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The COVID-19 pandemic has drastically changed global lifestyles. Some reports about lifestyle changes during this pandemic have been published. However, these studies have not assessed gender differences. Thus, we analyzed three lifestyle changes to determine gender differences.We analyzed physical activity, snacking habits, and drinking habits in 323 patients with diabetes. Gender differences in lifestyle habits were analyzed using the ꭓOf the 323 patients, 212 were male and 111 were female. When examined by quarter, the HbA1c values increased significantly in 2020 compared with that in 2019 in the July-September period. In terms of gender differences in the changes of lifestyle habits, decreased physical activity was higher in women. The factors that affected deterioration in HbA1c were snacking habits for the overall and the male populations.The lifestyle changes differed between the genders during the pandemic. A balanced diet is important for all patients with diabetes. Additionally, more attention should be paid to physical inactivity in women.

The renal protective effects of SGLT2 inhibitors are known to be due to the elimination of glomerular hypertension and improvement of hypoxia and oxidative stress in the proximal tubule. Therefore, this increased hematocrit (ΔHct) level has been hypothesized to indicate restored tubular function and improved renal prognosis. To analyze the relationship between ΔHct and decreased estimated glomerular filtration rate (eGFR) after SGLT2 inhibitor administration backward from medical record data. Data from 206 patients who continued SGLT2 inhibitors for3 years were analyzed. The decreased eGFR after administration of SGLT2 inhibitors was defined as Slope B. Factors statistically significantly associated with Slope B in multiple regression analysis were systolic blood pressure (sBP) (β -.211, P = .03), short-term decreased eGFR after SGLT2 inhibitor administration (initial dip) (β -.235, P = .003), ΔHct (β -.185, P = .026), and urine protein (β -.204, P = .015). These findings were the opposite of our hypothesis. ΔHct was not a marker indicating improved renal prognosis and may reflect the extent of the proximal tubular disorder before administering SGLT2 inhibitors.

Background An association between uric acid (UA) and cardiovascular diseases, including heart failure (HF), has been reported. However, whether UA is a causal risk factor for HF is controversial. In particular, the prognostic value of lowering UA in patients with HF with preserved ejection fraction (HFpEF) is unclear. Methods and Results We enrolled patients with HFpEF from the PURSUIT‐HFpEF (Prospective Multicenter Observational Study of Patients With Heart Failure With Preserved Ejection Fraction) registry. We investigated whether UA was correlated with the composite events, including all‐cause mortality and HF rehospitalization, in patients with hyperuricemia and HFpEF (UA >7.0 mg/dL). Additionally, we evaluated whether lowering UA for 1 year (≥1.0 mg/dL) in them reduced mortality or HF rehospitalization. We finally analyzed 464 patients with hyperuricemia. In multivariable Cox regression analysis, UA was an independent determinant of composite death and rehospitalization (hazard ratio [HR], 1.15 [95% CI, 1.03–1.27], P =0.015). We divided them into groups with severe and mild hyperuricemia according to median estimated value of serum UA (8.3 mg/dL). Cox proportional hazards models revealed the incidence of all‐cause mortality was significantly higher in the group with severe hyperuricemia than in the group with mild hyperuricemia (HR, 1.73 [95% CI, 1.19–2.25], P =0.004). The incidence of all‐cause mortality was significantly decreased in the group with lowering UA compared with the group with nonlowering UA (HR, 1.71 [95% CI, 1.02–2.86], P =0.041). The incidence of urate‐lowering therapy tended to be higher in the group with lowering UA than in the group with nonlowering UA (34.9% versus 24.6%, P =0.06). Conclusions UA is a predictor for the composite of all‐cause death and HF rehospitalization in patients with hyperuricemia and HFpEF. In these patients, lowering UA, including the use of urate‐lowering therapy, may improve prognosis.

Background: Little is known about factors associated with elevated N-terminal pro B-type natriuretic peptide (NT-proBNP) at the convalescent stage and their effects on 1-year outcomes in patients with heart failure with preserved ejection fraction (HFpEF). Methods and Results: This study included 469 patients with HFpEF. Elevated NT-proBNP was defined as the highest quartile. The first 3 quartiles (Q1-Q3) were combined together for comparison with the fourth quartile (Q4). Median NT-proBNP concentrations in Q1-Q3 and Q4 were 669 and 3,504 pg/mL, respectively. Multivariate logistic regression analysis revealed that low albumin (odds ratio [OR] 2.44; 95% confidence interval [CI] 1.35-4.39; P=0.003), low estimated glomerular filtration rate (OR 5.83; 95% CI 3.46-9.83; P

Background Pneumonia of unknown pathogenesis detected in Wuhan, Hubei Province, China, in December 2019 was later found to be a new coronavirus infection that causes the now-named coronavirus disease 2019 (COVID-19). The onset of the pandemic has drastically changed global lifestyles. Some reports about lifestyle changes in patients with diabetes during COVID-19 pandemic have been published. However, these studies have not assessed sex differences. Thus, we analyzed three lifestyle changes during the COVID-19 pandemic to determine any sex differences. Methods We analyzed physical activity, snacking habits, and drinking habits in 323 patients with diabetes. Sex differences in the changes in these three lifestyle habits were analyzed using the 𝜒2 test, and the factors that influenced the deterioration of HbA1c were analyzed by paired t-test. The factors that influenced the deterioration of HbA1c were determined by univariate and multivariate logistic regression analyses. Results Of the 323 diabetic, 212 were male (65.6%) and 111 were female (34.4%). HbA1c values were higher in 2020 compared with those in 2019. Increased physical activity was higher in men, whereas decreased physical activity was higher in women. The factors that affected the deterioration in HbA1c were snacking habits for the overall population and in the male sex. No factors significantly affected HbA1c in the female population. Conclusions Our study confirmed that lifestyle changes differed between the sexs during the COVID-19 pandemic. Snacks influenced the deterioration of HbA1c in the overall study population and in the male population. A balanced diet and regular exercise are important for all patients with diabetes during this pandemic. This study demonstrates that more attention should be given on men’s diet, whereas physical inactivity should be emphasized for women.

