Identification of Two Critical Neutralizing Epitopes in the Receptor Binding Domain of Hepatitis B Virus preS1.

Hepatitis B virus (HBV) infection is a major public health problem. Human hepatocytes are infected with HBV via binding between the preS1 region in the large envelope protein of HBV and sodium taurocholate cotransporting polypeptide. Although several monoclonal antibodies (MAbs) that recognize the receptor binding domain in preS1 and neutralize HBV infection have been isolated, details of neutralizing epitopes are not understood. In this study, we generated 13 MAbs targeting the preS1 receptor binding domain from preS1-specific memory B cells derived from DNA immunized mice. The MAbs were classified into three groups according to the epitope regions, designated epitopes I-III. A virus neutralization assay revealed that MAbs recognizing epitopes I and III neutralized HBV infection, suggesting that these domains are critical epitopes for viral neutralization. In addition, a neutralization assay against multiple genotypes of HBV revealed that epitope I is a semi-pangenotypic neutralizing epitope, whereas epitope III is a genotype-specific epitope. We also showed that neutralizing MAbs against preS1 could neutralize HBV bearing vaccine-induced escape mutation. These findings provide insight into novel immunoprophylaxis for the prevention and treatment of HBV infection.IMPORTANCE The HBV preS1 2-47 aa region (preS1/2-47) is essential for virus binding with sodium taurocholate cotransporting polypeptide. Several MAbs targeting preS1/2-47 have been reported to neutralize HBV infection; however, which region in preS1/2-47 contains the critical neutralizing epitope for HBV infection is unclear. Here, we generated several MAbs targeting preS1/2-47 and found that MAbs recognizing the N- or C-terminus of preS1/2-47 remarkably neutralized HBV infection. We further confirmed the neutralizing activity of anti-preS1 MAbs against HBV with vaccine escape mutation. These data clarified the relationship between the antibody epitope and the virus neutralizing activity and also suggested the potential ability of a vaccine antigen containing the preS1 region to overcome the weakness of current HB vaccines comprising the small S protein.

Dynamic rotation of the protruding domain enhances the infectivity of norovirus.

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the resting conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two similar P domain conformations were also found simultaneously in the human norovirus GII.3 capsid, although the mechanism of the conformational change is not yet clear. These results provide new insights into the mechanisms of non-enveloped norovirus transmission that invades host cells, replicates, and sometimes escapes the hosts immune system, through dramatic environmental changes in the gastrointestinal tract.

Surfeit 4 Contributes to the Replication of Hepatitis C Virus Using Double-Membrane Vesicles.

A number of positive-strand RNA viruses, such as hepatitis C virus (HCV) and poliovirus, use double-membrane vesicles (DMVs) as replication sites. However, the role of cellular proteins in DMV formation during virus replication is poorly understood. HCV NS4B protein induces the formation of a "membranous web" structure that provides a platform for the assembly of viral replication complexes. Our previous screen of NS4B-associated host membrane proteins by dual-affinity purification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and small interfering RNA (siRNA) methods revealed that the Surfeit 4 (Surf4) gene, which encodes an integral membrane protein, is involved in the replication of the JFH1 subgenomic replicon. Here, we investigated in detail the effect of Surf4 on HCV replication. Surf4 affects HCV replication in a genotype-independent manner, whereas HCV replication does not alter Surf4 expression. The influence of Surf4 on HCV replication indicates that while Surf4 regulates replication, it has no effect on entry, translation, assembly, or release. Analysis of the underlying mechanism showed that Surf4 is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs and the structural integrity of RNA replication complexes. Surf4 also participates in the replication of poliovirus, which uses DMVs as replication sites, but it has no effect on the replication of dengue virus, which uses invaginated/sphere-type vesicles as replication sites. These findings clearly show that Surf4 is a novel cofactor that is involved in the replication of positive-strand RNA viruses using DMVs as RNA replication sites, which provides valuable clues for DMV formation during positive-strand RNA virus replication.IMPORTANCE Hepatitis C virus (HCV) NS4B protein induces the formation of a membranous web (MW) structure that provides a platform for the assembly of viral replication complexes. The main constituents of the MW are double-membrane vesicles (DMVs). Here, we found that the cellular protein Surf4, which maintains endoplasmic reticulum (ER)-Golgi intermediate compartments and the Golgi compartment, is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs. Moreover, Surf4 participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated vesicles as replication sites. These results indicate that the cellular protein Surf4 is involved in the replication of positive-strand RNA viruses that use DMVs as RNA replication sites, providing new insights into DMV formation during virus replication and potential targets for the diagnosis and treatment of positive-strand RNA viruses.

