Search

Mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of mature cell types and are a promising source of regenerative medicine. The success of regenerative medicine using MSCs strongly depends on their differentiation potential. In this study, we sought to identify marker genes for predicting the osteogenic differentiation potential by comparing ilium MSC and fibroblast samples. We measured the mRNA levels of 95 candidate genes in nine ilium MSC and four fibroblast samples before osteogenic induction, and compared them with alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation after induction. We identified 17 genes whose mRNA expression levels positively correlated with ALP activity. The chondrogenic and adipogenic differentiation potentials of jaw MSCs are much lower than those of ilium MSCs, although the osteogenic differentiation potential of jaw MSCs is comparable with that of ilium MSCs. To select markers suitable for predicting the osteogenic differentiation potential, we compared the mRNA levels of the 17 genes in ilium MSCs with those in jaw MSCs. The levels of 7 out of the 17 genes were not substantially different between the jaw and ilium MSCs, while the remaining 10 genes were expressed at significantly lower levels in jaw MSCs than in ilium MSCs. The mRNA levels of the seven similarly expressed genes were also compared with those in fibroblasts, which have little or no osteogenic differentiation potential. Among the seven genes, the mRNA levels of IGF1 and SRGN in all MSCs examined were higher than those in any of the fibroblasts. These results suggest that measuring the mRNA levels of IGF1 and SRGN before osteogenic induction will provide useful information for selecting competent MSCs for regenerative medicine, although the effectiveness of the markers is needed to be confirmed using a large number of MSCs, which have various levels of osteogenic differentiation potential.

Intestinal ischemia is a rare complication of Takayasu arteritis (TAK), which often requires colectomy. We report the case of a 27 year-old man with ulcerative colitis (UC), who was admitted to our hospital due to abdominal pain. Computed tomography revealed an edematous wall of the ascending colon with ascites and a thickened aortic wall with mild stenosis of the superior mesenteric artery (SMA), suggesting large vessel vasculitis, especially TAK. Colonoscopy revealed acute ischemic colitis associated with mild stenosis of the SMA caused by TAK, but there was no worsening of UC. The patient was successfully treated with conservative therapy.

A 71-year-old woman presented with chest pain, cough, and back pain. A chest roentgenogram showed multiple nodular shadows in both lungs. She was diagnosed with granulomatosis with polyangiitis (GPA). The multiple nodular shadows in both lungs regressed spontaneously in a few months. There are few reports of spontaneous regression of GPA, and the underlying mechanism is unclear. Neutrophil extracellular traps (NETs) have been recently shown to be involved in GPA. NETs may also be related to the natural regression of GPA.

We have recently reported a new method for detecting T-cell-derived extracellular vesicles (EVs), CD3+CD4+EVs,CD3+CD8+EVs, and CD3+HLA-DR+EVs. In our previous study, CD3+HLA-DR+EVs were released profusely by CD8+T cells, only moderately by T helper1 (Th1) CD4+T cells, and very little from Th2 CD4+T cells in vitro. EVs were measured sequentially in patients undergoing hematopoietic stem cell transplantation (HSCT), and their relationship to GVHD was investigated in comparison with other conventional biomarkers. We analyzed peripheral blood samples from 20 patients (13 children and 7 adults) who underwent HSCT at Tokyo Medical and Dental University Hospital. CD3+CD4+EV and CD3+CD8+EV levels specifically correlated with the CD4+ and CD8+T lymphocyte counts, respectively. CD3+CD8+EVs and CD3+HLA-DR+EVs increased in GVHD and reflected the persistence of GVHD more specifically than soluble IL-2 receptor (sIL-2R). In engraftment syndrome, sIL-2R was markedly elevated, but CD3+HLA-DR+EVs were not. Furthermore, ferritin and sIL-2R markedly increased in hemophagocytic syndrome (HPS) that developed before engraftment; however, the change in CD3+HLA-DR+EVs was marginal. CD3+CD4+, CD3+CD8+, and CD3+HLA-DR+EVs efficiently reflect the cell-mediated immune response, and CD3+CD8+EVs and CD3+HLA-DR+EVs are more useful than other conventional biomarkers, such as sIL-2R, for monitoring and evaluation of acute GVHD.

A 72-year-old man diagnosed with stage 4 lung adenocarcinoma developed asymptomatic pneumatosis intestinalis while undergoing treatment with first-line chemotherapy, which included carboplatin, paclitaxel, and bevacizumab (BEV). He was treated conservatively. The pneumatosis recurred while the patient was undergoing treatment with the third-line chemotherapy, which included pemetrexed (PEM). His condition resolved after 4 weeks of supportive therapy. To our knowledge, this is the first case in which pneumatosis intestinalis was induced twice by two drugs in a patient with lung cancer. BEV and PEM are often administered to patients with lung cancer; thus, it should be noted that pneumatosis intestinalis may occur as an adverse event in patients treated with these drugs.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD). Smoking susceptibility is important for the onset and development of COPD. We previously reported an association between serum iron concentrations and pulmonary function in male smokers. However, the mechanism governing smoking susceptibility in relation to iron deficiency is unclear; this study aimed to elucidate this mechanism. C57BL/6 male mice were fed an iron-deficient or normal diet and then exposed to cigarette smoke. BAL, histological analysis, and pulmonary function tests were performed after cigarette smoke exposure. Human alveolar type II epithelial A549 cells were treated with an iron chelator. Subsequently, A549 cells were exposed to cigarette smoke extract. In mice exposed to cigarette smoke for 2 weeks, the concentration of alveolar macrophages in the BAL fluid recovered from iron-deficient mice was significantly higher than that in normal diet mice. IL-6 and MCP-1 (monocyte chemotactic protein 1) concentrations in the BAL fluid increased significantly from baseline in iron-deficient mice, but not in normal diet mice. In mice exposed to cigarette smoke for 8 weeks, the pathological mean linear intercepts, physiological total lung capacity, and functional residual capacity in the lungs of iron-deficient mice were significantly greater than in normal diet mice. Phosphorylation of NF-κB was enhanced in the lungs of iron-deficient mice exposed to cigarette smoke and in the iron-chelating A549 cells exposed to cigarette smoke extract. Iron deficiency exaggerated cigarette smoke-induced pulmonary inflammation, suggesting that it may accelerate COPD development.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) vaccination is progressing globally. Several adverse reactions have been reported with vaccination against COVID-19. It is unknown whether adverse reactions to COVID-19 vaccination are severe in individuals with allergies. We administered the COVID-19 vaccine to the medical staff at Yamagata University Hospital from March to August 2021. Subsequently, we conducted an online questionnaire-based survey to investigate the presence of allergy and adverse reactions after vaccination and examined the association between allergy and adverse reactions after immunization.Responses were collected from 1586 subjects after the first vaccination and 1306 subjects after the second administration of the BNT162b2 mRNA COVID-19 vaccine. Adverse reactions included injection site pain, injection site swelling, fever, fatigue or malaise, headache, chills, nausea, muscle pain outside the injection site, and arthralgia. The frequency and severity of most adverse reactions were higher after the second vaccination compared to the first. The frequency of some adverse reactions and their severity were higher, and the duration of symptoms was longer in subjects with allergies than in subjects without allergies. Although several participants visited the emergency room for treatment after the first and second vaccinations, nobody was diagnosed with anaphylaxis.Given the serious consequence of COVID-19 and the reported high efficacy of this vaccine against this disease, we conclude that vaccination of allergic individuals is generally recommended.

