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1.  Pneumococcal polysaccharide vaccination in rheumatoid arthritis patients receiving tacrolimus 
In rheumatoid arthritis (RA) patients receiving immunosuppressive treatments, vaccination against Streptococcus pneumoniae is recommended. The objective of the study was to evaluate the effects of tacrolimus (TAC) on immune response following administration of a 23-valent pneumococcal polysaccharide vaccine (PPSV23) in patients with established RA.
Patients with RA (n = 133) were vaccinated with PPSV23. Patients were classified into TAC (n = 29), methotrexate (MTX) (n = 55), control (n = 35), and TAC/MTX (n = 14) treatment groups. We measured the concentrations of pneumococcal serotypes 6B and 23F by using an enzyme-linked immunosorbent assay and determined antibody functionality by using a multiplexed opsonophagocytic killing assay, reported as the opsonization index (OI), before and 4 to 6 weeks after vaccination. A positive antibody response was defined as at least a twofold increase in the IgG concentration or as at least a 10-fold increase in the OI.
IgG concentrations and OIs were significantly increased in all treatment groups after PPSV23 vaccination. The TAC treatment group appears to respond in a manner similar to that of the RA control group in terms of 6B and 23F serotype concentration and function. In contrast, the MTX group had the lowest immune response. Patients who received a combination of TAC and MTX (TAC/MTX) also had a diminished immune response compared with those who received TAC alone.
TAC monotherapy does not appear to impair PPSV23 immunogenicity in patients with RA, whereas antibody production and function may be reduced when TAC is used with MTX. Thus, PPSV23 administration during ongoing TAC treatment should be encouraged for infection-prone TAC-treated patients with rheumatic diseases.
Trial registration
University Hospital Medical Information Network Clinical Trials Registry: UMIN000009566. Registered 12 December 2012.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0662-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4481124  PMID: 26036592
3.  Epstein–Barr Viral Load is Associated to Response in AIDS-Related Lymphomas 
AIDS-related lymphoma (ARL) development is associated to immunodeficiency state with proliferation of B-cells driven by HIV itself and EBV infection. However, Epstein–Barr DNA is not detected in malignant cells of all ARL subtypes. A prospective and controlled study to analyze EBV viral load (VL) in plasma and peripheral blood mononuclear cells (PBMC) of ARL patients was performed to analyze if Epstein–Barr VL could be related to response in these patients. Fifteen patients with ARL were included in this study with measurement of EBV VL at three different periods of time: at lymphoma diagnosis, upon completion of chemotherapy, and 3 months after. Two control groups composed by HIV-negative and HIV-positive patients were also evaluated for EBV VL comparison. In situ hybridization for EBER was performed on diagnostic samples of all ARL patients. Median EBV VL in PBMC and plasma had a significant decrease (p = 0.022 and p = 0.003, respectively) after ARL treatment. EBER was positive in 7 (46.7 %) cases. Median EBV VL in PBMC before lymphoma treatment in patients positive for EBER was significantly higher compared to EBER negative cases (p = 0.041). Reduction of EBV viral load during treatment of lymphoma could be predictive of response. EBER expression was associated to advanced stages of disease and worse immune status. Our study suggests that measurement of EBV VL during ARL treatment could be used as a marker for response, but further studies are needed to validate this association.
PMCID: PMC4115083  PMID: 25114406
Aids-related lymphoma; HIV; EBV
4.  Carboxyhemoglobin Formation in Preterm Infants Is Related to the Subsequent Development of Bronchopulmonary Dysplasia 
Disease Markers  2015;2015:620921.
Objective. To evaluate the usefulness of carboxyhemoglobin (CO-Hb) levels as a biomarker to predict the development and severity of bronchopulmonary dysplasia (BPD). Methods. Twenty-five infants born at <33 wk of gestational age or with a birth weight of <1,500 g were enrolled. CO-Hb levels were measured between postnatal days 5 and 8, 12 and 15, 19 and 22, and 26 and 29. Urinary levels of 8-hydroxydeoxyguanosine (8-OHdG), advanced oxidation protein products, and Nε-(hexanoyl) lysine were measured between postnatal days 5 and 8 and 26 and 29. Receiver operating characteristic (ROC) analysis was used to compare the biomarkers' predictive values. Results. Compared with infants in the no-or-mild BPD group, infants with moderate-to-severe BPD exhibited higher CO-Hb levels during the early postnatal period and higher 8-OHdG levels between postnatal days 5 and 8. Using ROC analysis to predict the development of moderate-to-severe BPD, the area under the curve (AUC) for CO-Hb levels between postnatal days 5 and 8 was higher than AUCs for the urinary markers. Conclusions. CO-Hb levels during the early postnatal period may serve as a practical marker for evaluating oxidative stress and the severity of subsequently developing BPD.
