Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2) is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1), but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines  Epigenetic alterations to the genome take place by covalent modifications to the DNA or histones and result in changes in gene expression that do not arise from changes in the underlying DNA sequence. Histone post-transcriptional modifications can occur at specific amino acids with a diverse set of chemical modifications including acetylation, methylation, phosphorylation, SUMOylation and ubiquitination . Among tG9a  and the closely related GLP  are HMTs that share 80% sequence identity in their catalytic domains and are believed to form homo- and hetero-dimers . H3K9-me50 of ~3 nM and >100-fold selectivity over other methyltransferases and other non-epigenetic targets [The first disclosed selective small molecule inhibitor of G9a was BIX01294, which was shown to decrease global H3K9me2 in mouse embryonic stem (ES) cells and fibroblasts . More re targets . We findAll tumor cell lines were obtained from American Type Culture Collection (ATCC). All tumor cell lines were grown according to manufacturer’s conditions in the recommended growth media in the presence of 10% fetal bovine serum (HyClone). Cell lines were tested and authenticated by short tandem repeat analysis using the GenePrint 10 System (Promega) and capillary electrophoresis (ABI Prism 3130). Primary human peripheral blood mononuclear cells (PBMCs) from two donors were obtained through BioreclamationIVT, pooled together and grown in AIM-V medium (Invitrogen). Viable cell numbers were measured using CellTiter-Glo reagent (Promega) as an endpoint assay. Inhibition was quantified as the ratio of treated cells compared to non-treated controls relative to cell free wells as 100% inhibition.2) for 72 hours. Cells were then fixed with 3% (w/v) formaldehyde/PBS for 15 minutes. After five washes with 0.1% (v/v) Triton X-100 in PBS, cells were blocked for 1 hour with 1% (w/v) BSA in PBS, then incubated with primary H3K9me2 mouse antibody at 1:500 and total Histone H3 rabbit antibody at 1:1000 in 0.3% BSA and PBS for 4 hours. Secondary fluorescence-conjugated antibodies (LiCor Biosciences) were added for 1 hour. After five washes with 0.1% Tween-20 in PBS, the plates were read on an Odyssey (LiCor Biosciences) scanner at 800 nm  and 700 nm . Fluorescence intensity was quantified using the Licor Odyssey imaging suite software.Antibodies used included anti-H3K9me2 , anti-total Histone H3 , anti-G9a , anti-H3K4me2 , anti-H3K4me3 , anti-H3K9me3 , anti-H4K20me1 , anti-H4K20me3 , anti-H3K36me2 , and anti-H3K27me3. Western blots were performed as previously described . For In-MV4;11 or HL-60 cells were incubated with the indicated concentrations of A-366 for the indicated number of days. Cells were collected and then washed with cold PBS before staining with 0.5ml PBS containing 50 μg/ml propidium iodide (PI), 0.1mg/ml RNase A, 0.1% BSA, and 0.1% Triton-X100 for 20 minutes. Cell cycle distribution was analyzed using a BD LSR-II flow cytometer (BD Biosciences). FACS analysis of CD11b expression was performed on duplicate cell samples using PE-conjugated human CD11b-specific mouse monoclonal antibody at a 1:5 dilution (BD Pharmingen) on a BD LSR11 FACS machine.Live cells were washed and seeded at 500 cells per well into six-well plates in drug-free medium. 24 hours later, compounds were diluted in the appropriate cellular media and added to the cells. Cells were cultured at 37°C for 7–10 days. Cells were then fixed and stained with 0.2% crystal violet to visualize and count colonies using a GelCount colony counter (Oxford Optronix).MV4;11 cells were treated with DMSO or 5 μM A-366 for 10 days. Following incubation, treated cells were stained using a Wright-Giemsa kit .Cells were treated for 48 hours with the indicated concentrations of compound and assessed for viability using CellTiter-Glo reagent (Promega). H3K9me2 levels were measured utilizing the H3K9me2 AlphaLISA assay system (PerkinElmer) and normalized to cell viability. All values were then normalized to non-treated cells as 0% inhibition of H3K9me2.6 cells per mouse were injected subcutaneously into the flank of 6–8 week old SCID-beige female mice . The tumors were allowed to grow to approximately 200 mm3, at which time mice were allocated by tumor volume into study groups (n = 10 mice/ group) so that the mean tumor volumes of the groups were statistically similar. Mice were then entered into the dosing phase of the study (described below) and tumor volumes were recorded 2 times per week. Tumor volumes were estimated by the formula V = (L x W2)/ 2, where V is the volume (mm3), L is the tumor length (mm) and W is the tumor width (mm), measured at right angles using a digital caliper. The effect of a treatment on tumor growth inhibition was determined as %TGI = mean tumor volume of control group—mean tumor volume of treatment group/ tumor volume of treatment control group x 100. A-366 was formulated for delivery by osmotic mini-pump in 98% PEG-400 and 2% Tween-80.All animal studies were conducted in accordance with the guidelines established by internal Institutional Animal Care and Use Committees at AbbVie, Inc. . Tumor cells were mixed with 50% Matrigel  and 5 x 10in vitro enzymatic IC50 of ~3 nM and >100-fold selectivity over other methyltransferases and other non-epigenetic targets [50 of ~300 nM  ultimately led to the generation of A-366  cell line MOLT-16, UNC0638 inhibited cell proliferation at concentrations consistent with its H3K9me2 EC50; however, A-366 had no impact on the proliferation of this cell line even though it inhibited H3K9me2 as potently as UNC0638  cell line HL-60 with A-366. As opposed to the differentiation and ultimate decrease in viability seen with A-366-treated MV4;11 cells, short or long-term treatment  of HL-60 cells resulted in a dose-dependent differentiation and a corresponding decrease in proliferation . Howevertostasis .in vivo setting we tested A-366 in a AML flank xenograft model using MV4;11 cells. However, initial MTD studies with A-366 dosed intraperitoneally (IP) showed toxicity in the animals at 10 mg/kg, which would limit exposures to a level not predicted to be efficacious for inhibiting tumor growth. To achieve a projected efficacious exposure while mitigating the presumed Cmax-driven toxicity, osmotic mini-pump (OMP) dosing was utilized. Mice treated with either vehicle control or A-366 administered via osmotic mini-pump at 30 mg/kg/day for two weeks showed no overt toxicity. We observed a modest 45% tumor growth inhibition resulting from A-366 treatment in this model Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(A) HL-60 cells were incubated with A-366 for 4 days. Cells were fixed, stained with an anti-CD11b antibody and analyzed by flow cytometry. (B) HL-60 cells from (A) were assessed for cellular proliferation (Cell Titer-Glo) and viability (trypan blue exclusion) following 4 days of treatment with A-366. (C) Cell cycle DNA content analysis was performed by propidium iodide staining in HL-60 cells treated for 4 days with A-366.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor ( Thus, statins are thought to exert their atheroprotective effects through KLF2 to some extent.Since the discovery of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors KLF2 functions as a regulator of inflammation, and also participates in vasodilatation and anti-coagulation KLF2 reportedly mediates the expression of a series of response genes KLF2 is of particular interest in atherogenesis, because cholesterol accumulation and low shear stress in the vascular wall are two major aspects of atherosclerotic plaque formation, suggesting that the KLF family exerts important biological effects on the cellular phenotype. Thus, we focused on KLFs to investigate the molecular mechanism of gene regulation in statin-treated vascular cells.Krüppel-like factors are zinc finger transcriptional factors that have been implicated in blood vessel development and T lymphocyte activation KLFs in HUVECs under statin treatment, we performed transcriptome analysis using a microarray and statistically identified the affected genes. Unexpectedly, KLF4 was induced more than the others, including KLF2. Since the importance of KLF4 in the regulation of other atheroprotective genes was thus suggested, we focussed on the molecular mechanisms of pitavastatin-dependent KLF4 induction.Human umbilical vein endothelial cells (HUVECs) are primary cultivated endothelial cells widely used in vascular biology research and provided a critical model for molecular mechanism of atherosclerosis, because umbilical vein carries oxygenated blood flow just as arteries KLF4, followed by KLF2  in either condition were selected for further study (384 genes). Hierarchical clustering analysis was performed after selecting the genes  ligand 2 (CCL2) was also included in this cluster.First, to evaluate the gene expression profiles of the KLF family members, we performed microarray analysis of HUVECs treated with pitavastatin for 4 hours. Based on the results of repeated experiments, the most highly induced gene was  by KLF2 . To confhe genes A. Figur present B. This KLF4 and THBD or NOS3, we performed quantitative real time PCR to determine the time course profile of mRNA expression. A significant induction of KLF4 was observable within 2 hours of pitavastatin treatment, followed by THBD and NOS3 mRNA induction after 4 hours , we focused on MEF2C, because it is expressed more potently than the two other MEF2 family members in HUVECs. To identify the transcription regulatory elements by which MEF2C contributes to the KLF4 induction under pitavastatin treatment, we made an effort to identify the binding sites in a whole genome manner. A newly established monoclonal antibody against MEF2C   A. SustahIP-qPCR . HistonehIP-qPCR A.MEF2C binding sites are functionally active, we carried out a series of luciferase reporter assays in HUVECs with constructs containing the human KLF4 promoter (between bp −994 and +594 bp) coupled to the MEF2C binding region from kb −98 (255 bp), or kb −148 (506 bp)  B. As shobserved C, suggeMEF2 binding motif in the kb −148 enhancer region. Based on this sequence, we generated the enhancer (−148 kb)-KLF4-luc containing a 6 base mutation (GAGATAAAAATAACC to GGTACCAACCGAGAT) of the MEF2 binding site to determine the importance of this motif for statin-mediated KLF4 promoter activation. As shown in MEF2C binding site. Taking these results together, statins increased the expression of KLF4 in ECs