Background Elderly patients who are hospitalized due to pneumonia experience deterioration of their activities of daily living (ADL) during this period; in some cases, this loss of ADL is not recovered at the end of antibiotic treatment. In this study, we examined whether erector spinae muscle cross-sectional area (ESMCSA) measured by computed tomography (CT) could predict a low level of ADL at the end of antibiotic treatment for pneumonia. Methods Eighty patients (mean age 74.8 years) with pneumonia, who were admitted to Yamagata university hospital between 2015 and 2016, were analyzed retrospectively. In all cases, chest CT was performed on admission and ESMCSA was measured at the level of the 12th thoracic vertebra. Patient levels of ADL were also measured, both on admission and at the end of treatment, using the Barthel Index. Results Patients with lower levels of ADL at the end of treatment were significantly older and tended to have a lower body mass index, poorer nutritional status, and more severe pneumonia than did patients who were self-reliant. Significantly smaller ESMCSAs were noted in patients who required assistance at the end of treatment than in those who were self-reliant. In multivariate logistic regression analysis, smaller ESMCSA was significantly associated with a lower level of ADL at the end of treatment, independent of age, sex, severity of pneumonia, nutritional status, or dehydration status. Conclusion These results suggest that ESMCSA can predict ADL level after antibiotic treatment of pneumonia.

To elucidate the host genetic loci affecting severity of SARS-CoV-2 infection, or Coronavirus disease 2019 (COVID-19), is an emerging issue in the face of the current devastating pandemic. Here, we report a genome-wide association study (GWAS) of COVID-19 in a Japanese population led by the Japan COVID-19 Task Force, as one of the initial discovery GWAS studies performed on a non-European population. Enrolling a total of 2,393 cases and 3,289 controls, we not only replicated previously reported COVID-19 risk variants (e.g., LZTFL1, FOXP4, ABO, and IFNAR2), but also found a variant on 5p35 (rs60200309-A at DOCK2) that was significantly associated with severe COVID-19 in younger (-8 (odds ratio = 2.01, 95% confidence interval = 1.58-2.55). This risk allele was prevalent in East Asians, including Japanese (minor allele frequency [MAF] = 0.097), but rarely found in Europeans. Cross-population Mendelian randomization analysis made a causal inference of a number of complex human traits on COVID-19. In particular, obesity had a significant impact on severe COVID-19. The presence of the population-specific risk allele underscores the need of non-European studies of COVID-19 host genetics.

Background Several studies have demonstrated the association between mean corpuscular hemoglobin concentration (MCHC), a hematological index used for the assessment of anemia, and the prognosis of patients with heart disease. While the red cell distribution width (RDW) is known to be related to the prognosis of patients with chronic obstructive pulmonary disease (COPD), few studies have focused on the association between the MCHC and COPD. Therefore, we examined the association between the MCHC and prognosis in patients with exacerbation of COPD. Methods We examined the association between the 30-day mortality and clinical findings in patients with COPD exacerbation who were hospitalized between October 2008 and December 2018. Results We enrolled 195 patients with COPD exacerbation (average age: 76.4 years; 181 men, 14 women). The MCHC was significantly lower, while the RDW was significantly higher in the 27 patients (13.8%) who died during the 30-day observation period compared to those in the patients who survived. Multivariate logistic regression analysis revealed that the MCHC was independently associated with 30-day mortality. The area under the curve calculated from the MCHC obtained from peripheral blood was 0.688 and the cutoff value was 31.6 g/dL, with a sensitivity of 0.593 and specificity of 0.810 (p = 0.0001). Conclusion The MCHC might be a valuable biomarker for evaluating the prognosis of patients with COPD exacerbation.

Background The deterioration of pulmonary function, such as FEV1-decline, is strongly associated with poor prognosis in patients with chronic obstructive pulmonary disease (COPD). However, few investigations shed light on useful biomarkers for predicting the decline of pulmonary function. We evaluated whether thymus and activation-regulated chemokine (TARC), a Th2 inflammation marker, could predict rapid FEV1-decline in COPD patients. Methods We recruited 161 patients with stable COPD and performed pulmonary function test once every six months. At the time of registration, blood tests, including serum levels of TARC were performed. We assessed the correlation between changes in parameters of pulmonary function tests and serum levels of TARC. The rapid-decline in pulmonary function was determined using 25th percentile of change in FEV1 or FEV1 percent predicted (%FEV1) per year. Results In the FEV1-rapid-decline group, the frequency of exacerbations, the degree of emphysema, and serum levels of TARC was higher than in the non-rapid-decline group. When using %FEV1 as a classifier instead of FEV1, age, the frequency of exacerbations, the degree of emphysema and serum levels of TARC in the rapid-decline group was significantly greater than those in the non-rapid-decline group. In univariate logistic regression analysis, TARC was the significant predictive factor for rapid-decline group. In multivariate analysis adjusted for emphysema, serum levels of TARC are independently significant predicting factors for the rapid-decline group. Conclusions TARC is an independent predictive biomarker for the rapid-decline in FEV1. Measuring serum TARC levels may help the management of COPD patients by predicting the risk of FEV1 decline.

