Real-time shear wave elastography (2D-SWE) is a two-dimensional transient elastography and a competitor as a biomarker of liver fibrosis in comparison with the standard reference transient elastography by M probe (TE-M). The aims were to compare several criteria of applicability, and to assess inflammation and steatosis impact on elasticity values, two unmet needs.We took FibroTest as the fibrosis reference and ActiTest and SteatoTest as quantitative estimates of inflammation and steatosis. After standardization of estimates, analyses used curve fitting, quantitative Lin concordance coefficient [LCC], and multivariate logistic regression.A total of 2,251 consecutive patients were included. We validated the predetermined 0.2 kPa cut-off as a too low minimal elasticity value identifying not-reliable 2D-SWE results  89.6%(88.2–90.8), was significantly higher than that of TE, 85.6%. In patients with non-advanced fibrosis (METAVIR F0F1F2), elasticity values estimated by 2D-SWE was less impacted by inflammation and steatosis than elasticity value estimated by TE-M: LCC (95%CI) 0.039  vs 0.090  and 0.105  vs 0.192  respectively. The three analyses methods gave similar results.Elasticity results including very low minimal signal in the region of interest should be considered not reliable. 2D-SWE had a higher applicability than TE, the reference elastography, with less impact of inflammation and steatosis especially in patients with non-advanced fibrosis, as presumed by blood tests.NCT01927133ClinicalTrials.gov  TM is a two-dimensional transient elastography technique, [Liver fibrosis evaluation using real-time shear wave elastography 2D-SWE) by Aixplorerchnique,  which isD-SWE by 2D-SWE estimates the speed of a shear wave to provide a quantitative estimate of tissue stiffness. 2D-SWE has the advantage over TE of being able to image liver stiffness in real time, not limited at a single location, and guided by a higher frame-rate B-mode image.,3 Two diS1 Table) The secondary aim was to better quantify the impact of inflammation and steatosis on elasticity values, independently of fibrosis value.The first aim was to better define quality criteria ,3,4,5,6 These aims were reachable more rapidly and in larger populations using validated blood tests as reference, rather than using liver biopsies.. We included patients aged 18 years or older who had undergone simultaneous serum sampling for FibroTest and attempted liver stiffness measurements with 2D-SWE and TE-M and TE-XL.. All co-authors had access to the study data and had reviewed and approved the final manuscript.TM ultrasound system . For each patient the mean, and the median of Qbox elasticity were assessed, as well as the lowest and the highest elasticity values. A single estimate of the Qbox elasticity was performed, as it has been previously validated using biopsy that less than five measures were sufficient for 2D-SWE in comparison with TE, three measures [2D-SWE was performed using the Aixplorermeasures ,8 and fimeasures .TM  according to the instructions and training provided by the manufacturer.[TE-M and TE-XL were performed using M and XL probes respectively, using FibroScanfacturer. Steatosifacturer. The measFibroTest, ActiTest and SteatoTest were performed according to the manufacturer’s’ recommendations, using the usual predetermined cutoffs ,12,13. FFor FibroTest, a measurement was classified as a failure when serum sampling was impossible; it was classified as not-reliable if one component in the measurement had an extreme value, which induced a change of more than 0.30 in the FibroTest value when calculated using the usual median instead.For TE-M and TE-XL, signal absence was considered a failure, and the standard reliability definition was the IQR/liver stiffness measurement (IQR/M) <0.30, at least 10 measurements and a success rate of 60% or greater.,14 Tables using the “strength of concordance” method detailed elsewhere.[For 2D-SWE, there were no standard,,4,5 and lsewhere.. In the The strength of concordance between 2D-SWE and FibroTest were estimated using the Lin concordance coefficient of correlation (LCC), stratified according to presence of significant steatosis and significant inflammation  and lower concordances .Secondly, we describe the association between elasticity values and fibrosis severity using curve fitting. We compared graphically and with squared correlation coefficient (R2)Thirdly, we used LCC with bilateral 95% confidence interval, for assessing the quantitative strength of concordance between elasticity estimates, FibroTest measures being taken as reference. LCC was also used to compare the impact of inflammation and steatosis on elasticity values. A quantitative multivariate regression analysis was performed using the diagnostic of F3F4 as the endpoint. We compared the regression coefficients as well as the area under the ROC curves (AUROC) of the logistic regression function, including elasticity values, inflammation and steatosis and the cause of liver disease as variability factors. Due to the relatively small number of patients, only NAFLD and CHC were entered in these models.The AUROC were estimated by the non-parametric method, and compared using the paired method of Zhou et al. RecommenBetween October 2013 and April 2015, 4,151 consecutive patients were assessed for fibrosis, 2,251 patients constituted the "intention-to-diagnose population", 1,720 patients constituted the "SWE reliability population", 1,558 patients the "concordance population", 1,270 patients the "concordance population with SteatoTest" and 663 patients were the "not-applicable population". Tables. Only 53/1588 (3.3%) patients had an interval between blood tests and elasticity measurements between one to six months.There were no major, or unexpected differences, between the different populations characteristics.) by using Log10 transformation  and limiting the maximum to 75 kPa and dividing by 75, permitted to obtain as for FibroTest a similar range between 0.00 to 1.00 as well as less skewed distribution .The standardization of elasticity values  S1 and  Figs.For the "minimal 2D-SWE value" we confirmed a discriminant cut-off at 0.2 kPa. The LCC was not significant between 2D-SWE and FibroTest, only in patients with minimal elasticity <0.2kPa (S10 Table), for elasticity measure depth , and for BMI (S12 Table), we did not found sufficiently discriminant cutoffs comparatively to the "minimal 2D-SWE value".For the elasticity CV (Table 2). In a total of 145 patients, elasticity measure was applicable using 2D-SWE and not applicable using TE-M., greater than that of TE-M , not different than TE-XL 88.2%  and lower than FibroTest 99.5% . TE-M applicability was lower than TE-XL (P = 0.008). than TE-M and TE-XL, and whatever the liver disease . 2D-SWE elasticity was less impacted by steatosis  than TE-M, but not than TE-XL, and whatever the liver disease . For 2D-SWE, R2 = 0.07 lower than that of TE-M . Curves fitting using SteatoTest  or CAP  were similar.Without stratification on fibrosis severity, 2D-SWE elasticity was less impacted by inflammation  was less impacted by inflammation than TE-M  and TE-XL . In patients F0F1F2 the 2D-SWE R2 was 0.04, lower than that of TE-M  and not different than TE-XL  respectively. In patients F3F4 the 2D-SWE R2 = 0.17, lower than those of TE-M (0.24) and TE-XL (0.21) respectively (P<0.01).2D-SWE  was less impacted by steatosis than TE-M , but not than TE-XL . In patients F0F1F2 the 2D-SWE the R2 was 0.04, lower than that of TE-M  and not different than TE-XL , respectively. In patients F3F4 the 2D-SWE the R2 was 0.17, lower than those of TE-M (0.24) and TE-XL (0.21) respectively .2D-SWE  vs 0.393(0.361–0.423) and vs 0.370(0.347–0.407) respectively (All P<0.01). After stratification by diseases, LCC remained significantly lower for 2D-SWE, only in patients with NAFLD.After stratification on inflammation severity, LCC remained significantly lower for 2D-SWE only in patients without significant inflammation, in comparison with TE-M and TE-XL. After stratification on steatosis severity, LCC remained significantly lower for 2D-SWE in patients without significant inflammation in comparison with TE-M, and in patients with significant steatosis in comparison with TE-XL.After stratification on both inflammation and steatosis, LCC remained significantly lower for 2D-SWE, only in patients without significant inflammation/steatosis. The exclusion of patients (3%) with interval greater than 30 days did not change significantly the LCC for activity or steatosis the results.2D-SWE were less associated (LLC) with inflammation than TE-M, and TE-TXL: 0.135(0.112–0.158), 0.220(0.193–0.246), 0.203(0.176–0.230), respectively . After stratification on severity of fibrosis, LCC remained significantly lower for 2D-SWE, than TE-M among patients without severe fibrosis..2D-SWE was less associated  with steatosis than TE-M . After stratification on severity of fibrosis, LCC remained significantly lower for 2D-SWE vs TE-M. There was no significant difference between strength of concordance (LLC) of elasticity values estimated by 2D-SWE, TE-M or TE-XL, and BMI (P>0.05)(S14 Table), as compared to 0.747 to 0.824 (S15 Table) for TE-M, and 0.747 to 0.824 (S16 