by activating the MEF2C binding element in the −148 kb region.By consensus motif search, we found one KLF4 gene functions, we sought to determine the localization of transcriptionally active RNA polymerase II (Pol II). To perform ChIP-seq, a monoclonal antibody was established against the C-terminal domain (CTD) of the largest subunit of Pol II, in which the tetramer repeats (Y1S2P3T4S5P6S7) contain two phosphorylation sites at the second and fifth Serine  based experiments. First, to test whether the distal enhancer directly communicates with the promoter region, we performed whole genome chromatin interaction analysis with paired end tag sequencing (ChIA-PET) KLF4 was detected in the serum-starved control, suggesting this enhancer may co-exist with the promoter in the Pol II rich transcription complex As was reported previously KLF4 and the −148 kb element was confirmed  exert certain beneficial effects, especially a reduction of the plasma cholesterol levels. Various reports have also suggested that direct effect on vascular wall activity might also contribute to the atheroprotectiveness of the statins KLF family members are reported to exert anti-atherogenic effects in both in vivo and in vitro models NOS3, THBD and a disintegrin and metalloproteinase with thrombospondin motif 1 (ADAMTS1), were identified CCL2)  C. TheseKLF4 induction is not well understood, although a few critical pathways have been reported MEK5/ERK5/MEF2C pathway was shown to participate in the maintenance of the vascular network KLF4 have been reported MEF2 family members, MEF2A, -2C and -2D, are expressed in ECs. While there was no vascular phenotype reported for MEF2D-deficient mice MEF2C-deficient mice suggests an essential role for MEF2CMEF2A was also reported in an inherited disorder with features of coronary artery disease MEF2C was demonstrated in this report, the relative importance and the interactions of the MEF2 family members involved in statin-dependent KLF4 induction await further elucidation.The signaling cascade elicited by statin treatment that exerts an effect on MEF2C binding sites were identified in a whole genome manner by ChIP-seq, and inducible MEF2C recruitment in the −98 and −148 kb regions of KLF4 (MEF2C compared with statin (−) was shown in the kb −148 region, and there was a 2.0 fold increase compared with statin (−) for the kb −98 region   were cultured in EGM2MV medium (Lonza), and cells were cultured at 37°C in 5% COPitavastatin was supplied by Nissan Chemical industries .Klf4, Thbd, Nos3 and β-actin, real-time quantitative PCR was performed. Regarding 2O. The same procedure was repeated for 5 min with periodate-lysine-paraformaldehyde (PLP) fixative solution. The heart and aorta were removed rapidly and fixed in PLP fixative solution. Thereafter, the aortic sinus was dissected and embedded in paraffin, and 3-µm cross sections were prepared for Victoria blue-HE staining. The internal elastic lamina was identified under light microscopy  after Victoria blue-HE staining, and quantification of the plaque lesions was performed using image analysis . All animal experiments were performed in the KOWA company and they were approved by Animal Ethics Committee of Kowa Company.C57BL/6J ApoE−/− mice were purchased from Jackson Laboratory . The animal room was controlled at 23 ± 3°C and a relative humidity of 50 ± 20%. Body weight was measured at the time of drug administration. Eight-week-old ApoE deficient mice (n = 12 for each group) were treated for 12 weeks with vehicle alone or pitavastatin at 3mg/kg/treatment with a normal chow diet . The mice were fed twice a day with vehicle or pitavastatin. The mice were anesthetized with an intraperitoneal injection of pentobarbital sodium  and the aortae were isolated. All of the aortae were homogenized with ISOGEN solution and the RNA extracted. Total RNA was extracted with a commercially available RNA extraction kit. To determine the levels of expression of 8 column with a mobile phase that consisted of acetic acid/methanol . Pitavastatin and IS were detected by LC/MS/MS with positive electrospray ionization (ESI). The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 422–290 for pitavastatin and m/z 406–318.1 for IS, respectively. The standard curve was 6 points (2∼200 ng/mL) and all samples were included in a standard range.An Agilent 1100 system consisting of a degasser, a binary pump, an ALS thermostat, a column compartment set at 30 °C , an autosampler HTC-PAL set at 20 °C  and a TSQ Quantum triple quadrupole mass spectrometry  were used for the LC/MS/MS analysis. Data acquisition and quantification were performed using LCquan . Each mouse was orally administrated pitavastatin once, and the serum average Cmax concentration of 3 mg/kg pitavastatin was 0.26 µM. A sample was extracted from mouse plasma and added to internal standard solution (IS) Life Technologies negative control siRNA were transfected with lipofectamine 2000 RNAiMAX  according to the manufacturer's protocol. Growth medium was replaced 4 hr after transfection. All of the validated siRNAs were purchased from Life Technologies/Thermo Fisher Scientific.KLF4, THBD, NOS3 and cyclophilin are shown in Klf4, Thbd and Nos3 were purchased from Life Technologies/Thermo Fisher Scientific.RNA was isolated from cells using Isogen reagent  according to the manufacturer's protocol. Real-time quantitative PCR was performed on a CFX96 detection system  using TaqMan and SYBR green. The sequences of the applied primers for Human Protein samples were separated by 10% SDS-PAGE and transferred electrophoretically onto nitrocellulose membranes . Membranes were blocked with 5% (wt/vol) non-fat milk in PBS containing 0.1% Tween for 1 hour, incubated with antibodies for 1 hour and detected by chemiluminescence using West Dura extended duration substrate  according to the manufacturer's protocol.MEF2A, MEF2C, KLF2 and KLF4 were established with a baculovirus expression system, as described previously MEF2A (IgG-Y0841), (aa) 161–248 of human MEF2C (IgG-Y1740), (aa) 2–34 of human KLF2 (IgG-N2212) and (aa) 26–90 of human KLF4 (IgG-Y6929) were generated. The recognition of MEF2A by Y0841, MEF2C by Y1740, KLF2 by N2212 or KLF4 by Y6929 was confirmed by immunoblotting using HUVEC proteins. The specificity of these antibodies was confirmed by the reduction of the protein levels using siRNA (1S2P3T4 (phospho-S)5P6 (phospho-S)Y7SPTSPSYSPC] (Sigma-Genosys) coupled to keyhole limpet hemocyanin, and the specificity was evaluated by ELISA and immunoblotting, as described in Antibodies recognizing β-actin and α-tubulin  as well as MEF2D and nucleoporin p-62  were purchased products. Monoclonal antibodies for human ng siRNA . Mouse mKLF4 starting at 2 hours and reached a plateau in 6 hours . Preparation of the cRNA and hybridization of the probe arrays were performed according to the manufacturer's instructions . Affymetrix GeneChip Human Genome U133 plus 2.0 arrays containing 54,675 probe sets were applied. The expression values for each mRNA were obtained by the Robust Multi-array Analysis (RMA) method KLF4 was compared with siControl, then the genes which had a fold change> = 2.0 or < = 0.5 in either comparison were selected (384 genes)  . Hierarc4 genes) . All ana6 HUVEC cells were plated on a 15 cm culture plate and cultivated for 3 days. They were treated with pitavastatin at a concentration of 1 µM for 4 hours and cells were crosslinked for 10 min using 1% paraformaldehyde. After neutralization using 0.2 M Glycine solution, cells were recollected and resuspended in SDS lysis buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% SDS, 1 mM EDTA). Cell suspensions, including the DNA-protein complexes, were subjected to fragmentation using a Sonifier-250 (Branson) for 10 min. MEF2C-bound chromatin was isolated from whole cell lysate using an established mouse monoclonal antibody to MEF2C in combination with magnetic beads (Life Technologies/Thermo Fisher Scientific). The prepared DNA was quantified using Qubit fluorometer (Life Technologies/Thermo Fisher Scientific) and more than 10 ng of DNA were processed for ChIP-seq. The remaining DNA was used for ChIP-quantitative PCR (qPCR). Primer pairs for ChIP-PCR are shown in The detailed experimental procedure for chromatin immunoprecipitation followed by deep sequencing is described elsewhere http://www.illumina.com/pages.ilmn?ID=203) All of the protocols for Illumina/Solexa sequencing preparation, sequencing and quality control were provided by Illumina . Primer pairs for construction of KLF4 promoters are shown in KLF4 promoter fragments were amplified by PCR using genomic DNA from HEK293 as the template. For construction of the KLF4-luc plasmid, the human Briefly, HUVECs were cultivated and serum starved for 16 hrs. The prepared cells in culture plates were crosslinked using ethylene glycol bis (succinimidylsuccinate) (EGS)  at a concentration of 10 mM in 50% glacial acetic acid for 45 min, followed by crosslinking with 1% of paraformaldehyde , then neutralized by 2.5 M glycine for 5 min. The harvested cell suspension was sonicated with a Branson sonifier for 4 min 40 sec. Chromatin immunoprecipitation was performed using an antibody for Pol II (Pd75C9) using magnetic beads (Life Technologies/Thermo Fisher Scientific). The detailed library construction procedures and the sequences of the half-linkers were previously reported The assay was performed utilizing TaqMan 3C Chromosome Conformation Kits  (Life Technologies) according to the manufacturer's instructions with certain modifications. Briefly, HUVECs were cross-linked with 1% paraformaldehyde for 10 min. The reaction was stopped by the addition of 125 mM glycine for 5 min. Nuclei were re-suspended with restriction enzyme buffer. Samples were treated with 400 U of Hind III at 37°C for 16 hours. After enzymatic digestion, the samples were diluted with ligation buffer and treated with DNA ligase at 16°C for 1 hour. Samples were finally reverse cross-linked and purified. Two human bacterial artificial chromosome (BAC) clones  were used to normalize the levels of 3C products which may have been PCR-amplified with different primer efficiencies, as mentioned previously KLF4 gene and the MEF2C binding enhancer) were selected based on a genome database (The UCSC Genome Browser on Human Feb. 2009 (GRCh37/hg19) Assembly). The BAC clones were purchased from Advanced GenoTechs Co.  and used as probes. Probe K includes the KLF4 gene (RP11-80F13) while probe M includes the MEF2C binding region (RP11-359L14)  equipped with a Plan-Apochromat 63×/1.4 Oil DIC objective lens. For each optical section, images were collected