Background Diabetes has been reported as a comorbidity of chronic obstructive pulmonary disease (COPD) in Western countries, but it has not been demonstrated in epidemiological reports in Japan. The purpose of this study was to clarify whether the relationship between airflow obstruction and diabetes can be confirmed in a Japanese general population. Methods From 2004 to 2006, blood sampling and pulmonary function tests were performed on 3045 people over the age of 40 years in annual health check-ups held in Takahata, Yamagata Prefecture, Japan. Pulmonary function was re-evaluated in 2009 and 2011. Results The prevalence of diabetes did not differ between subjects with and without airflow obstruction. Furthermore, although body mass index decreased, no increase in the prevalence of diabetes was observed with the progression of airflow obstruction. The annual changes in forced expiration volume in 1s (FEV1) did not differ depending on the presence or absence of diabetes in the study population. Conclusion There was no difference in the prevalence of diabetes between subjects with airflow obstruction and those without. As patients with COPD in Japan are thinner than in the West, diabetes may not be a common comorbidity in Japanese patients with COPD.

Extracellular vesicles (EVs) are known to contain unique proteins that reflect the cells of origins. Activated T cells are reported to secrete various EVs. To establish T cell subset-specific biomarkers, we performed proteomic analysis with Th1- and Th2-derived EVs and identified HLA-DR as a Th1-dominated EV membrane protein. We designed a measurement system for CD3+CD4+, CD3+CD8+, and CD3+HLA-DR+EVs to specifically determine EV subpopulations derived from CD4+, CD8+, and Th1-type T cells, respectively.In vitroanalysis showed that CD3+CD4+EVs and CD3+CD8+EVs were selectively secreted from activated CD4+and CD8+T cells, respectively, and that CD3+HLA-DR+EVs were actively secreted from not only Th1 but also activated CD8+T (probably mostly Tc1) cells. To evaluate the clinical usefulness of these EVs, we measured the serum levels in patients with inflammatory diseases, including Epstein-Barr virus (EBV,n=13) infection, atopic dermatitis (AD,n=10), rheumatoid arthritis (RA,n=20), and osteoarthritis (OA,n=20) and compared the levels with those of healthy adults (n=20). CD3+CD4+EVs were significantly higher in all of EBV infection, AD, RA, and OA while CD3+CD8+EVs were higher in EBV infection, lower in RA, and not different in AD and OA relative to the control. The levels of CD3+HLA-DR+EVs were markedly higher in EBV infection and significantly lower in AD. The results suggest that these EV subpopulations reflectin vivoactivation status of total CD4+, total CD8+, and Th1/Tc1-type T cells, respectively, and may be helpful in T cell-related clinical settings, such as cancer immunotherapy and treatment of chronic infection, autoimmune diseases, and graft-versus-host disease.

Summary In this study, a constitutive model of high damping rubber bearings (HDRBs) is developed that allows the accurate representation of the force–displacement relationship including rate-dependence for shear deformation. The proposed constitutive model consists of two hyperelastic springs and a nonlinear dashpot element and expresses the finite deformation viscoelasticity laws based on the classical Zener model. The Fletcher–Gent effect, manifested as high horizontal stiffness at small strains and caused by the carbon fillers in HDRBs, is accurately expressed through an additional stiffness correction factor α in the novel strain energy function. Several material parameters are used to simulate the responses of high damping rubber at various strain levels, and a nonlinear viscosity coefficient η is introduced to characterize the rate-dependent property. A parameter identification scheme is applied to the results of the multi-step relaxation tests and the cyclic shear tests, and a three-dimensional function of the nonlinear viscosity coefficient η with respect to the strain, and strain rate is thus obtained. Finally, to investigate the accuracy and feasibility of the proposed model for application to the seismic response assessment of bridges equipped with HDRBs, an improved real-time hybrid simulation (RTHS) test system based on the velocity loading method is developed. A single-column bridge was used as a test bed and HDRBs was physically tested. Comparing the numerical and RTHS results, advantage of the proposed model in the accuracy of the predicted seismic response over comparable hysteretic models is demonstrated. Copyright © 2016 John Wiley & Sons, Ltd.

Elastomeric bearings have been a mature and efficient technique to mitigate structural damage caused by earthquakes. This paper proposed a generalized mathematical model for accurately evaluating the rate-dependent stress–strain response of natural rubber bearing (NRB), high damping rubber bearing (HDRB) and super high damping rubber bearing (SHDRB). A novel strain energy function including an additional stiffness correction factor α is proposed to describe the Fletcher–Gent effect of elastomeric materials, which featured by high initial stiffness at small strain ranges. The proposed constitutive model is composed of two parts, the first part consisting of a hyperelastic spring represents the rate-independent equilibrium stress, while the second part consisting of a Maxwell element expresses the rate-dependent overstress. Parameter identification scheme is implemented based on the results of the multi-step relaxation tests and monotonic shear tests. Numerical simulations for monotonic shear tests were conducted to demonstrate the capacity of the proposed model in predicting the stress–strain relationship of elastomeric bearings at different strain rates. Finally, in order to investigate the accuracy and feasibility of the proposed model on the application to the seismic response assessment of isolated bridges, real-time hybrid simulation (RTHS) test was performed and favorable results were obtained.

The precise predictions of the differentiation direction and potential of mesenchymal stromal cells (MSCs) are an important key to the success of regenerative medicine. The expression levels of fate-determining genes may provide tools for predicting differentiation potential. The expression levels of 95 candidate marker genes and glycosaminoglycan (GAG) contents after chondrogenic induction in 10 undifferentiated ilium and 5 jaw MSC cultures were determined, and their correlations were analyzed. The expression levels of eight genes before the induction of chondrogenic MSC differentiation were significantly correlated with the GAG levels after induction. Based on correlation patterns, the eight genes were classified into two groups: group 1 genes (AURKB,E2F1,CDKN2D,LIF, andACLY), related to cell cycle regulation, and group 2 genes (CD74,EFEMP1, andTGM2), involved in chondrogenesis. The expression levels of the group 2 genes were significantly correlated with the ages of the cell donors. The expression levels ofCDKN2D,CD74, andTGM2were >10-fold higher in highly potent MSCs (ilium MSCs) than in MSCs with limited potential (jaw MSCs). Three-dimensional (3D) scatter plot analyses of the expression levels of these genes showed reduced variability between donors and confirmed predictive potential. These data suggest that group 2 genes are involved in age-dependent decreases in the chondrogenic differentiation potential of MSCs, and combined 3D analyses of the expression profiles of three genes, including two group 2 genes, were predictive of MSC differentiation potential.

