasing evidence suggests that HRV-C causes more severe pathogenic infections than HRV-A or -B; however, a large amount of epidemiological data is required to confirm this association in different clinical settings. Consequently, a simple and rapid method for identifying HRV-C is required to expedite such epidemiological studies. Here, two degenerate primer sets (HEV and HRVC) were designed based on bioinformatic analyses. The HEV set targeting the fifth IRES domain sequence within the 5′-UTR, which is highly conserved among enteroviruses, was designed to detect all enteroviruses, whereas the HRVC set, which targeted the VP2 coding region, was designed to detect HRV-C alone. Both primer sets were tested against a panel of standard enteroviruses and clinical lavage samples. HEV detected all enteroviruses tested whereas HRVC was specific for HRV-C. Although the primer design strategy was confirmed with a limited number of samples, extensive tests are required to be applied in clinical settings. [ABSTRACT FROM AUTHOR]

Background Chronic rhinosinusitis (CRS) can be aggravated by viral upper respiratory infections. We aimed to investigate whether any specific human rhinovirus (HRV) serotype is more common in the mucosa of CRS patients, and to find any defining clinical characteristics, according to the various HRV serotypes. Methods A prospective case-control study was conducted to determine HRV serotypes in 111 CRS patients and 51 non-CRS controls. No participant had a history of upper respiratory infection over a 4-week period. Nasal lavage fluids and turbinate epithelial cells were collected prospectively. When HRV was detected with multiplex polymerase chain reaction (PCR), strains were further characterized by sequencing the VP4/VP2 region of the HRV genome. Results HRV was detected in 40 CRS subjects (36%) and 11 non-CRS controls (21%). The overall detection rates of HRV in CRS patients were higher than in non-CRS controls ( p < 0.05). Of the 8 serotypes detected in CRS patients, 5 belonged to HRV-A and 3 belonged to HRV-B; HRV-C was not detected. In non-CRS controls, only HRV-A was identified, with only 2 serotypes detected (HRV-A13 and HRV-A16). HRV-B and C were not detected. Conclusion The high prevalence of HRV in CRS patients was confirmed in our study. However, we were unable to determine whether certain HRV serotypes are more predominant in CRS patients than non-CRS controls. HRV-A13 was the most common serotype in both CRS patients and non-CRS controls. We could not find any differences in the clinical characteristics according to the HRV serotypes in CRS patients. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are multicompetent lymphocytes of the innate immune system that play a central role in host defense and immune regulation. Although increasing evidence suggests that innate immunity plays a key role in the pathogenesis of chronic rhinosinusitis (CRS), the role of NK cells in CRS has been poorly studied. This study aimed to characterize the peripheral blood NK cells from patients with CRS, and to compare the functions of these cells with those from non-CRS controls. The correlation between NK cell functional activity and prognosis was also assessed. Eighteen CRS patients and 19 healthy non-CRS controls were included. The patients with CRS were classified into two subgroups, namely a treatment-responsive group and recalcitrant group. NK cell degranulation was determined by measuring the cell surface expression of CD107a against 721.221 and K562 cells. Intracytoplasmic cytokine production was determined by flow cytometry. Compared to the controls, the NK cells of CRS group had an impaired ability to degranulate and to produce cytokines such as IFN-γ and TNF-α. The recalcitrant subgroup showed the most severe defects in NK cell effector functions. Moreover, the decreased NK cell functions in patients with CRS were associated with poor prognostic factors such as concomitant asthma and peripheral blood eosinophilia. NK cells, which were originally named for their ability to mediate spontaneous cytotoxicity towards diseased cells including infected cells, may play an important role in regulating the inflammatory process in CRS pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: A major concern of antiviral therapy using small interfering RNAs (siRNAs) targeting RNA viral genome is high sequence diversity and mutation rate due to genetic instability. To overcome this problem, it is indispensable to design siRNAs targeting highly conserved regions. We thus designed CAPSID (Convenient Application Program for siRNA Design), a novel bioinformatics program to identify siRNAs targeting highly conserved regions within RNA viral genomes. From a set of input RNAs of diverse sequences, CAPSID rapidly searches conserved patterns and suggests highly potent siRNA candidates in a hierarchical manner. To validate the usefulness of this novel program, we investigated the antiviral potency of universal siRNA for various Human enterovirus B (HEB) serotypes. Assessment of antiviral efficacy using Hela cells, clearly demonstrates that HEB-specific siRNAs exhibit protective effects against all HEBs examined. These findings strongly indicate that CAPSID can be applied to select universal antiviral siRNAs against highly divergent viral genomes. [Copyright &y& Elsevier]