sequentially for three fluorochromes  using multi-argon (458/477/488/514 nm) and helium-neon (543/633 nm) lasers. To improve the signal-to-noise ratio, each sectional image obtained was an average of eight successive scans. The z-step between sections was 0.2 µm. Stacks of 12-bit gray scale two-dimensional images were obtained with 512×512 pixels in each channel. Confocal image stacks were processed with microscope operating software  and saved as LSM files. More than 50 nuclear images were captured from each of the cellular materials. Nuclei from cultured cells with singlet-singlet signals were adopted for calculation as in the G1 phase of the cell cycle, but with doublet-doublet or singlet-doublet signals for each probe, which were suspected of being in the S or G2 phase, were not selected for capture. The representative 3D-images shown in The image stacks were 3D-reconstructed using the program AVS/Express version 7.2 and the reasonable thresholds for each channel were determined by a 3D coordination of the whole volume of the gene signals. The 3D coordinates of two target regions in each cell were specified and the shortest distance between the gravity center of each gene was calculated using 3D image processing and analysis software CTMS (Chromosome Territory Measurement Software) (Cybernet Co. Ltd.).P-value was less than 0.05.Values are reported as the mean ± standard deviation (SD). Student's t-test, Dunnett's test, or Wilcoxon rank-sum test was used for comparisons between two groups. Differences were considered statistically significant when the All of the microarray data files, ChIP-seq data files and ChIA-PET data file are deposited. The GEO and accession numbers are: GSE32547, GSE32644, GSE32693, GSE41553.File S1Supporting Information. Figure S1. Flow chart for microarray analysis. Affymetrix GeneChip Human Genome U133 plus 2.0 arrays were applied for all analysis. The analysis was performed with GeneSpring GX 12.5 following the layout on this flow chart. The details are shown in the Methods section. Insignificant or unannotated probe data was eliminated at each step. The number on the right side shows the number of the remaining probe sets or genes at each step. The 384 selected genes were used for further analyses in Figure S2. Gene regulation by pitavastatin in HUVECs and the aortae of Apo-E-deficient mice. (A) HUVECs were treated with 1 µM pitavastatin for the indicated time. (B) ApoE deficient mice were orally administered pitavastatin twice daily at 3 mg/kg/treatment for 12 weeks before sacrifice. Total RNA was isolated and determined by real-time quantitative PCR, as described in Methods. The sequences of applied primers are shown in Table S2B in File S1. Vertical lines indicate the S.D. , * P<0.01, ** P<0.001, compared with the control sample, Dunnett's test in A and Student's t test in B. Figure S3. Histological examination of atherosclerotic regions in the aortic sinus. Eight-week-old ApoE deficient mice (n = 12 for each group) were treated twice daily for 12 weeks with vehicle alone (-) or pitavastatin at 3 mg/kg/treatment (+). The heart and aorta were removed rapidly and fixed and embedded in paraffin for Victoria blue-HE staining as described in Methods. Victoria blue-hematoxylin-eosin staining (A) revealed the atherosclerotic lesions (arrow). (B) shows the total plasma cholesterol and triglyceride levels. Note that pitavastatin reduced the plaque area without changing the plasma cholesterol and triglyceride levels. The vertical lines indicate the SEM (n = 12), * P<0.001 compared with the control sample, Student's t test. n.s. indicates not significant. Figure S4. Identification of the MEF2A, MEF2C, KLF2 and KLF4 antibodies. HUVECs were incubated with 1 µM pitavastatin for 4 hours. Before pitavastatin treatment, as described in the Methods section, cells were transfected with siRNA to KLF2 (A), KLF4 (B), MEF2A (C) and MEF2C (D).  The whole cell fraction was prepared for the Western blotting experiment to detect KLF2, MEF2A and MEF2C. Beta-actin was used as the internal control. (B) HUVECs were incubated with 1 µM pitavastatin for 24 hours. The nuclear extract fraction was prepared for further Western blotting to detect KLF4. Nucleoporin p62 was used as an internal control. NS; Non silencing for negative control. Note that loss of band detection was observable only by treatment specific siRNA, which demonstrates the specificity of new antibodies. N2212, Y6929, Y0841, Y1740 are clone numbers for each antibody. Figure S5. Triple knock down of MEF2A, C and D reduces KLF2 and KLF4. HUVECs were incubated with 1 µM pitavastatin for 4 hours. Before the treatment, as described in the Methods section, cells were transfected with siRNA to MEF2A, -2C and -2D. A whole cell fraction was prepared for a further Western blotting experiment to detect KLF2, and a nuclear extract fraction was prepared for KLF4. Alpha Tubulin and Nucleoporin p62 were used as the internal controls for the whole cell lysate and nuclear extract fractions, respectively. NS; Non silencing for negative control. Figure S6. Localization of the MEF2C binding sites in HUVECs. HUVECs were incubated with DMSO [statin(-)]or 1 µM pitavastatin for 4 hours. (A) Chromatin immunoprecipitation was performed using an MEF2C antibody, followed by deep sequencing. 4,878 MEF2C binding sites were detected in the control [pitavastatin (−)]. 42% of the MEF2C binding sites were located between the TSS and 3′UTR of the genes, while the remaining of 58% were found in intergenic regions. (B) Co-localization of MEF2C binding sites and H3K27ac in control [pitavastatin (−)] HUVECs. 25,477 binding sites were detected using anti-H3K27ac antibody ChIP-seq analysis in the control [pitavastatin (−)] HUVECs. Among them, 798 binding sites displayed co-localization of H3K27ac and MEF2C. (C) Distribution of the MEF2C binding sites in the pitavastatin-treated HUVECs. 13,030 MEF2C binding sites were detected in the treated HUVECs; 40% of them were located between the TSS and 3′UTR of the genes, while 60% were located in intergenic regions. Figure S7. ChIP-qPCR with an anti-MEF2C antibody against the KLF4 gene.MEF2C Binding sites detected in the KLF4 upstream region , *P<0.01, compared with the kb −147 KLF4, statin (−), ‡P<0.01 compared with the kb −98 or kb-148 KLF4, statin (−), Student's t test. Figure S8. The specificity of the Pol II antibody. A newly-developed monoclonal antibody (Pd75C9) was used to perform ELISA with RNA polymerase II C-terminal domain peptides containing different phosphorylation patterns. (A) The list of peptides used for ELISA. Phosphorylated amino acids are indicated in red. C-terminal repeat of RNA Polymerase II is comprises from 25 to 52 tandem copies of the consensus repeat heptad Y1S2P3T4S5P6S7 (Also shown in the right panel of figure A). And antibodies for a variety of phosphorylated CTD were raised using 19 AA peptides including phosphorylated Serine as is depicted. (B) ELISA. Microtiter plates coated with the peptides (the sequence shown in A) were incubated with 3-fold dilutions of the monoclonal antibody (starting from a 1∶27 dilution of a hybridoma culture supernatant). After incubation with a peroxidase-conjugated secondary antibody and washing, the colorimetric signal of tetramethylbenzidine was detected by measuring the absorbance at 405 nm (Abs) using a plate reader. Although Pd75C9 was generated using the Pd peptide to immunize mice, this clone reacted with all of the phospho-serine containing peptides, with preference for those containing phospho-S5 and phosphor-S2, but reacted only slightly with the unphosphorylated peptides. Figure S9. Sequence of the PCR product generated in the quantitative 3C assay. To validate the quantitative 3C assay, PCR products were directly sequenced and identified. The chromatogram shows the sequence of the target analysis (the fragments including KLF4 and 148 kb upstream from the TSS of KLF4) under pitavastatin treatment. As expected, the sequence of the TSS and that of the kb −148 enhancer were directly connected by a Hind III recognition sequence, and the measurement of the amount of this PCR product stands for the frequency of chromatin interaction. Figure S10. The sensitivity and specificity of the probes. To validate the probes, BAC clone DNA (RP11-80F13 and RP11-359L14) was tested for correct chromosomal location at 9q31.2 and the absence of other signals by 2D-FISH, respectively. The following datafrom (A) to (K) is the detailed information on probe K and (L) to (Q) is the detailed information of probe M. (A) Probe design for the 2D-FISH analysis of the target region on human Chr.9q31.2. The numbers in the middle indicate the location on Chr.9 using the hg19 build program. Probe K includes the KLF4 gene. Three-color 2D-FISH was carried out by a combination of a labeled Probe K and human Chr.9 arm-specific painting probes . (B) The p arm of Chr.9 is represented in purple. (C) The q arm of Chr.9 represented in green. (D) Probe K is represented in red. (E) Nuclear DNA was counterstained with DAPI  and is shown in blue. The merged image with all of the colors is shown in (F). Probe K in the interphase is shown in Figures G to K. All of the combinations of the labeled Probe K and human Chr.9 p and q arm-specific painting probes were the same as in B-F. The white arrows indicate the representative signals of Probe K in the interphase (K). (L) Probe design for the 2D-FISH analysis of the target region on human Chr.9q31.2. The numbers in the middle indicate the location on Chr.9 using the hg19 build program. Probe M includes the MEF2C binding region, which is located 148kb upstream from the TSS of KLF4. (M) The q arm of Chr.9 is represented in red. (N) The p arm of Chr.9 is represented in purple. (O) Probe M is represented in green. (P) Nuclear DNA was counterstained with DAPI  and is represented in blue. The merged image with all of the colors is shown in (Q). The white arrows indicate the representative signals of Probe M in the metaphase. The signals in the interphase are shown in the other nuclei. It was confirmed that the signal of the BAC clone was clearly detected on the Chr.9q region and absent from the other chromosomes. Figure S11. Statin effect on KLF4 expression. HUVECs were treated with pitavastatin at a concentration ranging from 0.01 to 10 µM for 12 hours. Total RNA was isolated and determined by Real-time quantitative PCR, as described in the Methods section. The vertical lines indicate the S.D. (n = 3), * P<0.001, compared with the sample of 0 µM, Dunnett's test. Table S1. Table S2.m region  were qua(PDF)Click here for additional data file.