Background The designation of some cardiac-specific proteins as prognostic biomarkers in chronic obstructive pulmonary disease (COPD) exacerbations suggest that the process of exacerbation involves cardiomyocyte injury. Among these cardiac biomarkers, heart-type fatty acid binding protein (h-FABP) is considered a very sensitive diagnostic marker for cardiomyocyte injury and a prognostic marker in chronic heart failure. However, the prognostic usefulness of h-FABP in patients with COPD remains unclear. Methods Sixty-six patients were enrolled in this study. Subjects who recovered from COPD exacerbation and were discharged without needing home oxygen therapy were defined as the improved group. Those who died of the COPD exacerbations, were discharged but needed home oxygen therapy, or were transferred to a rehabilitation hospital for respiratory failure and the remaining aftereffects of exacerbation were defined as the unimproved group. Results The improved and unimproved groups included 54 and 12 subjects, respectively. Compared with the improved group, the unimproved group had significantly higher white blood cell counts and alanine aminotransferase, lactate dehydrogenase, blood urea nitrogen (BUN), uric acid, potassium, and h-FABP levels, and significantly lower total protein and total cholesterol levels and estimated glomerular filtration rates, either at admission or during the early morning within 24h after admission. A multivariate analysis revealed that higher serum h-FABP and potassium levels were independently predictive of a poor prognosis following a COPD exacerbation, and a receiver operating characteristic curve analysis yielded a cutoff of 4.5ng/ml for predicting lack of improvement. Conclusion H-FABP may predict the outcomes of COPD exacerbation.

Background: Iron is essential metal for living. We reported association of serum iron and pulmonary function in smoking man. Further, several studies show hypoironemia and anemia were associated with pulmonary function or respiratory disease. However, it is not clear how iron play role in lungs. We used cigarette smoke exposure model to evaluate influence of iron deficiency. Methods: Iron deficient (ID) or normal diet (control) were fed to C57BL/6J male mice for 3 weeks. Later, mice were exposed cigarette smoke. Bronchoalveolar lavage (BAL) and pulmonary function tests were performed after 2 weeks of cigarette exposure or 8 weeks of cigarette exposure. Results: In mice exposed to CS for 2 weeks, concentration of alveolar macrophages in BAL fluid recovered from ID mice were significantly increased compared to those from control mice. Further, interleukin (IL)-6 levels in BAL fluid were significantly increased from baseline in ID mice, but this increase of IL-6 was not observed in control mice. In mice exposed to CS for 8 weeks, total lung capacity in ID mice was significantly larger than those in control mice. Conclusion: Iron deficiency may enhance airway inflammation and worsen pulmonary function in mice exposed to cigarette smoke.

Background Few studies are available regarding the annual decline of forced expiratory volume in 1s (FEV1) in chronic obstructive pulmonary disease patients with mild airflow obstruction. This study sought to clarify to what extent cigarette-smoking individuals with mild airflow obstruction lose pulmonary function annually. Methods From 2004 to 2006, pulmonary function tests were performed on people >40 years of age, during the annual health checkup held in Takahata, Yamagata, Japan (initial study population, n =3253). In 2011, pulmonary function tests were performed again on participants who agreed to undergo reexamination (follow-up study population, n =838). Results Smokers have decreased pulmonary function in terms of percent forced vital capacity (FVC), %FEV1, and FEV1/FVC; the stages of airflow obstruction were also more severe in smokers than never-smokers. The annual decline in FEV1 was significantly greater in smokers than in never-smokers. The median annual decline in FEV1 was most significant in individuals with mild airflow obstruction. The annual decline in FEV1 was greater in smokers with mild airflow obstruction than in smokers with moderate airflow obstruction. In analyzing the decline in %FEV1, the annual change in smokers with mild airflow obstruction was greater than that in smokers with normal spirometric values. Conclusion The annual decline in FEV1 was most significant in smokers with mild airflow obstruction in a Japanese general population. This highlights the importance of early detection of chronic obstructive pulmonary disease patients among the general population in order to prevent disease progression in undiagnosed patients.

Background: Several methods have been developed to detect allergen-specific IgE in sera. The passive IgE sensitization assay using human IgE receptor-expressing rat cell line RBL-2H3 is a powerful tool to detect biologically active allergen-specific IgE in serum samples. However, one disadvantage is that RBL-2H3 cells are vulnerable to high concentrations of human sera. Only a few human cultured cell lines are easily applicable to the passive IgE sensitization assay. However, the use of human induced pluripotent stem cells (iPSCs) to generate human mast cells (MCs) has not yet been reported. Methods: The nuclear factor-kappa B (NF-κB)-responsive luciferase reporter gene was stably introduced into a human iPSC line 201B7, and the transfectants were induced to differentiate into MCs (iPSC-MCs). The iPSC-MCs were sensitized overnight with sera from subjects who were allergic to cedar pollen, ragweed pollen, mites, or house dust, and then stimulated with an extract of corresponding allergens. Activation of iPSC-MCs was evaluated by β-hexosaminidase release, histamine release, or luciferase intensity. Results: iPSCs-MCs stably expressed high-affinity IgE receptor and functionally responded to various allergens when sensitized with human sera from relevant allergic subjects. This passive IgE sensitization system, which we termed the induced mast cell activation test (iMAT), worked well even with undiluted human sera. Conclusions: iMAT may serve as a novel determining system for IgE/allergens in the clinical and research settings. Keywords: Allergen-specific IgE, Allergy test, Human mast cells, Induced pluripotent stem cells, Luciferase

Background: Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) is a hyaluronic acid receptor that is selectively expressed in the endothelia of lymphatic capillaries. The density of lymphatic vessels expressing LYVE-1 on immunohistochemistry negatively correlates with prognosis of patients with non-small-cell lung cancer. However, the relationship between LYVE-1 serum levels and lung cancer staging is unknown. Methods: We collected blood samples from 58 lung cancer patients before treatment and measured LYVE-1 serum levels using an enzyme-linked immunosorbent assay. Results: Mean serum LYVE-1 levels were 1,420 pg/mL. Serum LYVE-1 levels correlated positively with serum albumin levels, but inversely with primary tumor size, leukocyte counts, and platelet counts by Pearson's product-moment correlation coefficient. A high cancer staging, occurrence of lymph-node metastases, and occurrence of distant metastases were significantly associated with low LYVE-1 levels. Moreover, multiple logistic regression analyses revealed that LYVE-1 levels were predictive of the presence of lymph node and distant metastases, independently of the other factors. Kaplan-Meier analysis showed that the survival of patients with serum LYVE-1 ≤1,553 pg/mL was significantly poorer than that of patients with serum LYVE-1 >1,553 pg/mL. This survival difference relative to LYVE-1 levels remained statistically significant after adjusting for age and gender by the Cox proportional-hazard analysis. Conclusion: Serum LYVE-1 is significantly low in lung cancer patients with metastasis, compared with those without. Measuring LYVE-1 levels in lung cancer patients may be useful for evaluating lung cancer progression.

