Combinations of eight key mutations in the X/preC region and genomic activity of hepatitis B virus are associated with hepatocellular carcinoma.

Accumulation of eight key mutations located in the X/preC regions of the hepatitis B virus (HBV) genome (G1613A, C1653T, T1753V, A1762T, G1764A, A1846T, G1896A and G1899A) is a risk marker for the development of hepatocellular carcinoma (HCC). In this study, we analysed the 8 key mutations in 442 serum samples collected from 310 non-HCC and 132 HCC patients to identify the combinations linked to HCC. After the patients were stratified according to the age groups and mutation combinations, clinical parameters were compared between the HCC and the non-HCC groups. Analyses were focused on patient ≥40 years of age infected by HBV genotype C with A1762T and G1764A mutations in the basal core promoter region (BCP double mutation). In patients with ≥6 mutations, the combination of [G1613A + C1653T + A1846T + G1896A] mutations was closely linked to HCC, whereas no specific single or double mutation combination was associated with HCC. In patients with ≤5 mutations, HBeAg and HBV DNA serum titres were lower in the HCC group than those in the non-HCC group. Unlike the number of mutations, no specific combination correlated with advanced clinical stage in HCC. Of the BCP double mutation-based HBV mutant types, combinations of ≥6 mutations that include G1613A + C1653T + A1846T + G1896A, and combinations of ≤5 mutations with reduced HBeAg production, may be more specific indicators of HCC risk than only the number of mutations or any specific combination(s).

Pro-IL-16 is associated with MHC class II-mediated negative regulation of mouse resting B cell activation through MAP kinases, NF-κB and Skp2-dependent p27kip regulation.

MHC class II molecules, in addition to their essential role as antigen-presenting molecules to CD4(+) T cell receptor, have a signal-transducing role related to B cell function. We identified pro-IL-16 as one of the proteins associated with MHC class II-mediated signalling in an analysis of MHC class II-associated molecules using immunoprecipitation and proteomics data obtained from the 38B9 resting B cell line, and investigated the role of pro-IL-16 in resting B cell activation. We found that pro-IL-16, rather than mature form of IL-16, is present both in the cytoplasm and nucleus of resting B cells, and its expression is influenced by MHC class II-mediated signalling. In addition, overexpression of pro-IL-16 impaired resting B cell proliferation and this inhibitory effect was mediated through regulating nuclear factor (NF)-κB activation. Knock-down of pro-IL-16 expression using siRNA decreased the level of cell-cycle inhibitor p27(kip) and increased the level of Skp2. In addition, knock-down of pro-IL-16 modulated mitogen-activated protein kinase activation. Given that IL-16 acts as an immunomodulator by impairing antigen-induced T cell activation and its precursor, pro-IL-16, plays a role in regulating the cell cycle in T lymphocytes and T cell lymphoma, we concluded that pro-IL-16 is involved in resting B cell proliferation, similar to its function in T lymphocytes.

Reactive oxygen species-dependent necroptosis in Jurkat T cells induced by pathogenic free-living Naegleria fowleri.

Naegleria fowleri, a free-living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N. fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N. fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N. fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N. fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP-ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase-3 was observed in Jurkat T cells co-incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin-1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba-induced ROS generation and Jurkat cell death, whereas pan-caspase inhibitor z-VAD-fmk did not. N. fowleri-derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP-induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin-1. These results suggest that N. fowleri induces ROS-dependent necroptosis in Jurkat T cells.

A case report of ectopic pregnancy arising in a unicornuate uterus, accompanied by the undescended tube and ovary with double inferior vena cava.

