Nectin-2 Acts as a Viral Entry Mediated Molecule That Binds to Human Herpesvirus 6B Glycoprotein B.

Human herpesvirus 6B (HHV-6B) is a T-lymphotropic virus and the etiological agent of exanthem subitum. HHV-6B is present in a latent or persistent form after primary infection and is produced in the salivary glands or transmitted to this organ. Infected individuals continue to secrete the virus in their saliva, which is thus considered a source for virus transmission. HHV-6B primarily propagates in T cells because its entry receptor, CD134, is mainly expressed by activated T cells. The virus then spreads to the host's organs, including the salivary glands, nervous system, and liver. However, CD134 expression is not detected in these organs. Therefore, HHV-6B may be entering cells via a currently unidentified cell surface molecule, but the mechanisms for this have not yet been investigated. In this study, we investigated a CD134-independent virus entry mechanism in the parotid-derived cell line HSY. First, we confirmed viral infection in CD134-membrane unanchored HSY cells. We then determined that nectin cell adhesion molecule 2 (nectin-2) mediated virus entry and that HHV-6B-insensitive T-cells transduced with nectin-2 were transformed into virus-permissive cells. We also found that virus entry was significantly reduced in nectin-2 knockout parotid-derived cells. Furthermore, we showed that HHV-6B glycoprotein B (gB) interacted with the nectin-2 V-set domain. The results suggest that nectin-2 acts as an HHV-6B entry-mediated protein.

Magnetically responsive antibacterial nanocrystalline jute cellulose nanocomposites with moderate catalytic activity.

Nanocrystalline jute cellulose (NCJC) particles were prepared from bleached jute pulp by a modified acid hydrolysis. The surface of NCJC particles were first modified with iron oxide nanoparticles and then with Ag nanoparticles to prepare antibacterial NCJC/Fe3O4/Ag nanocomposite particles. The successive structural modification of NCJC particles with Fe3O4 and Ag nanoparticles was confirmed. NCJC/Fe3O4/Ag nanocomposite particles responded well in external magnetic field. The SEM and TEM images of NCJC particles were in the nanometer range. NCJC/Fe3O4/Ag nanocomposite particles (0.005 mg mL-1) completely degraded 20 mL of 0.1 mM congo red aqueous solution within 13 min aided by NaBH4 reducing agent. NCJC/Fe3O4/Ag nanocomposite particles were moderately active against both Gram positive and Gram negative bacteria. A maximum inhibition zone of 21 mm was observed against Gram negative Shigella boydii bacteria with 60 mg/disc of nanocomposite particles. The antioxidant property of nanocomposite particles was also positive.

Risk assessment for hepatitis E virus infection from domestic pigs introduced into an experimental animal facility in a medical school.

Hepatitis E virus (HEV) is known to cause zoonotic infections from pigs, wild boars and deer. Domestic pigs have been used as an experimental animal model in medical research and training; however, the risks of HEV infection from pigs during animal experiments are largely unknown. Here, we retrospectively investigated the seroprevalence and detection rates of viral RNA in 73 domestic pigs (average 34.5 kg) introduced into an animal experimental facility in a medical school during 2012-2016. We detected anti-HEV immunoglobulin G antibodies in 24 of 73 plasma samples (32.9%), though none of the samples were positive for viral RNA. Plasma samples of 18 pigs were sequentially monitored and were classified into four patterns: sustained positive (5 pigs), sustained negative (5 pigs), conversion to positive (6 pigs) and conversion to negative (2 pigs). HEV genomes were detected in 2 of 4 liver samples from pigs that were transported from the same farm during 2016-2017. Two viral sequences of the overlapping open reading frame (ORF) 2/3 region (97 bp) were identical and phylogenetically fell into genotype 3. A 459-bp length of the ORF2 region of an amplified fragment from a pig transported in 2017 was clustered with the wbJYG1 isolate (subgenotype 3b) with 91.5% (420/459 bp) nucleotide identity. Based on our results, we suggest that domestic pigs introduced into animal facilities carry a potential risk of HEV infection to researchers, trainees and facility staff. Continuous surveillance and precautions are important to prevent HEV infection in animal facilities.