Table) for TE-XL. Most of the improvement was associated with the inflammation adjustment. Steatosis adjustment was significant only for TE-M.The multivariate analyses showed that taking into account the inflammation, the steatosis, and the cause of liver disease improved all the elasticities performances for the diagnosis of F3F4. The 2D-SWE AUROC was the most improved from 0.716 to 0.816Using FibroTest as a reference, the prevalence of F3F4 was 35.4%(31.6–39.4), higher than those presumed by 2D-SWE , TE-M  and TE-XL (All P<0.001).Using the worst elasticity value among the 3 elasticity values, the presumed prevalence of F3F4 was 27.4%(23.8–31.1), and using only patients with the three concordant values, the prevalence of F3F4 was only 10.5%(8.2–13.5)(P<0.0001).According to elastography method, inflammation and steatosis, the presumed prevalence of F3F4 varied from 9.6%(6.7–13.3) using TE-XL in patients without significant inflammation and without significant steatosis, to 72.0% using TE-M in patients with significant inflammation and significant steatosis..In intention to diagnose, when applicability and inflammation were taken into account, 2D-SWE had higher performance for the diagnosis of F3F4 versus TE-M,  vs , difference which was not identified in standard per-protocol comparisons  An ideal study would have been to obtain a large surgical biopsy for each patient included, a perfect reference. In the absence of such perfect references, the standard method was to use biopsies, a non-perfect reference, with its own limitations including sampling error. Even 25 mm length biopsies had 25% of false positive/negative rates for the diagnosis of fibrosis stage, activity grade and steatosis grade ,21. The The first main concern was that the reference utilized to assess fibrosis, inflammation and steatosis were suboptimal. We acknowledge that FibroTest had limitations, but had been extensively and independently validated with a low risk of non-reliable results, around 2% . Even if One example of omitted evidence based was the largest histological validation of ActiTest in 1,459 patients of a prospective trial in CHC with biopsies [ActiTest also has been extensively histologically validated 5,326 patients) and is the only blood tests with diagnostic performance greater than transaminases for the prediction of necro-inflammatory histologic activity ,26. Vali patientsbiopsies . One example of omitted evidence based was the largest validation of SteatoTest in 1,415 patients of a prospective trial in CHC with biopsies [We acknowledge that SteatoTest had much less studies available , than FibroTest and ActiTest ,26. Vali We acknowledge that despite statistical significance, the difference in curve-fitting between TE and 2D-SWE were moderate and could be viewed as not clinically relevant. The measurements were not blinded as the same operator performed successively TE-M, TE-XL and 2D-SWE. We utilized the same cut-off values for 2D-SWE and TE-XL as of TE-M in the absence of consensual cut-offs. There was a low prevalence of patients with decompensated cirrhosis as one previously observed advantage of 2D-SWE was its higher applicability in patients with ascites than for TE-M.[Table 3) There was missing data for SteatoTest in 318 patients, but these patients were similar to those with non-missing SteatoTest.The following other limitations were detailed in for TE-M. We did nfor TE-M. Few patiThe first original result was that logarithmic transformation but also standardization according to range had a direct impact for concordance analyses. 2D-SWE has a possible range up to 300kPa compared to a 75kPa maximum value for TE-M. A method with larger range of elasticity values will have an artificial decrease in quantitative concordance coefficient if not standardized as the reference method. These rules should be discussed in specific guidelines.The second original result was the clarification of the relative interest of four quality criteria proposed for 2D-SWE. Among these criteria only the minimal value of the elasticity in the ROI minimal (0.2kPa) seemed useful as a cutoff to identify and exclude unreliable results. As this cutoff was predetermined and validated in a previous study, it could be recommended as a simple criterion for clinicians.The better applicability rate of 2D-SWE versus TE-M was confirmed. These paFor the first time to our knowledge it was possible, in a large number of patients with different liver diseases, to assess the relative impact of inflammation and steatosis on the elasticity value, independently of fibrosis severity. The influence of inflammation on elasticity measured by TE-M has been well validated, but few studies were published for 2D-SWE.,12 The i. In patients with high risk of inflammation or steatosis, 2D-SWE had an advantage for being more applicable and more specific for staging fibrosis than the TE-M, the present standard elastography method. Further studies are necessary to compare other new elastography methods such as ARFI or other real time elastography [As already described, the comparison between blood tests and elastography performances should be performed using direct comparisons, and intention to diagnose analyses. In intentography ,30.In patients with CHC, according to the severity of inflammation and steatosis, and to the elastography method, the presumed prevalence of F3-F4 varied from 9.6% to 72.0%. This spectrum variability should be taken into account for the prioritization of reimbursement of DAA.Elasticity results 2D-SWE including minimal signal <0.2 kPa in the ROI should be considered as not reliable. 2D-SWE had a higher applicability than TE-M the reference elastography, with less impact of inflammation and steatosis especially in patients with non-advanced fibrosis, as presumed by blood tests.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 FigCurve fitting in all patients.(DOCX)Click here for additional data file.S4 FigCurve fitting according to the 5 causes of liver disease.(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.S8 Fig(DOCX)Click here for additional data file.S9 Fig(DOCX)Click here for additional data file.S10 Fig(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 TableReliability population, n = 1720.(DOCX)Click here for additional data file.S11 TableDepth (mm) measured from probe surface to the top of the region of interest.(DOCX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S13 Table(DOCX)Click here for additional data file.S14 Table(DOCX)Click here for additional data file.S15 Table(DOCX)Click here for additional data file.S16 Table(DOCX)Click here for additional data file.S17 TableImpact of inflammation and steatosis severities, presumed by ActiTest (n = 599) and SteatoTest (n = 477).(DOCX)Click here for additional data file.S18 TableAnalyses performed in "Intention to diagnose" in 2,239 patients, and "Per protocol" in 1,588 patients with applicable elasticity data.(DOCX)Click here for additional data file.


However, the extent of NK cell–mediated Dk-dependent resistance to infection varies in different mouse strains. The molecular genetic basis of this variation remains unclear. Previous work to examine the Dk effect on MCMV resistance in MA/My × C57L offspring discovered multiple quantitative trait loci (QTL) that may serve to modify NK cells or their capacity to respond during MCMV infection. One QTL in particular, Cmv5, was found to regulate the frequency of NK cells and secondary lymphoid organ structure in spleen during MCMV infection. Cmv5 alleles, however, have not been identified. We therefore sequenced and analyzed genome-wide exome (GWE) variants, including those aligned to the critical genetic interval, in Cmv5-disparate mouse strains. Their GWE variant profiles were compared to assess strain-specific sequence data integrity and to analyze mouse strain relatedness across the genome. GWE content was further compared against data from the Mouse Genomes Project. This approach was developed as a platform for using GWE variants to define genomic regions of divergence and similarity in different mouse strains while also validating the overall quality of GWE sequence data. Moreover, the analysis provides a framework for the selection of novel QTL candidate sequences, including at the Cmv5 critical region.Host resistance to murine cytomegalovirus (MCMV) varies in different strains of laboratory mice due to differences in expression of determinants that control and clear viral infection. The major histocompatibility complex class I D While MA/My.L-H2b/k (M.H2het) mice are more resistant to MCMV than their MA/My.L-H2b (M.H2b) congenic counterparts, they are also more susceptible to infection than either MA/My or C57L.M-H2b/k (L.H2het) mice  class I DK) cells . Ly49G2-H-2D has a profound and specific effect on the frequency of Ly49G2-positive NK cells and inhibitory receptor expression on individual NK cells in naive mice  on immune cell and response features before and after infection in MA/My × C57L mice  and then further compared to the relevant strains. We assessed GWE sequence quality, reliability, and overall extent of variability among the strains. Cmv5-disparate GWE profiles were further compared against a panel of common laboratory