RATIONALE: The incidence of a unicornuate uterus is 0.2% to 0.3% of the whole population. A unicornuate uterus is closely associated with obstetrical complications such as early miscarriages, ectopic pregnancy, and malpresentation. PATIENT CONCERNS: A 32-year-old patient developed a rare ectopic pregnancy arising at a distal, fimbriated end of the undescended fallopian tube. DIAGNOSES: A transvaginal ultrasound scan revealed hemoperitoneum and no gestational sac in the uterine endometrium. A laparoscopic finding showed that high up in the right abdomen, just below the liver, an ectopic mass could be seen arising at a distal, fimbriated end of the fallopian tube, which was developed adjacent to the undescended right ectopic ovary. INTERVENTIONS: After laparoscopic removal of the right salpinx, we removed it with a bag. OUTCOMES: One day after the operation, she was discharged without problems. Postoperative hysterosalpingography showed the unicornuate uterus with patent left and some right salpinx. Magnetic resonance imaging revealed a unicornuate uterus, right ovary at the right inferior hepatic area, a bilateral normal kidney, and double inferior vena cava. LESSONS: This is the first reported case of its type. It demonstrated that ectopic pregnancy may occur in the upper abdomen, not in the pelvic cavity, in uterine anomaly, and double inferior vena cava; hence, we must thoroughly check the whole abdominal cavity. Additional imaging tests are needed after treatment to see if there are any abnormalities.

Hemodialysis catheter placement via a persistent left superior vena cava.

Persistent left superior vena cava (PLSVC) is the most common thoracic venous circulation anomaly and discovered incidentally during hemodialysis catheter insertion. PLSVC may have some clinical implications for nephrologists. PLSVC can create difficulties during catheter insertion and cause serious complications; it can be mistaken for placement into other sites and cause inappropriate response such as catheter removal. Nephrologists who place hemodialysis catheters in the left jugular or subclavian vein should be aware of the existence of PLSVC.

Self-optimization and auto-stabilization of receiver in DPSK transmission system.

We propose a self-optimization and auto-stabilization method for a 1-bit DMZI in DPSK transmission. Using the characteristics of eye patterns, the optical frequency transmittance of a 1-bit DMZI is thermally controlled to maximize the power difference between the constructive and destructive output ports. Unlike other techniques, this control method can be realized without additional components, making it simple and cost effective. Experimental results show that error-free performance is maintained when the carrier optical frequency variation is approximately 10% of the data rate.

Cell cycle arrest and lytic induction of EBV-transformed B lymphoblastoid cells by a histone deacetylase inhibitor, Trichostatin A.

Latent infection of the Epstein-Barr virus (EBV) is strongly associated with the pathogenesis of several human tumor types. The restricted expression of the latent EBV antigens is critical for EBV-associated tumors to escape from immune surveillance. EBV lytic replication can be triggered by various treatments and the induced lytic genes cause strong cytotoxic T lymphocyte (CTL) responses. Histone acetylation or deacetylation is associated with chromatin remodeling and regulates gene expression. Histone deacetylase (HDAC) inhibitors affect cell cycle progression as well as gene expression in a wide variety of transformed cells. We examined whether an HDAC inhibitor, TSA, can affect cell cycle progression and induce EBV lytic replication in EBV-transformed lymphoblastoid cell lines (LCLs). TSA caused cell cycle arrest at low concentrations and induced apoptosis at higher (>300 nM) concentrations in the LCLs and EBV negative BJAB cells. To clarify the underlying mechanism of TSA-induced cell cycle arrest, expression of cell cycle regulatory factors was examined by RNase protection assay and Western blot analysis. Following TSA treatment, a reduced expression of cyclin D2 and an induction of p21 may have played an essential role for G1 arrest in LCLs, while p21 induction might have arrested BJAB cells in G1 phase. A Cdk inhibitor, p57, was increased by 300 nM TSA in both LCLs and BJAB cells, indicating its role in apoptosis. Moreover, immunofluorescene assay and Western blotting showed that TSA induced EBV lytic replication in LCL cells. These results suggest that TSA may exert an enhanced anti-tumor effect for EBV-associated tumors not only by inducing a cell cycle arrest and apoptosis, but also by triggering an EBV lytic cycle.

Combination drug therapy using edaravone and Daio-Orengedoku-to after transient focal ischemia in rats.