This paper presents the experimental as well as analytical study conducted on layer-bonded scrap tire rubber pad (STRP) isolators to develop low-cost seismic isolators applicable to structures in developing countries. The STRP specimen samples were produced by stacking the STRP layers one on top of another with the application of adhesive. In unbonded application, the STRP bearings were placed between the substructure and superstructure without fastening between the contact surfaces which allows roll-off of the contact supports. The vertical compression and horizontal shear tests were conducted with varying axial loads. These results were used to compute the different mechanical properties of the STRP isolators including vertical stiffness, horizontal effective stiffness, average horizontal stiffness and effective damping ratios. The load-displacement relationships of STRP isolators obtained by experimental and finite element analysis results were found to be in close agreement. The tested STRP samples show energy dissipation capacity considerably greater than the natural rubber bearings. The layer-bonded STRP isolators serve positive incremental force resisting capacity up to the shear strain level of 150%.

Background:Plasma fibrinogen is considered a biomarker of respiratory disease, owing to the relationship between plasma fibrinogen and pulmonary function established in Western populations. However, such a relationship has not yet been confirmed in an Asian population. We assessed this relationship in the general Japanese population. Methods:Totally, 3,257 men and women aged ≥40 years who participated in a community-based annual health checkup in Takahata, Japan, from 2004 to 2006, underwent spirometry, and their plasma fibrinogen levels were determined. Results:We found an inverse relationship between spirometric measures (percent predicted forced vital capacity [%FVC] and forced expiratory volume in 1s [%FEV1], and FEV1/FVC) and plasma fibrinogen levels in men, but not in women. The plasma fibrinogen levels were significantly higher in subjects with restrictive, obstructive, and mixed ventilatory disorders than in those with normal spirometry results. Multiple linear regression analysis revealed that in men, plasma fibrinogen levels were predictive for %FVC and %FEV1 (independent of age, body mass index, and cigarette smoking) but not for FEV1/FVC. Conclusions:Plasma fibrinogen was significantly associated with pulmonary function in Japanese men, and as such, plasma fibrinogen might be a potent biomarker for pulmonary dysfunction in men.

Background: Metabolic syndrome (Mets) is reportedly associated with chronic obstructive pulmonary disease (COPD). However, the relationship between abdominal circumference (AC) and decline in FEV1 has not been elucidated. We aimed to investigate this relationship among male current smokers. Methods: Spirometry was performed on subjects (n = 3,257) ≥ 40 years of age, who participated in a community-based annual health check in Takahata, Japan, from 2004 through 2006 (visit 1). Spirometry was re-evaluated, and AC was assessed in 147 of the male current smokers in 2009 (visit 2). The diagnosis of Mets was based on the criteria used in the Hisayama Study. Results: No significant relationships were observed between AC and spirometric parameters such as % predicted forced vital capacity (FVC), % predicted forced expiratory volume in 1 s (FEV1) and FEV1/FVC. However, decline in FEV1 was significantly correlated with AC. Multivariate logistic regression analysis showed that AC was a significant discriminating factor for decline in FEV1, independently of age, Brinkman index and change in body mass index from visit 1 to visit 2. At visit 2, there was a greater prevalence of decline in FEV1 among subjects with Mets (n=17) than among those without Mets. Although there were no differences in % predicted FVC, % predicted FEV1 or FEV1/FVC between subjects with or without Mets, the rate of decline in FEV1 was significantly greater in subjects with Mets than in those without. Conclusions: This retrospective analysis suggested that measuring AC may be useful for discriminating male smokers who show a decline in FEV1.

Introduction or background: E-Selectin (ES) is one of the adhesion molecules expressed on the vascular endothelial cells. We previously demonstrated that ES predicts the occurrence of acute lung injury in the pneumonic patients. Aims and objectives: To assess whether ES predicts the outcome of the patients with inflammatory lung diseases (IDs). Methods: We analyzed serum levels of ES in the 118 patients who were admitted to our hospital due to IDs including interstitial pneumonia (n=41), and lower respiratory tract infection (n=69), and pneumothorax, as non-inflammatory disease control (n=8), from January 2013 to December 2013, retrospectively. One hundred eleven patients were alive during hospital stay, and seven patents were died due to respiratory failure (n=7). Results: Subjects9 mean age was 71.8 years (78 men and 40 women). Mean hospital stay was 18.8 days. ES levels in the IDs tended to be higher compared to control group (P =0.09). Among the patients with the IDs, circulating levels of ES were significantly higher in patients who were died than those who were alive (alive: 5241 ± 389 pg/mL, died: 11012 ± 1486 pg/mL, P Conclusions: ES may be a predictive factor for outcome of patients with IDs.

Background Low-grade exercise tolerance is associated with a poor prognosis in patients with chronic obstructive pulmonary disease (COPD). The 6 min walk test (6MWT) is commonly used to evaluate exercise tolerance in patients with COPD. However, little is known regarding the relationship between cardiac function and exercise tolerance in patients with COPD. The aim of this study was to identify predictive factors in cardiac function for low-grade exercise tolerance in patients with stable COPD. Methods We recruited 57 patients with stable COPD (men 54, women 3) to perform the 6MWT. Patients with underlying orthopaedic disease or heart failure were excluded. Cardiac function was evaluated by echocardiography and contrast-enhanced cardiac CT. We also measured pulmonary function and the 6MWT distance. Results Forced expiratory volume in 1 s (FEV1) and per cent predicted FEV, along with left ventricular end diastolic volume and left ventricular cardiac output as measured by cardiac CT, were significantly related to the 6MWT distance. On multivariate analysis, left ventricular stroke volume was the factor most closely associated with a decreased walked distance in the 6MWT. Conclusions Decreased left ventricular stroke volume was associated with low-grade exercise tolerance in patients with stable COPD without heart failure.