strains in the Mouse Genomes Project (MGP) to glean additional insight about the quality of GWE sequences, strain-dependent relatedness, and putative Cmv5 candidates.As one approach toward discovering genetic variants of interest, we examined genome-wide exome (GWE) sequences in b congenic mice were generated previously (MA/My and C57L mice were purchased from The Jackson Laboratory and maintained at the University of Virginia (UVA). M.H2b (spleen and lung) female tissues (14–22 wk old) using a kit (Gentra Systems). GWE sequencing was performed at Hudson-Alpha Institute for Biotechnology . Briefly, 2 μg of intact genomic DNA was fragmented by sonic shearing using a Covaris LE220. DNA fragments were hybridized to the NimbleGen SeqCap EZ Target Enrichment System Exome Library. Complexes of library oligos and DNA fragments were captured, purified, and then enriched by PCR. Samples were run on the Illumina HiSequation 2000. The DRAGEN Genome sequencing analysis pipeline was used to perform sequence alignment and variant calling based on the B6 GRCm38/mm10 reference genome (http://www.edicogenome.com/pipelines/dragen-genome-pipeline/). Average depth of coverage across MA/My and C57L exomes was ∼10 times, and the M.H2b exome was ∼17 times. Binary alignment map (BAM) files with raw sequence data have been uploaded to the NCBI Sequence Read Archive (accession no.: SRP077658). BAM file comparisons to the B6 reference genome yielded exome variant call files (VCFs) for each strain, also provided by Hudson Alpha.To identify high probability gene candidates for previously mapped QTL, genome-wide sequencing of mouse exomes was performed. DNA was extracted from MA/My (liver), C57L (liver), and M.H2File S1). After filtering, 496407, 228732, and 387357 variants were detected in the MA/My, C57L, and M.H2b strains, respectively. C57L had the fewest variants, which is indicative of its close genealogical relatedness to B6  were loaded as data frames into R using [read.csv]. A source file containing R code is also provided (File S3). Low and modifier impact variants, duplicate variants, and mitochondria DNA variants were removed using [subset]. Chromosome “X” and deletions “-” were renamed “20” and “D,” respectively, and bases were converted to Mb. VEP data frame comparisons were performed in R, using [sqldf] to identify common and strain-specific exome variants. Variant numbers per 10-Mb bin were enumerated for all bins on each chromosome [data.frame)))]. Data frames of enumerated bins were merged using [list], [reduce], and [merge], resulting in a master data frame with both total and strain-specific variant numbers per bin. The sum of specific variants for combinations of two strains  was calculated using [transform]. Heatmaps depicting total variant numbers per strain and the sum of strain-specific variants per two strain combinations were created in R using [ggplot2] and [gridExtra]. Histograms of the frequency distribution of total variants per 10-Mb bin for each strain were generated by using [melt] in the [reshape package] on total variant numbers per bin to create a data frame with columns for strain and number of variants per bin. Rows with 0 variants/bin were removed using [subset]. Log2(variants/ bin) calculated in R using [transform] and a data frame containing median value for each GWE distribution was generated using [aggregate]. Figures depicting the results were created in R using [ggplot2].VCFs were analyzed using Ensembl Variant Effect Predictor (VEP) to assess variation in MA/My, C57L, and M.H2Cmv5 interval (Rrp1-Sgol1) using [subset(Chr == 17 & Pos >32.05 & Pos <53.68)]. Allele columns in each data frame were renamed to the strain names using [paste]. Additional data frames were created in R using [sqldf], including variants  common to MA/My and C57L for C57L; variants common to MA/My and C57L for MA/My; variants common to MA/My, C57L, and M.H2b; MA/My-specific variants for MA/My; MA/My-specific variants for M.H2b; C57L-specific variants for C57L; C57L-specific variants for M.H2b; and M.H2b-specific variants. All eight data frames were necessary for figure creation. Nucleotide calls in each data frame were assigned a variant number based on type: those common to MA/My and C57L, and those that are MA/My-, C57L-, or M.H2b-specific. Data frames were then merged in R, which resulted in three data frames containing variants common to MA/My and C57L in addition to those that are MA/My-, C57L-, or M.H2b-specific and their variant type number. These were further merged into a single data frame and converted using [melt] to contain columns for chromosome, position, gene, strain, and variant type number. A figure depicting MHC-linked exome (MLE) variants in the Cmv5-disparate strains was also created in R using [ggplot2] and color-coded by variant type.Original data frames were reduced to the Cmv5 interval for all MGP strains were acquired and loaded into R as a data frame. Duplicates were removed, and bases were converted to Mb. MA/My, L-UVA, and M.H2b data frames were reduced to the same interval using [subset(Chr == 17 & Pos >32.05 & Pos <53.68)]. Columns were reordered to reflect MGP data frame organization with unnecessary columns removed. Allele columns for each data frame were renamed strain names using [paste]. The data frames were then merged into one. All “NA” text descriptors were converted to “0,” and all variants were converted to “1.” The data frame was then reduced to MA/My-specific variants  using [subset]. MA/My-defining MLE variants were compared to all other strains in R and new data frames were created to report on each strain and the frequency of MLE variants that were in common with MA/My. A bar graph depicting the percentage match with MA/My was created in R using [ggplot2].For MLE sequence comparisons to genomic content of MGP strains, variants aligned to the File S1 provides a workflow for GWE sequence variant filtering. File S2 provides a zip folder of VEP files for the sequenced strains. File S3 provides the R code used to analyze VEP files.Mouse strains are available upon request. BAM files with raw sequence data and VCFs are available at the NCBI Sequence Read Archive (accession no.: SRP077658). b strains  distinguished MA/My from B6 , chr-2 (β2m-Sirpα), chr-4 (Tnfrsf multigene locus), chr-6 (NK gene complex), chr-11 (Itgae-Hic1), chr-14 (Tcr α/δ locus), and chr-15 (Ly6 multigene locus) also distinguished MA/My or C57L from B6 and/or each other  for 36 different mouse strains, including C57L, with an average depth of coverage of the genome of 64 times (Cmv5 (Rrp1b-Sgol1) interval on chr-17 in MA/My, L-UVA, and M.H2b. In total, MA/My had 670 MLE variants relative to B6, while L-UVA and M.H2b had 319 and 306, respectively , which suggests that GWE sequence artifacts, undetected cross-over events, and/or strain-specific sequence variants may have been acquired.We next compared MLE profiles corresponding to the ectively . Of the ch other . Only a b to MGP MLE profiles using the statistical computing program R. We found that the MHC region in the RF strain of William E. Castle’s group B mice  features. On the other hand, strains 129, BTBR, C57BL, C57L/J (MGP), KK, LP, C57L (L-UVA), and M.H2b display much greater MLE variability in comparison to MA/My  and H2bc  strains. MA/My MLE sequences also differ substantially from H2b strains, including BTBR_T_ltpr3tf_J, C57L/J , C57L-UVA, and M.H2b; however, the extent of variation differs when compared to the H2b strains C57BL/6NJ and C57BL/10J, which suggests that MHC-linked loci have further diversified in different H2b-related strains since strain separation. A viable approach for selection and analysis of GWE sequences as QTL candidates, and a genomic framework and context for discovery of Cmv5 alleles is established. We envisage leveraging it with precision QTL mapping and complementary gene expression and sequencing analyses, which could lead to Cmv5’s discovery.We further assessed www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.042531/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.