Immune-Focusing Properties of Virus-like Particles Improve Protective IgA Responses.

Virus-like particles (VLPs) provide a well-established vaccine platform; however, the immunogenic properties acquired by VLP structure remain poorly understood. In this study, we showed that systemic vaccination with norovirus VLP recalls human IgA responses at higher magnitudes than IgG responses under a humanized mouse model that was established by introducing human PBMCs in severely immunodeficient mice. The recall responses elicited by VLP vaccines depended on VLP structure and the disruption of VLP attenuated recall responses, with a more profound reduction being observed in IgA responses. The IgA-focusing property was also conserved in a murine norovirus-primed model under which murine IgA responses were recalled in a manner dependent on VLP structure. Importantly, the VLP-driven IgA response preferentially targeted virus-neutralizing epitopes located in the receptor-binding domain. Consequently, VLP-driven IgA responses were qualitatively superior to IgG responses in terms of the virus-neutralizing activity in vitro. Furthermore, the IgA in mucosa obtained remarkable protective function toward orally administrated virus in vivo. Thus, our results indicate the immune-focusing properties of the VLP vaccine that improve the quality/quantity of mucosal IgA responses, a finding with important implications for developing mucosal vaccines.

Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells.

Norovirus is the leading cause of acute gastroenteritis worldwide. Since the discovery of human norovirus (HuNoV), an efficient and reproducible norovirus replication system has not been established in cultured cells. Although limited amounts of virus particles can be produced when the HuNoV genome is directly transfected into cells, the HuNoV cycle of infection has not been successfully reproduced in any currently available cell-culture system. Those results imply that the identification of a functional cell-surface receptor for norovirus might be the key to establishing a norovirus culture system. Using a genome-wide CRISPR/Cas9 guide RNA library, we identified murine CD300lf and CD300ld as functional receptors for murine norovirus (MNV). The treatment of susceptible cells with polyclonal antibody against CD300lf significantly reduced the production of viral progeny. Additionally, ectopic CD300lf expression in nonsusceptible cell lines derived from other animal species enabled MNV infection and progeny production, suggesting that CD300lf has potential for dictating MNV host tropism. Furthermore, CD300ld, which has an amino acid sequence in the N-terminal region of its extracellular domain that is highly homologous to that of CD300lf, also functions as a receptor for MNV. Our results indicate that direct interaction of MNV with two cell-surface molecules, CD300lf and CD300ld, dictates permissive noroviral infection.

Evaluation of the use of various rat strains for immunogenic potency tests of Sabin-derived inactivated polio vaccines.

Slc:Wistar rats have been the only strain used in Japan for purpose of evaluating a national reference vaccine for the Sabin-derived inactivated polio vaccine (sIPV) and the immunogenicity of sIPV-containing products. However, following the discovery that the Slc:Wistar strain was genetically related to the Fischer 344 strain, other "real" Wistar strains, such as Crlj:WI, that are available worldwide were tested in terms of their usefulness in evaluating the immunogenicity of the past and current lots of a national reference vaccine. The response of the Crlj:WI rats against the serotype 1 of sIPV was comparable to that of the Slc:Wistar rats, while the Crlj:WI rats exhibited a higher level of response against the serotypes 2 and 3. The immunogenic potency units of a national reference vaccine determined using the Slc:Wistar rats were reproduced on tests using the Crlj:WI rats. These results indicate that a titer of the neutralizing antibody obtained in response to a given dose of sIPV cannot be directly compared between these two rat strains, but that, more importantly, the potency units are almost equivalent for the two rat strains.

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B.

UNLABELLED: It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE: The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors.

Effects of atorvastatin on renal function in patients with dyslipidemia and chronic kidney disease: assessment of clinical usefulness in CKD patients with atorvastatin (ASUCA) trial.