In this study, we investigated the effect of Daio-Orengedoku-to (DOT) on ischemic brain damage in a rat model of focal ischemia-reperfusion and attempted to identify synergistic effects for the combination of edaravone and DOT against ischemic insult. Ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 2 h and reperfusion followed for 22 h. To determine the neuroprotective effect of DOT, it was administered orally just before reperfusion and then 2 h after reperfusion. To examine the effects of combination therapy on survival, rats were divided into groups treated with edaravone, DOT, and edaravone and DOT. Microglial activation, neutrophil infiltration and brain-derived neurotrophic factor (BDNF) expression were examined in surviving animals. Infarct volume was significantly reduced by DOT (100, 200 and 400 mg/kg; P < 0.05), and edaravone plus DOT markedly improved the survival rate after transient ischemia (P = 0.0133). Microglial activation was reduced by edaravone and DOT and their combination (P < 0.05), and neutrophil infiltration was lowered in these groups (P < 0.05). BDNF-positive cells were increased in the combination edaravone and DOT group (P < 0.05). It appears that the neuroprotective mechanisms of combined therapy involve inhibition of microglial activation, reduction of invading neutrophils and enhancement of BDNF expression.

Oral efficacy of Vero cell attenuated porcine epidemic diarrhea virus DR13 strain.

A Vero cell attenuated porcine epidemic diarrhea virus (PEDV) strain, DR13, was distinguished from wild-type PEDV using restriction enzyme fragment length polymorphism (RFLP). Cell attenuated DR13 was orally or intramuscularly (IM) administered to late-term pregnant sows, and mortality resulting from the highly virulent PEDV challenge was investigated in passively immunized suckling piglets of the two different groups. The mortality rate of the oral group (13%) was lower than that of the IM group (60%). In particular, the concentration of IgA against PEDV was higher in piglets of sows in the oral group, compared to the IM group. The attenuated DR13 virus remained safe, even after three backpassages in piglets. The findings of this study support the theory that the Vero cell attenuated DR13 virus may be applied as an oral vaccine for inducing specific immunity in late-term pregnant sows with a high margin of protection against PEDV infection.

Factors Delaying Organ Procurement After Declaration of Brain Death in Korea.

BACKGROUND: Organ donation after brain death is a major source for obtaining transplantable organs for patients with end-stage organ disease. However, the time from declaring brain death to organ procurement is often longer than expected. Analyzing factors that delay organ procurement may help to prevent damage to organs from marginal and unstable donors and aid in preparation for recipient operation. The aim of this study was to examine factors associated with the interval between the time of declaring brain death and organ procurement. METHODS: Medical records of patients who underwent organ procurement after brain death from February 2009 to April 2015 were retrospectively reviewed. RESULTS: Of the 77 patients which were scheduled to undergo organ procurement, 68 eventually underwent procurement of ≥1 organ. The average time interval from 1st exam for brain death to organ procurement decreased from 1,248 minutes in 2009 to 910 minutes in 2015. Although not statistically significant, during the 6-year period, the time interval decreased from 1,105 minutes to 1,075 minutes in the latter half of the period (P = .623). Organ procurement was extensively delayed most commonly owing to false negative electroencephalogram (EEG; 62.5%). CONCLUSIONS: With increasing experience in dealing with brain death donors, the time interval from declaring brain death to organ procurement decreased. We suggest that an EEG be performed during the initial stages of examination for brain death to prevent unnecessary preparation of recipient operation owing to a false EEG test.

Renal denervation for treatment of uncontrolled hypertension in an Asian population: results from the Global SYMPLICITY Registry in South Korea (GSR Korea).