HER2). The insertion re-targeted the virus tropism to the HER2-positive cancer cells. This was unexpected since gB is known to be a fusogenic glycoprotein, not a tropism determinant. The gB-retargeted recombinant offered the possibility to investigate how HER2 mediated entry. In contrast to wt-gB, the activation of the chimeric gBHER2 did not require the activation of the gD and of gH/gL by their respective receptors. Furthermore, a soluble form of HER2 could replace the membrane-bound HER2 in mediating virus entry, hinting that HER2 acted by inducing conformational changes to the chimeric gB. This study shows that (i) gB can be modified and become the major determinant of HSV tropism; (ii) the chimeric gBHER2 bypasses the requirement for receptor-mediated activation of other essential entry glycoproteins.Herpes simplex virus (HSV) entry into the cells requires glycoproteins gD, gH/gL and gB, activated in a cascade fashion by conformational modifications induced by cognate receptors and intermolecular signaling. The receptors are nectin1 and HVEM (Herpes virus entry mediator) for gD, and αvβ6 or αvβ8 integrin for gH. In earlier work, insertion of a single chain antibody (scFv) to the cancer receptor HER2  in gD, or in gH, resulted in HSVs specifically retargeted to the HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Here, the scFv to HER2 was inserted in gB (gB Herpesviridae family. In herpesviruses other than HSV, but not in HSV, gB also contributes to determine the virus tropism. We took advantage of retargeting studies to investigate the process of HSV glycoprotein activation, and the specific roles played by the glycoproteins. When a heterologous ligand is engineered in gB, the virus tropism is retargeted to the ligand receptor. gB becomes the major determinant of HSV tropism, and does not any longer need the receptor-mediated activation of glycoproteins gD and gH/gL.Herpes simplex virus encodes an entry apparatus made of the glycoproteins gD, gH/gL and gB. gD is the major determinant of HSV tropism. Receptor-induced modifications to gD and gH/gL activate in a cascade fashion gB, the conserved fusogenic glycoprotein across the  Herpesviridae family. The quartet assembles in complexes [in vivo, was not investigated. The lack of contribution of gB receptors to overall viral tropism is highlighted by the finding that abrogation of the gD interaction with one of its receptors abrogates virus entry in virtually any cell.Herpes simplex virus encodes a multipartite entry apparatus made of four essential glycoproteins, named gD, the heterodimer gH/gL and gB, with distinct functions –4. gD, womplexes , 16–18. omplexes ,17–20. Tomplexes –24. gH/gomplexes ,26. αvβ6omplexes –30. Littomplexes . The effThe current model of HSV entry envisions that the four glycoproteins switch from an inactive to a fusion-active conformation –4,31. AlGM-CSF, for the treatment of metastatic melanoma [The HSV glycoproteins are of interest in the design of oncolytic HSVs. Recently, this field received much attention upon Federal Drug Administration and European Medicines Agency approval of the oncolytic HSV, originally named Oncovexmelanoma ,34. For melanoma ,36. In emelanoma –39. The melanoma . The strmelanoma –42. The melanoma . In our melanoma . Retargemelanoma –47. Recemelanoma . Thus, tvia gB was unexpected, since gB is the fusogenic glycoprotein, and was not known to be a determinant of HSV tropism. Inasmuch as the scFv to HER2 mediates entry when engineered in gD, gH, or gB we asked how can a same ligand, engineered in one or the other of the three glycoproteins—gD, gH or gB—enable entry through the HER2 receptor.Here, we asked whether gB is a suitable glycoprotein for retargeting. We engineered the scFv to HER2 between AA 43 and 44 of gB, thus generating R-903. By further deletion of AA 6–38 in gD, the recombinant could be detargeted from natural receptors. The retargeting to HER2 The scFv to HER2 was engineered in gB between AA 43–44, thus generating R-903 . This povia gB and detargeted from natural gD receptors.The modifications to tropism were assayed in J cells which transgenically express the receptor of choice, HVEM, nectin1, or HER2. J cells are negative for HSV gD receptors and cannot be infected by wt-HSV . The celvia transmission to gH/gL and then to gB.Three main conclusions can be drawn from this set of experiments. First, the insertion of a scFv in gB modifies HSV tropism. This was a surprising result since gB is known for its ability to carry out virion-cell fusion, but not as determinant of HSV tropism. Second, the finding that R-909 infected J-HER2 cells, but not J cells, suggests that the scFv in gB enabled gB activation upon interaction with HER2, and rules out that gB activation occurred independently of the interaction with HER2. Lastly, the infection of J-HER2 cells with R-909 occurs in the absence of a gD receptor, or with a deleted gD unable to bind its natural receptor. Essentially, it is independent of receptor-mediated gD activation. These properties imply that the cascade of glycoprotein activation that ultimately leads to gB activation does not occur in a canonical manner in R-909, i.e. starting from receptor-bound gD and Fundamental properties for any candidate oncolytic virus are the extent of replication and the ability to kill the infected cells. SK-OV-3 cells were infected with R-909 at 0.1 PFU/cell. The gH-retargeted R-809, the gD-retargeted R-LM113, and the wt HSV-1(F) were included for comparison. Virus yields were determined at 0 time (3 h), 24 and 48 h after infection. The killing ability of R-909 for the HER2-pos MDA-MB-453 and SK-OV-3 cancer cells is reported in HER2) with HER2, we verified whether gBHER2 binds HER2, and whether R-909 infection was inhibited by trastuzumab, the MAb to HER2 from which the scFv was derived [HER2 from R-909, gHHER2 from R-803. 293T cells were transfected with gBHER2, gHHER2, or their wt counterpart, and reacted with a soluble truncated form of HER2. HER2, and gHHER2 reacted with soluble HER2, whereas the wt-gB and wt-gH did not. This result shows that the binding of gBHER2, or gHHER2 to HER2 occurs in the absence of the other glycoproteins. For the infection assay, we infected J-HER2 or SK-OV-3 cells in the presence of trastuzumab. A wt HSV was not included as it does not infect J-HER2 cells  and gB (gBHER2) offered the opportunity to dissect these two functions of gD. We set up a cell-cell fusion assay, whereby the donor cells express the glycoproteins and T7 Polymerase, and the target cells express the receptor—HER2 or nectin1—along with T7 promoter driven luciferase. gBHER2 was cloned from R-909, and the gHHER2 was cloned from R-803. To mimic the situation in R-909 and R-809 recombinants, we also cloned gDΔ6–38. These glycoproteins, or their wt versions, were transfected in appropriate combinations. HER2 or gHHER2 promoted fusion with CHO-nectin1 cells in the presence of wt-gD. These results imply that gBHER2 and gHHER2 maintain the fusogenic, or pro-fusogenic, activity of their wt counterparts, although at somewhat reduced extent, in agreement with the infectivity of R-909 and R-809. Importantly, in the absence of gD, the fusion activity dropped by at two-three logs and reached the background level , suggesting that gD can not be omitted. HER2, or gHHER2 exhibited a significant fusion activity with CHO-HER2 cells, again somewhat lower than that of their wt counterparts with CHO-nectin1 cells. wt-gB and wt-gH did not induce fusion with CHO-HER2 cells. The fusion activity with receptor-negative CHO cells was at the background level , or by endocytosis into acid endosomes –59. αvβ3 R-LM113 . Inhibit R-LM113 . In thes R-LM113 , in agrePreviously, our laboratory showed that αvβ6 or αvβ8 integrins serve as receptors for HSV entry, bind gH and contribute to its activation ,62. HencThe ligand to αvβ6 or αvβ8 integrins is gH/gL . Earlierattachment/entry of R-909 to J-HER2 or J-HER2+αvβ6 cells did not result in a decrease in 53S reactivity relative to 52S reactivity, irrespective of the overexpression of αvβ6 integrin.By contrast, attachment/entry of R-LM113 and R-LM249 to J-HER2+αvβ6, but not to J-HER2 cells resulted in a sharp decrease in 53S reactivity, as previously observed for the wt-HSV, herein exemplified by R-LM5.Attachment/entry of the gH-retargeted R-809 did result in decrease in 53S reactivity; however, this decrease took place independently of whether αvβ6 was present or absent; hence, it was induced by HER2 and not by integrin.In the samples where the 53S reactivity was decreased, there was a concomitant release of gL in the medium . We concThe gL displacement mediated by αvβ6 integrin takes place with R-LM113 and R-LM249; in this case, in contrast to the wt-virus, the gD-activation is mediated by HER2 and not by nectin1.The gL displacement takes place with R-809; in this case it is independent of αvβ6-integrin, and is mediated by HER2, which directly targets gH.The gL displacement does not take place in R-909, either in the absence or in the presence of αvβ6 integrin. The data argue that attachment/entry of R-909 is independent of β-integrins, does not lead to gL displacement, and therefore the integrin-mediated gH activation does not appear to be a critical step.In a simplistic view, a receptor can mediate virus entry by promoting juxtaposition of the cell and virion membranes. Alternatively, the receptor induces conformational changes and promotes activation of the cognate glycoprotein. Experimentally, the two mechanisms can be differentiated since, in the latter, but not in the former case, a soluble form of the receptor (or of the glycoprotein), can substitute for the membrane-form of the receptor (or of the glycoprotein) ,64. The Herpesviridae family. Second, we asked how does the novel HER2 receptor enable entry of the recombinant carrying the HER2-retargeted gB. We deleted a portion of gD, so that the virus lost the ability to use and be activated by the natural gD receptors; we further depleted cells of β6 and β8 integrins, which activate gH. We found that the entry of the gB-retargeted recombinant occurred in the absence of the cascade of gD and gH activation. Third, retargeting through the chimeric gBHER2 was intriguing since, the same scFv ligand to HER2 mediates entry when engineered in gD [In this study we addressed the question whether gB is a suitable glycoprotein for HSV retargeting. The aim was