PMCID: PMC4532888  PMID: 26294808
5.  Fluorescence Polarization Switching from a Single Silicon Vacancy Colour Centre in Diamond 
Scientific Reports  2015;5:12244.
Single-photon emitters with stable and uniform photoluminescence properties are important for quantum technology. However, in many cases, colour centres in diamond exhibit spectral diffusion and photoluminescence intensity fluctuation. It is therefore essential to investigate the dynamics of colour centres at the single defect level in order to enable the on-demand manipulation and improved applications in quantum technology. Here we report the polarization switching, intensity jumps and spectral shifting observed on a negatively charged single silicon-vacancy colour centre in diamond. The observed phenomena elucidate the single emitter dynamics induced by photoionization of nearby electron donors in the diamond.
PMCID: PMC4511871  PMID: 26202940
6.  Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases 
Scientific Reports  2015;5:11674.
NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.
PMCID: PMC4495429  PMID: 26154028
7.  Heat-Denatured Lysozyme Inactivates Murine Norovirus as a Surrogate Human Norovirus 
Scientific Reports  2015;5:11819.
Human norovirus infects humans through the consumption of contaminated food, contact with the excrement or vomit of an infected person, and through airborne droplets that scatter the virus through the air. Being highly infectious and highly viable in the environment, inactivation of the norovirus requires a highly effective inactivating agent. In this study, we have discovered the thermal denaturing capacity of a lysozyme with known antimicrobial activity against gram-positive bacteria, as well as its inactivating effect on murine norovirus. This study is the first report on the norovirus-inactivating effects of a thermally denatured lysozyme. We observed that lysozymes heat-treated for 40 min at 100 °C caused a 4.5 log reduction in infectivity of norovirus. Transmission electron microscope analysis showed that virus particles exposed to thermally denatured lysozymes were expanded, compared to the virus before exposure. The amino acid sequence of the lysozyme was divided into three sections and the peptides of each artificially synthesised, in order to determine the region responsible for the inactivating effect. These results suggest that thermal denaturation of the lysozyme changes the protein structure, activating the region responsible for imparting an inactivating effect against the virus.
PMCID: PMC4488868  PMID: 26134436
8.  Spatio-temporal Dynamics and Mechanisms of Stress Granule Assembly 
PLoS Computational Biology  2015;11(6):e1004326.
Stress granules (SGs) are non-membranous cytoplasmic aggregates of mRNAs and related proteins, assembled in response to environmental stresses such as heat shock, hypoxia, endoplasmic reticulum (ER) stress, chemicals (e.g. arsenite), and viral infections. SGs are hypothesized as a loci of mRNA triage and/or maintenance of proper translation capacity ratio to the pool of mRNAs. In brain ischemia, hippocampal CA3 neurons, which are resilient to ischemia, assemble SGs. In contrast, CA1 neurons, which are vulnerable to ischemia, do not assemble SGs. These results suggest a critical role SG plays in regards to cell fate decisions. Thus SG assembly along with its dynamics should determine the cell fate. However, the process that exactly determines the SG assembly dynamics is largely unknown. In this paper, analyses of experimental data and computer simulations were used to approach this problem. SGs were assembled as a result of applying arsenite to HeLa cells. The number of SGs increased after a short latent period, reached a maximum, then decreased during the application of arsenite. At the same time, the size of SGs grew larger and became localized at the perinuclear region. A minimal mathematical model was constructed, and stochastic simulations were run to test the modeling. Since SGs are discrete entities as there are only several tens of them in a cell, commonly used deterministic simulations could not be employed. The stochastic simulations replicated observed dynamics of SG assembly. In addition, these stochastic simulations predicted a gamma distribution relative to the size of SGs. This same distribution was also found in our experimental data suggesting the existence of multiple fusion steps in the SG assembly. Furthermore, we found that the initial steps in the SG assembly process and microtubules were critical to the dynamics. Thus our experiments and stochastic simulations presented a possible mechanism regulating SG assembly.