Risk factors for death from respiratory failure in the general population are not established. The aim of this study was to determine the characteristics of individuals who die of respiratory failure in a Japanese general population. In total, 3253 adults aged 40 years or older participated in annual health check in Takahata, Yamagata, Japan from 2004 to 2006. Subject deaths through the end of 2010 were reviewed; 27 subjects died of respiratory failure (pneumonia, n = 22; COPD, n = 1; pulmonary fibrosis, n = 3; and bronchial asthma, n = 1). Cox proportional hazard analysis revealed that male sex; higher age, high levels of D-dimer and fibrinogen; lower body mass index (BMI) and total cholesterol; and history of stroke and gastric ulcer were independent risk factors for respiratory death. On analysis with C-statistics, net reclassification improvement and integrated discrimination improvement, addition of the disease history and laboratory data significantly improved the model prediction for respiratory death using age and BMI. In conclusion, we identified risk factors for mortality from respiratory failure in a prospective cohort of a Japanese general population. Men who were older, underweight, hypocholesterolemic, hypercoagulo-fibrinolytic and had a history of stroke or gastric ulcer had a higher risk of mortality due to respiratory failure.

Hyperhomocysteinaemia is associated with chronic obstructive pulmonary disease. However, the relationship between plasma homocysteine levels and spirometric measures has not been investigated in a general population. We aimed to determine whether homocysteine levels are predictive for a rapid decline in lung function among healthy current smokers. Blood sampling and spirometry were performed on subjects participating in a community-based annual health check in Takahata, Japan, from 2004 to 2006 (n=3257). Spirometry was re-evaluated in 147 male current smokers in 2009. On initial assessment, forced vital capacity (FVC) % predicted and forced expiratory volume in 1 s (FEV1) % predicted correlated inversely with homocysteine levels and were predictive for homocysteine levels, independent of various clinical factors. Homocysteine levels were higher in subjects with restrictive, obstructive or mixed ventilatory disorders. In addition, homocysteine levels were higher in subjects with mixed ventilatory disorders, compared with restrictive or obstructive disorders. On follow-up, subjects showing a decline in FEV1 had higher homocysteine levels than those who did not. Logistic regression analysis indicated that homocysteine levels were predictive for a decline in FEV1. FVC % pred and FEV1 % pred were significantly associated with homocysteine levels, and hyperhomocysteinaemia predicted the annual rate of decline in FEV1 among male smokers.

A new base isolation system using scrap tire rubber pads (STRP) has been introduced for seismic mitigation of ordinary residential buildings. The rubber and the steel reinforcing cords used in manufacturing the tire are the alternative materials of the proposed base isolation system. The steel reinforcing cords represent the steel plates used in conventional laminated rubber bearings. These steel reinforcing cords shall prevent the lateral bulging of the rubber bearing. The proposed base isolation system has no bonding between the superstructure and the foundation beam which allows for rollover deformation. In the first part of the study, the STRP layers were just stacked one on top of another without applying the adhesive. This paper presents loading test as well as finite element analysis (FE analysis) of strip STRP isolators that are subjected to any given combination of static vertical and lateral loads. The results of the static vertical and horizontal loading test conducted on STRP isolators were used to calculate the mechanical properties of the isolators, including stiffness and damping values. The load–displacement relationship of STRP isolators were compared between experimental and FE analysis results and the results were found to be in close agreement. The stress state within the STRP isolators was also analyzed in order to estimate the maximum stress demand in the rubber and steel reinforcing cords. These STRP isolators have several advantages over conventional laminated rubber bearings including superior damping properties, lower incurred cost, light weight and easily available material. This study suggests that using the STRP as low cost base isolation device for ordinary residential buildings is feasible.

Pulmonary innate immunity is impaired in cigarette smokers, because the abundant oxidants present in cigarette smoke (CS) cause injury to lung cells. Pulmonary surfactant is a unique material that is important roles in reducing surface tension in the lung and defending against invading pathogens. Oxidants reportedly cleave surfactant phospholipids, resulting in the production of oxidized phospholipids, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (PON-GPC). Although oxidation of surfactant lipids is thought to be involved in the pathogenesis of smoking-related lung disease, there are no reports on the effect of oxidized surfactant lipid on the immune function of macrophages. We hypothesized that cigarette smoking elevates PON-GPC levels in the lung, and that PON-GPC impairs the innate immune function of macrophages.The levels of PON-GPC in bronchoalveolar lavage fluid (BALF) recovered from mice exposed to CS for 2 weeks (n = 7) were measured by liquid chromatography with electrospray-ionization tandem mass spectrometry. The effects of PON-GPC on inducibility of tumor necrosis factor (TNF)-α, nitric oxide (NO), and nicotinamide adenine dinucleotide phosphate (NADP(+)) production, as well as bactericidal activity, were investigated in RAW264.7 cells or primary alveolar macrophages.The levels of PON-GPC in BALF of mice exposed to CS were significantly elevated, compared with those of control mice. PON-GPC attenuated TNF-α, NO, and NADP(+) production in macrophages on stimulation with LPS plus IFN-γ. PON-GPC treatment attenuated the phosphorylation of p38 mitogen-activated protein kinase (MAPK). In addition, PON-GPC reduced the bactericidal activity of RAW264.7 cells.CS may attenuate innate immunity in the lungs through oxidization of surfactant phospholipids.