twofold, i.e., to better understand the biology of HSV entry through the lesson of retargeting studies, and to expand and improve the toolkit for the design of retargeted oncolytic HSVs. We report on three findings. First, the insertion of a scFv to HER2 in gB re-addresses HSV tropism to the HER2 receptor. In essence, when gB carries a ligand, the ligand receptor contributes to define the host range of the virus. This was an unexpected result since gB is responsible to execute the virion-cell fusion in HSV and across the ed in gD ,46, gH [ed in gD , or gB . The transfected cells were allowed to fuse with target cells expressing HER2 or nectin1. In all combinations the fusion by HER2-retargeted gB, HER2-retargeted gH, or their wt counterparts required gD, be it wt or the non-activable gDΔ6–38. This provides formal evidence for gD activity other than triggering, most likely for a structural role.With respect to gD, earlier and current work hinted that gD may encode activities other than triggering of glycoprotein activation upon receptor binding ,48,54,55HER2 by HER2 bypassed the need for activation by receptor-activated gD and gH.To shed further light on the mechanism of HER2-mediated entry, we compared three sets of recombinants: the gD-retargeted R-LM113 and R-LM249, the gH-retargeted R-809, and the gB-retargeted R-909. For all three recombinants the soluble HER2 was able to replace the membrane-bound HER2 in mediating entry. Thus, a common feature was that HER2 did not serve the function of anchoring the viruses to the cell; rather it induced conformational modifications to the respective targeted glycoprotein. Interestingly, HER2 exerted differential effects on the entry apparatus of the three sets of recombinants. While the wt R-LM5 required nectin1/HVEM for gD activation and integrin for gL displacement, the gD-retargeted viruses required HER2 for gD activation, and integrins for gH activation and gL displacement . In the It is possible to engineer a chimeric gB which carries two topologically and functionally distinct domains: the scFv that enables gB activation upon binding to the HER2, and the fusogenic machine, intrinsic to the glycoprotein. The chimeric gB does not need the activation signaling by receptor-bound gD and gH. Functionally, the entry machine of the recombinant R-909 is a monopartite apparatus.The question arises as to why herpesviruses evolved to have a multipartite entry system. The advantages of a multipartite entry system were in part addressed in a recent review . Briefl(negative for HSV receptors) and their derivatives expressing HER2, nectin1 or HVEM were previously described [The J cells escribed ,68. J ceescribed . The R6 escribed . The RS escribed . The aboescribed . The recescribed ,46,48.R8 polyclonal antibody (PAb) to gD and BD80 monoclonal antibody (MAb) to gD were generously provided by Dr. G. H. Cohen and Dr. R. Eisenberg. MAbs HD1 and H126 were a gift from Dr. L. Pereira. MAb H1817 was purchased from Goodwin Institute. MAbs 52S and 53S were described . Bovine 4)2SO4, 100 mM KH2PO4, 1.8 μg FeSO4·H2O, adjusted to pH7) supplemented with 1 mg/L D-biotin, 0,2% galactose, 45 mg/L L-leucine, 1 mM MgSO4·7H2O and 12 μg/ml chloramphenicol. In order to exclude galK false positive bacterial colonies, the positive recombinant clones were streaked also on MacConkey agar base plates supplemented with 1% galactose and 12 μg/ml chloramphenicol and checked by colony PCR with primer galK_129_f ACAATCTCTGTTTGCCAACGCATTTGG and galK_417_r CATTGCCGCTGATCACCATGTCCACGC. Next, the trastuzumab scFv cassette with the downstream Ser-Gly linker and bracketed by homology arms to gB was amplified with primers gB43_sc4D5_for GGTGGCGTCGGCGGCTCCGAGTTCCCCCGGCACGCCTGGGGTCGCGGCCGCGTCCGATATCCAGATGACCCAGTCCCCG and gB43_sc4D5_rev GGCCAGGGGCGGGCGGCGCCGGAGTGGCAGGTCCCCCGTTCGCCGCCTGGGTACCGGATCCACCGGAACCAGAGCC using pSG-scFvHER2-SG plasmid as template [2O and 12 μg/ml chloramphenicol. Bacterial colonies were also checked for the presence of sequence of choice by means of colony PCR with primers gB_ext_for GAGCGCCCCCGACGGCTGTATCG and gB_431_rev TTGAAGACCACCGCGATGCCCT. The starting material for R-909 was the R-903 BAC genome. To engineer the AA 6–38 deletion in gD, galK cassette flanked by homology arms to gD was amplified with primers gD5_galK_f TTGTCGTCATAGTGGGCCTCCATGGGGTCCGCGGCAAATATGCCTTGGCGCCTGTTGACAATTAATCATCGGCA and gD39_galK_r ATCGGGAGGCTGGGGGGCTGGAACGGGTCCGGTAGGCCCGCCTGGATGTGTCAGCACTGTCCTGCTCCTT. Next, we replaced galK sequence with a synthetic double-stranded oligonucleotide gD_aa5_39f TTGTCGTCATAGTGGGCCTCCATGGGGTCCGCGGCAAATATGCCTTGGCGCACATCCAGGCGGGCCTACCGGACCCGTTCCAGCCCCCCAGCCTCCCGAT. To reconstitute the recombinant virus R-903, 500 ng of BAC DNA was transfected into SK-OV-3 cells by means of Lipofectamine 2000 (Life Technologies). Alternatively, for R-909, the corresponding BAC was first transfected in gD-complementing cell line R6 (a rabbit skin cell derivative expressing glycoprotein D under the control of HSV late promoter UL26.5) [HER2, SK-OV-3 cells were infected at 3 PFU/cell with R-909 and R-LM5, and harvested 72 h after infection. Cell lysates were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes and immunoblotted with MAb H1817 to gB.First, we engineered R-903 by insertion of the sequence encoding the trastuzumab scFv between AA 43 and 44 of immature gB, corresponding to AA 13 and 14 of mature gB, after cleavage of the signal sequence, which encompasses AA 1–30. The starting genome was the BAC LM5 BG, which carries LOX-P-bracketed pBeloBAC11 and eGFP sequences inserted between UL3 and UL4 of HSV-1 genome . The engtemplate . The rec UL26.5) . After oThe indicated J cell derivatives were infected with R-909, R-903, R-809 and R-LM5 at an input multiplicity of infection of 3 PFU/cell for 90 min at 37°C and monitored by fluorescence microscopy 24 h post infection. The SK-OV-3, BT-474, MDA-MB-453 HER2-pos cancer cells, and theHER2-neg HeLa and MDA-MB-231 cancer cells, and the HER2-neg non-cancer HaCaT cells were infected at an input multiplicity of infection of 3 PFU/cell (as titrated in SK-OV-3) for 90 min at 37°C with R-909 and R-903. Pictures were taken 24 h after infection by fluorescence microscopy.SK-OV-3 cells were infected at 0.1 PFU/cell for 90 min at 37°C. Unabsorbed virus was inactivated by means of an acidic wash . Replicate cultures were frozen at the indicated times  after infection and the progeny was titrated in SK-OV-3. For plaque size determinations, the indicated viruses were plated onto SK-OV-3 cells with agar overlay. Pictures of 10 plaques were taken for each virus. Plaque areas (pxE2) were measured with Nis Elements-Imaging Software (Nikon). Each result represents average areas ± SD.3 cells/well, and infected with the indicated viruses or mock-infected for 90 min at 37°C. The input multiplicity of infection (as titrated in the correspondent cell line) was 2 PFU/cell for the SK-OV-3 and MDA-MB-453. MDA-MB-231 cells were infected with the recombinant viruses R-909 and R-809 at approximately 0.1 PFU/cell . MDA-MB-231 cells were infected with the wt HSV-1(F) at 0.05 PFU/cell. AlamarBlue (Life Technologies) was added to the culture media (10 μl/well) at the indicated times after virus exposure and incubated for 4 h at 37°C. Plates were read at 560 and 600 nm with GloMax Discover System (Promega). For each time point, cell viability was expressed as the percentage of AlamarBlue reduction in infected versus uninfected cells, after subtraction of the background value . Each point represents the average of at least three triplicate samples ± SD.SK-OV-3, MDA-MB-453 and MDA-MB-231 cells were seeded in 96 well plates 8 x 10HER2/gL, wt gB or gBHER2 was transfected into 293T cells by means of Lipofectamine 2000 (Life Technologies). 24 h later cells were fixed with paraformaldehyde, incubated with a soluble truncated form of HER2 tagged with 6x His tag , subsequently incubated with mouse anti-His antibody , and with Anti-Mouse IgG (Fc specific)-Alkaline Phosphatase  in presence of BCIP  and NBT  substrate.100 ng of DNA encoding wt gH/gL, gHReplicate monolayers of J-HER2 cells, or SK-OV-3 cells in 12-well plates were preincubated with trastuzumab, the MAb to HER2 from which the scFv-HER2 was derived or with non–immune mouse IgG . After 1 h at 37°C, the cells were infected at an input multiplicity of infection of 5 PFU/cell (as titrated in SK-OV-3) with R-909 and R-LM113 or R-LM5. Alternatively, where indicated, virions were pre-incubated with MAb HD1 to gD , MAb H126 to gB , MAb 52S to gH (ascites fluid 1:25) for 1 h at 37°C, and then allowed to adsorb to cells for 90 min. In the case of MAb HD1, the combination of HD1 plus trastuzumab was also tested. Viral inocula were then removed, and cells were overlaid with medium containing the indicated antibodies. Infection was quantified by fluorescent activated cell sorter (FACS).NM_017003) and is deleted of the tyrosine kinase domain [Expression plasmids for wt-gD, gB, gH, and gL were described . EGFR2Δ e domain . Plasmide domain . Plasmide domain ,76.HER2, gHHER2, and gDΔ6–38 were PCR amplified from R-909 or R-803, and cloned into pcDNA3.1(-) (Thermo Fisher Scientific), as follows. gBHER2 was amplified with primers gB5_XbaIf CCCCGTAGTTCTAGAGGCACGACGGGCCCCCGTAGTCCCGCCATGC