BACKGROUND: Dyslipidemia is a risk factor for the progression of chronic kidney disease (CKD). While conventional lipid lowering therapy provides a benefit to CKD management, the effect of statins on eGFR remains unclear. METHODS: A prospective, multi-center, open-labeled, randomized trial. Total of 349 CKD patients with hyperlipidemia were randomized into 2 groups, and followed for 2 years. Group A included patients who were treated with atorvastatin. Group C were treated with conventional lipid lowering drugs other than statin. Primary endpoint was changes in eGFR. Secondary endpoints included changes in urinary albumin excretion, serum LDL-C, serum triglyceride, cardio-vascular events and all-cause mortality. RESULTS: As the primary endpoint, eGFR decreased by 2.3 ml/min/1.73 m2 in Group A and by 2.6 ml/min/1.73 m2 in Group C, indicating that there was no difference in change of eGFR between the two groups. As secondary endpoints, atorvastatin succeeded to reduce serum LDL-C level significantly and rapidly, but conventional therapy did not. In fact, mean LDL-C level did not reach the target level of 100 mg/dl in Group C. Serum triglyceride was lowered only by atorvastatin, but not conventional drugs. The number of cardiovascular events and all-cause mortality did not differ between in two groups. CONCLUSION: The ASUCA (Assessment of Clinical Usefulness in CKD Patients with Atorvastatin) trial demonstrated that atorvastatin failed to exhibit reno-protections compared to conventional therapy in Japanese patients with dyslipidemia and CKD. It would be due in part to the ability of atorvastatin to more potently reduce serum LDL and triglycerides compared to conventional therapy.

Relationships between estimation errors and falls in healthy aged dwellers.

BACKGROUND: Although fall predictions using motor ability have been well reported in elderly people, there are few reports on physical cognitive ability. OBJECTIVE: To examine the relationship of the results of motor function tests that include physical cognitive ability on the ability to predict falls and to determine which test is the most appropriate. METHODS: We studied 174 community-dwelling elderly adults (mean age 75.7 ± 5.7, 41 males and 133 females), and measured grip strength, one-leg standing time (OLS), timed up and go test (TUG), functional reach test, sit and reach test, and maximal step length (MSL). The estimation error (EE), which was defined as the difference between the predicted and actual values, was calculated in all motor ability tests. Other assessments included the number of falls in the previous year, BMI, frequency of going out, Mini-Mental State Examination score, and Falls Efficacy Scale. In the baseline study, we divided the subjects into a fall group (n = 33) and a nonfall group (n = 141) and compared motor ability and EE for the two groups. During a 1-year follow-up, the nonfall group (baseline study) was assessed for the same measurements by using the same methods. RESULTS: In the baseline study, the fall group had significantly lower values of OLS and MSL. Furthermore, the fall group significantly overestimated their OLS, TUG, and MSL. In logistic regression analysis, EE of TUG (OR = 1.27) and EE of MSL (OR = 1.08) were detected as risk factors for falls. During follow-up, 11 subjects (7.8%) experienced falls. In logistic regression analysis, TUG (OR = 1.89) and EE of MSL (OR = 1.06) were detected as significant risk factors for falls. Since EE of MSL had higher values of both the area under the receiver operating characteristic curve and the sum of sensitivity and specificity than EE of TUG, the nonfall group was divided into two groups with a cutoff value of 2 cm for EE of MSL. A significant distribution disparity in falls between the two groups was found during follow-up and showed a relative risk of 18.78 for EE of MSL. CONCLUSIONS: We suggest that EE of MSL is a potent predictor for falls among healthy elderly adults.

Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP.

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood-borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.

Effects of atorvastatin on renal function in patients with dyslipidemia and chronic kidney disease: rationale and design of the ASsessment of clinical Usefulness in CKD patients with Atorvastatin (ASUCA) trial.