An automotive photovoltaic (PV) system using an isolated modular boost converter (IMBC) on an electric vehicle is fabricated. The PV system prototype can not only keep generating power from the sun under partial shade, but also charge low-voltage PV power to the high voltage battery. The PV system prototype generated 4.11 kWh in 8 hours under actual sunlight. In addition, partial shade experiment was performed to clarify the influence to distributed maximum power point tracking (MPPT) control that deployed to the IMBC. It was confirmed that more power was generated under a shadow covering the long side of the PV strings than under a shadow covering the short side of the PV strings since the voltage of optimal operating point of PV module changes less.

An isolated modular boost converter (IMBC) consisting of plural phase-shifted full bridge converters is proposed as a solution for an automotive photovoltaic system. The IMBC not only controls multiple photovoltaic (PV) modules around their maximum power points individually, but also supplies distributed power from each PV module to the high voltage battery used for propelling the vehicle through boosting by the cascaded structure. This paper shows that 4M-IMBC prototype consisting of four boost modules can operate with distributed maximum power point tracking control under both a solar irradiance imbalance and partial shade transitions. In addition, 12M-IMBC prototype consisting of twelve boost modules charged power by actual photovoltaic modules to the high voltage battery. The average measured power conversion efficiency during sunny weather was 84.8%, which is similar to the efficiency of a single boost module.

Background: Secondary prevention in patients with myocardial infarction (MI) is critically important to prevent ischaemic heart failure and reduce social burden. Pioglitazone improves vascular dysfunction and prevents coronary atherosclerosis, mainly via anti-inflammatory and antiatherogenic effects by enhancing adiponectin production in addition to antihyperglycemic effects, thus suggesting that pioglitazone attenuates cardiovascular events in patients with mild (HbA1c levels < 6·5%) diabetes mellitus (DM). Therefore, we evaluated the effects of pioglitazone on cardiovascular events in patients with both previous MI and mild DM. Methods: In this multicentre, prospective, randomised, open, blinded-endpoint trial of PPAR study, we randomly assigned 630 patients with mild DM with a history of MI to undergo either DM therapy with (pioglitazone group) or without (control group) pioglitazone. DM was diagnosed using the 75-g oral glucose tolerance test, and mild DM was defined if HbA1c level was < 6·5%. The primary endpoint was the composite of cardiovascular death and hospitalisation caused by acute MI, unstable angina, coronary revascularisation (including percutaneous coronary intervention and cardiac bypass surgery), and stroke. HbA1C levels were 5·9 and 5·8% (p = 0.71) at baseline and 6·0 and 5·8% (p < 0.01) at 2 years for the control and pioglitazone groups, respectively. Findings: The primary endpoint was observed in 14·2% and 14·1% patients in the control and pioglitazone groups during two years, respectively; the incidence of MI and stroke was 0·3% and 2·2% and 1·0% and 0·3%, respectively. Post-hoc analyses of the 7-year observation period showed that these trends became clearer. Interpretation: Pioglitazone could not reduce the occurrence of cardiovascular events in patients with mild DM and previous MI. This result contradicts the results of the IRIS trial that pioglitazone decreased cardiovascular events in stroke patients or the results of the PROactive trial that pioglitazone decreased cardiovascular events in patients with moderate DM and macrovascular disease. Funding: The Grants of Japan Heart Foundation for PPAR study. Conflict of Interest: Dr. Asakura reports grants from Acterion Pharmaceutical Japan, grants from Boehringer Ingelheim Japan, Inc., personal fees from Otsuka Pharmaceutical Co., Ltd., personal fees from Sanofi K.K., personal fees from Bayer Yakuhin, Ltd., personal fees from MSD K.K., personal fees from Takeda Pharmaceutical Company Limited., personal fees from ONO PHARMACEUTICAL CO., LTD., personal fees from Mitsubishi Tanabe Pharma Corporation, personal fees from DAIICHI SANKYO COMPANY, LIMITED , personal fees from Pfizer Japan Inc., personal fees from Astellas Amgen Biopharma, outside the submitted work. Dr. Kim has nothing to disclose. Dr. Asanuma reports grants from Japan Heart Foundation, during the conduct of the study; grants from Japan Society For The Promotion Of Science (Grants-in-Aid For Scientific Research), grants from Novartis Pharmaceuticals Corporation, grants from Mitsubishi Tanabe Pharma Corporation, personal fees from Otsuka Pharmaceutical Co,Ltd, personal fees from Kyowa Hakko Kirin Co, Ltd, personal fees from Daiichi Sankyo Company, Limited, outside the submitted work. Dr. Hamasaki has nothing to disclose. Dr. Nakama has nothing to disclose. Dr. Tsukahara reports grants from Japan Heart Foundation, during the conduct of the study. Dr. Higashino reports grants from Japan Heart Foundation, during the conduct of the study. Dr. Ishikawa reports grants from Japan Heart Foundation during the conduct of the study. Dr. Koba reports grants from Japan Heart Foundation during the conduct of the study; grants from Daiichi Sankyo Company, Ltd, personal fees from MSD K.K. outside the submitted work. Ethical Approval Statement: The study was performed following the principles of the Declaration of Helsinki and the Japanese ethical guidelines for clinical research.