Metabolic pathway catalyzed by Vanin-1 pantetheinase plays a suppressive role in influenza virus replication in human alveolar epithelial A549 cells.

Our previous analysis of gene expression profiles in the peripheral blood from patients with influenza A (H1N1) pdm09 pneumonia revealed elevated transcription levels of the vanin-1 (vascular non-inflammatory molecule 1, VNN1) gene, which encodes an epithelial ectoenzyme with pantetheinase activity involved in recycling coenzyme A. Here, to elucidate the role of VNN1 in influenza A virus (IAV) H1N1 infection, we investigated the change of VNN1 expression in the context of IAV infection and the effects of its related substances, i.e., its direct substrate pantetheine and its two metabolites pantothenic acid and cysteamine on the replication of IAV in the human alveolar epithelial carcinoma cell line A549. The messenger RNA expression of VNN1 in A549 cells was significantly increased (by 4.9-fold) after IAV infection under an elevated concentration of pantetheine. Moreover, VNN1 mRNA levels were elevated by > 100-fold in response to pro-inflammatory cytokines, especially TNF-α and IL-1ß. Pantetheine significantly reduced the IAV replication and IAV Matrix 1 (M1) mRNA levels when it was administered prior to and during infection. In addition, cysteamine treatment during IAV infection significantly reduced the viral replication and IAV M1 mRNA levels, whereas pantothenic acid did not. These findings suggest that the metabolic pathway catalyzed by VNN1 pantetheinase plays a suppressive role in IAV infection in the respiratory tract, especially in severe conditions under hypercytokinemia.

Monitoring of human herpesviruses-6 and -7 DNA in saliva samples during the acute and convalescent phases of exanthem subitum.

The amounts of the DNAs of human herpesviruses-6 (HHV-6) and -7 (HHV-7) in saliva samples were monitored during the acute and convalescent phases of exanthem subitum (ES) to elucidate the kinetics of virus shedding after ES. A total of 247 saliva samples were collected from 17 children (5 males and 12 females: 8-31 months old at onset). The monitoring period ranged from 152 to 721 days after onset, and in 15 children it was longer than 1 year. Among the 17 cases, 16 were attributed to HHV-6B, while a single case was attributed to HHV-7. Detection rates and average amounts of HHV-6 DNA in saliva samples after ES attributed to HHV-6B were low in the acute phase, increased to the maximum in the convalescent phase at 3-7 months, and then decreased. In addition, to investigate the source of infection, saliva samples from the older siblings (age 3-9 years) and parents of ES patients and children with a history of ES were also examined. The detection rate of HHV-6 DNA in saliva samples from 3- to 9-year-old children was significantly higher than the rate in adult saliva samples. Taken together, these findings suggest that the saliva of children in the convalescent phase of ES might be a more likely source of HHV-6 infection than that of adults. J. Med. Virol. 89:696-702, 2017. © 2016 Wiley Periodicals, Inc.

Inhibitory Effects of Edaravone, a Free Radical Scavenger, on Cytokine-induced Hyperpermeability of Human Pulmonary Microvascular Endothelial Cells:A Comparison with Dexamethasone and Nitric Oxide Synthase Inhibitor.

Lung hyperpermeability affects the development of acute respiratory distress syndrome (ARDS), but therapeutic strategies for the control of microvascular permeability have not been established. We examined the effects of edaravone, dexamethasone, and N-monomethyl-L-arginine (L-NMMA) on permeability changes in human pulmonary microvascular endothelial cells (PMVEC) under a hypercytokinemic state. Human PMVEC were seeded in a Boyden chamber. After monolayer confluence was achieved, the culture media were replaced respectively by culture media containing edaravone, dexamethasone, and L-NMMA. After 24-h incubation, the monolayer was stimulated with tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). Fluorescein-labeled dextran was added. Then the trans-human PMVEC leak was measured. Expressions of vascular endothelial-cadherin (VE-cadherin) and zonula occludens-1 protein (ZO-1) were evaluated using real-time quantitative polymerase chain reaction and immunofluorescence microscopy. The results showed that TNF-α＋IL-1ß markedly increased pulmonary microvascular permeability. Pretreatment with edaravone, dexamethasone, or L-NMMA attenuated the hyperpermeability and inhibited the cytokine-induced reduction of VE-cadherin expression on immunofluorescence staining. Edaravone and dexamethasone increased the expression of ZO-1 at both the mRNA and protein levels. Edaravone and dexamethasone inhibited the permeability changes of human PMVEC, at least partly through an enhancement of VE-cadherin. Collectively, these results suggest a potential therapeutic approach for intervention in patients with ARDS.