Abstract: Coxsackievirus A24 (CVA24) is responsible for acute hemorrhagic conjunctivitis, a highly contagious eye disease for which no prevention or treatment is currently available. We thus assessed the antiviral potential of a small interfering RNA (siRNA) targeting CVA24. HeLa cells with or without four different siRNAs complementary to 2C or 3D genome region, were challenged with various CVA24s. Among several siRNAs, a siRNA targeting the highly conserved genome region called the cis-acting replication element (CVA24-CRE), was the only siRNA that decreased virus replication and subsequent cytotoxicity by both CVA24 variant and clinical isolates. Furthermore, CVA24-CRE had effective antiviral activity against CVA24 in primary human conjunctival cells. In addition, CVA24-CRE was highly resistant to the emergence of genetically altered escape mutants. Collectively, the present study provides evidence that CVA24-CRE targeting a conserved viral genome region had universal, prolonged anti-CVA24 activity. This siRNA may thus hold a potential to act clinically as a novel anti-CVA24 agent. [Copyright &y& Elsevier]

Advanced gastric cancer cannot be treated with surgery or conventional cancer therapy, which has prompted a search for new therapeutic modalities. Previously, we and other groups showed that E1B 55 kDa-deleted recombinant adenoviruses, such as YKL-1, effectively replicate and induce cytotoxicity in p53-deficient cancer cells while sparing normal cells. Here, we investigated selective YKL-1 replication and resultant cytolysis in human gastric cancer cells. The cytopathic effects were obvious in all five gastric cancer cell lines we examined. Evaluation of p53 expression indicated that only the AGS cell line retained functionally normal p53. Nevertheless, AGS was 10-fold more sensitive to YKL-1 than the other cell lines. Transmission electron microscopy showed typical morphological alterations along with efficient replication of YKL-1 in AGS cells. Therefore, YKL-1 induces preferential cytotoxic effects in human gastric cancer cells in a p53-independent manner, making YKL-1 a promising therapeutic agent for human gastric cancers. [Copyright &y& Elsevier]

Selectively replicating recombinant adenovirus has emerged as a novel strategy for the treatment of incurable human cancers. One of the major characteristics of hepatocellular carcinoma is the transcriptional reactivation of α-fetoprotein (AFP). In this study, we evaluated the liver cancer-specific oncolytic potential of E1B 55kDa-deleted recombinant adenovirus (YKL-1001), which retained other E1 genes driven by the AFP promoter. Transient transfection study using luciferase indicated the selective activation of the AFP promoter only in human liver cancer cells secreting AFP (HepG2, Hep3B). YKL-1001 induced both cytopathic effects exclusively in AFP-positive liver cancer cells and the growth inhibition of pre-established Hep3B xenografts. Finally, hematoxylin–eosin staining and the immunohistochemistry to the adenoviral hexon showed a large distributed necrotic area and this implied a wide spread of YKL-1001. Therefore, the present study demonstrated that YKL-1001 holds significant promise as an oncolytic agent for hepatocellular carcinoma. [Copyright &y& Elsevier]

The assessment of the biological activity of capsaicin, the compound responsible for the spicy flavor of chili pepper, produced controversial results, showing either carcinogenicity or cancer prevention. The innate immune system plays a pivotal role in cancer pathology and prevention; yet, the effect of capsaicin on natural killer (NK) cells, which function in cancer surveillance, is unclear. The present study found that capsaicin inhibited NK cell-mediated cytotoxicity and cytokine production (IFN-γ and TNF-α). Capsaicin impaired the cytotoxicity of NK cells, thereby inhibiting lysis of standard target cells and gastric cancer cells by modulating calcium mobilization in NK cells. Capsaicin also induced apoptosis in gastric cancer cells, but that effect required higher concentrations and longer exposure times than those required to trigger NK cell dysfunction. Furthermore, capsaicin inhibited the cytotoxicity of isolated NK cells and of an NK cell line, suggesting a direct effect on NK cells. Antagonists of transient receptor potential vanilloid subfamily number 1 (TRPV1), a cognate capsaicin receptor, or deficiency in TRPV1 expression failed to prevent the defects induced by capsaicin in NK cells expressing functional TRPV1. Thus, the mechanism of action of capsaicin on NK cells is largely independent of TRPV1. Taken together, capsaicin may have chemotherapeutic potential but may impair NK cell function, which plays a central role in tumor surveillance. [ABSTRACT FROM PUBLISHER]