We evaluated the effects of an alpha-glucosidase inhibitor, voglibose, on cardiovascular events in patients with a previous myocardial infarction (MI) and impaired glucose tolerance (IGT). This prospective, randomized, open, blinded-endpoint study was conducted in 112 hospitals and clinics in Japan in 3000 subjects with both previous MI and IGT receiving voglibose (0.6 mg/day, n = 424) or no drugs (n = 435) for 2 years. The Data and Safety Monitoring Board (DSMB) recommended discontinuation of the study in June 2012 after an interim analysis when the outcomes of 859 subjects were obtained. The primary endpoint was cardiovascular events including cardiovascular death, nonfatal MI, nonfatal unstable angina, nonfatal stroke, and percutaneous coronary intervention/coronary artery bypass graft. Secondary endpoints included individual components of the primary endpoint in addition to all-cause mortality and hospitalization due to heart failure. The age, ratio of males, and HbA1C were 65 vs. 65 years, 86 vs. 87%, and 5.6 vs. 5.5% in the groups with and without voglibose, respectively. Voglibose improved IGT; however, Kaplan–Meier analysis showed no significant between-group difference with respect to cardiovascular events [12.5% with voglibose vs. 10.1% without voglibose for the primary endpoint (95% confidence interval, 0.82–1.86)]; there were no significant differences in secondary endpoints. Although voglibose effectively treated IGT, no additional benefits for cardiovascular events in patients with previous MI and IGT were observed. Voglibose may not be a contributing therapy to the secondary prevention in patients with MI and IGT. Clinicaltrials.gov number: NCT00212017

Glycosylation of Env defines pathogenic properties of simian immunodeficiency virus (SIV). We previously demonstrated that pathogenic SIVmac239 and a live-attenuated, quintuple deglycosylated Env mutant (Δ5G) virus target CD4 + T cells residing in different tissues during acute infection. SIVmac239 and Δ5G preferentially infected distinct CD4 + T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323–9336, 2012, https://doi.org/10.1128/JVI.00948-12 ). Here, we studied the host responses relevant to SIV targeting of CXCR3 + CCR5 + CD4 + T cells in SLOs. Genome-wide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239-infected animals. Consistent with robust expression of CXCL10, CXCR3 + T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs was secondary to the induction of CD14 + CD16 + monocytes and MAC387 + macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387 + macrophages, T cells, dendritic cells (DCs), and residential macrophages near high endothelial venules, the results highlight critical roles of innate/inflammatory responses in SIVmac239 infection. Restricted infection in SLOs by Δ5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DC lineages. IMPORTANCE We previously demonstrated that a pathogenic SIVmac239 virus and a live-attenuated, deglycosylated mutant Δ5G virus infected distinct CD4 + T cell subsets in SLOs and the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323–9336, 2012, https://doi.org/10.1128/JVI.00948-12 ). Accordingly, infections with SIVmac239, but not with Δ5G, deplete CXCR3 + CCR5 + CD4 + T (Th1) cells during the primary infection, thereby compromising the cellular immune response. Thus, we hypothesized that distinct host responses are elicited by the infections with two different viruses. We found that SIVmac239 induced distinctly higher levels of inflammatory Th1 responses than Δ5G. In particular, SIVmac239 infection elicited robust expression of CXCL10, a chemokine for CXCR3 + cells, in CD14 + CD16 + monocytes and MAC387 + macrophages recently infiltrated in SLOs. In contrast, Δ5G infection elicited only modest inflammatory responses. These results suggest that the glycosylation of Env modulates the inflammatory/Th1 responses through the monocyte/macrophage subsets and elicits marked differences in SIV infection and clinical outcomes.

J-GRID is an undertaking to enhance international research collaboration across national borders, which has been commissioned by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to Japanese universities and research institutions. The undertaking includes the construction of bilateral collaboration centers in the counterpart countries and their networking. As the headquarters of this network, Center of Research Network for Infectious Diseases (CRNID) was set up at RIKEN. Since its initiation in 3 countries in FY2005, J-GRID has expanded to include 13 research collaboration centers in 8 countries (6 in Asia and 2 in Africa). J-GRID was highly appreciated as an ideal model of Japan’s science and technology diplomacy and an important platform to mitigate the risk of infectious diseases in the counterpart countries, our own country and the world. Recently, the Japanese government decided to create a new funding and managing body named Japan Agency for Medical Research and Development (AMED), which is expected to start in 2015. The AMED selected 9 special topics of high priority. One of them is “The Control of Emerging and Reemerging Infectious Diseases” and J-GRID is expected to enhance research capacity and make a great deal of contribution in this scheme. Here, I look back on the 10 years journey of J-GRID development since its initiation and discuss the future perspective.

Since infectious diseases heed no national borders, international research collaboration across borders must be enhanced. The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan launched the J-GRID program in the fiscal year (FY) 2005, which consists of the two elements; (1) the construction of collaboration centers in Asian and African countries on a reciprocal basis between a Japanese university/institution and an overseas partner university/institution and (2) the networking of those collaboration centers and setting up its headquarters at RIKEN. J-GRID initiated with 5 collaboration centers in 3 Asian countries has expanded to include 13 centers in 8 countries (6 in Asia and 2 in Africa). The aims of J-GRID include conducting high quality research on infectious diseases of regional and global importance, advancing relevant technologies and developing human resources in the field. In this way, J-GRID is expected to contribute to the public health of the host countries, Japan and the rest of the world. After the completion of the first start-up phase, Term I (2005-2009), J-GRID has stepped up its activity for the second step-up phase, Term II (2010-2014). While the first term was just like an incubation period, the second term should be the exponential growth phase, maximizing its research activities. Indeed, J-GRID is now generating remarkable research outcomes with an increasing number of publications. The mid-term evaluation made by the MEXT in FY2012 commended J-GRID as an ideal model to demonstrate Japan's leadership, in science and technology, and strongly recommended its extension in years to come after Term II terminates in FY 2014.