Silencing of the paternal X chromosome (Xp), a phenomenon known as imprinted X-chromosome inactivation (I-XCI), characterises, amongst mouse extraembryonic lineages, the primitive endoderm and the extraembryonic endoderm (XEN) stem cells derived from it.Using a combination of chromatin immunoprecipitation characterisation of histone modifications and single-cell expression studies, we show that whilst the Xp in XEN cells, like the inactive X chromosome in other cell types, globally accumulates the repressive histone mark H3K27me3, a large number of Xp genes locally lack H3K27me3 and escape from I-XCI. In most cases this escape is specific to the XEN cell lineage. Importantly, the degree of escape and the genes concerned remain unchanged upon XEN conversion into visceral endoderm, suggesting stringent control of I-XCI in XEN derivatives. Surprisingly, chemical inhibition of EZH2, a member of the Polycomb repressive complex 2 (PRC2), and subsequent loss of H3K27me3 on the Xp, do not drastically perturb the pattern of silencing of Xp genes in XEN cells.The observations that we report here suggest that the maintenance of gene expression profiles of the inactive Xp in XEN cells involves a tissue-specific mechanism that acts partly independently of PRC2 catalytic activity. This Venn diagram includes only X-linked genes that are expressed in all three cell types and that are common to the present study as well as to analyses of liver cells [E:20617]  and subjE:20617]  compareder cells . Expresser cells . XEN celClick here for fileDistribution of H3K4me2 along the X chromosome in XEN cells. (A) Boxplots showing the H3K4me2 distribution along expressed X-linked genes (Exp.) or along genes that are not significantly expressed (Not exp.) in male and female XEN cells (GHP7/7 and GHP7/9 cell lines). Similar results were obtained with female XEN cells . Expression data were extracted from Gene Expression Omnibus ID [GSE:15519] [n = 642 X-linked genes. *P < 0.05 by Kolmogorov–Smirnov test. (B) Representative examples of H3K4me2 distribution along [K27-high] and [K27-low] genes in male and female XEN cells. mm9 UCSC screenshots. (C) Scatterplots of H3K4me2 percentages along the body of expressed X-linked genes in female (y-axis) relative to male (x-axis) XEN cells. Each dot represents a single gene and its respective percentage of H3K4me2 in the corresponding cell line. [K27-low] genes are shown in blue. The dotted line marks equal H3K4me2 percentages in male and female cells. Genes below the line are depleted in female compared to male cells, as expected for genes subject to XCI. In contrast, genes located around the line show similar levels of H3K4me2 in male and female cells, suggesting a biallelic enrichment in H3K4me2. [K27-low] genes are significantly enriched in H3K4me2 compared to [K27-high] genes in female XEN cells (P < 0.05 by Χ2 test).E:15519] . n = 642Click here for fileSingle-cell RT-qPCR analysis on XEN cells. (A) Representative heatmap of a single-cell RT-qPCR BioMark chip from Fluidigm. Each row represents a single XEN cell , and each column a specific PCR assay. This Chip shows the quantification (cycle threshold (Ct) values) of two different reporter genes, twelve different allelic assays for X-linked genes  and fifteen different assays for lineage-specific markers. Negative controls include reactions in which the reverse transcriptase has been omitted (RT−), reactions in the absence of cell (TE control) and internal controls for allele specificity of each X-linked assay performed on genomic DNA (gDNA) of either paternal (Pgk1a) or maternal (129) origin. (B) Cumulative histograms showing allelic gene expression levels of Xist, Atp7a, Jpx, Ftx and Pbdc1 on mixed populations of XEN cells (GHP7/9 cell line). Expression levels are standardised by the expression level of the reporter gene Rplp0. AU, Arbitrary unit. Note that these levels of expression are in agreement with single-cell expression of the same genes shown in Figure Click here for fileH3K27me3 abrogation upon treatment of female XEN cells with the EZH2 inhibitor GSK126. (A) Representative images of female XEN (GHP7/9) cells treated or not with 2 μM GSK126 for 5 days. No morphological differences are apparent after 5 days of treatment with 2 μM GSK126. (B) Western blot analysis of H3K27me3 in XEN female cells (GHP7/9) treated with increasing concentrations of GSK126 for 1, 3 or 5 days. Total histone H3 detection is shown as a loading control. −, untreated; +, treated with GSK126. (C) RT-qPCR analysis of lineage markers in XEN cells (GHP7/9) treated with 2 μM GSK126 for 3 or 5 days. Mean ± standard deviation values of the relative expression levels of each indicated gene are shown. Values have been standardised by the ubiquitously expressed Rplp0 gene. n = 2 independent experiments. ICM, Inner cell mass marker; PE, Parietal endoderm marker; PrE, Primitive endoderm marker; TE, Trophectoderm marker; VE, Visceral endoderm marker. No significant differences in the expression of most lineage markers were observed before and after treatment with GSK126, indicating that XEN cells retained their PrE identity. Only Fst was significantly downregulated after GSK126 treatment (P < 0.05 by Χ2 test). We do not have any explanation for this result. (D) Representative images of RNA-FISH following H3K27me3 immunostaining (green) analysis in XEN female cells (GHP7/9 cell line) treated with GSK126 (2 μM) for 5 days. The inactive X chromosome is detected by Xist RNA accumulation (red). Primary transcription at the indicated X-linked gene is codetected in yellow. Scale bar = 5 μm. (E) Cumulative histograms of the percentages of nuclei with the depicted expression pattern. Only nuclei showing a complete lack of H3K27me3 accumulation at the inactive X territory (Xist-coated X) are scored. X-linked genes are grouped according to H3K27me3 level. On the left of the histogram, the diagram shows the localisation of RNA-FISH probes along the X chromosome. No significant difference in the expression pattern could be detected between treated and untreated cells (P < 0.05 by Χ2 test). n > 50.Click here for fileX-linked gene expression in differentiating female TS cells. (A) Representative photographs illustrating the different cell morphologies observed after 3 days (left panel) and after 6 days (right panel) of differentiation of female TS cells (F3 cell line). At day 6, TGCs (*) are clearly visible. (B) Heatmap of single-cell, steady-state RNA levels for the 20 most stringent discriminants of differentiated and undifferentiated TS cells (P < 10−3 by F-test). Hierarchical clustering has been applied. Complete data set is given in Additional file n = 65 female TS cells (green), n = 54 TS differentiated for 3 days (light blue) and n = 46 female TS cells differentiated for 6 days (dark blue). The expected tissue specificity of each gene is indicated. PE, Parietal endoderm; VE, Visceral endoderm; ICM, Inner cell mass; EPI, Epiblast; TE, Trophectoderm; Pl. Lab, Placental labyrinth; TGCs, Trophoblast giant cells; EPC, Ectoplacental cone. The colour scale is the same as that in Figure (C) Three-dimensional projections of principal components (PCs) of single-cell expression profiles of genes shown in (B). (D) Two-colour RNA-FISH analysis of the indicated genes in TS cells differentiated for 3 days. The arrowhead points to a Kdm5c transcription signal on the Xist-coated inactive X chromosome that is not readily visible. Scale bar = 5 μm. (E) Three-colour RNA-FISH analysis of the indicated genes in TS cells differentiated for 5 days. Arrowheads point to Rlim and Kdm5c transcription signals on the Xist-coated inactive X chromosome that are especially difficult to see. Scale bar = 5 μm. (F) Cumulative histogram of the percentage of nuclei with the depicted expression pattern in TS cells differentiated for 3 days (D3) or for 5 days (D5). Results obtained in undifferentiated TS cells (D0) are also shown to facilitate the comparison. The asterisks mark significant differences between X-linked gene expression profiles in differentiated compared to undifferentiated TS cells (P < 0.05 by Χ2 test). n > 100.Click here for fileAdditional Methods.Click here for file


The current Japanese encephalitis (JE) vaccine derived from G3 JE virus (JEV) can induce protective immunity against G1–G4 JEV genotypes. However, protective efficacy against the emerging G5 genotype has not been reported.in vitro and in vivo tests, biological phenotype and cross-immunoreactions were compared between G3 JEV and G5 JEV (wild strains). The PRNT90 method was used to detect neutralizing antibodies against different genotypes of JEV in JE vaccine-immunized subjects and JE patients. In JE vaccine-immunized mice, the lethal challenge protection rates against G3 and G5 JEV wild strains were 100% and 50%, respectively. The seroconversion rates (SCRs) of virus antibodies against G3 and G5 JEV among vaccinated healthy subjects were 100% and 35%, respectively. All clinically identified JE patients showed high levels of G3 JEV neutralizing antibodies (≥1:10–1280) with positive serum geometric mean titers (GMTs) of 43.2, while for G5 JEV, neutralizing antibody conversion rates were only 64% with positive serum GMTs of 11.14. Moreover, the positive rate of JEV neutralizing antibodies against G5 JEV in pediatric patients was lower than in adults.Using Low levels of neutralizing/protective antibodies induced by the current JE vaccine, based on the G3 genotype, were observed against the emerging G5 JEV genotype. Our results demonstrate the need for more detailed studies to reevaluate whether or not the apparent emergence of G5 JEV can be attributed to failure of the current vaccine to induce appropriate immune protectivity against this genotype of JEV. The human disease Japanese encephalitis (JE) can be prevented by vaccination, although it is not entirely clear if the emerging JEV G5 genotype can be controlled using the vaccine based on the G3 genotype. Consequently, we systematically compared G3 and G5 cross-neutralizing immune responses in vaccinated humans and, separately, cross-protective immune responses in mice using the current G3 JE vaccine to induce the immunity. Based on these results, we propose that the current JE vaccine derived from G3 JE virus (JEV) does not provide adequate levels of protection against the emerging G5 JEV genotype. Japanese encephalitis (JE), probably the world’s most frequently occurring viral encephalitis, is a neurological infectious disease caused by Japanese encephalitis virus (JEV), transmitted via mosquito bite . The morSince there is no effective therapeutic antiviral treatment, JE vaccination is the most effective prevention and control measure . StudiesHere, using wild-type strains of G3 and G5 JEV genotypes, we report comparative studies of neutralizing antibody levels against G5 JEV in both JE-vaccinated subjects and clinically diagnosed JE patients.2 and cultured in minimum essential medium (MEM)  supplemented with 10% fetal bovine serum (FBS)  and Penicillin (1000 unit/mL)-Streptomycin  . JEV-infected BHK cells were cultured in MEM containing 2% FBS and PS.BHK-21 cells (newborn hamster kidney cells) were maintained at 37°C with 5% COAll JEV strains used in this study were isolated from field samples using cell culture and stored at low passage level in our laboratory: Department of Viral Encephalitis and Arbovirus, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention . G1 JEV was GZ56 strain (GenBank: HM366552) , G3 JEV −1 to 10−6) were added to BHK-21 cells in 6-well plates (0.1 mL/well). After 1 h of adsorption at 37°C, the cells were overlaid with 1.3% methylcellulose-MEM medium containing 2% FBS (5 mL/well). After culturing for 3 ~ 5 days, the cells were stained with crystal violet and virus titres expressed as plaque-forming units (pfu) were calculated. For each virus, the diameter of 8 plaques was measured and the mean plaque size in mm and standard error (SEM) was calculated.BHK-21 cells were inoculated at a multiplicity of infection (MOI) of 0.01 and the cytopathic effects (CPE) of the cells were observed. Simultaneously, aliquots of the infected media were collected every 12 h and measured using plaque assays to generate a viral reproduction curve . AliquotSerum samples from JE-vaccinated children (3 mL of blood) were collected from two-year-old children 28 days before and after vaccination SA14-14-2 JE live attenuated vaccine (LAV), . Serum samples (5 mL of blood) were collected from patients diagnosed with JE. All serum samples were obtained from whole blood after clotting at room temperature and centrifuged at 5,000 rpm for 5 min. Aliquots of serum were stored at -80°C until use.The implementation agencies of the present study were the National Reference Laboratory of Japanese Encephalitis and the World Health Organization JE Regional Reference Laboratory, China . Therefore, all the human serum samples used in this study were from those collected and maintained during the JE epidemiological monitoring period by these JE reference laboratories. All adult subjects provided written informed consent, and a parent or guardian of any child participant provided informed consent on their behalf. This research project was approved by the Ethics Committee of the IVDC, China CDC (No.2014112509).Two kits were used to duplicate test serum samples collected from JE patients: the JE-Dengue IgM Combo ELISA , and the JEV IgM-Capture ELISA kit . Methods and result interpretation were performed according to manufacturers’ instructions . Both deBALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.  