Author Summary
Cells suffer from various environmental stresses such as heat shock and viral infection. In response to a stress, small non-membranous cytoplasmic aggregates, stress granules (SGs), are assembled. SGs contain mRNAs and related proteins. Hippocampal CA1 neurons located in the brain, which are vulnerable to ischemia, do not assemble SGs, while CA3 neurons, which are resilient to ischemia, assemble SGs. The dysfunction of SGs has been reported in human diseases including pathogenic viral infection. These observations led to a hypothesis that SGs play an important role in cell fate decisions, and the dynamics of SG assembly would regulate cell fate. However, the conditions that determine the number and distribution of SGs in a cell in response to a stress are largely unknown. We approached this problem by experiments and simulations. Our stochastic simulations replicated the observations. Furthermore, we found that initial steps in the SG assembly process were important to the dynamics of SG assembly, and that SG size resembled the gamma distribution both in simulations and experiments, suggesting the existence of multiple steps in the SG assembly process. To the best of our knowledge, this work was the first to show SG assembly in a whole cell by stochastic simulations.
PMCID: PMC4482703  PMID: 26115353
9.  Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes 
PLoS ONE  2015;10(6):e0129855.
Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed “patterned culture”), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed “non-patterned cultures”). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.
PMCID: PMC4483150  PMID: 26115194
10.  mTOR Complex Signaling through the SEMA4A–Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells 
Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4+ T cell responses, the involvement of mTOR in CD8+ T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8+ T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A−/− CD8+ T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8+ T cell responses were significantly impaired in SEMA4A−/− mice. Furthermore, SEMA4A−/− CD8+ T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8+ T cells. IFN-γ production and mTORC1 activity in SEMA4A−/− CD8+ T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8+ T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8+ T cells, but also reveal a novel link between a semaphorin and mTOR signaling.
PMCID: PMC4505953  PMID: 26116513
11.  Atom–Photon Coupling from Nitrogen-vacancy Centres Embedded in Tellurite Microspheres 
Scientific Reports  2015;5:11486.
We have developed a technique for creating high quality tellurite microspheres with embedded nanodiamonds (NDs) containing nitrogen-vacancy (NV) centres. This hybrid method allows fluorescence of the NVs in the NDs to be directly, rather than evanescently, coupled to the whispering gallery modes of the tellurite microspheres at room temperature. As a demonstration of its sensing potential, shifting of the resonance peaks is also demonstrated by coating a sphere surface with a liquid layer. This new approach is a robust way of creating cavities for use in quantum and sensing applications.
PMCID: PMC4476097  PMID: 26095793
Efficacy and safety of intracerebral gene therapy for brain disorders, like Parkinson’s disease, depends on appropriate distribution of gene expression.
To assess if the distribution of gene expression is affected by vector titer and protein type.
Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received in the right and left ventral postcommisural putamen 30μl inoculation of a high or low titer suspension of AAV5 encoding glial derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Inoculations were performed using convection enhanced delivery and intraoperative MRI (IMRI).
IMRI confirmed targeting and infusion cloud irradiating from the catheter tip into surrounding area. Postmortem analysis six weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection side that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the Substantia Nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many SNpc and SNpr neurons.
After controlling for target and infusate volume, intracerebral distribution of gene product is affected by vector titer and product biology.
PMCID: PMC4127999  PMID: 24943657
13.  Regulation of NF-κB Oscillation by Nuclear Transport: Mechanisms Determining the Persistency and Frequency of Oscillation 
PLoS ONE  2015;10(6):e0127633.