Reports detailing the response of hypertensive patients to renal denervation (RDN) in Asian patients are limited. We evaluated 6- and 12-month outcomes after RDN in an Asian population and compared outcomes to a primarily Caucasian population. The Global SYMPLICITY Registry (GSR) is a prospective, all-comer, worldwide registry that evaluates the safety and effectiveness of RDN and includes the Korean registry substudy (GSR Korea) and a Caucasian subset (GSR Caucasian). Given differences in baseline characteristics among GSR Korea (n=93) as compared with GSR Caucasian (n=169) patients, including lower baseline office systolic blood pressure (SBP), lower body mass index and differences in medications, propensity score adjustment was performed when comparing the change in SBP between subsets. The 6- and 12-month change in SBP in GSR Korea was -19.4±17.2 and -27.2±18.1 mm Hg, respectively (P<0.001 for both vs baseline). GSR Caucasian had a SBP change similar to GSR Korea at 6 months (-20.9±21.4 mm Hg, unadjusted P=0.547, adjusted P=0.998), whereas at 12 months the change was significantly less pronounced (-20.1±23.9 mm Hg, unadjusted P=0.004, adjusted P=0.002). There were no protocol-defined procedure-related adverse events and no chronic adverse events associated with the device in an Asian population. RDN provided a significant reduction in 6- and 12-month office SBP among Asian patients, with a favorable safety profile. The 12-month SBP reduction was larger than that observed in Caucasian patients.

Plant-originated glycoprotein, G-120, inhibits the growth of MCF-7 cells and induces their apoptosis.

We characterized the biological function of G-120, glycoprotein isolated from the ethanol extract of the herb, Ulmus davidiana Nakai (UDN). G-120 has anti-tumor activity and significantly inhibited proliferation of MCF-7 cells, as measured by the thymidine uptake assay. In addition, MTT and trypan blue exclusion experiments showed that the G-120-mediated inhibition of DNA synthesis may be due to a cytostatic, rather than a cytotoxic effect. Further studies of DNA analysis and propidium iodide staining revealed that G-120 induces apoptosis in MCF-7 cells. Interestingly, G-120 (100 microg/ml) completely suppressed the binding of NF-kappaB to DNA and increased the cytosolic level of IkappaBalpha which prevented nuclear translocation of NF-kappaB. In addition, G-120 increased the expression of c-Jun, Fra-1, and Fra-2, but did not affect the expression of c-Fos. Collectively, it is believed that G-120 exerts an important role in the induction of apoptosis, suppression of NF-kappaB activation, and induction of c-Jun/Fra-1 or c-Jun/Fra-2 dimerization in MCF-7 cells. Consequently, G-120 could be considered as an anti-cancer agent, although further detailed experiments should be performed.

T cell epitope recognition involved in the low-responsiveness to a region of hen egg lysozyme (46-61) in C57BL/6 mice.

The predominant T cell epitope of hen egg lysozyme (HEL) in high-responder C3H mice has been previously identified as the HEL 46-61 region. In contrast, this region is poorly recognized by T cells from low-responder C57BL/6 mice upon immunization with HEL. In previous studies, we have demonstrated that several C57BL/6 derived T cell hybridomas reactive to this epitope and other HEL epitopes preferentially recognize phosphorylcholine (PC)-conjugated HEL over unconjugated HEL. To understand the mechanisms involved in this difference of T cell recognition, we have further analysed the reactivity of T cells and T cell hybridomas from low-responder C57BL/6 mice. T cells from HEL-immunized mice were preferentially reactive to HEL 47-60. These results suggest a potential deficiency in generating an appropriate T cell epitope from the 46-61 region of native HEL in low-responder C57BL/6 mice. The minimal T cell epitope of this region was defined as HEL 51-60 using the PCH4.1 T hybridoma clone. This minimal epitope represents a single amino acid shift from the minimal epitope of HEL high-responder C3H mice (HEL 52-61). Various peptides representing this region were synthesized with single alanine substitutions at each position. The residues at positions 51, 52, 53 and 57 of HEL appear to be involved in Ia binding and the residues at 55 and 56 in contracting the TCR. T cell reactivity to HEL 51-61 peptides with various substitutions at position 61 strongly suggest that primarily the size of the C-terminal residue interferes with binding to the Ia molecules of low-responder mice. In addition, substitutions of the TCR contacting residues at positions 55 and 56 with similar residues (isoleucine-->leucine or leucine-->isoleucine) significantly increased the T cell reactivity, suggesting a low reactivity with the native residues. Therefore, the requirement of many residues in the T cell epitope for interaction with Ia, the necessity for additional Ag processing to facilitate Ia binding, and the low affinity of the TCR contacting residues may together render C57BL/6 mice unresponsive to the HE 46-61 region.