Gene expression analysis in children with complex seizures due to influenza A(H1N1)pdm09 or rotavirus gastroenteritis.

Viral infections have been implicated as a cause of complex seizures in children. The pathogenic differences in complex seizures due to influenza A(H1N1)pdm09 or rotavirus gastroenteritis remain unclear. This study analyzed the gene expression profiles in the peripheral whole blood from pediatric patients with complex seizures due to influenza A(H1N1)pdm09 or rotavirus gastroenteritis. The gene expression profiles of ten patients (five with seizures and five without) with influenza A(H1N1)pdm09 and six patients (three with seizures and three without) with rotavirus gastroenteritis were examined. Gene expression profiles in the whole blood were different in complex seizures due to influenza A(H1N1)pdm09 or rotavirus gastroenteritis. Transcripts related to the immune response were significantly differentially expressed in complex seizures with influenza A(H1N1)pdm09, and transcripts related to the stress response were significantly differentially expressed in complex seizures with rotavirus gastroenteritis. Pathway analysis showed that the mitogen-activated protein kinases in the T cell receptor signaling pathway were activated in complex seizures due to influenza A(H1N1)pdm09. Dysregulation of the genes related to immune response or stress response could contribute to the pathogenic differences of the complex seizures due to influenza A(H1N1)pdm09 or rotavirus gastroenteritis.

Pathogenic mechanisms of influenza A(H1N1)pdm09 infection elucidated on gene expression profiling.

BACKGROUND: The pathogenic mechanisms underlying influenza A(H1N1)pdm09-associated central nervous system (CNS) manifestations and pneumonia remain unclear. This study examined A(H1N1)pdm09 host responses using gene expression profiles of patients' peripheral blood. METHODS: Sixteen A(H1N1)pdm09-infected children in three groups were examined: a CNS group, with convulsion and altered consciousness (n = 6); a pneumonia (Pneu) group (n = 5); and a group of infected control patients (n = 5). The signal ratios of the acute to recovery phases in CNS or Pneu were analyzed versus those of the control. RESULTS: The CNS (619 transcripts) and Pneu (656 transcripts) groups had significantly increased signal ratios compared to the control group. Regarding the increased ratios of transcripts shown by multiple probes, contactin-associated protein-like 3 transcripts, oleoyl-ACP hydrolase transcripts, and interleukin 1 type 1 receptor were observed in CNS and Pneu. Increased ratios of prostaglandin-endoperoxide synthase 2 and α-synuclein were characteristic of CNS. Alkaline phosphatase and the Fc fragment of IgA receptor were characteristic of Pneu. Regarding enriched gene ontology terms, 'response to lipopolysaccharide', 'innate immune response', and 'intrinsic to membrane' were observed commonly in CNS and Pneu. Enriched gene ontology terms related to 'hemoglobin' and 'hemostasis' were, respectively, characteristic of CNS and Pneu. CONCLUSION: These symptom-associated transcripts might be some clues to the pathogenesis of the A(H1N1)pdm09 infection.

Redox-active protein thioredoxin-1 administration ameliorates influenza A virus (H1N1)-induced acute lung injury in mice.