and gBB_EcoRI_r ACAACAAACGAATTCGATGCACATGCGGTTTAACACCCGTGG, then digested with XbaI and EcoRI (New England Laboratories). The 3498 bp XbaI/EcoRI fragment was ligated into the XbaI/EcoRI MCS of pcDNA3.1(-). The gHHER2 was amplified with primers gH803_XbaI_f GGGACGGGGTCTAGAATGGGGAATGGTTTATGGTTCG and gH803_NotI_r CCGAAGCCAGCGGCCGCTTATTCGCGTCTCCAAAAAAACGGG, then digested with XbaI and NotI (New England Laboratories). The 3325 bp XbaI/NotI fragment was ligated into XbaI/NotI MCS of pcDNA3.1(-). gDΔ6–38 was amplified with primers gD_XbaI_f GTGGTGCGTTCTAGAATGGGGGGGGCTGCCGCCAGG and gD_NotI_r CCATTAAGGGCGGCCGCCTAGTAAAACAAGGGCTGGTGCG, then digested with XbaI and NotI (New England Laboratories). The 1093 bp XbaI/NotI fragment was ligated into XbaI/NotI MCS of pcDNA3.1(-). Inserts were verified by sequencing and by immunofluorescence of the encoded glycoproteins in transfected cells.gBThe luciferase-based cell-cell fusion assay was performed as described ,78. DonoA 160 mM stock solution of Bafilomycin A (BFLA) (Sigma Aldrich) was prepared in dimethyl sulfoxide, and further diluted in medium. Cells were exposed to BFLA for 1 h at 37°C and then infected with R-LM5, R-LM113, R-LM249, R-809 and R-909 (5 PFU/cell) for 90 min in the presence of BFLA. The viral inoculum was removed and the cells were overlaid with medium containing BFLA for 18 h. The extent of infection of R-809 and of the GFP-expressing viruses was determined by FACS, or by GloMax Discover System (Promega), respectively.Integrins were silenced in SK-OV-3 cells by means of ON-TARGET plus, smart polls (Dharmacon), as previously described . ControlJ cells transfected with indicated receptors were exposed for 30 min at 37°C to R-LM5, R-LM113, R-LM249, R-809, and R-909 at 10 PFU/cell, fixed with 4% (wt/vol) paraformaldehyde and incubated with 53S or 52S MAbs and secondary antibody. At the end of virus absorption, the culture medium was concentrated; devoid of serum IgGs by Protein A-Sepharose and subjected to SDS/PAGE. Immunoblot was performed by means of PAb to gH/gL, MAb H1817 to gB and MAb BD80 to gD.The receptor-negative J cells were exposed to R-LM5, R-LM113, R-LM249, R-809 and R-909 for 3 h at 37°C. Soluble form of HER2 (150 nM) and bovine serum albumin (150 nM), used as negative control, were added to cell-virions mixture for additional 3 h. As positive control, soluble nectin1 (150 nM) was added to J cells exposed to R-LM5. 24 h later pictures were taken by fluorescence microscopy and the number of fluorescent cells was determined by flow cytometry.


However, we observed that F proteins of the virulent strain F48E9 and avirulent strain LaSota substituted with an identical cleavage motif, “112RRQRR↓L117”, induced extensive and slight syncytium formation, respectively. Accordingly, we hypothesized that the difference in syncytium formation is caused by other regions of the fusion protein.Newcastle disease virus (NDV) causes severe diseases in avian species. Its fusion protein cleavage site (Fcs) is a major contributor to virulence and membrane fusion. Previous studies showed that a change from phenylalanine (F) to lysine (L) at position 117 of the virulent strain fusion protein, which has the polybasic amino acid Fcs motif “The exchanged regions between the fusion proteins of two strains, F48E9 and LaSota, showed that the region from amino acid 118–499 plays an important role in modulation of fusogenic activity in transfected cells. Further dissection of this region indicated that replacement of two amino acids  in heptad repeat 2 (HR2) of the avirulent fusion protein by the virulent counterpart resulted in fusion promotion. Moreover, the role of these two amino acids in fusion is dependent on the unique Fcs sequence “RRQRR↓L”.Our results demonstrated that two amino acids  of the virulent strain F protein with this unique Fcs were critical for promoting fusogenic activity, and residue F or L at position 117 did not affect syncytium formation. These findings provide novel insights into fusogenic triggering by the fusion protein and may be useful for designing antiviral peptides. Avulavirus, family Paramyxoviridae and order Mononegavirales. It possesses an enveloped, non-segmented, single-stranded, negative-sense RNA genome, which encode six major proteins—the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and large RNA-directed RNA polymerase protein (L) [0), which is cleaved into two disulfide-linked polypeptides, F1 and F2, by host proteases at the Fcs. This cleavage results in exposure of the fusion peptide at the N terminus of the F1 subunit, which triggers fusogenic activity.Newcastle disease is a highly contagious disease caused by Newcastle disease virus (NDV) that results in severe economic losses in the poultry industry. The NDV belongs to the genus tein (L) , 2. NDV tein (L) . The F ptein (L) . Importatein (L) –7. The F112R/K-R-Q-K/R-R↓F117”, at the C terminus of the F2 protein and a phenylalanine (F) at position 117, which is located at the N terminus of the F1 protein that is cleaved by ubiquitous intracellular proteases. In contrast, the lentogenic Fcs possesses the monobasic amino acid motif “112G/E-K/R-Q-G/E-R↓L117” at the C terminus of the F2 protein and a leucine (L) at position 117 that is cleaved by extracellular trypsin-like proteases restricted to the respiratory and intestinal tract [NDV strains are generally classified into three pathotypes based on their pathogenicity in chickens: lentogenic (low virulent or avirulent), mesogenic (moderately virulent) and velogenic (highly virulent) , 9. The al tract . Alteratal tract , 11–14. al tract –17. In oThe unique cleavage motif “RRQRR↓L” was found in a natural isolate (99–0868-2) in Australia. Although this isolate had an intracerebral pathogenicity index (ICPI) of 1.38, indicative of a virulent virus, inoculation of the virus into 7-week-old birds demonstrated an avirulent phenotype, suggesting that the motif “RRQRR↓L” is a deduced avirulent Fcs . In our 2 subunit (1–111 aa) of the ectodomain (1–499 aa), transmembrane domain  and cytoplasmic tail  of F48-F* with the corresponding regions of La-F* in the F48-F* backbone    Fig.  and b. T01) Fig.  and d. T01) Fig.  and d, s01) Fig.  and b. T1 subunit in the transfected cells, but there were no significant differences between them  of the F protein. The virulent Fcs usually possesses residue F at position 117 (F117), which is required for membrane fusion [117 or L117 at the F48-F* enables induction of fusogenic activity. Furthermore, the mutated F48-F with the Fcs “RRQKR↓L” could also induce syncytium formation (data not shown), indicating that the basic residue K or R at position 115 of Fcs has no effect on fusion. These findings may be due to the low amino acid identity (92%) of the F protein between strains F48E9 and BC compared to the strikingly high amino acid identity (98%) between strains LaSota and BC. This suggests that some amino acids in the F proteins of different strains affect the membrane fusion in addition to the Fcs.Generally, virulent F protein with a virulent Fcs can trigger the fusion activity, and those with an avirulent Fcs fail to induce membrane fusion. In our study, the Fcs “RRQRR↓L” is a deduced avirulent Fcs composed of four basic residues with residue L at position 117  did not affect membrane fusion compared to F48-F* in transfected BHK-21 cells. Furthermore, the mutants F48-F  and La-F  did not significantly change the fusogenic activity compared to wild-type F48-F and La-F (data not shown). The results demonstrated that the two mutations affecting fusogenic activity depended on the unique motif “RRQRR↓L”. In addition, although the single mutations La-F*(N479D) and La-F*(R486S) increased membrane fusion activity, their fusion index did not reach that induced by F48-F*. However, the fusion level caused by a substitution construct La-F*/F48(118–499) was similar to that of F48-F* (data not shown), suggesting that except for residues D479 and S486, other amino acids in the 118–499 region of F48-F* might act synergistically to achieve the high level of fusion induced by La-F*. A recent study reported that the mutation N403D in the F protein of mesogenic strain BC with the avirulent Fcs “GRQGR↓L” substantially reduced the fusion activity . Another