and were female except for the neonatal mice. All mice were housed under pathogen-free conditions at the China CDC animal facility. All animal experiments were conducted in strict compliance with the regulations set by the Animal Ethics Committee of China CDC (No.2014112509).5 pfu/mL JEV (0.01 mL/each); 5 ~ 6-week-old BALB/c (5 mice/group) were intraperitoneally (i.p.) inoculated with 10-fold diluted JEV , followed by a sham i.c. injection. The incidence and mortality of mice were observed and recorded every day and the Reed-Muench method was used to calculate the 50% lethal dose (LD50) of JEV to adult mice.BALB/c neonatal mice  were intracerebrally (i.c.) inoculated with 1×10G3 and G5 JEV (2000 pfu/300 μL each) were thermally inactivated at 56°C for 30 min and then 4-week-old BALB/c mice were separately immunized (7 mice/group) by multiple subcutaneous injections (300 μL/each) with an equal volume of mixed Freund’s complete adjuvant at day 0 followed by direct i.p. injection (300 μL/each) every two weeks for a total of three times. Two weeks after the third immunization, sera were collected to test the cross-immunoreaction between G3 and G5 JEV.−2 (2510 pfu), 10−3 (251 pfu) or 10−4 (25 pfu) LAV or with 2 i.p. doses of the IPV  7 days apart. Fourteen days after the initial immunization, mice were challenged i.p. with a dose containing an estimated 500 LD50 of the G3 or G5 JEV strain (0.3 mL/each). The mice were monitored frequently mortality rates were recorded until 21 days post-challenge [The two vaccines used for immunization of mice were SA14-14-2 live attenuated JE vaccine (LAV)  and P3 lyophilized JE inactivated purified vaccine (IPV) . Animals were vaccinated i.p. with either a single dose of 10hallenge .90) compared to an unvaccinated control serum [Serum neutralizing antibody to JEV was detected using the plaque reduction neutralization test (PRNT) method. The inactivated serum was diluted two-fold (from 1:5 to 1:1280) and mixed with an equal volume of 200 pfu JEV, incubated in 37°C for 1 h. The mixture was then added to 6-well BHK-21 plates for 1 h followed by overlaying with 1.3% methylcellulose-MEM medium containing 2% FBS for 3 ~ 5 days. The plaques were stained with crystal violet and counted. Neutralizing antibody titers were calculated as the reciprocal of the highest dilution resulting in 90% plaque reduction . All data were processed and drawn using GraphPad Prism 5.0 software , Student’s t-tests were used for all analyses and a P-value <0.05 was considered statistically significant.In this study, the positive cut-off value of neutralizing antibody titer was defined as PRNTCPE due to virus infection showed that G5 JEV (XZ0934 strain) and G3 JEV (P3 strain) can cause BHK cells to shrink and shed . The pla3 pfu/mouse; 12/group) all died in the first 5 days. The 5 ~ 6-week-old BALB/c adult mice were i.p. inoculated with the two JEV genotypes (5/group) and the LD50 of each virus was calculated to assess the virulence of JEV. The LD50 values of the G5 and G3 virus titers (logarithmic) were 3.5 and 4.1, respectively, demonstrating no significant difference in virulence  and 60% in the low dose group  vaccination. Among the 26 serum samples collected before immunization, one showed 1:10 neutralizing antibody titer against the G1 JEV strain and two showed 1:10 against the G3 JEV strain but all were negative against the G5 JEV strain. After LAV vaccination, neutralizing antibodies against G3 JEV showed 100% positive seroconversion (GMT = 125.9), with the highest neutralizing antibody titer reaching 1:1280  and the 90 (expressed as GMTs) against G3 JEV and SCRs were analyzed and the results showed that when neutralizing antibodies against G3 JEV serum were ≥ 1:40, SCRs against G1 JEV were as high as 100%, but none were seropositive against G5 JEV (0% SCRs). When neutralizing antibodies against G3 JEV serum were 1:160–1:320, SCRs against G5 JEV were only 50%. Protective antibodies were produced against G5 JEV (SCRs up to 100%) only when the dilution of G3 JEV serum was higher than 1:320. The results are shown in In serum samples after immunization with the current JE vaccine, neutralizing antibody titers estimated using PRNTThis study further examined levels of neutralizing antibody against G5 JEV in serum samples from clinically diagnosed JE patients. All 45 serum samples were confirmed IgM-positive when using two JEV IgM antibody detection kits. Neutralizing antibodies against G1, G3 and G5 JEV were detected in all 45 serum samples. The results showed that neutralizing antibodies (≥ 1:10–1280) against G3 JEV were detected in all serum samples from JE patients with serum GMT of 43.2. Neutralizing antibodies (≥ 1:10–80) against G1 JEV were detected in 87% (39/45) of serum samples with serum GMT of 16.37. Additionally, neutralizing antibodies against G5 JEV were detected in only 64% (29/45) of serum samples with serum GMT of 11.14. Evaluation of JE patient’s age and neutralizing antibodies against G5 virus showed that 41% (9/22) of JE patients 1–15 years of age and 87% (20/23) of patients over 16 years of age had positive neutralizing antibodies against G5 JEV .G5 JEV and G3 JEV wild-type isolates were used in this study. Comparative studies of wild strains showed that the biological characteristics of G5 JEV, such as CPE, virus titers and virus lethality to animals, were similar to those of G3 JEV. However, cross-neutralization studies showed that antigenic cross-reactivity between G3 and G5 JEV was low  and this90 assays were used to detect neutralizing antibodies against JE viruses in specimens collected from JE-vaccinated healthy children. The results showed that after receiving JE vaccines, SCRs of neutralizing antibodies against G3, G1 and G5 JEV in the serum samples were 100%, 96% and 35%, respectively  . Previouectively . SCRs ofectively . This st90≥1:10). However, antibodies against G5 were all negative (PRNT90<1:10). These results indicate that within 11–26 days after being infected with JEV, patients failed to produce protective antibodies against G5 JEV. Therefore, pediatric patients with natural JEV infection still had significant potential risk of G5 virus re-infection. Based on our results, whether patients infected with JEV can produce protective antibodies against G5 JEV in the recovery period was inconclusive. The results did, however, imply that JE pediatric patients may not produce protective antibodies against G5 JEV after infection with non-G5 strains of JEV.In this study, JEV IgM-positive serum samples (n = 45) collected from clinically diagnosed JE patients were used to evaluate them for the presence of G5 JEV-specific neutralizing antibodies. The positive rates of neutralizing antibodies against G3 and G1 JEV in the serum were 100% (45/45) and 89% (39/45), respectively. However, the positive rate of neutralizing antibodies against G5 JEV was only 64% (29/45). Further analysis showed that the positive rates of neutralizing antibodies against G5 JEV in JE patients 1–15 years of age were 41% (9/22) and 87% (20/23) in JE patients over 15 years of age, indicating that JE patients of different ages showed different levels of G5 JEV protective antibodies. This possibly reflects the greater number of exposures to JEV in the older age-group. Additionally, in this study, the serum sample of a 9-year-old child collected on the 26th day after onset of JE  of JEV in these subjects.26 serum samples from JE-vaccinated children were collected from two-year-old children 28 days before and after vaccination SA14-14-2 JE live attenuated vaccine (LAV), . The PRNT(XLS)Click here for additional data file.S2 Table90 method was used to detect neutralizing antibodies against different genotypes  of JEV in these serum samples.45 serum samples were collected from patients diagnosed with JE and two kits of Capture ELISA were used to duplicate test these serum samples for confirmed JE patients. Then The PRNT(XLS)Click here for additional data file.


However, current BOLD CMR methods  are limited by poor spatial coverage and image artifacts . To address these limitations, we developed a fast, free breathing 3D T2 mapping technique at 3T that utilizes near 100% imaging efficiency. This quantitative BOLD approach, which can be performed within 5 minutes, permits full LV coverage during adenosine stress. In this study, we tested our approach in a canine model and validated our findings with simultaneously acquired 3 with 14 partitions, adiabatic T2 prep pulses  every other heart beat. Total acquisition time for whole LV coverage was 5 mins. For validation, dynamic 13N-ammonia PET scans were acquired along with MR data. PET images were analyzed using qPET . Epi and endo-cardial contours were used to segment the myocardium and mean myocardial T2 (T2avg) values were measured from basal, mid and apical slices at rest and adenosine stress. The corresponding slices were matched to 13N-ammonia PET images and mean myocardial perfusion (Qavg) values were measured. Myocardial BOLD reserve (T2avg (stress):T2avg(rest)) and perfusion reserve (T2avg (stress):T2avg(rest)) were computed on a slice basis and regressed.Mongrel dogs (n=3) were studied with a state of the art PET-MR system . After scouting and whole-heart shimming, fast 3D free-breathing T2 mapping sequence were prescribed at rest and under adenosine stress (140 mg/min/kg) with the following scan parameters: TR/TE =3.4/1.7 ms, flip angle = 15°, imaging resolution = 2 x 2 x 6 mm2=0.67, p<0.05).A representative set of BOLD and PET images acquired from a canine under rest and adenosine stress are collected in Fig. The proposed fast 3D free breathing T2 mapping at 3T permits whole LV assessment of BOLD changes between rest and adenosine stress. The BOLD responses were very closely correlated with PET perfusion, suggesting that the proposed BOLD CMR method is a viable approach for imaging myocardial perfusion. Further studies are required to examine its utility in the setting of ischemic heart disease.This work was supported in part by National Heart, Lung, and Blood Institute HL091989.The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, And Blood Institute or the National Institutes of Health.