BACKGROUND: Since dyslipidemia has been shown to be an independent risk factor for the progression of chronic kidney disease (CKD), low-density lipoprotein cholesterol (LDL-C)-lowering therapy can be potentially associated with inhibition of CKD progression. The ASsessment of clinical Usefulness in CKD patients with Atorvastatin (ASUCA) trial was designed to determine whether atorvastatin has protective effects on renal function in patients with dyslipidemia and CKD. METHODS: We decided to carry out a prospective multi-center, open-labeled, randomized trial to compare the reno-protective effects between diet therapy alone and atorvastatin plus diet therapy in patients with dyslipidemia (LDL-C ≥ 140 mg/dL if not treated or LDL-C ≥ 100 mg/dL if treated with lipid-lowering drugs in subjects taking dyslipidemia-treating agents other than statins) and CKD [estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m(2)]. The primary endpoint is the change in eGFR (mL/min/1.73 m(2)) as calculated by the modified MDRD equation for Japanese after 2 years of treatment. RESULTS: Enrollment began in April 2009 and was completed in March 2011. A total of 334 patients (213 male and 121 female) were randomly assigned to either diet therapy alone or atorvastatin plus diet therapy and included in an intent-to-treat population. In the atorvastatin and control groups, the mean ages were 63.2 and 63.1 years, mean eGFRs were 55.9 and 54.0 mL/min/1.73 m(2), and median urinary albumin/creatinine ratios were 24.9 and 29.1 mg/g, respectively. CONCLUSIONS: This study distinguishes itself from similar studies by increasing statistical accuracy derived from its significantly larger sample size and longitudinal magnitude. The results of this study will help to determine whether atorvastatin has reno-protective effects in patients with dyslipidemia and CKD.

[Clinical implications from the results of the CASE-J extension].

The Candesartan Antihypertensive Survival Evaluation in Japan (CASE-J) trial was conducted to compare the effects of the angiotensin II receptor blocker (ARB) candesartan and the calcium channel blocker (CCB) amlodipine on the incidence of cardiovascular events in Japanese high-risk hypertensive patients. The CASE-J Extension (CASE-J Ex) was an observational study designed to evaluate the long-term effects of ARB candesartan and CCB amlodipine, incorporating an additional 3-year follow-up of the CASE-J trial. As in the CASE-J trial, no statistically significant difference was observed in the incidence of primary cardiovascular events, all-cause mortality, or cardiovascular death between the two groups. The superiority of candesartan over amlodipine in reducing new-onset diabetes was sustained in the three-year-long CASE-J Ex, thereby corroborating the results of the CASE-J trial.

Influence of coronary risk factors on coronary events in Japanese high-risk hypertensive patients. - Primary and secondary prevention of ischemic heart disease in a subanalysis of the Candesartan Antihypertensive Survival Evaluation in Japan (CASE-J) trial-.

BACKGROUND: The Candesartan Antihypertensive Survival Evaluation in Japan (CASE-J) trial was conducted to compare the effects of candesartan and amlodipine on cardiovascular events in Japanese high-risk hypertensive patients. The aim of the present subanalysis was to evaluate the influence of coronary risk factors on coronary events in these patients as an observational study irrespective of allocated drugs. METHODS AND RESULTS: The adjusted hazard ratios (HRs) of the association of baseline risk factors including gender, age, allocated drugs, body mass index, systolic/diastolic blood pressure (SBP/DBP), diabetes mellitus (DM), hyperlipidemia (HL), smoking, left ventricular hypertrophy, previous ischemic heart disease (IHD), previous cerebrovascular events, and chronic kidney disease (CKD) with coronary events in 4,703 patients who were enrolled in the CASE-J trial, were examined. The coronary events occurred in 83 patients, and were significantly associated with previous IHD, DM, male sex, CKD, and low DBP. Significant predictors were previous IHD (HR, 3.89), DM (HR, 3.10), male sex (HR, 1.81), CKD (HR, 1.60), and low DBP (HR, 1.36), respectively. In 4,107 patients without previous IHD, DM (HR, 4.88), HL (HR, 2.67), and DBP (HR, 1.39) were significantly associated with the risk of coronary events, while male sex (HR, 3.03), CKD (HR, 2.44), and DM (HR, 2.15) were in 596 patients with previous IHD. CONCLUSIONS: DM is the important factor in both primary and secondary prevention of coronary events. Comprehensive risk management including surveillance of DM, CKD and HL is needed for preventing coronary events, in addition to blood pressure control.

Impact of left ventricular hypertrophy on the time-course of renal function in hypertensive patients ­ a subanalysis of the CASE-J trial ­.