Forced expiratory volume in 6 seconds (FEV(6)) is becoming a substitute of forced vital capacity (FVC). However, the Japanese predictive equation for FEV(6) has not been established, and the validity for the use of FEV(1)/FEV(6) for diagnosing airflow limitation in Japanese has not been confirmed.Subjects aged 40 or older, who had participated in a community-based health check in Takahata, Japan, from 2004 through 2005, were enrolled. The smoking histories of these subjects were investigated using a self-reporting questionnaire. FVC, FEV(1), and FEV(6) were measured using spirometric machines. Predictive equations of FEV(6) were obtained from never-smoking subjects without history of pulmonary diseases by multiple linear regression assay.FEV(6) and FEV(1)/FEV(6) were significantly correlated with FVC (r=0.998, p0.001) and FEV(1)/FVC (r=0.989, p0.001), respectively. The cutoff values of percent predicted (%) FEV(6) and FEV(1)/FEV(6) for discrimination of having the restrictive lung disorder determined by %FVC0.8 and having the airflow limitation determined by FEV(1)/FVC0.7 were 0.80 and 0.72, respectively (%FEV(6): sensitivity=0.995, specificity=0.983, positive predictive valuePPV=0.832, negative predictive valueNPV=1.000; FEV(1)/FEV(6): sensitivity=0.942; specificity=0.971; PPV=0.787; NPV=0.993). When the 5th percentile the lower limit of normal values was used as criterion for discrimination of having airflow limitation, sensitivity, specificity, PPV, and NPV of FEV(1)/FEV(6) were 0.932, 0.985, 0.808, and 0.995, respectively.The results of the present study suggest that %FEV(6) and FEV(1)/FEV(6) are excellent substitutes for %FVC and FEV(1)/FVC, respectively. We confirmed the validity of the use of FEV(6) and FEV(1)/FEV(6) for identifying pulmonary diseases in Japanese individuals.

Background: Pneumonia is still one of the most frequent causes of death in the elderly. Complication of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) by pneumonia makes patients very ill due to severe respiratory failure. Biomarkers that can discriminate the presence of complicating ALI/ARDS are required for early detection. The aim of this research was to investigate whether soluble endothelial selectin (sES) could be a biomarker for ALI. Methods: Serum sES levels were measured in 27 pneumonia patients, who were enrolled between April 2006 and September 2007. Among these patients, six had ALI or a condition that was clinically comparable to ALI (cALI). All patients who were enrolled were successfully treated and survived. Results: Circulating sES levels were elevated in pneumonia patients with ALI/cALI, and sES levels decreased following treatment of their pneumonia. Univariate and multivariate logistic regression analyses showed that sES was the only significant factor for identifying complicating ALI/cALI, independently of C-reactive protein (CRP) and lactate dehydrogenase (LDH). By receiver operating characteristic (ROC) curve analysis, the cut-off value for sES was 40.1 ng/mL, with a sensitivity of 0.8 and a specificity of 0.8. Conclusion: sES may be a useful biomarker for discriminating complicating ALI/cALI in patients with severe pneumonia.

本研究では，非線形復元力特性を持つ多自由度系の逐次の接線剛性行列を推定する手法として最小更新法に着目し，推定精度や推定更新公式の特性について検討を行った．応答塑性率，降伏耐力，入力波，緩和バイリニアモデルの設定など振動モデルの種々の条件設定に基づいて，得られた変位および復元力データを入力データと見なして各ケースにおける接線剛性行列の推定計算を行った．入力データにノイズが存在する場合の影響についても検討した．最小更新法は，非線形復元力特性を持つ多自由度系の接線剛性行列の低次の固有値の変化が良好な精度で推定できることを確認した．また，精度が最も高い更新式が振動モデルの条件により異なることを示した．

Background Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation. The prevalence of airflow limitation in Japan is 10.9% (16.4% of males and 5.0% of females). Cigarette smoking is well known as a major cause of COPD. However, few epidemiological studies have evaluated the effects of cigarette smoking on pulmonary function in healthy subjects. Methods Subjects aged 40 years or older (n=2,917), who had participated in a community-based annual health check in Takahata, Japan, from 2004 through 2005, were enrolled in the study. The smoking histories of these subjects were investigated using a self-reported questionnaire. Forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and forced expiratory flow at 25-75% of FVC (FEF25-75) were measured by standard procedures using spirometric machines. Results There were 554 current smokers (18.6%) and 403 former smokers (13.8%). The prevalence of airflow limitation defined by FEV1/FVC

Azithromycin (AZM), a 15-member macrolide antibiotic, possesses anti-inflammatory activity. Macrophages are important in innate and acquired immunity, and produce pro-inflammatory cytokines such as interleukin (IL)-12, which are composed of subunit p40 and p35. The key function of IL-12 is the induction and maintenance of T-helper-1 responses, which is associated with the pathogenesis of chronic inflammatory diseases. We investigated the effect of azithromycin on IL-12p40 production in macrophages after lipopolysaccharide (LPS)/interferon (IFN)-gamma stimulation. RAW264.7 macrophage cell line was pre-treated with vehicle or AZM, followed by the stimulation with LPS/IFN-gamma. We measured IL-12 production by RT-PCR and ELISA. IL-12 transcriptional regulation was assessed by electrophoretic mobility shift assay and reporter assay. Phosphorylation of activator protein (AP)-1 and interferon consensus sequence binding protein (ICSBP) was assessed by immunoprecipitation using phosphotyrosine antibody, and immunoblotting using specific antibodies against JunB and ICSBP. AZM reduced the induction of IL-12p40 by LPS/IFN-gamma in a dose dependent manner. AZM inhibited the binding of AP-1, nuclear factor of activated T cells (NFAT), and ICSBP, to the DNA binding site in the IL-12p40 promoter. AZM also reduced LPS/IFN-gamma-induced IL-12p40 promoter activity. Phosphorylation of JunB and ICSBP was inhibited by azithromycin-treatment in stimulated cells. In conclusion, AZM reduced IL-12p40 transcriptional activity by inhibiting the binding of AP-1, NFAT, and ICSBP to the promoter site. This may represent an important mechanism for regulating the anti-inflammatory effects of AZM in macrophages.