OBJECTIVES: Influenza virus infections can cause severe acute lung injury leading to significant morbidity and mortality. Thioredoxin-1 is a redox-active defensive protein induced in response to stress conditions. Animal experiments have revealed that thioredoxin-1 has protective effects against various severe disorders. This study was undertaken to evaluate the protective effects of recombinant human thioredoxin-1 administration on influenza A virus (H1N1)-induced acute lung injury in mice. DESIGN: Prospective animal trial. SETTING: Research laboratory. SUBJECTS: Nine-week-old male C57BL/6 mice inoculated with H1N1. INTERVENTION: The mice were divided into a vehicle-treated group and recombinant human thioredoxin-1-treated group. For survival rate analysis, the vehicle or recombinant human thioredoxin-1 was administered intraperitoneally every second day from day -1 to day 13. For lung lavage and pathological analyses, vehicle or recombinant human thioredoxin-1 was administered intraperitoneally on days -1, 1, and 3. MEASUREMENTS AND MAIN RESULTS: Lung lavage and pathological analyses were performed at 24, 72, and 120 hrs after inoculation. The recombinant human thioredoxin-1 treatment significantly improved the survival rate of H1N1-inoculated mice, although the treatment did not affect virus propagation in the lung. The treatment significantly attenuated the histological changes and neutrophil infiltration in the lung of H1N1-inoculated mice. The treatment significantly attenuated the production of tumor necrosis factor-α and chemokine (C-X-C motif) ligand 1 in the lung and oxidative stress enhancement, which were observed in H1N1-inoculated mice. H1N1 induced expressions of tumor necrosis factor-α and chemokine (C-X-C motif) ligand 1 in murine lung epithelial cells MLE-12, which were inhibited by the addition of recombinant human thioredoxin-1. The recombinant human thioredoxin-1 treatment started 30 mins after H1N1 inoculation also significantly improved the survival of the mice. CONCLUSIONS: Exogenous administration of recombinant human thioredoxin-1 significantly improved the survival rate and attenuated lung histological changes in the murine model of influenza pneumonia. The protective mechanism of thioredoxin-1 might be explained by its potent antioxidative and anti-inflammatory actions. Consequently, recombinant human thioredoxin-1 might be a possible pharmacological strategy for severe influenza virus infection in humans.

Preparation of hemispherical polymer particles by cleavage of a Janus poly(methyl methacrylate)/polystyrene composite particle.

Micrometer-sized, hemispherical particles were successfully prepared as a result of the cleavage of Janus PMMA/PS composite particles by dispersion into acetone/water (9/1-10/0 v/v) media or a THF/water (8/2 v/v) medium. The spherical composite particles having a Janus structure were prepared by the slow evaporation of toluene from homogeneous PMMA/PS/toluene droplets dispersed in an aqueous medium in advance. It was clarified that the difference in affinity between PMMA and PS phases with respect to the media caused the cleavage of the composite particles. This method is expected to be a novel approach to the preparation of nonspherical polymer particles.

[A case of malignant paraganglioma presenting with skull metastases].

A non-functioning paraganglioma is usually benign, however, it may cause distant metastases. There is no histological appearance for the diagnosis of malignancy or absolute criteria for predicting malignant potential. Bony metastases from paraganglioma are known to occur, but, skull metastases are very rare. We report a case of intracranial metastases from a renal paraganglioma. A 61-year-old male presented with temporal headache and exophthalmos on the left side. Seven years prior, he underwent surgery to remove a mass in the right renal hilum, which was diagnosed as renal cell carcinoma at that time. Computed tomography and magnetic resonance imaging showed a ring-like enhanced mass in the left middle fossa, which destroyed the sphenoid bone and the lateral wall of the orbit. Another osteolytic lesion was revealed in the occipital bone. The fragile tumor was totally resected. Histopathological study revealed the Zellballen pattern with extensive coagulation necrosis. No apparent nuclear atypia or mitosis were present. Immunohistochemistry showed reactivity for synaptophysin and chromogranin A in the tumor cells. Review of the surgical specimen of the previously resected renal tumor revealed the same pathological and immunohistochemical findings as those of chief cells in the middle fossa tumor. Thus, this tumor was diagnosed as a malignant paraganglioma metastasized from renal paraganglioma. After six cycle chemotherapy with cyclophosphamide and vincristine, his condition was stable for two years, however, he died four years after the diagnosis of malignancy.