Background: Long-term outcomes of patients with bifurcated lesions and the restenotic response of the side branches after sirolimus-eluting stent (SES) implantation, comparing 1-stent with 2-stent treatment, are still under discussion. Methods and Results: Japan Post-Marketing Surveillance Registry (J-PMS) is a prospective registry designed to evaluate the safety and efficacy of the SES in routine clinical practice. Angiograms of 1,063 patients with 1,250 lesions were analyzed at the independent core lab. Of these, 324 patients with bifurcation lesions were enrolled. Clinical endpoints were assessed at 3 years. Both main and side branches were evaluated by quantitative coronary angiography at post-procedure (n=349) and 8-month follow up (n=293). Two-stent treatment was performed in 12% of the cases. In-segment restenosis rates at 8 months were 25.6% in the side branch, but newly developed restenosis was seen in only 6.8%. Late loss at the carina of the side branch was -0.11mm in the 1-stent group. Major adverse cardiovascular events rate was 18.3% at 3 years. Target-lesion revascularization rate up to 3 years was 21.6% in the 2-stent group and 8.7% in the 1-stent group (P=0.037). Stent thrombosis occurred in 6 cases (2.0%) until 3 years. Of these, 4 cases were treated with 2-stent (10.81% vs. 0.76% in 1-stent, P=0.003, respectively). Conclusions: In a real-world setting, treatment of coronary bifurcation lesions using SES demonstrated favorable long-term outcomes as long as the side branch was not stented. (Circ J 2011; 75: 306-314)

This paper presents experimental results of lightning surges incoming into a customer due to lightning to an antenna of the customer, a pole and a ground nearby the customer, and briefly discusses lightning current distribution in the customer, a distribution line and a telephone line. Based on experimental results, modeling of each component is explained, and EMTP simulations are carried out. The ground voltage rise is represented by a mutual resistance between grounding electrodes. EMTP simulation results have been observed to agree qualitatively with the measured results, and it becomes possible to investigate lightning surges and current distribution in a customer by an EMTP simulation.

Background: The accuracy for the detection of coronary stenosis by multidetector row computed tomography (MDCT) has been getting more recognition. However, the usefulness of MDCT in patients with chronic kidney disease (CKD) has not been confirmed. Methods: Weanalyzed 19 consecutive patients with asymptomatic diabetic CKD who underwent both MDCT and coronary angiography (CAG) at the initiation of dialysis. The definition of stenosis in this study was lesions with ≧50% stenosis by CAG. Results: CAG revealed stenosis in 35 of 76 branches in 19 patients. Vessel diameter could not be evaluated by MDCT in 11 (14%) major vessels because of motion artifacts, pericardial effusion, pleural effusion, and severe calcification. Almost all of such lesions were located in the right coronary (4/11; 36%) or left circumflex (5/11; 45%) artery. The sensitivity, specificity, positive and negative predictive values of MDCT for a diagnosis of stenosis in the 65 evaluable major vessels were 86, 81, 78, and 88%, respectively. The severity of vessel calcification was increased in a stepwise manner with increments in the proportion of major vessels with ≧50% stenosis (p = 0.004 for trend). Conclusion: MDCT seemed to be an effective non-invasive method of screening patients with diabetic CKD for CAD.

Background. The high prevalence of asymptomatic coronary artery stenosis (CAS) in chronic kidney disease (CKD) has emerged as an important predictor of outcome. However, diagnostic tools that can identify asymptomatic CAS have not yet been established. We investigated whether asymptomatic patients at the initiation of renal replacement therapy (RRT) could be screened using cardiac troponin T (cTnT) and atherosclerotic surrogate markers such as ankle-brachial blood pressure index (ABPI) and intimamedia thickness (IMT). Methods and results. Among 142 patients who were about to start RRT, 60 who were asymptomatic underwent coronary evaluation by multi-slice computed tomography (MSCT) and/or coronary angiography (CAG). CAG diagnosed 35 patients (43.8%) as CAS positive and 27 of them had multi-vessel disease. Factors associated with CAS were smoking, elevated cTnT, low ABPI and high IMT. Moreover, the severity of CAS was associated with smoking, cTnT and ABPI. Stepwise logistic regression analyses revealed that cTnT was a powerful predictor of asymptomatic multi-vessel CAS. Receiver operating characteristic analysis documented the usefulness of cTnT as a screening tool with a cut-off point 0.05 ng/ml. The optimal screening tool for multi-vessel CAS was cTnT (sensitivity, 92.6%; 95% CI, 82.7–99.9; specificity, 63.6%; 95% CI, 47.2–80.0). Conclusion. We concluded that cTnT should be measured as part of a strategy for detecting asymptomatic CAS, especially multi-vessel disease in patients with CKD at the start of RRT.

Thirteen Streptococcus dysgalactiae subsp. equisimilis isolates possessing Lancefield's group A antigen recovered from people in Japan during 2000 to 2004 were genotyped. The results indicate that a conserved clone has persisted and spread within Japan, and two different emm types were observed within members of this clone.

A current promising AIDS vaccine strategy is to elicit CD8(+) cytotoxic T lymphocyte (CTL) responses that broadly recognize highly-diversified HIVs. In our previous vaccine trial eliciting simian immunodeficiency virus (SIV) mac239 Gag-specific CTL responses, a group of Burmese rhesus macaques possessing a major histocompatibility complex haplotype 90-120-Ia have shown vaccine-based viral control against a homologous SIVmac239 challenge. Vaccine-induced Gag(206-216) epitope-specific CTL responses exerted strong selective pressure on the virus in this control. Here, we have evaluated in vivo efficacy of vaccine-induced Gag(206-216)-specific CTL responses in two 90-120-Ia-positive macaques against challenge with a heterologous SIVsmE543-3 that has the same Gag(206-216) epitope sequence with SIVmac239. Despite efficient Gag(206-216)-specific CTL induction by vaccination, both vaccinees failed to control SIVsmE543-3 replication and neither of them showed mutations within the Gag(206-216) epitope. Further analysis indicated that Gag(206-216)-specific CTLs failed to show responses against SIVsmE543-3 infection due to a change from aspartate to glutamate at Gag residue 205 immediately preceding the amino terminus of Gag(206-216) epitope. Our results suggest that even vaccine-induced CTL efficacy can be abrogated by a single amino acid change in viral epitope flanking region, underlining the influence of viral epitope flanking sequences on CTL-based AIDS vaccine efficacy.