P = 0.009 and P = 0.046, respectively) and low SNAIL expression . High PD-L1 expression was associated with poor histopathological classification (P = 0.034), and low E-cadherin (P = 0.001), high N-cadherin (P = 0.044), high vimentin (P < 0.001) and high ZEB1 (P = 0.036) expression. Multivariate analysis showed that CD4+ TILs, PD-L1 expression and N-cadherin expression were independent prognostic factors (hazard ratio (HR) = 0.61; 95% confidence interval (CI) = 0.38–1.00; HR=4.27; 95% CI = 1.82–9.39; HR = 2.20; 95% CI = 1.18–3.92, respectively). These findings could help to identify potential biomarkers for predicting not only the prognosis, but also the therapeutic response to immunotherapy for eCCA.Extrahepatic cholangiocarcinoma (eCCA) has a poor prognosis. Although the possibility of immunotherapy has been studied, immune checkpoint molecules such as programmed death ligand 1 (PD-L1) in eCCA are not well understood. Epithelial-mesenchymal transition (EMT) has recently been shown to regulate PD-L1 expression. Our aims were to assess the clinicopathological significance of tumor-infiltrating lymphocytes (TILs) and tumor PD-L1 expression in eCCA and to compare these immune responses with EMT marker expression. In this retrospective study, we conducted immunohistochemical analyses for 117 patients with eCCA. We stained for CD4, CD8, Foxp3, and PD-L1 as markers reflecting local immune responses, and for E-cadherin, N-cadherin, vimentin, ZEB1, ZEB2, SNAIL, and TWIST as markers associated with EMT. High numbers of CD4+ and CD8+ TILs correlated with node-negative ( Extrahepatic cholangiocarcinoma (eCCA) arises from the epithelium of the main bile duct . The incRecent research has focused on the role which the immunological tumor microenvironment plays in the regulation of tumor growth. Tumor-infiltrating lymphocytes (TILs) such as CD4+ and CD8+ T lymphocytes play important roles in host immune response. The level of local infiltration of CD4+ and CD8+ T lymphocytes has been reported as a good prognostic factor in several cancers, including biliary tract cancer . HoweverPD-L1 expression on tumor cells can result in constitutive oncogenic signaling and responsiveness to the inflammatory signals produced by active tumor-infiltrating immune cells , 22. FurAlthough there have been two reports related to the prognostic significance of PD-L1 expression in eCCA, the significance of PD-L1 expression and its association with EMT remain unclear , 32 due The aim of this study was to clarify the prognostic impact of TILs and the expression of immune escape molecules in eCCA. Moreover, we determined the association of these immune responses with EMT-related marker expression in eCCA.Surgical specimens were obtained from 117 patients with a median age of 71 years . The median follow-up period was 27 months  and 98 patients (84%) died during follow-up. Of the patients who died, 31 patients (26%) died of hepatic recurrence, 27 patients (23%) of peritonitis carcinomatosa, 21 patients (18%) of local recurrence and 15 patients (13%) of lymph node metastasis. No patient received chemotherapy before surgery. Table P = 0.028) and negative for lymph node metastasis (P = 0.009). High infiltration of CD8+ T lymphocytes also correlated with negative results for lymph node metastasis (P = 0.046).Using immunohistochemical analysis, we counted the numbers of CD4+, CD8+, and Foxp3+ T lymphocytes both in the invasive front and tumor bulk Figure  . No sigWe immunohistochemically analyzed PD-L1 expression on tumor cells utilizing 2 types of monoclonal antibodies against PD-L1: SP142 and E1L3N Figure . When PDTo explore the best method for evaluating PD-L1 expression compared to methods previously used, we performed multivariate analysis of overall survival according to PD-L1 expression as determined using the 2 types of antibodies (SP142 or E1L3N) with 5 different cutoff values. To determine a cutoff value for the H-score of PD-L1, we used a receiver operating characteristic (ROC) curve. The H-score of PD-L1 as a continuous variable, and median survival as a binary variable, were subjected to ROC analysis. The other four cut-off values used were set based on the percentage of stained tumor cells: 1%, 5%, 10% or 50%. These analyses showed that evaluation using the H-score of SP142 reflected prognosis better than most of the other evaluations . TherefoP = 0.034). PD-L1 expression was not associated with the infiltration of CD4+, CD8+ or Foxp3+ T lymphocytes.The correlation between PD-L1 expression and clinicopathological factors including TILs is shown in Table P = 0.016, and P = 0.022, respectively). On the other hand, high infiltration of Foxp3+ T lymphocytes was correlated with high vimentin expression (P = 0.006). We also examined correlations between PD-L1 expression and EMT-related proteins (Table P = 0.001), high expression of N-cadherin (P = 0.044), high expression of vimentin (P < 0.001) and high expression of ZEB1 (P = 0.036).Representative staining patterns of EMT-related proteins are shown in Figure ns Table . High exP = 0.032), venous invasion (P = 0.024), T (P = 0.031), N (P = 0.001) and M (P = 0.001) classification, the infiltration of CD4+ lymphocytes (P = 0.009), and PD-L1 (P < 0.001), E-cadherin (P = 0.033), N-cadherin (P = 0.002) and vimentin (P = 0.024) expression as significant prognostic indicators. Multivariate analysis using Cox regression modeling showed that the infiltration of CD4+ T lymphocytes , the expression of PD-L1  and the expression of N-cadherin  were independent prognostic factors.Overall survival according to clinicopathological features including TILs, PD-L1 expression and EMT-related protein expression was examined Table . UnivariOver the past few years, the tumor microenvironment has been intensively investigated, with a focus on the tumor and the host immune response, particularly from the perspective of immune checkpoint molecules, including PD-L1, PD-1 and CTLA-4 , 33, 34.First, to examine the prognostic significance of PD-L1 expression in eCCA, we validated two different anti-PD-L1 antibodies. The use of the optimal antibody in IHC analyses, demonstrated that high PD-L1 expression in eCCA was an independent poor prognostic factor. The interaction of PD-L1 with its receptor PD-1 is an immune evasion mechanism that suppresses activation of the immune system . Thus, PRegarding the clinical significance of tumor PD-L1 expression and CD4+ T lymphocyte infiltration in eCCA, our results demonstrated, for the first time to the best of our knowledge, that high PD-L1 expression on the tumor and low tumor infiltration of CD4+ T lymphocytes were independent poor prognostic factors by multivariate analysis, although a correlation between these two factors was not identified. A previous study in eCCA also showed that tumor PD-L1 expression was not correlated significantly with the infiltration of CD8+ T lymphocytes . In termOn the other hand, in intrahepatic cholangiocarcinoma (iCCA), a retrospective study showed that the level of CD8+ TILs inversely correlated with PD-L1 expression, and it was hypothesized that this result was due to PD-L1 expression acting as a negative regulator of T lymphocytes . In termRegarding the underlying mechanism for the regulation of PD-L1 expression, at least two possibilities have been described. The first mechanism is the upregulation of PD-L1 expression by constitutive oncogenic signaling, such as that seen in Hodgkin lymphoma . The secWe also found that high tumor infiltration of CD4+ and CD8+ T lymphocytes significantly correlated with low tumor expression of SNAIL. This result is consistent with a previous report that SNAIL accelerates cancer metastasis not only through enhanced invasion, but also through induction of immunosuppression such as reduction of TILs by multiple mechanism including immunosuppressive cytokines, regulatory T cells, impaired dendritic cells and cytotoxic T lymphocyte resistance .A key limitation of this study was that the tissue microarray (TMA) method used may not represent the entire tumor specimen due to tissue heterogeneity. Our data showed that a higher number of CD4+ TILs in the invasive front was significantly associated with higher survival rate. These data were consistent with the previous literature on lung cancers and breast cancers, which has reported that high infiltration of CD4+ T cells into the stroma showed better prognosis than that into cancer cell nests , 51. WakIn conclusion, we found that CD4+ T lymphocyte tumor infiltration and PD-L1 expression on tumor cells were independent prognostic factors in eCCA. Furthermore, we provided the first evidence that high tumor PD-L1 expression correlates with a mesenchymal phenotype. Collectively, the present data together with previous findings suggest that the tumor microenvironment in eCCA consists of a complex balance between PD-L1 expression status, levels of TILs and EMT-related protein expression. These findings may help to identify potential biomarkers predictive of not only prognosis, but also therapeutic response in eCCA.n = 122) underwent surgical resection in the Department of Gastroenterological Surgery II at Hokkaido University Hospital between January 1995 and November 2006 and eCCA tumors were confirmed histopathologically. Five patients were excluded from analysis because insufficient tumor tissue was available for analysis. Ultimately, a total of 117 specimens were evaluated. We categorized eCCA into two groups, perihilar or distal, based on the predominance of the main tumor ..53].The numbers of CD4+, CD8+ and Foxp3+ T lymphocytes were calculated by counting the number of stained infiltrating cells in four high-powered fields in each of the invasive front and the tumor bulk . Semi-quA ROC curve was used to determine the cutoff values of continuous variables such as the numbers of CD4+, CD8+ or Foxp3+ T lymphocytes or the H-scores of EMT-related proteins or PD-L1 . The valP < 0.05 in univariate analysis were included in multivariate models. The threshold for significance was P < 0.05. All statistical analyses were conducted using the JMP for Windows version 12.0 software package .The correlation of each factor such as the tumor infiltration of CD4+, CD8+ or Fop3+ lymphocytes, PD-L1 expression, EMT-related protein expression and clinicopathological factors was analyzed using the Pearson Chi-squared method. Survival was estimated with the Kaplan-Meier method, and survival estimates were compared using the log-rank test. Overall survival was calculated from the date of surgery to the date of death from any cause or last contact/follow-up. Multivariate analysis was conducted using Cox proportional hazards regression modeling. Baseline variables with