Lactoferrin causes IgA and IgG2b isotype switching through betaglycan binding and activation of canonical TGF-ß signaling.

Lactoferrin (LF), a pleiotropic iron-binding glycoprotein, is known to modulate the humoral immune response. However, its exact role in Ig synthesis has yet to be elucidated. In this study, we investigated the effect of LF on Ig production by mouse B cells and its underlying mechanisms. LF, like transforming growth factor (TGF)-ß1, stimulated B cells to produce IgA and IgG2b, while downregulating other isotypes. Using limiting dilution analysis, LF was shown to increase the frequency of IgA-secreting B-cell clones. This was paralleled by an increase in Ig germ-line α (GLα) transcripts, indicating that LF plays a role as an IgA switch factor. Interestingly, LF directly interacted with betaglycan (TGF-ß receptor III, TßRIII) and in turn induced phosphorylation of TßRI and Smad3 through formation of the TßRIII/TßRII/TßRI complex, leading to IgA isotype switching. Peroral administration of LF increased intestinal/serum IgA production as well as number of IgA plasma cells in lamina propria. Finally, we found that LF has an adjuvant activity when nontoxigenic Salmonella typhimurium was inoculated perorally, conferring protection against intragastrical infection of toxigenic S. typhimurium. These results suggest that LF has an important effect on the mucosal/systemic IgA response and can contribute to protection against intestinal pathogens.

Up-regulation of the expression of major histocompatibility complex class I antigens by plasmid DNA transfection in non-hematopoietic cells.

The effect of DNA on the surface expression of major histocompatibility (MHC) class I antigens was examined in non-hematopoietic tumor cell lines. Transfection with plasmid DNA via liposome or electroporation significantly increased the surface expression of MHC class I molecules in a transient manner. Northern blot analysis showed that levels of MHC class I mRNA were increased by DNA transfection, probably via transcriptional activation. In contrast, the expression of the MHC class II and beta-actin genes was not affected, suggesting that the up-regulation of MHC class I expression by plasmid DNA works in a gene-specific manner.

Coronary stenting (Cordis) without anticoagulation.

We evaluated the effect of antithrombotic regimens on subacute thrombosis and short-term clinical courses after successful implantation of the Cordis coronary stent, which is a flexible, balloon expandable, radiopaque tantalum stent. Two hundred seventy-five consecutive patients with 290 lesions were treated with 356 Cordis stent implantations. According to poststent antithrombotic regimen, patients were divided into 3 groups; 165 patients with 175 lesions received aspirin 200 mg/day, ticlopidine 500 mg/day, and warfarin for 1 month (group 1), 66 patients with 69 lesions received aspirin and ticlopidine (group 2), and 44 patients with 46 lesions received aspirin alone (group 3) after successful Cordis stenting. The overall procedural success rates were 97.7% in group 1, 98.6% in group 2, and 100% in group 3. More than 65% of the patients were eligible for elective stenting. The overall rate of stent thrombosis was 1.8%: 1.2% in patients assigned to the treatment with aspirin, ticlopidine, and warfarin; 0% in patients with aspirin and ticlopidine; and 6.8% in patients assigned to the treatment with aspirin alone. In conclusion, the Cordis coronary stent is an effective endovascular stent in various clinical indications including unstable angina and acute myocardial infarction. Antiplatelet therapy using aspirin and ticlopidine after successful Cordis coronary stenting is a promising alternative to anticoagulation therapy to overcome the drawbacks of stenting. However, poststent antithrombotic therapy with aspirin alone is associated with a significant rate of stent thrombosis.