Nucleotide sequencing of the entire genomes was completed in the 1980s for most members of the Paramyxoviridae. It then became a new common task with challenge for researchers in the field to establish a system to recover the virus entirely from cDNA, thereby allowing reverse genetics (free manipulation of the viral genome). Using Sendai virus, we established a system of incomparable virus recovery efficiency early on. This technology was then fully exploited in answering a series of long-held questions. In particular, two accessory genes whose functions had remained enigmatic were demonstrated to encode special functions critical in viral in vivo pathogenesis producing fatal pneumonia in mice, although dispensable in virus replication at the in vitro cellular level. Their in vivo functions were found to counteract the two respective facets of the antiviral state induced by interferons and an interferon regulatory factor 3-dependent but yet unknown effector. These achievements appear to have facilitated a scientific trend where the accessory genes are a focus of active investigation in studies on other paramyxoviruses and opened up a new common ground shared between virology and immunology.

Summary Background Patients who have acute myocardial infarction remain at major risk of cardiovascular events. We aimed to assess the effects of either human atrial natriuretic peptide or nicorandil on infarct size and cardiovascular outcome. Methods We enrolled 1216 patients who had acute myocardial infarction and were undergoing reperfusion treatment in two prospective, single-blind trials at 65 hospitals in Japan. We randomly assigned 277 patients to receive intravenous atrial natriuretic peptide (0·025 μg/kg per min for 3 days) and 292 the same dose of placebo. 276 patients were assigned to receive intravenous nicorandil (0·067 mg/kg as a bolus, followed by 1·67 μg/kg per min as a 24-h continuous infusion), and 269 the same dose of placebo. Median follow-up was 2·7 (IQR 1·5–3·6) years for patients in the atrial natriuretic peptide trial and 2·5 (1·5–3·7) years for those in the nicorandil trial. Primary endpoints were infarct size (estimated from creatine kinase) and left ventricular ejection fraction (gauged by angiography of the left ventricle). Findings 43 patients withdrew consent after randomisation, and 59 did not have acute myocardial infarction. We did not assess infarct size in 50 patients for whom we had fewer than six samples of blood. We did not have angiographs of left ventricles in 383 patients. Total creatine kinase was 66 459·9 IU/mL per h in patients given atrial natriuretic peptide, compared with 77 878·9 IU/mL per h in controls, with a ratio of 0·85 between these groups (95% CI 0·75–0·97, p=0·016), which indicated a reduction of 14·7% in infarct size (95% CI 3·0–24·9%). The left ventricular ejection fraction at 6–12 months increased in the atrial natriuretic peptide group (ratio 1·05, 95% CI 1·01–1·10, p=0·024). Total activity of creatine kinase did not differ between patients given nicorandil (70 520·5 IU/mL per h) and controls (70 852·7 IU/mL per h) (ratio 0·995, 95% CI 0·878–1·138, p=0·94). Intravenous nicorandil did not affect the size of the left ventricular ejection fraction, although oral administration of nicorandil during follow-up increased the left ventricular ejection fraction between the chronic and acute phases. 29 patients in the atrial natriuretic peptide group had severe hypotension, compared with one in the corresponding placebo group. Interpretation Patients with acute myocardial infarction who were given atrial natriuretic peptide had lower infarct size, fewer reperfusion injuries, and better outcomes than controls. We believe that atrial natriuretic peptide could be a safe and effective adjunctive treatment in patients with acute myocardial infarction who receive percutaneous coronary intervention.

The rhesus macaque exhibits individual differences in susceptibility and resistance to infectious agents such as simian immunodeficiency virus (SIV) under experimental conditions, and these may be genetically determined at least in part by major histocompatibility complex (MHC) class I polymorphism. Although the importance of defining MHC class I polymorphism is well recognized, development of a generic and comprehensive molecular typing method of MHC class I alleles of the rhesus macaque has been hampered because, during the evolution of this species, multiple copies of similar DNA sequences have been generated by duplication events including the coding sequences of Mamu-A and Mamu-B loci. We report here a newly developed reference strand-mediated conformation analysis (RSCA)-based typing method of multiple Mamu-A and Mamu-B cDNAs that allowed us to estimate the number of expressed alleles. This technique detected 1-7 Mamu-A signals and 2-12 Mamu-B signals in a single sample, indicating that the number of functional alleles may vary. By comparing the data from the parents with those from the descendants in the breeding colony, several MHC class I haplotypes consisting of variable numbers of functional Mamu-A and Mamu-B alleles could be assigned.

Background Sendai virus (SeV) is a new class of cytoplasmic RNA vector that is free from genotoxicity that infects and multiplies in most mammalian cells, and directs high-level transgene expression. We improved the vector by deleting all of the envelope-related genes from the SeV genome and thus reducing its immunogenicity. Methods The matrix (M), fusion (F) and hemagglutinin-neuraminidase (HN) genes-deleted SeV vector (SeV/ΔMΔFΔHN) was recovered in a newly established packaging cell line. Then, the generated SeV/ΔMΔFΔHN vector was characterised by comparing with single gene-deleted type SeV vectors. Results This SeV/ΔMΔFΔHN vector carrying the green fluorescent protein gene in place of the envelope-related genes could be propagated to a titer of more than 108 cell infectious units/ml. This vector showed an efficient transduction capability in vitro and in vivo, and the cytopathic effect and induction of neutralizing antibody in vivo were greatly reduced compared with those of single gene-deleted type SeV vectors. No activity of neutralizing antibody or anti-HN antibody was seen when SeV/ΔMΔFΔHN was transduced ex vivo. Additional introduction of amino acid mutations that had been identified from SeV strains causing persistent infections was also effective for the reduction of cytopathic effects. Conclusions The deletion of genes from the SeV genome and the additional mutation are very effective for reducing both the immunogenic and cytopathic reactions to the SeV vector. These modifications are expected to improve the safety and broaden the range of clinical applications of this new class of cytoplasmic RNA vector. Copyright © 2006 John Wiley & Sons, Ltd.