HR expression was quantified in esophageal biopsies from non-EoE control (N = 23), inactive EoE  and active EoE  subjects using qRT-PCR and immunofluorescent localization. HR expression and histamine-mediated cytokine secretion were evaluated in human primary and telomerase-immortalized esophageal epithelial cells. H1R, H2R, and H4R expression were increased in active EoE biopsies compared to inactive EoE and controls. H2R was the most abundantly expressed receptor, and H3R expression was negligible in all 3 cohorts. Infiltrating eosinophils expressed H1R, H2R, and H4R, which contributed to the observed increase in HR in active subjects. H1R and H2R, but not H3R or H4R, were constitutively expressed by primary and immortalized cells, and epithelial histamine stimulation induced GM-CSF, TNFα, and IL-8, but not TSLP or eotaxin-3 secretion. Epithelial priming with the TLR3 ligand poly (I:C) induced H1R and H2R expression, and enhanced histamine-induced GM-CSF, TNFα, and IL-8 secretion. These effects were primarily suppressed by H1R antagonists, but unaffected by H2R antagonism. Histamine directly activates esophageal epithelial cytokine secretion in vitro in an H1R dependent fashion. However, H1R, H2R and H4R are induced in active inflammation in EoE in vivo. While systemic antihistamine (anti-H1R) therapy may not induce clinical remission in EoE, our study suggests that further study of histamine receptor signaling in EoE is warranted and that targeting of additional histamine receptors may lead to novel treatment strategies for this important disease.Eosinophilic Esophagitis (EoE) is a chronic allergic disorder, whose pathobiology is incompletely understood. Histamine-producing cells including mast cells and basophils have been implicated in EoE. However, very little is currently known about the role of histamine and histamine receptor (HR) expression and signaling in the esophageal epithelium. Herein, we characterized HR  expression in human esophageal biopsies and investigate the role of histamine signaling in inducible cytokine expression in human esophageal epithelial cells  Eosinophilic esophagitis (EoE) is a chronic food antigen-mediated disease characterized by eosinophil infiltration into the esophageal epithelium . EoE is The pathophysiology of EoE is incompletely understood, though both genetic predisposition and complex interactions between many inflammatory cells and pathways ,4 are liIn addition to their ability to release proteases and prime host Th2-type responses both mast cells and basophils produce and secrete histamine –12, a biAntihistamines have been used for several decades for the treatment of allergic disorders, and H1R antagonists are used as first-line therapy for several conditions including allergic rhinitis, allergic conjunctivitis, and urticaria . H1R antin vitro and identify the specific HRs important for this response in the esophagus.In this study, we characterize the expression of H1R, H2R, H3R and H4R in the human esophagus and show that expression of H1R, H2R and H4R are upregulated in active EoE. Using immortalized and primary human esophageal epithelial cell lines, we investigate the role of histamine signaling in upregulation of physiologically relevant esophageal epithelial cytokine secretion All research involving human subjects was approved by the Institutional Review Board at The Children’s Hospital of Philadelphia (CHOP Protocol #7737). Following written informed consent obtained from each subject’s parents or legal guardians, 2–4 additional pinch biopsies were obtained from the distal esophagus during routine diagnostic esophagogastroduodenoscopy (EGD). Consistent with recently published clinical guidelines, the diagnosis of EoE was made by the histologic presence of 15 or more esophageal epithelial eosinophils per high powered field (hpf), hyperplasia of the basal epithelium, and the absence of tissue eosinophilia in the distal GI tract [Peak eosinophil numbers, mean +/- sd  in non-EoE controls (n = 23), inactive EoE (n = 26), and active EoE (n = 23) were 0.65+/-1.94 , 1.77 +/- 2.9 , and 48.14 +/- 26.62  respectively. Mean age +/- sd (range) of non-EoE, inactive EoE and active EoE cohorts were 8.6 +/- 5.07 (1.1–16.6), 9.12 +/- 4.01 (3.3–16.8), and 9.45 +/- 4.68 (1.8–16.8) respectively. 74% non-EoE, 96% inactive and 100% active subjects had a history of atopy such as allergic rhinitis (AR), atopic dermatitis (AD) and allergic airway disease (RAD). EoE subjects were managed with dietary restrictions (54.2%), swallowed steroid treatment (10.4%), or a combination of both (18.8%). Approximately 43.5% of the non-EoE control subjects were also on steroid treatments such as nasal or inhaled steroids for other atopic condition.Biopsy samples were placed in ice cold Hank’s buffer, for isolation of esophageal epithelial cells within 30 minutes of harvest. Samples were transferred to dispase  at 37° C for 20 minutes, followed by trypsinization at 37°C for 20 minutes. Trypsin was inactivated using soybean trypsin inhibitor  and biopsy samples were transferred into a cell strainer. Epithelial cells were washed through the strainer with additional PBS. Cell suspensions were centrifuged at 1000 RPM for 5 minutes. Cell pellets were re-suspended in complete KSFM containing fungizone  (1:500), and seeded in tissue culture dishes.2 incubator and were maintained in keratinocyte serum-free media  supplemented with epidermal growth factor (1ng/mL), bovine pituitary extract (50μg/mL), penicillin (100 units/mL), and streptomycin (100μg/mL) .EPC2-hTERT and primary esophageal epithelial cells (EPC) were grown at 37°C in a humidified 5% COFormalin fixed paraffin embedded human esophageal biopsy sections were rehydrated in sodium citrate buffer and blocked with Startingblock T20 blocking buffer . Sections were then incubated with rabbit anti-H1R , goat anti-H2R , or rabbit anti-H4R  diluted in PBT overnight at 4°C. Mouse anti-MBP  was used to co-stain for eosinophils. After washing with PBS, Cy3 or Cy2-conjugated secondary antibody was applied for 2 hours at room temperature . Cells were washed in PBS and stained with 4,6-diamidino-2-phenylindole  according to manufacturer’s recommendations. Coverslips were applied after the application of mounting media , and sections were imaged using Olympus BX51 fluorescent microscope.5 cells/mL in 12 well plates one day prior to stimulation. Confluent cells were cultured in 100uM histamine (Sigma) for 24 hrs and the supernatants were assayed for target protein secretion. For priming experiments, cells were incubated in 10ug/ml poly (I:C)  for 24 hrs prior to stimulation with histamine. For inhibition studies, cells were pre-treated for 1 hr with the H1R antagonist pyrilamine , or H2R antagonists cimetidine , or ranitidine (100uM) (GlaxoSmithKline) prior to histamine stimulation.Esophageal epithelial cells were seeded at a density of 1 X 10T method of relative quantitation, where CT is the threshold cycle.RNA was isolated from human esophageal biopsies using Mirvana miRNA isolation kit . RNA was isolated from EPC2-hTERT and primary esophageal epithelial cells using an RNeasy kit  according to manufacturer’s recommendations. RNA samples were reverse transcribed using a high capacity cDNA reverse transcriptase kit according to manufacturer’s instructions . Pre-formulated Taqman Gene Expression Assays were purchased from Applied Biosystems for H1R (Hs00185542_m1), H2R (Hs00254569_s1), H3R (Hs00200610_m1), H4R (Hs00222094_m1), and GAPDH (4352934E). Quantitative RT-PCR was performed using the Taqman Fast Universal PCR Master Mix kit (Applied Biosystems) and reactions were performed in triplicate using 96-well optical plates on a StepOnePlus Real-Time PCR System (Applied Biosystems). GAPDH was used as an endogenous control to normalize the samples using the ΔΔCFollowing stimulation, cell supernatants were collected and stored at -80°C until later use. Cytokine levels in supernatants were quantified using ELISA kits purchased from R&D systems   and eBiosciences  (TSLP). Assays were performed according to manufacturer recommendations.Kruskal Wallis analysis with Dunn’s post test and Mann Whitney U test were used to analyze mRNA data from biopsy samples. Two tailed student t-test and One way ANOVA with Bonferroni comparison was used for in-vitro studies. All analyses were performed in GraphPad Prism 5.Fig. 1A). Both H3R and H4R mRNA expression was minimal, or not detected in the majority of esophageal biopsies. Overall, H2R was the most abundantly expressed receptor subtype in the esophageal biopsies  .Although HR expression has been studied in epithelial cells in other models such as the bronchial epithelium  and epidFig. 1B). Consistent with its mRNA expression in esophageal biopsies, H2R was the most abundantly expressed HR throughout the esophageal epithelium . Immunofluorescent detection of H4R staining was faint, and was notably clustered within the epithelial papilla .We localized esophageal H1R, H2R, and H4R expression with immunofluorescence. H1R was faintly distributed throughout the esophageal epithelium (Table 1).HR expression is altered in the epithelium during allergic conditions  suggestiFig. 2A, B, and D). Additionally, expression of all HRs in inactive EoE biopsies was similar to that in non-EoE control biopsies. H3R expression was unchanged in active EoE subjects compared to either non-EoE control or inactive EoE subjects .Interestingly, H1R, H2R, and H4R expression levels were significantly elevated in active EoE subjects compared to non-EoE control and inactive EoE subjects . Subsets of MBP positive cells also expressed H1R, H2R and H4R as evident from their co-localization . Based upon these results, we next determined whether tissue eosinophilia in EoE correlated with HR expression in esophageal biopsies. Shown in Fig. 3J-L, peak eosinophil numbers per hpf correlated positively with H1R, H2R, and H4R expression. H1R had the strongest correlation to eosinophils numbers among the three receptor subtypes, followed by H4R and H2R. These findings suggest that a significant portion of upregulated histamine receptor expression detected in active EoE biopsies is derived from infiltrating inflammatory cells.HRs are also expressed by various inflammatory cells including eosinophils, dendritic cells, and T cells ,28. To dFig. 4A), non-EoE control (n = 8) , and EoE (n = 13)  cell lines, neither H3R nor H4R were detected in any of the studied cells (data not shown). Consistent with histamine expression patterns within esophageal biopsy samples, expression of H2R was significantly greater than H1R in all cell lines . Importantly, relative expression of H1R and H2R was not significantly different in EoE compared to non-EoE control cell lines. . Further stratification of EoE cells into inactive EoE (N = 4) and active EoE (N = 9) did not show a significant difference in receptor expression either (data not shown). Based on these similarities in HR expression between primary and EPC2-hTERT esophageal epithelial cells, subsequent experiments were performed in EPC2-hTERT and one representative cell line from each of the EoE and non-EoE cohorts.Recognizing that the quantification of HR expression in mucosal biopsies may reflect HR expression by a number of cell types , we next sought to elucidate the specific role of esophageal epithelial cells in histamine signaling in EoE. Constitutive expression of HRs in the immortalized human esophageal epithelial cell line, EPC2-hTERT and in primary esophageal epithelial cell lines derived from EoE and non-EoE esophageal biopsies was quantified by quantitative RT-PCR. While both H1 and H2 receptors were constitutively expressed by EPC2-hTERT , and primary EoE and non-EoE cells (data not shown). Surprisingly, histamine stimulation of EPC2-hTERT and primary cells did not induce either thymic stromal lymphopoetin (TSLP) or eotaxin-3 (CCL26) secretion, which are epithelial derived cytokines prominently linked to EoE pathogenesis [Histamine stimulation induced robust secretion of GM-CSF and IL-8 and a mild induction in TNFα secretion by EPC2-hTERT cells . TNFα expression was mildly induced by H1R inhibition while H2R inhibition did not have any effect . Interestingly, neither H1R nor H2R inhibition suppressed histamine mediated IL-8 expression in EPC2-hTERT cells .In order to determine the specific HRs involved in inducible cytokine expression in esophageal epithelial cells, we pre-treated EPC2-hTERT cells with pharmacological inhibitors of H1R and H2R prior to histamine stimulation. H3 and H4 receptor inhibitors were not studied based upon the absence of these receptors in primary or immortalized esophageal epithelial cells. Pretreatment with H1 inhibitor pyrilamine suppressed histamine-mediated GM-CSF expression, whereas H2 receptor antagonists cimetidine and ranitidine had a very mild inducible effect  in human coronary artery endothelial cells [in vitro.Based on the increased HR expression observed in active EoE biopsies, and histamine mediated cytokine expression in epithelial cells al cells . We haveal cells , and othal cells . We therFig. 