The activated transcription factor NF-κB shuttles between the cytoplasm and the nucleus resulting in the oscillation of nuclear NF-κB (NF-κBn). The oscillation pattern of NF-κBn is implicated in the regulation of gene expression profiles. Using computational models, we previously reported that spatial parameters, such as the diffusion coefficient, nuclear to cytoplasmic volume ratio, transport through the nuclear envelope, and the loci of translation of IκB protein, modified the oscillation pattern of NF-κBn. In a subsequent report, we elucidated the importance of the “reset” of NF-κBn (returning of NF-κB to the original level) and of a “reservoir” of IκB in the cytoplasm. When the diffusion coefficient of IκB was large, IκB stored at a distant location from the nucleus diffused back to the nucleus and “reset” NF-κBn. Herein, we report mechanisms that regulate the persistency and frequency of NF-κBn oscillation by nuclear transport. Among the four parameters of nuclear transport tested in our spatio-temporal computational model, the export of IκB mRNA from the nucleus regulated the persistency of oscillation. The import of IκB to the nucleus regulated the frequency of oscillation. The remaining two parameters, import and export of NF-κB to and from the nucleus, had virtually no effect on the persistency or frequency. Our analyses revealed that lesser export of IκB mRNA allowed NF-κBn to transcript greater amounts of IκB mRNA, which was retained in the nucleus, and was subsequently exported to the cytoplasm, where large amounts of IκB were synthesized to “reset” NF-κBn and drove the persistent oscillation. On the other hand, import of greater amounts of IκB led to an increase in the influx and the efflux of NF-κB to and from the nucleus, resulting in an increase in the oscillation frequency. Our study revealed the importance of nuclear transport in regulating the oscillation pattern of NF-κBn.
PMCID: PMC4456371  PMID: 26042739
14.  Limitation of immune tolerance–inducing thymic epithelial cell development by Spi-B–mediated negative feedback regulation 
The Journal of Experimental Medicine  2014;211(12):2425-2438.
Akiyama et al. show that transcription factor Spi-B is up-regulated by RANKL to trigger mTEC differentiation. Osteoprotegerin is also induced by this signaling pathway and acts as a negative feedback loop to attenuate mTEC development and thymic T reg cells.
Medullary thymic epithelial cells (mTECs) expressing the autoimmune regulator AIRE and various tissue-specific antigens (TSAs) are critical for preventing the onset of autoimmunity and may attenuate tumor immunity. However, molecular mechanisms controlling mTEC development remain elusive. Here, we describe the roles of the transcription factor Spi-B in mTEC development. Spi-B is rapidly up-regulated by receptor activator of NF-κB ligand (RANKL) cytokine signaling, which triggers mTEC differentiation, and in turn up-regulates CD80, CD86, some TSAs, and the natural inhibitor of RANKL signaling, osteoprotegerin (OPG). Spi-B–mediated OPG expression limits mTEC development in neonates but not in embryos, suggesting developmental stage–specific negative feedback regulation. OPG-mediated negative regulation attenuates cellularity of thymic regulatory T cells and tumor development in vivo. Hence, these data suggest that this negative RANKL–Spi-B–OPG feedback mechanism finely tunes mTEC development and function and may optimize the trade-off between prevention of autoimmunity and induction of antitumor immunity.
PMCID: PMC4235644  PMID: 25385757
15.  Prognostic significance of amino-acid transporter expression (LAT1, ASCT2, and xCT) in surgically resected tongue cancer 
British Journal of Cancer  2014;110(10):2506-2513.
Amino-acid transporters are necessary for the tumour cell growth and survival, and have a crucial role in the development and invasiveness of cancer cells. But, it remains unclear about the prognostic significance of L-type amino-acid transporter 1 (LAT1), system ASC amino-acid transporter-2 (ASCT2), and xCT expression in patients with tongue cancer. We conducted the clinicopathological study to investigate the protein expression of these amino-acid transporters in tongue cancer.
Eighty-five patients with surgically resected tongue cancer were evaluated. Tumour sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67, and microvessel density (MVD) determined by CD34, and p53.
L-type amino-acid transporter 1 and 4F2hc were highly expressed in 61% (52 out of 85) and 45% (38 out of 47), respectively. ASC amino-acid transporter-2 and xCT were positively expressed in 59% (50 out of 85) and 21% (18 out of 85), respectively. The expression of both LAT1 and ASCT2 was significantly associated with disease staging, lymph-node metastasis, lymphatic permeation, 4F2hc expression and cell proliferation (Ki-67). xCT expression indicated a significant association with advanced stage and tumour factor. By univariate analysis, disease staging, lymphatic permeation, vascular invasion, LAT1, ASCT2, 4F2hc, and Ki-67 had a significant relationship with overall survival. Multivariate analysis confirmed that LAT1 was an independent prognostic factor for predicting poor prognosis.
L-type amino-acid transporter 1 and ASCT2 can serve as a significant prognostic factor for predicting worse outcome after surgical treatment and may have an important role in the development and aggressiveness of tongue cancer.