Human immunodeficiency virus type 1 (HIV-1) efficiently enters cells of Old World monkeys but encounters a block before reverse transcription. This restriction is mediated by a dominant repressive factor. Recently, a member of the tripartite motif (TRIM) family proteins, TRIM5α, was identified as a blocking factor in a rhesus macaque cDNA library. Among Old World monkey cell lines, the African green monkey kidney cell line CV1 is highly resistant to not only HIV-1 but also simian immunodeficiency virus SIVmac infection. We analyzed TRIM5α of CV1 cells and HSC-F cells, a T-cell line from a cynomolgus monkey, and found that both CV1- and HSC-F-TRIM5αs could inhibit CD4-dependent HIV-1 infection, as well as vesicular stomatitis virus glycoprotein-mediated infection. CV1-TRIM5α could also inhibit SIVmac infection, whereas HSC-F-TRIM5α could not. In the SPRY (B30.2) domain of CV1-TRIM5α, there was a 20-amino-acid duplication that was not present in HSC-F-TRIM5α. A chimeric TRIM5α containing 37 amino acid residues from CV1-TRIM5α, which spanned the 20-amino-acid duplication, in the background of HSC-F-TRIM5α fully gained the ability to inhibit SIVmac infection. Conversely, the mutant CV1-TRIM5α lacking the 20-amino-acid duplication completely lost the ability to restrict SIVmac infection. These findings clearly indicated that a specific region of 37 amino acid residues in the SPRY domain of CV1-TRIM5α contained a determinant of species-specific restriction of SIVmac.

A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli (EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli (EPEC); invE for enteroinvasive E. coli (EIEC); elt, estp, and esth for enterotoxigenic E. coli (ETEC); CVD432 and aggR for enteroaggregative E. coli (EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.