Activation of Akt is associated with poor prognosis and chemotherapeutic resistance in pediatric B-precursor acute lymphoblastic leukemia.

BACKGROUND: Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway, a pro-survival pathway, plays important roles in tumor cell growth. However, the role of Akt in the pathogenesis of pediatric B-precursor acute lymphoblastic leukemia (B-pre ALL) remains to be clarified. This study was undertaken to explore the clinical relevance and molecular mechanisms underlying the activation of Akt (i.e., phosphorylated Akt, P-Akt) in pediatric B-pre ALL. PROCEDURE: We evaluated the activation status of Akt in bone marrow samples from 21 children with newly diagnosed B-pre ALL and correlated the expression level of P-Akt with clinicopathologic and prognostic features. Additionally, we transfected the myristoylated Akt cDNA into the B-pre ALL cell line, Nalm-6, and examined the effect, in vitro, of Akt activation on the response to antitumor drugs. RESULTS: P-Akt expression in B-pre ALL blast cells at diagnosis was associated significantly with poor response to induction chemotherapy including prednisolone, dexamethasone, vincristine, and adriamycin in B-pre ALL patients. Both overall survival and relapse-free survival in patients with P-Akt expression were reduced significantly more than in patients without P-Akt expression. Activation of Akt reduced the extent of apoptosis induced by the antitumor drugs in Nalm-6 listed above. Activation of Akt did not induce expression of P-glycoprotein, a drug transporter that is capable of conferring multidrug resistance. CONCLUSION: These results support the contention that Akt activation is a mechanism of chemotherapeutic resistance in B-pre ALL and suggest that Akt can be a therapeutic target for the treatment of relapsed or refractory pediatric B-pre ALL.

Tumor necrosis factor-α can induce Langhans-type multinucleated giant cell formation derived from myeloid dendritic cells.

The formation of the rich cellular features of MGCs, where the nuclei are arranged circularly at the periphery of the cell (morphologically epithelioid; Langhans-type), is assumed to be associated with any granulomatous disease. The mechanism by which TNF controls the formation of human MGCs in vitro was investigated, focusing on the effect of the TNF-neutralizing antibody. Peripheral blood monocytes were isolated with mAb-coated immunologic magnetic beads and cultured for 10 days in the presence of 20 ng/mL GM-CSF and 10 ng/mL IL-4. These cells were further incubated in the presence of TNF-α with/without its blockade antibodies for 14 days. Myeloid DCs can be generated from peripheral blood monocytes, and both IL-4 and GM-CSF can provide sufficient stimulus for their differentiation. The formation of MGC can be induced in the presence of TNF-α. This reaction was prohibited by the presence of the TNF-neutralizing antibody but not by the presence of anti-TNF receptor II antibody. The activation of Rho and focal adhesion kinases induced by TNF-α stimulation might be linked to cell assembling and the formation of Langhans-type MGCs. MGCs can produce only small amounts of superoxide anions compared to isolated macrophages such as myeloid DCs.

Increase of tumor necrosis factor-alpha in the blood induces early activation of matrix metalloproteinase-9 in the brain.

Increases of cytokine in the blood play important roles in the pathogenesis of influenza-associated encephalopathy. TNF-alpha was administered intravenously to wild-type mice, after which blood, CSF and brain tissue were obtained, and changes in BBB permeability, the amounts of MMP-9 and TIMP-1, and the localization of activated MMP were assessed. There was a significant increase in BBB permeability after 6 and 12 hr. MMP-9 was increased after 3 hr in the brain and cerebrospinal fluid, which was earlier than in the serum. TIMP-1 protein in the brain increased significantly after MMP-9 had increased. Activation of MMP-9 was observed in neurons in the cerebral cortex and hippocampus, and in vascular endothelial cells. These findings suggest that an increase in blood TNF-alpha promotes activation of MMP-9 in the brain, and may also induce an increase in permeability of the BBB. Early activation of MMP-9 in the brain may contribute to an early onset of neurological disorders and brain edema prior to multiple organ failure in those inflammatory diseases associated with highly increased concentrations of TNF-alpha in the blood, such as sepsis, burns, trauma and influenza-associated encephalopathy.