6A). Neither H3R nor H4R mRNA was induced by poly (I:C). To determine whether poly (I:C) mediated induction in HR expression resulted in enhanced sensitivity to histamine stimulation, esophageal epithelial cells were primed with poly (I:C) for 24 hrs prior to stimulation with histamine, followed by quantification of GM-CSF, TNFα, IL-8, eotaxin 3, and TSLP secretion. Poly (I:C) stimulation alone induced robust secretion of GM-CSF, TNFα, and IL-8, which was synergistically enhanced by subsequent histamine stimulation . Again, neither eotaxin-3 nor TSLP was induced by combined poly (I:C) and histamine stimulation (data not shown). We next determined the roles of specific HRs in poly (I:C)/histamine mediated cytokine induction using pharmacological inhibitors of H1R and H2R. H3R and H4R inhibitors were not used based upon lack of poly (I:C) inducible expression of these receptors in esophageal epithelial cells in vitro. The secretion of GM-CSF, TNFα, and IL-8 in poly (I:C) primed epithelial cells was significantly suppressed by H1R antagonist pyrilamine . The H2R antagonist cimetidine suppressed poly (I:C)-histamine induced TNFα secretion , though to a lesser degree than pyrilamine alone. The effects of combined pyrilamine with either cimetidine or ranitidine were not significantly different from that of pyrilamine alone, indicating the effect is primarily mediated via H1R.Stimulation of EPC2-hTERT cells with poly (I:C) led to robust induction in H1R and H2R mRNA expression  enhances epithelial sensitivity to histamine stimulation through primarily H1R and to a lesser degree H2R-mediated mechanisms.In the current study, we now show for the first time that H1R, H2R, and H4R, but not H3R, are expressed in human esophageal epithelial biopsies, and their expression is induced in the setting of active inflammation in EoE. Importantly, we demonstrate that H1R and H2R, but not H3R or H4R, are constitutively and inducibly expressed by human esophageal epithelial cell lines in vitro were not statistically different and esophageal epithelial cells from EoE and non-EoE subjects had similar cytokine secretion patterns upon histamine stimulation. Together with our observation that HRs are expressed by infiltrating eosinophils in active EoE samples, these observations suggest that increased esophageal epithelial histamine receptor expression in active EoE biopsies may reflect the inflammatory milieu and may not represent an intrinsic defect in HR expression or histamine signaling in epithelial cells.Mucosal expression of H1R, H2R, and H4R was significantly increased in biopsies with active inflammation, compared to both non-EoE control and inactive EoE biopsies. This is consistent with previous reports describing similar alterations in HR patterns in allergic diseases including allergic rhinitis  and inteThe precise factors involved in inducing esophageal epithelial histamine receptor expression remain unknown. Mirzahosseini et al. have recently shown that IgE stimulation results in upregulation of H2R in murine bone marrow derived mast cells . Howeverin vitro. Paradoxically, competitive inhibition of H2R signaling did not significantly suppress histamine-mediated GM-CSF expression. Alonso et al showed a negative cross-regulation mechanism between H1R and H2R in human and CHO cells [Yersinia enterocolitica infection [H2R was the most highly expressed HR in biopsies and epithelial cells HO cells . This manfection  and histnfection . It is tH3R is expressed by myocytes and in the central and peripheral nervous system . In the in vitro, upregulation of H4R in the esophageal epithelium in vivo suggests a potential role for H4R signaling in EoE. Further studies are required to understand the role of H4 receptor signaling in EoE pathobiology, particularly since current antihistamine therapies (H1 and H2) are already utilized in approximately 10% of patients diagnosed with EoE patients [Although there was no detectable H4R expression in esophageal epithelial cell lines, H4R expression was detectable in biopsies from EoE subjects with active disease. We noted co-localization of H4R and MBP, suggesting that infiltrating eosinophils and basophils may partially account for the detectable H4R signal. Other inflammatory cells including mast cells and T cells  may conterapies H and H2 ain vivo are incompletely understood, we recapitulated elevated HR expression in vitro using the TLR3 ligand and viral mimetic, poly(I:C), which strongly induced H1R and H2R expression. Viral infection is linked to other diseases such as allergic asthma [in vitro leads to the expression of various cytokines relevant to esophageal inflammation including TSLP[Recognizing that the biological triggers of increased epithelial HR expression c asthma  and perec asthma . This isding TSLP RANTES [ding TSLP, and IL-ding TSLP,53. Thouding TSLP.in vitro. GM-CSF, though not previously quantified in EoE, plays a critical role in promoting eosinophil migration, survival, activation, and degranulation [Histamine stimulation induced esophageal epithelial secretion of GM-CSF, IL-8, and TNFα nulation –56 and inulation ,58. Bothnulation ,32. In mnulation , which cnulation ,61. Thounulation , to our nulation . Interesnulation . This crIn summary, we show that H1R, H2R, and H4R expression is altered in the esophageal epithelium during active inflammation in EoE, possibly enhanced by the inflammatory microenvironment including infiltrating immune cells. Despite the lack of clinical data supporting the use of H1R antagonists in EoE, our findings suggest that H1R signaling may contribute to local epithelial cytokine responses in the esophagus, highlighting the complexity of the host immune response in EoE. Although H4R was neither constitutively or inducibly expressed by human esophageal epithelial cells, its upregulation in mucosal biopsies warrant future functional studies using animal models to further define the role of H4R in EoE associated inflammation.


Objective. A novel avian influenza A virus (AIV) H7N9 subtype which emerged in China in 2013 caused worldwide concern. Deletion of amino-acids 69 to 73 in the neuraminidase stalk was its most notable characteristic. This study is aimed to discuss the tropism and virulence effects of this deletion. Methods: Neuraminidase gene sequences of N9 subtype were collected from NCBI and GISAID. MEGA6.0, Stata12.0, and UCSF Chimera were employed for sequence aligning, significance testing, and protein tertiary structure homology modeling. Results: A total of 736 sequences were obtained; there were 81 human isolates of the novel AIV H7N9, of which 79 had the deletion. Among all the 654 avian origin sequences, only 43 had the deletion (p < 0.001). Tertiary structure displayed that the deletion obviously changed the spatial direction of neuraminidase. Conclusions: The deletion in neuraminidase stalk could have strengthened human tropism of the novel AIV H7N9, as well as its virulence. A novel avian influenza A virus (AIV) H7N9 subtype was detected in China on 19 February 2013, which caused worldwide concern .For a long time, haemagglutinin and polymerase subunit PB2 are believed to be decisive factors for host tropism of influenza virus, and neuraminidase (NA) to be responsible for virulence and interaction with drugs ,6. In thNeuraminidase gene sequences of N9 subtype were obtained from NCBI Influenza Virus Sequence Database and the Global Initiative on Sharing Avian Influenza Database (GISAID)  ,8. Sequeet al. and Xiong et al. [MEGA6.0 software was used for sequence aligning and phylogenetic tree constructing; after W method alignment, a phylogenetic tree was constructed by p-distance. Chi-square test in Stata12.0 software were used for analysis of significance. UCSF Chimera was employed for protein tertiary structure homology modeling. As in the procedure of protein homology modeling, according to Li p < 0.001).A total of 736 neuraminidase gene sequences of N9 subtype were obtained, including H1N9 (53), H2N9 (54), H3N9 (8), H4N9 (25), H5N9 (19), H6N9 (13), H7N9 (173), H8N9 (0), H9N9 (10), H10N9 (13), H11N9 (306), H12N9 (4), H13N9 (19), H14N9 (0), H15N9 (6), H16N9 (1), H17N9 (0), H18N9 (0), and mixed HA (32). Of these H7N9 subtype consisted of 124 novel isolates established in 2013 and 49 established beforehand. There were 81 human isolates of the novel AIV H7N9, of which 79 had a deletion of amino-acids 69 to 73 in the neuraminidase stalk. Among all the 654 avian origin sequences however, only 43 had the deletion , is a whale origin H13N9 established in 1984). The difference in the percentage of this deletion was statistically significant  virus was replaced with that of A/duck/Shantou/5540/01 (H6N2) isolated from wild aquatic birds and they found that chimeras of PB2 and M genes, encoding the C-terminal region of the PB2 protein and M2 protein from W312 were required for efficient replication in canine-derived (MDCK) cells and in chicken trachea. These results indicate that host range may be determined by some types of internal proteins such as PB2 and M2, as well as by surface glycoproteins like hemagglutinin [The host range of influenza A virus is determined by amino acid residue substitutions or receptor binding specificity. The HA molecule primarily contributes to the determination of host range by receptor specificity . In contglutinin . Other fglutinin ,12. Baseet al. observed that among 162 NA sequences of human H5N1 isolates from 1996 to 2007, only two viruses isolated in Hong Kong 2003 contained complete length stalk NA. The short-stalk NA of H5N1 viruses circulating in Asia were conceived to be of adaptability in human infection [N9 subtype influenza virus was first found in Canada in 1966 and later in USA in 1968. There were no deletion in neuraminidase stalk and no spillover to humans until the 2013 epidemic in China. Possible impact of the deletion in N9 subtype AIV has not been previously reported, but deletions in N1, especially H5N1, could significantly influence AIVs’ host species specificity. Matsuoka nfection . The H5Nnfection . Upon trnfection ,18. Seriet al. and Matsuoka et al. above-mentioned found that virulence in mice was enhanced for viruses with a truncated NA stalk compared to their equivalents with a long stalk. Viruses with a short-stalk NA showed a decreased capacity to elute from red blood cells and an increased virulence in mice [et al. indicates that a shortened NA stalk is a strong determinant of virulence and adaptation of waterfowl influenza viruses in chickens [i.e., 2–6 or 2–3 glycosides) around it, which subsequently results in more substrates cleaving and less novel infecting, in return less virulence [NA stalk length also affects other biological properties of influenza viruses. Both Zhou  in mice ,14. Datachickens . Possiblirulence ,20. In vConsidering subtypes of AIVs carried by poultry in eastern and southern China and its possible reassortments ,22, epidAlthough there is no experimental proof, statistical significance highlights the fact that there exists a remarkable difference in the host tropism of AIVs H7N9 with or without the deletion of amino-acids 69 to 73 in the neuraminidase stalk; tertiary structure homology modeling displays that the deletion notably changed the spatial direction of neuraminidase. We hence put forward an opinion that the deletion in neuraminidase stalk could have strengthened human tropism of the novel AIV H7N9, as well as its virulence. The results of this study should be interpreted with caution due to the limitations identified. And thus, further research warrants more experimental proof for these conclusions.