PMCID: PMC4021522  PMID: 24762957
tongue cancer; LAT1; ASCT2; xCT; prognostic factor
16.  A fatal case of acute progression of generalized edema and simultaneous flash pulmonary edema in a patient with idiopathic systemic capillary leak syndrome: a case report 
Idiopathic systemic capillary leak syndrome is a rare and fatal disease due to the unexplained episodic attacks of capillary leakage of plasma from the intravascular into the interstitial space. The attack consists of three phases, a prodromal phase, peripheral leak phase and recruitment phase. During the peripheral leak phase, generalized edema, mainly in the trunk and extremities, with hemoconcentration and hypoalbuminemia occurs, while usually the visceral organs like lungs, brain, heart and kidneys seem not to be involved. Treatment of the acute phase is supportive, focusing on adequate but not overzealous fluid resuscitation, because pulmonary edema usually occurs in the recruitment phase.
Case presentation
A 65-year-old Japanese woman was admitted to our hospital because of severe hypovolemic shock with metabolic acidosis and hemoconcentration and hypoalbuminemia. Although she was considered to be in the peripheral leak phase of idiopathic systemic capillary leak syndrome, which could not be diagnosed during the treatment, the generalized edema worsened further, severe flash pulmonary edema progressed rapidly after fluid resuscitation and she died. The autopsy showed generalized edema, especially alveolar pulmonary edema without endothelial apoptosis.
Because hypovolemic shock and fatal pulmonary edema may progress rapidly together even in the peripheral leak phase of idiopathic systemic capillary leak syndrome, we should keep in mind this rare and fatal disease and recognize the pathophysiology to treat it effectively when the patient has hypovolemia with metabolic acidosis.
PMCID: PMC4422423  PMID: 25928289
Systemic capillary leak syndrome; Hypovolemic shock; Pulmonary edema; Autopsy findings
17.  Mature Cystic Teratoma in Douglas' Pouch 
Case Reports in Pathology  2015;2015:202853.
Mature cystic teratoma is one of the most common ovarian neoplasms, but extragonadal teratoma is rare. Teratoma in Douglas' pouch is extremely rare, and only 12 cases have been reported since the first case was described in 1978. We report a 20-year-old woman with a multicystic mass in Douglas' pouch that was treated via laparoscopic resection. The tumor consisted of cysts lined by stratified squamous epithelium with an accumulation of keratin debris and various mature tissues. No immature elements or malignancy was found in the tumor, confirming the pathologic diagnosis of a mature cystic teratoma. The teratoma contained no ovarian tissues and both of the ovaries were intact on laparoscopy. These findings suggest that the teratoma originated primarily in Douglas' pouch rather than being caused by autoamputation of a previously existing ovarian teratoma. This is the first case that simultaneously showed normal ovaries and a teratoma in Douglas' pouch on laparoscopy.
PMCID: PMC4427846  PMID: 26060593
18.  Correlation between plaque vulnerability of aorta and coronary artery: an evaluation of plaque activity by direct visualization with angioscopy 
This study investigated the relationship between the degree of atherosclerotic changes in the descending thoracic aorta (TA) and the coronary artery using angioscopy. Twenty-five consecutive patients undergoing angioscopy of the TA and coronary angiography were enrolled in this study. Participants were divided into three groups according to the angioscopic grading of the TA: white plaque group (W-group), yellow plaque group (Y-group) and intensive yellow, ruptured plaque with ulceration and/or thrombus group (RP-group). The maximum plaque grade, plaque score, number of yellow plaques, frequency of yellow-plaque grades by coronary angioscopy, and SYNTAX score by coronary angiography were evaluated. Brachial-artery pulse wave velocity and high-sensitivity C-reactive protein level tended to be higher in the RP-group than in the other groups, although the differences were not statistically significant. The SYNTAX score was significantly higher in the RP-group than in the W-group (W-group 4.0 ± 3.6 vs. RP-group 17.5 ± 10.0, P = 0.045). In addition, the angioscopic maximum plaque grade, plaque score, and number of yellow plaques in the RP-group were significantly higher than in the W-group (maximum plaque grade W-group 0.8 ± 0.4 vs. RP-group 1.8 ± 0.8, P = 0.026; plaque score W-group 1.0 ± 1.2 vs. RP-group 4.0 ± 1.4, P = 0.014; and number of yellow plaques W-group 1.0 ± 1.2 vs. RP-group 2.5 ± 0.5, P = 0.023). The yellow-plaque grade in the coronary artery was correlated significantly with the plaque grading of TA (P = 0.043). Our study suggests that the angioscopic progression of aortic atherosclerosis is closely associated with vulnerability to and the extent of coronary stenosis, indicating that vulnerability toward atherosclerotic plaque development occurs simultaneously in the coronary tree and systemic arteries.