BACKGROUND: In this subanalysis of the CASE-J, which was conducted to compare the effects of candesartan and amlodipine in Japanese high-risk hypertensive patients, THE ASSOCIATION OF LEFT VENTRICULAR HYPERTROPHY (LVH) WITH RENAL FUNCTION IS CLARIFIED. METHODS AND RESULTS: Patients were divided into 2 groups: 1,082 patients with LVH and 2,119 patients without LVH. The primary endpoint was the change in the estimated glomerular filtration rate (eGFR). The eGFRs were increased from 63.6 to 65.1 ml · min(-1) · 1.73 m(-2) in patients with LVH and from 63.6 to 68.5 ml · min(-1) · 1.73 m(-2) in those without LVH. The improvement in the eGFR was greater in patients without LVH than in those with LVH (P=0.004). In patients with chronic kidney disease (CKD) patients, the eGFR increased from 52.7 to 60.5 ml · min(-1) · 1.73 m(-2) in patients without LVH, but from 53.1 to 57.2 ml · min(-1) · 1.73 m(-2) in those with LVH (P<0.001, patients without LVH vs patients with LVH). Furthermore, because the eGFR changed from 76.5 to 75.4 ml · min(-1) · 1.73 m(-2) in patients without CKD but with LVH, and from 76.5 to 77.5 ml · min(-1) · 1.73 m(-2) in those without either CKD or LVH, the final eGFR was higher in patients without LVH than in those with LVH (P=0.048). CONCLUSIONS: LVH related to the time-course of renal function in Japanese hypertensive patients.

Improvement of the light extraction efficiency of top-emitting organic light-emitting diodes by a two-dimensional diffraction layer fabricated using self-assembled nanoparticles.

The light extraction efficiency of top-emitting organic light-emitting diodes (OLEDs) was improved by insertion of a two-dimensional (2D) diffraction layer. The 2D diffraction layer was fabricated by our original nanofabrication technique, the embedded particle monolayer method, which could form a self-assembled particle monolayer. As a result, the electroluminescence intensity of the device with the 2D diffraction layer was improved by 1.67 times (in total luminous flux) and 2.07 times (in peak wavelength). High luminance top-emitting OLEDs were fabricated using the potentially low-cost self-assembling technique.

Fractional quantum Hall effects in In0.75Ga0.25As bilayer electron systems observed as "Finger print".

Observations of fractional quantum Hall (FQH) plateaus are reported in bilayer electron gas system in wide (>80 nm) In0.75Ga0.25As wells. Several q/p (p = 5, 3, and 2, q > 5) QH states are confirmed at high temperatures (~1.6 K) when the critical conditions including an electron density imbalance as well as a dynamical resistance behavior at the bilayer-monolayer transition are properly satisfied. The former leads to a quantum limit in either of the layers and the latter might bring a meta-stable nature into FQH phenomena. Such a behavior occurs as a probability process associating with impurities or defects in the wells, they inevitably reflect the local structural landscapes of each sample. This is verified by the new finding that the kinds of fractional plateaus (what set of fractional filling factors) appeared are different depending on the samples, that is, they are the "finger print" in each sample.

The optimal target blood pressure for antihypertensive treatment in Japanese elderly patients with high-risk hypertension: a subanalysis of the Candesartan Antihypertensive Survival Evaluation in Japan (CASE-J) trial.

For hypertensive patients, it has been recommended that antihypertensive treatment strategies be chosen on the basis of the patients' conditions and age. In this sub-analysis of the Candesartan Antihypertensive Survival Evaluation in Japan (CASE-J) trial, we aimed to compare the effects of candesartan and amlodipine on cardiovascular mortality and morbidity in Japanese elderly patients with high-risk hypertension and to determine their optimal target blood pressures (BPs). The effect of the two drugs on cardiovascular events was compared across different age subgroups (<65, 65-74, and 75-84 years) by use of Cox regression analysis. We also evaluated the associations between the achieved BP and the incidence of cardiovascular events, irrespective of the allocated drugs in multiple Cox regression analyses. The incidence of cardiovascular events was independent of the assigned treatment for each of the age subgroups. For systolic BP (SBP), cardiovascular risk increased steeply when control of SBP was inadequate (higher than 140 mmHg) for patients younger than 65 years old and those between 65 and 74 years old. Patients aged 75 to 84 years old showed a significantly increased risk when their SBP was >or=150 mmHg. For diastolic BP (DBP), the risk significantly increased for the subgroup aged 75 to 84 years when the DBP was >or=85 mmHg. The present results show that candesartan and amlodipine are equally effective in Japanese elderly patients with high-risk hypertension. Moreover, it is important to control BP levels to less than 150/85 mmHg for patients 75-84 years old.

Amino-imino tautomerization reaction of the 4-aminopyrimidine/acetic acid system.