Background and objective: Pulmonary emphysema is associated with frequent respiratory infections but little is known about the reasons for this susceptibility to bacterial infection. We previously demonstrated an impaired inflammatory response to Streptococcus pneumoniae in an experimental emphysema mouse model at 24 h, or longer following bacterial inoculation. Toll-like receptors (TLR) have been recognized as regulators in the inflammatory response. We examined the expression of TLR on alveolar macrophages in experimental emphysema mice and evaluated the immediate inflammatory response of the emphysematous lung to streptococcal infection. Methods: Elastase was administered once into mice trachea to induce pulmonary emphysema. Three weeks later, expression of TLR-2 and TLR-4 in the BAL cells was examined by immunostaining. Following the intratracheal inoculation of Streptococcus pneumoniae, pro-inflammatory cytokine concentrations were measured in the BAL fluids of the control and emphysema mice. Results: The expression of TLR-2 and TLR-4 was significantly elevated in the alveolar macrophages of emphysema mice. Six hours after infection, neutrophils in the BAL fluid of emphysema mice were significantly increased, and the levels of tumour necrosis factor-α, IL-1β and IL-6 were significantly elevated, compared with the control mice. At 3 h post inoculation, macrophage inflammatory protein-2 levels were significantly elevated. Conclusions: The immediate inflammatory response in the emphysematous lung is significantly enhanced in response to streptococcal infection. This may be partly attributed to the increased expression of TLR in the alveolar macrophages of emphysema mice.

The forced expiratory volume in the first second (FEV1.0)/the forced vital capacity (FVC) is an important index of a single forced expiration. Ectopic expression of the human olfactory receptor (OR) gene family in the lungs has suggested its potential involvement of respiratory physiology. We hypothesized that the individual variability of FEV1.0/FVC value may be attributed to the genetic variance of the OR gene family caused by the nonfunctioning SNPs (nSNPs). We conducted quantitative trait locus (QTL) analyses of population having the 7 OR gene nSNPs and FEV1.0/FVC values by ANOVA, in 2970 samples in the Yamagata Takahata cohort. We found significant association of one nSNP [rs10838851, OR, family 4, subfamily X, member 1 (OR4X1) gene, Tyr273Ter*] with FEV1.0/FVC (%) (P = 0.008). The FEV1.0/FVC value (%) of population having OR4X1 gene nSNP Ter*/Ter*, Ter*/Tyr, and Tyr/Tyr were 78.9 +/- 0.2, 78.2 +/- 0.2, and 77.7 +/- 0.4, respectively. Haplotype-based analysis of the OR4X1 gene with FEV1.0/FVC values demonstrated that two exclusive haplotypes [Hap-1/Hap-2 (frequency 0.669/0.330): SNP1 (rs7106648)T/A-SNP2 (rs871249)G/A-SNP3 (rs713325)G/A-SNP4 (rs10838851)A (Ter*)/T (Tyr)-SNP5 (rs4752923)G/A-SNP6 (rs960640)G/A] were significantly associated with FEV1.0/FVC values (global P = 0.005). These results suggest that OR4X1 may be one of the genes that contribute to the individual variability of FEV1.0/FVC value in pulmonary function test.

Background and Objective: Molecular gene markers, which can distinguish human bone marrow mesenchymal stem cells from human fibroblasts, have recently been reported. Messenger RNA levels of tissue factor pathway inhibitor-2, major histocompatibility complex-DR-α, major histocompatibility complex-DR-β, and neuroserpin are higher in human bone marrow mesenchymal stem cells than in human fibroblasts. However, human bone marrow mesenchymal stem cells express less apolipoprotein D mRNA than human fibroblasts. Periodontal ligament cells are a heterogeneous cell population including fibroblasts, mesenchymal stem cells, and progenitor cells of osteoblasts or cementoblasts. The use of molecular markers that distinguish human bone marrow mesenchymal stem cells from human fibroblasts may provide insight into the characteristics of human periodontal ligament cells. In this study, we compared the molecular markers of human periodontal ligament cells with those of human bone marrow mesenchymal stem cells and human gingival fibroblasts. Material and Methods: The mRNA expression of the molecular gene markers was analyzed using real-time polymerase chain reaction. Statistical differences were determined with the two-sided Mann–Whitney U-test. Results: Messenger RNA levels of major histocompatibility complex-DR-α and major histocompatibility complex-DR-β were lower and higher, respectively, in human periodontal ligament cells than in human bone marrow mesenchymal stem cells or human gingival fibroblasts. Human periodontal ligament cells showed the lowest apolipoprotein D mRNA levels among the three types of cells. Conclusion: Human periodontal ligament cells may be distinguished from human bone marrow mesenchymal stem cells and human gingival fibroblasts by the genes for apolipoprotein D, major histocompatibility complex-DR-α, and major histocompatibility complex-DR-β.

Background The forced oscillation technique (FOT) can measure respiratory system resistance and reactance under tidal volume respiration. MostGraph is a device that incorporates the FOT and enables the immediate, three-dimensional visualization of resistance and reactance parameters. The aim of this study was to establish MostGraph reference values for middle-aged and elderly Japanese individuals. Methods From 2004 to 2006, 3253 subjects living in Takahata, Yamagata underwent spirometry. Of these, 872 again underwent spirometry in 2011, and 784 (368 men, ages 46–89 years; 416 women, ages 47–90 years) underwent FOT examinations using MostGraph-01. Results In this study population, 19.0% of the men and 91.5% of the women were life-long never smokers. Abnormal spirometric findings were observed in 30.2% of the men and 14.6% of the women. Although the respiratory system resistance and reactance parameters obtained using MostGraph were not distributed normally, normal distribution was achieved via natural logarithm (R5, R20, Fres, and ALX), square root (R5-R20), or exponential (X5) transformation. Furthermore, the transformed values were converted back to the actual values after determining the values representing one and two standard deviations from the mean. Conclusion Respiratory system resistance and reactance reference values were determined using MostGraph in middle-aged and elderly Japanese individuals who participated in annual health checkups.

The Japanese encephalitis (JE) zoonotic vaccine strain ML-17 was sequenced and compared to related JE virus strains to identify genomic attenuation markers. Relative to its parental strain, JaOH0566, 25 nucleotide alterations and 10 amino acid changes to, prM/M(2), NS2A(1), NS4B(3) and NS5(4) proteins were recorded. Both structural-gene changes were in the prM/M region (127Met-->Ile and 274Asn-->Thr). To study the effects of these prM/M changes, mutants bearing the changes were prepared using an infectious clone of JaOArS982 previously established at this lab. Compared with JaOArS982, mutant 127(Met-->Ile) showed marked reduction in murine neuroinvasiveness. Mutant 274(Asn-->Thr), showed slight reduction. Neither mutant recorded ML-17-equivalent attenuation, implying that prM/M changes need to combine with other recorded genomic differences to cause attenuation. Importantly, ML-17 with its unchanged E region, presents a possible backbone candidate for preparation of "E-replacement" type live attenuated flavivirus chimeric vaccines.