During the 56 weeks of studies, no fever nor local redness and swelling at sites of injections was observed in the immunized macaques. After single immunization, 100% seroconversion based on 4-fold increased in neutralization titer (Nt) was detected in EV71vac immunized monkeys but not PBS controls. A dose-dependent IgG antibody response was observed in monkeys receiving EV71vac immunization. The Nt of EV71vac immunized macaques had reached the peak after 3 vaccinations, then decreased gradually; however, the GMT of neutralizing antibody in the EV71vac immunized macaques were still above 100 at the end of the study. Correspondingly, both dose- and time-dependent interferon-γ and CD4+ T cell responses were detected in monkeys receiving EV71vac. Interestingly, similar to human responses, the dominant T cell epitopes of macaques were identified mainly in VP2 and VP3 regions. In addition, strong cross-neutralizing antibodies against most EV71 subgenotypes except some C2 and C4b strains, and Coxsackievirus A16 were observed. In summary, our results indicate that EV71vac elicits dose-dependent T-cell and antibody responses in macaques that could be a good animal model for evaluating the long-term immune responses elicited by EV71 vaccines.Enterovirus 71 (EV71) has caused epidemics of hand, foot and mouth diseases in Asia during the past decades and no vaccine is available. A formalin-inactivated EV71 candidate vaccine (EV71vac) based on B4 subgenotype has previously been developed and found to elicit strong neutralizing antibody responses in mice and humans. In this study, we evaluated the long-term immunogenicity and safety of this EV71vac in a non-human primate model. Juvenile macaques were immunized at 0, 3 and 6 weeks either with 10 or 5 µg doses of EV71vac formulated with AlPO Picornaviridae, genus Enterovirus was first identified in California 45 years ago and subsequently reported in many parts of world Enterovirus 71 (EV71), a non-enveloped RNA virus of the family EV71 has an icosahedral viral particle containing a single, positive-sense RNA (7.5–8.5 kb) and four structural capsid proteins, including VP1, VP2 and VP3 on the external surface of the virion and VP4 within the interior of the viral particle Several types of EV71 vaccine candidates, including inactivated virus vaccine, attenuated virus vaccine All experiments were conducted in accordance with NHRI Laboratory Animal Center guidelines and approved by the NHRI Institutional Animal Care and Use Committee . An US patent (US20120045468 A1) has been filed with the results presented in this study.Macaca cyclopis) using in this study were obtained from the Animal Health Research Institute of Taiwan, and pre-screened for no anti-EV71 IgG antibody (titer <200) and neutralizing antibody (Nt <8). Animals were housed in a secured SPF room with locked double-doors and cameras to monitor the animal activity. Fresh fruit with fixed-formula diets (∼4% of the body weight) on the basis of two times a day are used to feed the monkeys. At the beginning of this study, monkeys are moved into individual cage (62×62×72 cm) with the double-door, squeeze-back design to allow the monkeys to contact each other in vision but avoiding physical touch. To enrich the environment, some toys and different fresh fruit are provided to the animals. If an animal appears to be in poor condition, an adequate medical treatment or environment improvement will be used to relieve the pain or distress based on the recommendation of certificated veterinarians. All monkeys are moved back to the community cages with bigger space and better interaction with other monkeys in the end of this study.Ten young (1–3 years old) Taiwan macaques  before immunization or bleeding. The vital sign was monitored by breath and heart beating during the anesthesia. For bleeding, about 5 ml of heparinized blood was drawn from femoral veins each 3 weeks for the first 3 months, then every 3 months until the end of this study. After drawn, monkeys were placed back into original cages for recovery, which usually needs 15∼20 minutes and under the veterinarian's supervision. A humane euthanasia is performed by pentobarbital intravenous injection (100 mg/Kg) under the certificated veterinarian's recommendation, if animal is out of control such as unknown fever, severe diarrhea , moribund or not eating/drinking and doesn't show improvement following medication or environmental enrichment.4 . The low dose group  was im vaccinated with 5 µg of EV71vac formulated with alum. The control group (A20 and A21) was im vaccinated with PBS plus 300 µg alum. All groups received two boosts with the same vaccine and dose three and six weeks later.For immunization, monkeys were randomly divided into three groups. The high dose group  was vaccinated by intramuscular (im) injection with 10 µg of EV71vac formulated with AlPOAll EV71 strains used for the in vitro neutralization assay were grown in Vero cells. Vero cells were kindly provided by the Taiwan Centers of Disease Control (Taiwan CDC). The EV71/E59 strain (B4 subgenotype), a clinical isolate of the EV71 virus, and C4D (C4a subgenotype) were obtained from the Taiwan CDC. EV71 clinical isolates 0204/TW86 (B1 subgenotype), N0692/TW08 (B5 subgenotype), N0448/TW08 (B5 subgenotype), 5746/TW98 (C2), 4643/TW98 (C2), 1757/TW98 (C4a), N3340/TW02 (C4b), and Coxsackievirus A16 5079/TW98 were obtained from Nation Cheng Kung University, Taiwan.4 in 1 mL of PBS) were stored at 4°C for the animal study.The inactivated EV71vac vaccine made from EV71/E59 strain (B4 subgenotype) was produced and fully characterized as previously reported Specific IgG titer against EV71/E59 was determined by ELISA 50 of virus (400 µL) was added to a tube containing diluted sera (400 µL). After incubation at 4°C for 18–24 hours, 100 µL of virus-serum mixture was added to Vero cells cultured in 96-well plates and incubated for seven days at 37°C, and TCID50 values were measured by counting cytopathic effects. The 50% neutralization inhibition dose was obtained by calculating the geometric reciprocal of the serum dilution yielding 50% reduction using the Reed-Muench method.Sera in a two-fold serial dilution with cell culture medium were added to microtubes. A suspension of 200 TCID5 fresh PBMCs were added with concanavalin A , inactivated EV71/E59 (50 µg/ml) or 5 µg/mL of VP1, VP2 or VP3 peptide mixture prepared from a series of 15-mer peptides with 10-mer overlapping to cover whole VP1 (1–295 aa), VP2 (1–254 aa) or VP3 (1–242 aa), respectively 6 PBMCs.Multiscreen plates (Millipore) were coated with anti-human IFN-γ antibody (2 µg/ml) or anti-human IL-4 antibody . After washing and blocking with culture medium, duplications of 5×106) from monkeys that received EV71vac immunization twice (week 6) were cultured in 96-well round-bottomed plates at 37°C in a 5% CO2 incubator and stimulated with inactivated EV71, PMA plus ionomycin or medium alone. Cytokines were retained in the cytoplasm by adding Golgi-plug and Golgi-stop inhibitors 4 hours after stimulation. After 18 hours of incubation, cells were harvested, washed with staining buffer (PBS with 0.5% BSA) and blocked with anti-CD16 and anti-CD32 antibodies for 20 min at 4°C. The surface markers were stained with anti-monkey CD3, CD4 and CD95 conjugated with APC, PerCP and PE, respectively  for 20 min at 4°C. After washing, cells were permeabilized with Cytofix buffer (BD Bioscience) and stained with FITC-conjugated anti-monkey TNF for 20 min at 4°C. Finally, cells were resuspended in 0.5 ml fixing solution (eBioscience) and assayed by FACS Caliber. Data were analyzed by FlowJo software (Treestar), and the CD3+ CD4+ T cell population was gated and presented.Fresh PBMCs , if there is no additional description.Ten juvenile macaque monkeys (1–3 years old) with a body weight range between 1.7–5.0 kg (Median: 2.8 kg) were divided into three groups, and every group had an equal number of male and female monkeys. Two groups (4 macaques/group) were vaccinated three times at 0, 3 and 6 weeks either with high (10 µg) or low (5 µg) doses of EV71vac; and the control PBS group (two macaques) was immunized with PBS/alum. Physiological conditions and adverse side effects were monitored by daily routine examination. During the 56 weeks of the study, no vaccine-related side effect such as fever, local specific redness or swelling at the site of injections was observed in the immunized monkeys . The fulp<0.05). Anti-EV71 IgG titers were found to decline quickly after the peak but still remained detectable at 56 weeks, the end of the experiment. The GMT of anti-EV71 IgG at week 56 was tested to be 476 and 283 for high- and low-dose immunized monkey groups, respectively. The current results suggest that EV71vac could elicit strong and quick anti-EV71 IgG antibody responses in monkeys, but the IgG antibody titers were surprisingly found to decline close to the baseline level 1 year later.After a single immunization, the EV71vac regardless dosage elicited 100% seroconversion (a 4-fold increase in IgG antibody titer) in the immunized monkeys as shown in rd dose > Nt of post-2nd post > Nt of post 1st dose. In contrast to the dose-dependent anti-EV71 IgG responses, the GMT of neutralizing antibody was found to be similar in both high- and low-dose immunized monkey groups. Similar to IgG responses, as shown in p<0.01). Although the Nt of each EV71vac immunized macaques had dropped significantly every month after the third vaccinations, 100% (4/4) of high-dose immunized monkeys still had Nt >100 at week 56. In contrast, 50% (2/4) of the low-dose group had Nt >100.It is of interest to investigate whether EV71 specific IgG could neutralize the vaccine strain. The EV71-specific neutralization titer (Nt) in the pre-immune serum of all monkeys was below the detection limit (<8). After first vaccination, both high- and low dose immunized monkeys generated neutralizing antibodies and the GMT was found to be 140 and 154 for the high- and low-dose monkey groups, respectively . The Nt + T cells in the high- and low-dose EV71vac-immunized monkeys (n = 3) and one PBS control monkey at week 6 . The EV71-specific TNF-producing CD4+ T cells for high dose, low dose and PBS control were shown to be 0.398±0.073, 0.205±0.075 and 0.146, respectively.To investigate the T cell response, we further measured EV71-specific IFN-γ and IL-4 production by ELISPOT assay. None of the monkeys showed EV71-specific IFN-γ or IL-4 responses before immunization. After immunization, the EV71-specific IFN-γ response was initially detected in one high-dose immunized monkey at week 3 and reached to a peak at week 6 after 1 booster dose . A dose-t week 6 . Without control ; p<0.05.nd immunization (week 6), 25% (1/4) of the low-dose immunized group (monkey A27) and 75%  of the high-dose immunized group but none of the PBS control group developed specific IFN-γ production  strain were used for the cross-neutralization test. No cross-neutralizing antibody responses (<8) were detected in any pre-immune sera. After immunization, dose-dependent cross-neutralizing antibody responses against EV71-0204 (B1), EV71-0692 (B5), EV71-0448 (B5), EV71-4643 (C2), EV71-C4D (C4a) and EV71-1757 (C4a) were found in EV71vac immunized monkeys and remained detectable even 56 weeks after vaccination . In contTo clarify the difference in cross-neutralization, we also performed a bioinformatics analysis by aligning the amino acid sequence of P1 protein between these strains and the vaccine strain, EV71/E59. The similarity between EV71/E59 and CAV16-5079N was the lowest. The sequence homology between EV71/E59 and other strains such as EV71-4643, EV71-5746, EV71-N3340 and CVA16-5079N was found to be 96.87%, 96.87%, 97.10% and 80.05%, respectively . Even thp<0.05 by Two-way ANOVA test).To understand the boosting effect of EV71vac-specific memory immunity, at 56 weeks all EV71vac immunized monkeys were revaccinated two times of 5 µg of EV71vac 3 weeks apart. Before the booster dose, the GMT of neutralizing antibody titer was below 500 in all EV71vac immunized monkeys. The neutralizing antibodies increased in all immunized monkeys after the first booster dose and the GMT of Nt were elevated from 450 to 1090 and 106 to 661 for the high- and low-dose groups, respectively . CompareSeveral inactivated EV71 whole-virion vaccine candidates have been developed and being tested in human clinical trials rd dose > Nt of post-2nd post > Nt of post 1st dose. These results are different from the human trial where no significant Nt elicited by the second dose of EV71vac Other EV71 vaccine studies in monkeys Although the protective level of neutralizing antibody against EV71 infection in human is unclear, the neutralizing antibodies with Nt >100 have been shown to protect against EV71 infection in mouse challenge model + T cells, was associated with a dose-dependent antibody response. The induced EV71-specific CD4+ T cells contribute to the differentiation of memory B cells and maintenance of long-term protective neutralizing antibodies. This is supported by the findings that a dramatic increase (ranging from 2 to 30-fold) in neutralizing antibody responses when the immunized macaques were re-vaccinated again after 56 weeks  between different enteroviruses. All of the P1 protein sequences were obtained from NCBI protein database and aligned by clustalW alignment. The consensus amino acid sequence was shown in the bottom line and the changed amino acids were indicated. The similarity of sequences was also represented in the bottom table.(DOC)Click here for additional data file.Table S1Physiological information of 10 monkeys involved in this study and the side effect results after each vaccination.(DOC)Click here for additional data file.Table S2The blood count data in monkeys received immunization.(DOC)Click here for additional data file.Checklist S1NC3Rs ARRIVE Guidelines Checklist for this study.(PDF)Click here for additional data file.