Sendai virus (SeV) is an enveloped virus with a linear, nonsegmented, negative-sense RNA genome of 15,384 nucleotides and belongs to the genus Respirovirus of the subfamily Paramyxovirinae. SeV contains six genes in the order 3′-(leader)-N-P-M-F-HN-L-(trailer)-5′ on the genome. The monocistronic mRNAs are transcribed by the viral RNA polymerase composed of L and P proteins (16). However, the P gene is exceptional in that it gives rise to multiple protein species by a process known as RNA editing and by the use of an overlapping open reading frame (ORF). The P protein is translated from the unedited mRNA, which is the exact copy of the P gene, while the V protein is translated from the edited mRNA, in which one nontemplated G residue is cotranscriptionally inserted to the editing position. The P and V proteins, therefore, have a common N terminus but have different C termini because the reading frame for V changes by +1 relative to the P frame (for a review, see references 33 and 38). The V protein is encoded by almost all viruses that are members of the subfamily Paramyxovirinae. The unique C-terminal region of V contains seven cysteine residues highly conserved among paramyxoviruses, forms zinc finger-like motifs, and, indeed, binds Zn2+ (7, 20, 35, 44, 51). The C protein is translated from the −1 reading frame relative to the frame common to P and V. The SeV P and V proteins are initiated at the AUG codon at position 104, whereas the SeV C reading frame produces a nested set of C′, C, Y1, and Y2 proteins initiating, respectively, at a non-AUG (ACG) codon at position 81 and at AUG codons at positions 114, 183, and 201 (4, 15, 45). The C′, C, Y1, and Y2 proteins are terminated at the same position 726 and are collectively called C proteins. Among them, the C protein is the major species expressed in infected cells, at a molar ratio severalfold higher than that of the other three proteins (32). The C proteins are expressed by the viruses belonging to three genera, Respirovirus, Morbillivirus, and Henipahvirus, and by Tupaia paramyxovirus-like viruses, but they are not expressed by the viruses belonging to two genera, Rubulavirus and Avulavirus of Paramyxovirinae (reviewed in reference 37). The amino acid sequence of the C proteins is well conserved within each genus but poorly conserved (less than 20%) between different genera. The C proteins are all relatively small (150 to 220 amino acids [aa]) and highly basic, with an isoelectric point of around 10 (38). Both the V and C proteins of SeV are categorized as accessory proteins that may not always be essential for the viral life cycle, because there is at least one virus which does not have or does not express these proteins in its close relatives (the genus Respirovirus) (33). We have employed two complementary approaches, SeV reverse genetics to delete the V and C proteins and plasmid-based expression of these proteins, to address how these proteins contribute to actual viral replication and pathogenesis (reviewed in references 37, 38, and 39). The C proteins are indeed dispensable for SeV replication in cultured cells, but the lack of C proteins profoundly affects the viral life cycle. Compared with the wild type, mutant SeV with the four C proteins (C′/C/Y1/Y2) knocked out produced nearly 1/104 of the progeny virus in ovo, 1/103 in tissue cultured cells, and an undetectable level in mouse lungs (32). The SeV C protein is extremely versatile. The first finding about the role of SeV C proteins involves the inhibition of viral RNA synthesis; C proteins supplied from the plasmid inhibit minigenome RNA synthesis in a dose-dependent manner in vitro (3). A larger quantity of viral RNA is synthesized in cells infected with the four-C knockout SeV than in the parental wild-type SeV-infected cells at the late stage of infection (17). Moreover, all the C-, Y1-, or Y2-expressing cell lines suppress SeV multiplication at the transcriptional level (24). Analysis of the stable transformants constitutively expressing various C-truncates from the N and C termini indicated that the 106-residue C-terminal half of the C proteins is sufficient for viral RNA inhibition (23). In the cells constitutively expressing SeV C protein, SeV growth is significantly suppressed because of the inhibition of viral RNA synthesis. This growth inhibition by SeV C proteins is also found in closely related human parainfluenza virus type I (hPIVI) but not in the more distantly related hPIV3 and measles virus. Based on these findings, C proteins are thought to down-regulate viral RNA synthesis specifically. This inhibition is suggested to occur via binding of the C proteins with the L protein (14, 19). The second finding about the role of SeV C proteins concerns the promotion of viral assembly and/or budding. SeV C proteins are expressed abundantly in infected cells but are incorporated in trace amounts in the virions and, thus, are practically considered to be nonstructural proteins having nothing to do with virion assembly (34, 46). However, as described above, production of the progeny viruses was hampered greatly in the cells infected with the four-C knockout SeV even enough viral genomic RNA and proteins were accumulated in the cells. More surprisingly, in the culture supernatant of cells infected with the SeV mutant with the four C proteins knocked out, there were many noninfectious particles with a sedimentation profile distributing widely from light to heavy fractions in the sucrose gradient centrifugation and with a highly anomalous morphology in the electron microscope observation (17). The role of the C proteins in this process remains to be clarified. The third finding about the role of SeV C proteins is that they counteract the antiviral activity of interferons (IFNs). Studies performed in the 1960s showed that preinfection or persistent infection of cells with SeV or hPIV3 enhanced the growth of heterologous postinfecting IFN-sensitive viruses, such as Newcastle disease virus and vesicular stomatitis virus (VSV), suggesting that preinfection with SeV and hPIV3 somehow rendered cells unresponsive to autocrine IFNs (18, 36). The anti-IFN capacity of these viruses was recently established (5, 13). HeLa cells preinfected with the parental wild-type and C′/C-, C′/C/Y1-, and V-knockout SeVs circumvented the anti-VSV action of IFN-β, whereas the virus with the four C proteins knocked out lost this IFN antagonism (13). The C-, Y1-, or Y2-expressing cell lines were all capable of circumventing the activation of IFN-stimulated genes (ISGs) and the induction of an antiviral state by IFN-α or -β and IFN-γ (24). These observations suggested that IFN signaling was somehow blocked by the SeV C′, C, Y1, or Y2 protein without the aid of other viral proteins. An analysis of the stable transformants expressing various truncates from the N and C termini indicated that the 106-residue C-terminal half of C protein was sufficient for IFN antagonism (23). The C protein with a spontaneous mutation at residue 170 of phenylalanine to serine (CF170S) (22), within the 106-residue region, neither antagonized IFNs (10) nor, compared to the effect of the parental C in vitro, inhibited viral RNA synthesis (14). IFN antagonism is fully generated even in cells expressing trace amounts of the C proteins, in which the inhibition of viral RNA synthesis is no longer observed. The fourth finding about the role of the SeV C proteins is that they suppress apoptosis. The parental wild-type SeV grows without any significant cytopathic effect in Hep-2 cells, and infected cells tolerate the apoptotic stimulus caused by VSV (29). Though cells infected either with C′-, C/C′-, or C′/C/Y1-knockout viruses tolerate the apoptosis signals as do the wild-type SeV-infected cells, cells infected with the four-C knockout SeV clearly showed the condensation of nuclei and the fragmentation of chromosomal DNA, indicating that SeV C proteins had antiapoptotic activity (28). In this report, we attempted to determine which regions of the SeV C protein were responsible for IFN antagonism and for the down-regulation of viral RNA synthesis by using cells expressing the wild-type C protein and several mutant C (Cm) proteins with charged amino acid substitutions for alanine residues, most of which were within the C-terminal half. A substitution at positions 151, 153, and 154 of the C protein abolished the anti-IFN and RNA synthesis-inhibiting activities simultaneously, but all of the other substitutions except the mutation at positions 114 and 115 abolished only the RNA synthesis-inhibiting activity. From these results, we conclude that the amino acids scattered throughout the C-terminal half of the C protein govern the down-regulation of viral RNA synthesis, while more limited residues are critical for playing a role in IFN antagonism. Using the various Cm proteins, we further found that SeV C proteins participate in the inhibition of IFN signaling through the unusual phosphorylation and dephosphorylation of STAT1 and STAT2, which play key roles in an IFN-α/β system.

Recently, encouraging AIDS vaccine trials in macaques have implicated cytotoxic T lymphocytes (CTLs) in the control of the simian human immunodeficiency virus SHIV89.6P that induces acute CD4+ T cell depletion. However, none of these vaccine regimens have been successful in the containment of replication of the pathogenic simian immunodeficiency viruses (SIVs) that induce chronic disease progression. Indeed, it has remained unclear if vaccine-induced CTL can control SIV replication. Here, we show evidence suggesting that vaccine-induced CTLs control SIVmac239 replication in rhesus macaques. Eight macaques vaccinated with DNA-prime/Gag-expressing Sendai virus vector boost were challenged intravenously with SIVmac239. Five of the vaccinees controlled viral replication and had undetectable plasma viremia after 5 wk of infection. CTLs from all of these five macaques rapidly selected for escape mutations in Gag, indicating that vaccine-induced CTLs successfully contained replication of the challenge virus. Interestingly, analysis of the escape variant selected in three vaccinees that share a major histocompatibility complex class I haplotype revealed that the escape variant virus was at a replicative disadvantage compared with SIVmac239. These findings suggested that the vaccine-induced CTLs had “crippled” the challenge virus. Our results indicate that vaccine induction of highly effective CTLs can result in the containment of replication of a highly pathogenic immunodeficiency virus.