Immune induction and modulation in mice following immunization with DNA encoding F protein of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is one of the principal agents of bronchiolitis and pneumonia in young children. Thus, there is a strong need to make a safe and effective vaccine against the RSV infection. DNA immunization is very effective at inducing both cellular and humoral immune responses. In this study, we inserted the RSV-F gene into expression vectors, pcDNA3.1 and pQE. These constructs were transformed into C2C12 and E. coli M15 cells, respectively. The expression of the RSV-F protein was confirmed by SDS-PAGE, followed by Western blot analyses. The immunization of pcDNA3.1-RSV-F elicited both anti-RSV-F titer in mouse sera and CTL activities with mouse splenocytes. Especially, the co-administration of IL-4, or the GM-CSF gene with the RSV-F gene construct, enhanced the production of anti-RSV-F Ab. However, this enhancement disappeared by the simultaneous injection of the Th1 and Th2 type cytokine genes. The CTL activities were affected by the co-delivery of the IFN-gamma gene, but not by Th2-type cytokines.

DNA-mediated immunization of glycoprotein 350 of Epstein-Barr virus induces the effective humoral and cellular immune responses against the antigen.

Epstein-Barr virus (EBV) is a human pathogen that is involved in numerous diseases and tumors. Since the EBV infection occurs in the early ages of life, and most of the population is subsequently exposed to EBV, the conventional method of vaccination to induce the prophylactic immunity cannot be considered effective in coping with the virus infection. In this study, we tested whether the injection of a plasmid vector that contained the gene for glycoprotein 350 (gp350), which had been identified as a ligand for virus' adsorption and a target for virus neutralizing antibodies, could induce effective immune responses against the antigen. As a result, the injection of the constructed plasmid vector into mice induced the production of gp350-specific antibodies. A major isotype of the gp350-specific antibodies was IgG1. The antibodies efficiently mediated the antibody-dependent cellular cytotoxicity against the cells expressing the gp350 antigen. In addition, the injection of the constructed plasmid vector stimulated the precursor T cell population that was specific to the gp350 antigen. In addition, gp350-specific cytotoxic T lymphocytes were efficiently stimulated by the injection of the constructed plasmid vector. These results suggested that the injection of the plasmid vector, containing the gp350 gene of Epstein-Barr virus, could be one of the most effective ways to induce both prophylactic and therapeutic vaccinations against the virus infection.

Cross-linking of MHC class II molecules with anti-MHC class II antibody or epitope peptide prevents resting B lymphocyte differentiation by inhibiting NF-kappaB-dependent gene expression.

To understand the mechanism(s) involved in anti-MHC class II antibody-mediated inhibition of B lymphocyte differentiation, we investigated the influence of anti-MHC class II antibody treatment on the gene expression of IL-6 in resting B lymphocytes, which had been known to be one of the most important cytokines involved in B cell physiology. The level of the IL-6 mRNA expression in the LPS-stimulated resting B cells was remarkably reduced by treatment of the corresponding anti-MHC class II antibodies. The inhibition was exerted in haplotype-specific and dose-dependent manners. Similarly, MHC class II-restricted epitope peptides, when applied as a dimer form, revealed haplotype-specific and dose-dependent inhibitory effects on the IL-6 gene expression by the LPS-stimulated B cells. In addition, treatment of the anti-MHC class II antibody and MHC class II-restricted epitope peptide inhibited, in haplotype-specific and dose-dependent manners, the activation of NF-kappaB, which had been known to be one of the critical transcription factors involved in the IL-6 gene expression. Interestingly, however, exogenous addition of the recombinant IL-6 did not reverse this inhibitory effect by the anti-MHC class II antibody. These results suggest that conjugation of the MHC class II molecules by the anti-MHC class II antibody inhibited B cell differentiation, possibly through the interruption of signaling pathways leading to the IL-6 gene expression via NF-kappaB activation in B lymphocytes.