Electronic supplementary material
The online version of this article (doi:10.1007/s10554-015-0669-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4486772  PMID: 25916323
Atherosclerosis; Angioscopy; Thoracic aorta; Coronary artery; Atherosclerotic plaque
19.  Biological significance of fluorine-18-α-methyltyrosine (FAMT) uptake on PET in patients with oesophageal cancer 
British Journal of Cancer  2014;110(8):1985-1991.
18F-FAMT as an amino-acid tracer for positron emission tomography (PET) is useful for detecting human neoplasms. 18F-FAMT is accumulated in tumour cells solely via L-type amino-acid transporter 1 (LAT1). This study was conducted to investigate the biological significance of 18F-FAMT uptake in patients with oesophageal cancer.
From April 2008 to December 2011, 42 patients with oesophageal cancer underwent both 18F-FAMT PET/CT and 18F-FDG PET/CT before surgical treatment. The immunohistochemical analysis of LAT1, CD98, Ki-67, CD34, p53, p-Akt and p-mTOR was performed on the primary lesions. In vitro experiments were performed to examine the mechanism of 18F-FAMT uptake.
High uptake of 18F-FAMT was significantly associated with advanced stage, lymph node metastasis and the expression of LAT1, CD98, Ki-67 and CD34. LAT1 expression yielded a statistically significant correlation with CD98 expression, cell proliferation, angiogenesis and glucose metabolism. In vitro experiments revealed that 18F-FAMT was specifically transported by LAT1.
The uptake of 18F-FAMT within tumour cells is determined by the LAT1 expression and correlated with cell proliferation and angiogenesis in oesophageal cancer. The present experiments also confirmed the presence of LAT1 as an underlying mechanism of 18F-FAMT accumulation.
PMCID: PMC3992502  PMID: 24667647
18F-FAMT; PET; LAT1; oesophageal cancer; BCH; biology
20.  Receptor mimicry by antibody F045-092 facilitates universal binding to the H3 subtype of influenza virus 
Nature communications  2014;5:3614.
Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012-2013. Here, we describe an antibody, F045-092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045-092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045-092 extends its recognition to divergent subtypes, including H1, H2, and H13, using the enhanced avidity of its IgG to overcome lower affinity Fab binding, as observed with other receptor-binding site antibodies. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small molecule therapeutics.
PMCID: PMC4358779  PMID: 24717798
21.  Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6 
The Journal of Experimental Medicine  2014;211(10):2013-2032.
Ma et al. show that the Toxoplasma gondii polymorphic dense granule protein GRA6 triggers the activation of the host transcription factor NFAT4, thus affecting the host immune response and maximizing parasite virulence.
Toxoplasma gondii infection results in co-option and subversion of host cellular signaling pathways. This process involves discharge of T. gondii effector molecules from parasite secretory organelles such as rhoptries and dense granules. We report that the T. gondii polymorphic dense granule protein GRA6 regulates activation of the host transcription factor nuclear factor of activated T cells 4 (NFAT4). GRA6 overexpression robustly and selectively activated NFAT4 via calcium modulating ligand (CAMLG). Infection with wild-type (WT) but not GRA6-deficient parasites induced NFAT4 activation. Moreover, GRA6-deficient parasites failed to exhibit full virulence in local infection, and the treatment of WT mice with an NFAT inhibitor mitigated virulence of WT parasites. Notably, NFAT4-deficient mice displayed prolonged survival, decreased recruitment of CD11b+ Ly6G+ cells to the site of infection, and impaired expression of chemokines such as Cxcl2 and Ccl2. In addition, infection with type I parasites culminated in significantly higher NFAT4 activation than type II parasites due to a polymorphism in the C terminus of GRA6. Collectively, our data suggest that GRA6-dependent NFAT4 activation is required for T. gondii manipulation of host immune responses to maximize the parasite virulence in a strain-dependent manner.