Although RSV causes serious pediatric respiratory disease, an effective vaccine does not exist. To capture the strengths of a live virus vaccine, we have used the murine parainfluenza virus type 1 (Sendai virus [SV]) as a xenogeneic vector to deliver the G glycoprotein of RSV. It was previously shown (J. L. Hurwitz, K. F. Soike, M. Y. Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, Vaccine 15:533-540, 1997) that intranasal SV protected African green monkeys from challenge with the related human parainfluenza virus type 1 (hPIV1), and SV has advanced to clinical trials as a vaccine for hPIV1 (K. S. Slobod, J. L. Shenep, J. Lujan-Zilbermann, K. Allison, B. Brown, R. A. Scroggs, A. Portner, C. Coleclough, and J. L. Hurwitz, Vaccine, in press). Recombinant SV expressing RSV G glycoprotein was prepared by using reverse genetics, and intranasal inoculation of cotton rats elicited RSV-specific antibody and elicited protection from RSV challenge. RSV G-recombinant SV is thus a promising live virus vaccine candidate for RSV.

Objective: A valine to isoleucine substitution at position 64 of CCR2 (CCR2-64I) is associated with a delay in progression to AIDS in HIV-1-infected individuals. The aim of the present study is to elucidate the molecular mechanism underlying the effect of this allele. Design: We analysed the effect of the 641 substitution on levels of expression of CCR2A and CCR2B, two CCR2 isoforms produced by alternative splicing. Methods: Sendai virus vector was used to express CCR2 molecules. Results: While CCR2B trafficked well to the cell surface, CCR2A, which differs from CCR2B only by the sequence of its C-terminal cytoplasmic tail, was detected predominantly in the cytoplasm. The level of expression of CCR2A-641 was significantly higher than that of CCR2A without the substitution. On the other hand, the 641 substitution did not affect levels of CCR2B expression. Pulse-chase experiments revealed that the 641 substitution increased the half-life of CCR2A in cells. When co-expressed with CCR5, CCR2A-641 interfered more severely with cell surface expression of CCR5 than did wild-type CCR2A. Furthermore, immunoprecipitation experiments showed that CCR2A co-precipitated with an immature form of CCR5. Conclusion: These results suggest that CCR2A hinds to CCR5 in the cytoplasm and down-modulates its surface expression. We propose that the increased ability of CCR2A-641 to down-modulate CCR5 expression might be a possible cause of a delay in HIV-1 disease progression in patients with this allele.

PCR法を水と食品の大腸菌および大腸菌群検査に適用するための検討を行った.PCR法では, 大腸菌のtransferase遺伝子 (wecF), β-galactosidase遺伝子 (lacZ) およびE.coli 16S rRNA遺伝子 (E.coli 16S rRNA) を標的としたプライマーを使用した.結果は次のようであった.1) ヒトおよび食品由来大腸菌のwecF, lacZおよびE.coli 16S rRNA遺伝子保有率はそれぞれ12/12 (100%), 99/99 (100%), 99/99 (100%), 食品由来Klebsiella属, Citrobacter属などの大腸菌群のwecF, lacZ E. coli16S rRNA遺伝子保有率はそれぞれ28/63 (44%), 70/177 (39%), 0/177 (0%) であった.2) 食品69検体 (食肉23検体, 野菜46検体) のBGLB培養液からのwecF, lacZ E. coli 16S rRNA遺伝子検出率はそれぞれ, 73%, 65%, 37%であった.E. coli 16S rRNA遺伝子検出と大腸菌検出との間には, 統計的に有意な相関が認められた.しかしE.coli 16S rRNA遺伝子検出と大腸菌群検出との間に有意な相関は認められなかった.wecFあるいはlacZ遺伝子検出と大腸菌群検出との間にも, 有意な相関は認められなかった3) 水道の原水など153検体の水の大腸菌汚染をコリラート培地を用いた特定酵素基質法で調べたところ, E.coli 16S rRNA遺伝子検出と大腸菌検出との間に有意な相関が認められた.以上の結果から, E.coli 16S rRNA遺伝子を標的としたPCR法は, 水および食品中の大腸菌を迅速, かつ特異的に検出する目的に使用できることが示唆された.

Background Sendai virus (SeV) is a new type of cytoplasmic RNA vector, which infects and replicates in most mammalian cells, directs high-level expression of the genes on its genome and is free from genotoxicity. In order to improve this vector, both the matrix (M) and fusion (F) genes were deleted from its genome. Methods For the recovery of the M and F genes-deleted SeV (SeV/ΔMΔF), the packaging cell line was established by using a Cre/loxP induction system. SeV/ΔMΔF was characterized and compared with wild-type and F or M gene-deleted SeV vectors in terms of transduction ability, particle formation, transmissible property and cytotoxicity. Results SeV/ΔMΔF was propagated in high titers from the packaging cell line. When this vector was administered into the lateral ventricle and the respiratory tissue, many of the ependymal and epithelial cells were transduced, respectively, as in the case of wild-type SeV. F gene-deletion made the SeV vector non-transmissible, and M gene-deletion worked well to inhibit formation of the particles from infected cells. Simultaneous deletions of these two genes in the same genome resulted in combining both advantages. That is, both virus maturation into particles and transmissible property were almost completely abolished in cells infected with SeV/ΔMΔF. Further, the cytopathic effect of SeV/ΔMΔF was significantly attenuated rather than that of wild type in vitro and in vivo. Conclusions SeV/ΔMΔF is an advanced type of cytoplasmic RNA vector, which retains efficient gene transfer, gains non-transmissible properties and loses particle formation with less cytopathic effect. Copyright © 2004 John Wiley & Sons, Ltd.

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