The amino-imino tautomerization of the 4-aminopyrimidine (4APM)/acetic acid (AcOH) system through dual hydrogen bonding in n-hexane at room temperature was investigated using UV absorption and fluorescence spectroscopies, fluorescence time-profile measurements, and molecular orbital calculations, with those of the imino-model compound of 3-methyl-4(1H)-pyrimidinimine (3M4PMI). From the experimental results, it was confirmed that the imino-tautomer was formed in the first excited singlet state (S1) state through the double-proton transfer of the dual hydrogen-bonded complex of 4APM with AcOH. The fluorescences of the free 4APM monomer (band maximum at 353nm), imino-tautomer (at 414nm), and 3M4PMI monomer (at 437nm) exhibit the single-exponential decays of 98, 73, and 19ps time constants, respectively. The shorter decay time of the imino-tautomer fluorescence compared with the free monomer one is suggestive of the low activation energy process in the S1 state. From the result of the shortest decay time of the 3M4PMI fluorescence, it can be deduced that 3M4PMI has a non-planar structure in the S1 state. The theoretical calculations on the S0 and S1 state double-proton transfer for the 4APM/AcOH dual hydrogen-bonded system were performed with the use of formic acid (FoOH) in place of AcOH for the sake of simplicity. Only one peak of transition state was resolved in the S0 and S1 states. The energy barrier for the S1 state double-proton transfer of the 4APM/FoOH complex-->3H-4(1H)-pyrimidinimine/FoOH tautomer was estimated to be approximately 2kJmol(-1) using the CIS/6-31G(d) methods. On the other hand, the energy barrier for the S0 state reverse proton transfer of the 3H-4(1H)-pyrimidinimine/FoOH tautomer-->4APM/FoOH complex was estimated to be almost zero kJmol(-1) at B3LYP/6-31G(d) level.

Factors affecting premature plantarflexor muscle activity during hemiparetic gait.

In hemiparetic stroke survivors, premature plantarflexor muscle activity (PPF) often appears as a gait abnormality from terminal swing to the loading response on the paretic side. This study aimed to discern factors giving rise to PPF. Lower extremity function, spasticity magnitude, and gait electromyograms were assessed in 31 hemiparetic stroke survivors. Mean amplitudes during gait phases were determined for the paretic soleus, tibialis anterior, rectus femoris, and biceps femoris. The subjects were classified into PPF and non-PPF groups based on their relative soleus amplitude at different phases of gait, and group differences in each measurement were calculated and subjected to logistic regression. The PPF group showed less activity of the tibialis anterior during the swing phase but greater activity of the rectus femoris during the swing phase and of the biceps femoris, both prematurely and during the loading response. Logistic regression revealed premature activity of the biceps femoris to be a significant variable related to presence of PPF (odds ratio = 1.054). PPF in hemiparetic gait may work with the biceps femoris to supplement compromised lower extremity extension strength. PPF might be reduced by attaining enhanced strength of the hip and knee extensors at the time of initial contact during gait.

Neutralizing Antibodies Induced by Gene-Based Hydrodynamic Injection Have a Therapeutic Effect in Lethal Influenza Infection.

The influenza virus causes annual epidemics and occasional pandemics and is thus a major public health problem. Development of vaccines and antiviral drugs is essential for controlling influenza virus infection. We previously demonstrated the use of vectored immune-prophylaxis against influenza virus infection. We generated a plasmid encoding neutralizing IgG monoclonal antibodies (mAbs) against A/PR/8/34 influenza virus (IAV) hemagglutinin (HA). We then performed electroporation of the plasmid encoding neutralizing mAbs (EP) in mice muscles and succeeded in inducing the expression of neutralizing antibodies in mouse serum. This therapy has a prophylactic effect against lethal IAV infection in mice. In this study, we established a new method of passive immunotherapy after IAV infection. We performed hydrodynamic injection of the plasmid encoding neutralizing mAbs (HD) involving rapid injection of a large volume of plasmid-DNA solution into mice via the tail vein. HD could induce neutralizing antibodies in the serum and in several mucosal tissues more rapidly than in EP. We also showed that a single HD completely protected the mice even after infection with a lethal dose of IAV. We also established other isotypes of anti-HA antibody (IgA, IgM, IgD, and IgE) and showed that like anti-HA IgG, anti-HA IgA was also effective at combating upper respiratory tract IAV infection. Passive immunotherapy with HD could thus provide a new therapeutic strategy targeting influenza virus infection.