PMCID: PMC4172224  PMID: 25225460
22.  The Inhibitory Effect of Kakkonto, Japanese Traditional (Kampo) Medicine, on Brain Penetration of Oseltamivir Carboxylate in Mice with Reduced Blood-Brain Barrier Function 
Oseltamivir phosphate (OP) is used to treat influenza virus infections. However, its use may result in central nervous system (CNS) adverse effects. In Japan, OP is used with Kampo formulations to improve clinical effectiveness. We evaluated the potential for using Kampo formulations to reduce CNS adverse effects by quantifying the CNS distribution of oseltamivir and its active metabolite oseltamivir carboxylate (OC) when administered with maoto and kakkonto. We administered lipopolysaccharide (LPS) by intraperitoneal injection to C57BL/6 mice to reduce blood-brain barrier function. Saline, maoto, and kakkonto were administered orally at the same time as LPS. OP was orally administered 4 hours after the last LPS injection and the migration of oseltamivir and OC was examined. Additionally, we examined the brain distribution of OC following intravenous administration. Changes in OC concentrations in the brain suggest that, in comparison to LPS-treated control mice, both Kampo formulations increased plasma levels of OC, thereby enhancing its therapeutic effect. Additionally, our findings suggest kakkonto may not only improve the therapeutic effect of oseltamivir but also reduce the risk of CNS-based adverse effects. Considering these findings, it should be noted that administration of kakkonto during periods of inflammation has led to increased OAT3 expression.
PMCID: PMC4350872  PMID: 25788966
23.  Crmp4 deletion promotes recovery from spinal cord injury by neuroprotection and limited scar formation 
Scientific Reports  2015;5:8269.
Axonal outgrowth inhibitors and scar formation are two major obstacles to central nervous system (CNS) repair. No target molecule that regulates both axonal growth and scarring has been identified. Here we identified collapsin response mediator protein 4 (CRMP4), a common mediator of inhibitory signals after neural injury, as a crucial factor that contributes to both axonal growth inhibition and scarring after spinal cord injury (SCI). We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord. Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection. Crmp4−/− mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation. This permissive environment for axonal growth due to CRMP4 deletion restored locomotor activity at an unusually early phase of healing. These results suggest that deletion of CRMP4 is a unique therapeutic strategy that overcomes two obstacles to CNS repair after SCI.
PMCID: PMC4317684  PMID: 25652774
Nagoya Journal of Medical Science  2015;77(1-2):307-313.
Aneurysms within the internal acoustic canal are rare. We report the case of a 71-year-old female with subarachnoid hemorrhage resulting from a ruptured distal anterior inferior cerebellar artery which was not detected on initial radiological examination. A second rupture was detected by contrast-enhanced computed tomography and successfully treated by endovascular coil embolization. The patient recovered without neurological deficits. To the best of our knowledge, this is the first report of an intrameatal aneurysm treated by endovascular coil embolization. We suggest endovascular coil embolization as an alternative to open surgery, even in cases of deep intrameatal aneurysm.
PMCID: PMC4361533  PMID: 25797996
coil embolization; endovascular treatment; intrameatal aneurysm; subarachnoid hemorrhage
25.  Residual Structure of Streptococcus mutans Biofilm following Complete Disinfection Favors Secondary Bacterial Adhesion and Biofilm Re-Development 
PLoS ONE  2015;10(1):e0116647.
Chemical disinfection of oral biofilms often leaves biofilm structures intact. This study aimed to examine whether the residual structure promotes secondary bacterial adhesion. Streptococcus mutans biofilms generated on resin-composite disks in a rotating disc reactor were disinfected completely with 70% isopropyl alcohol, and were again cultured in the same reactor after resupplying with the same bacterial solution. Specimens were subjected to fluorescence confocal laser scanning microscopy, viable cell counts and PCR-Invader assay in order to observe and quantify secondarily adhered cells. Fluorescence microscopic analysis, particularly after longitudinal cryosectioning, demonstrated stratified patterns of viable cells on the disinfected biofilm structure. Viable cell counts of test specimens were significantly higher than those of controls, and increased according to the amount of residual structure and culture period. Linear regression analysis exhibited a high correlation between viable and total cell counts. It was concluded that disinfected biofilm structures favored secondary bacterial adhesion.
PMCID: PMC4312048  PMID: 25635770

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