Prognostic factors in influenza-associated encephalopathy.

BACKGROUND: Recently, reports of influenza-associated encephalopathy have increased worldwide. Given the high mortality and morbidity rates attributable to this severe neurologic complication of influenza, we conducted a nationwide study in Japan to identify the prognostic factors. METHODS: We retrospectively evaluated 442 cases of influenza-associated encephalopathy that were reported to the Collaborative Study Group on Influenza-Associated Encephalopathy, which was organized by the Japanese Ministry of Health, Labor, and Welfare in collaboration with hospitals, clinics, and local pediatric practices in Japan between 1998 and 2002. The outcome for each patient was classified as either survival or death. Predictors of death were identified using logistic regression analysis. RESULTS: Four major prognostic factors for death were found to be significant by multivariate analysis (P < 0.05) in the 184 patients for whom we had complete data: elevation of aspartate aminotransferase, hyperglycemia, the presence of hematuria or proteinuria, and use of diclofenac sodium. CONCLUSIONS: We identified patients who had factors associated with a poor prognosis, and these findings might be clinically useful for the management of this illness.

Two cases of chronic active Epstein-Barr virus infection in which EBV-specific cytotoxic T lymphocyte was induced after allogeneic bone marrow transplantation.

CAEBV is a high mortality and morbidity disease with life-threatening complications. Nevertheless, the treatment regimens for CAEBV have not yet been established. Although some reports have described CAEBV therapy involving treatments such as antiviral drugs, immunomodulatory agents, and immunochemotherapy, none of these treatments have been demonstrated to be effective. The only treatment reported to be effective is allogeneic SCT. However, the complications of SCT are severe, so treatment results have been poor. Recently, immunotherapy has been devised, but this is still in the developmental stage. In this report, two cases of CAEBV in which allogeneic SCT was performed soon after diagnosis are reported. In both cases, a high EBV genome titer in the peripheral blood was detected at onset. After SCT, the EBV genome titer decreased as CTL activity gradually increased. This fact suggested that not only high-dose chemotherapy as a preconditioning treatment of SCT but also increased CTL activity which could eliminate virus-infected cells might be effective, although additional cases should be studied in order to establish effective treatments.

HHV-6 and seizures.

Human herpes virus-6 (HHV-6) is a ubiquitous virus, but one that can induce various neurological diseases. Recently, several seizures have been reported as new HHV-6-associated diseases based on virological analysis. Neonates who are perinatally infected with HHV-6 can develop afebrile seizures, which are considered to be exanthem subitum (ES) in the neonatal period. Infants with ES also tend to develop atypical febrile seizures. After primary infection, HHV-6 commonly establishes latency in the central nervous system (CNS) and sometimes reactivates in the hippocampus, causing limbic encephalitis and temporal lobe epilepsy. These HHV-6-associated CNS diseases due to virus reactivation can occur in both immunocompromised and immunocompetent hosts. This article summarizes HHV-6-associated seizures during childhood.

Virological aspects of Epstein-Barr virus infections.

Epstein-Barr virus (EBV) is usually maintained in an asymptomatic and latent form by the host immune system, and primarily by EBV-specific cytotoxic T cells (CTLs). However, EBV has been linked to several refractory diseases such as EBV-associated hemophagocytic syndrome(EBV-AHS) and chronic active EBV infection (CAEBV). In these ectopic diseases, EBV infects T/NK cells, causing severe immunodeficiency with a very high EBV load. In recent years, the laboratory procedure to assess these types of EBV infections has been improved. In particular, real-time polymerase chain reaction (PCR) has been used to quantify the EBV load, and the MHC: peptide tetramer assay has been used to quantitate EBV-specific CTLs; these tests have been employed for the management of the illnesses associated with EBV infection. Here, we have reviewed the recent progress in the clinical application of these assays. The pathogenesis of EBV-infected T/NK cells, and the host immune response to infection, including the roles carried out by innate immunity and inflammatory cytokines, are likely to be revealed in the future.