Surface modification with oxide nanostructures is one of the efficient ways to improve physical or biomedical properties of shape memory alloys. This work reports a fabrication of highly ordered Ti-Ni-O nanotube arrays on Ti-Ni alloy substrates through pulse anodization in glycerol-based electrolytes. The effects of anodization parameters and the annealing process on the microstructures and surface morphology of Ti-Ni-O were studied using scanning electron microscope and Raman spectroscopy. The electrolyte type greatly affected the formation of nanotube arrays. A formation of anatase phase was found with the Ti-Ni-O nanotube arrays annealed at 450 °C. The oxide nanotubes could be crystallized to rutile phase after annealing treatment at 650 °C. The Ti-Ni-O nanotube arrays demonstrated an excellent thermal stability by keeping their nanotubular structures up to 650 °C. It can only absorb in the UV regime and thus significantly limits its widespread application [2 and thus broaden its application field, various kinds of decoration techniques have been carried out. Among the diverse approaches, doping of the oxide is one of the most efficient ways to obtain improved physical and chemical properties. For example, many metal elements  [etc.) [TiOications –4. Howevlication ,6. In orr, etc.) –9, and n) [etc.) –12, haveet al. [2 nanotube arrays through one-step anodization of alloy substrates. Mor et al. [et al. [et al. [One-dimensional nanostructured materials, especially nanotubes, offer great potential for various applications such as sensing, water photolysis, solar energy conversion and electrochemical energy storage . The anoet al.  reportedr et al.  fabricat [et al.  fabricat [et al.  also fabUnlike the anodization of other Ti alloys, anodization of Ti-Ni films in traditional electrolytes usually could not yield nanotube arrays because Ti-Ni-O could easily dissolve in aqueous solutions. To date, few works have been reported on anodic fabrication of Ti-Ni-O nanotube arrays on Ti-Ni shape memory alloy substrates. In this work, highly ordered Ti-Ni-O nanotube arrays were successfully grown on Ti-Ni alloy substrates through pulse anodization in glycerol-based electrolytes. The influence of anodization voltages on the microstructures and thermal stability of Ti-Ni-O nanotube arrays was investigated.2.2.1.4)2SO4 and 0.4 M NH4F. When the anodization voltage was 30 V, the surface of the Ti-Ni-O presented an irregular nanoporous structure. When the anodization voltage was increased to 40 V, the nanotubes with a small diameter formed. Some regions could not have complete nanotubes because of corrosion of the nanotubes. When the anodization voltage was 50 V, the nanotubes were completely corroded.4)2SO4 and 0.2 M NH4F. When the Ti-Ni substrate was anodized at 30 V, the top surface of the oxide layer presented uniform nanoporous structures. When the anodization voltage was increased to 40 V, highly ordered Ti-Ni-O nanotube arrays could form. As shown in In order to investigate the effect of fluoride concentration on the formation of nanotube arrays, non-aqueous electrolyte containing lower fluoride species was also evaluated. 2.2.4)2SO4 and 0.2 M NH4F and further annealed at different temperatures. In comparison with the anodic Ti-Ni-O nanotube arrays, a heat treatment at 500 °C in air did not change the surface morphology of the Ti-Ni-O nanotube arrays , 197 (Eg), 399 (B1g), 513 (A1g), 519 (B1g) and 639 (Eg) cm−1. Typical Raman peaks of rutile TiO2 were detected at 143 (B1g), 235(Eg), 447(Eg) and 612 (A1g) cm−1.Structural properties of the Ti-Ni-O nanofilms were analyzed using Raman spectroscopy. 2 because the Ni-doping had changed the vibrational modes of TiO2. For the as-annealed Ti-Ni-O nanotubes, the Raman peaks around 146, 244, 288, 406, 437 and 607 cm−1 could be detected  could be also found. This reveals that the anatase phase could gradually transform to rutile phase with increase of the heat-treatment temperature. The rutile modes became stronger while the anatase modes got weaker.−1 appeared distinctly. The Ti-Ni-O demonstrated nearly all of the Raman features of the rutile phase, which indicated that the anatase–rutile transformation was almost completely finished. As the heat-treatment temperature increased to 700 and 800 °C, all of the Raman peaks corresponding to rutile phase became stronger. For the nanotube samples annealed at 800 °C, the titanate peaks around 288 and 706 cm−1 were much stronger than those of the samples annealed at 500 °C [After annealing at 650 °C, a new peak around 244 cmt 500 °C –25.−1) in −1 decreased and the red-shift of frequency was remarkable. The change for the Raman mode may be attributed to the phase transition. In addition, the gradual relaxation of residual stress during the heat-treatment process could be another factor to cause a frequency red-shift of the Raman mode [From the red-shift of the anatase peak  of the rutile phase were observed when the heat-treatment temperature was gradually changed from 450 to 800 °C. With increase of the heat-treatment temperature the intensity of the Eg and A1g modes increased, accompanied by a frequency blue-shift and a decrease of the peak linewidth. As higher heat-treatment temperature could improve the crystalline structure and enhance the degree of crystallinity of the rutile phase, the peak linewidth decreased and the intensity of the Eg and A1g modes increased [g and A1g modes of the Ti-Ni-O should be related to a change in the nature of residual stress in the oxide layer [The Encreased . The blude layer ,28. In ade layer –31.To investigate the deformation capability of the Ti-Ni-O nanotubes grown on the shape memory alloy, the substrate with as-annealed nanotubes was bent about 80° for five cycles. The Ti-Ni-O nanotube arrays grown on the shape memory alloy are expected to find wide applications in both industrial and biomedical occasions. For a real application, mechanical stability of the nanotube arrays has to be considered since a final deformation (such as 5% or 8%) of the shape memory alloy (NiTi substrate) will usually occur during applications. Although the nanotublular oxide film could demonstrate unusual plastic deformation capability due to a nanoscale or nanoporous effect of nanoceramics, deformation behavior of the Ti-Ni-O nanotubes should be further investigated to address such a potential mechanical stability problem. Optimization of the oxide thickness, heat-treatment temperature as well as substrate type (plate or wire) should be realized to minimize a mechanical failure of the nanotube/NiTi alloy system without sacrificing the favorable shape memory effect.3.3.1.2 stream. Anodic samples were fabricated with different pulse voltages with a constant frequency of 4000 Hz and duty cycle of 50% for 180 min. We used two kinds of electrolytes, i.e., 5% ethylene glycol/glycerol containing 0.30 M (NH4)2SO4 and 0.4 M NH4F, and 5% ethylene glycol/glycerol containing 0.30 M (NH4)2SO4 and 0.2 M NH4F. The as-anodized Ti-Ni-O samples were annealed at different temperatures varying from 450 to 800 °C for 1 h to investigate phase transformation of the Ti-Ni-O.Prior to anodization, all of the Ti-Ni alloy (atomic ratio of Ti and Ni elements was 1:1) samples were ground and polished with 2000# SiC emery papers, and then ultrasonically cleaned with absolute alcohol. Finally, they were rinsed with deionized water and dried in a N3.2.Surface morphology and composition of the Ti-Ni-O were investigated using a scanning electron microscope  equipped with energy dispersive X-ray analysis . Phase structures of the as-annealed Ti-Ni-O samples were characterized with a Raman microscope system  using an argon ion laser operating at 532 nm.4.4)2SO4 and 0.2 M NH4F. The concentration of fluoride in the electrolyte was an important factor for the formation of Ti-Ni-O nanotube arrays. Lower fluoride concentration in the electrolyte was helpful in the formation of Ti-Ni-O nanotube arrays because Ti-Ni-O had a higher corrosion rate for high-concentration electrolyte. This indicated that Ni oxide could easily dissolve in the electrolyte solution. The Raman analysis showed that the Ti-Ni-O annealed at 450 °C, was anatase phase, and the oxide could transform to rutile phase after the annealing treatment at 650 °C. The Ti-Ni-O nanotube arrays had an excellent thermal stability by preserving their nanotubular structures up to 650 °C.In conclusion, highly ordered Ti-Ni-O nanotube arrays were fabricated through pulse anodization of the Ti-Ni shape memory alloy substrates, in a non-aqueous electrolyte of 5% ethylene glycol/glycerol containing 0.30 M (NH


We investigated the relationship between these two variants and the risk of PCOS, evaluated the genotypic effects on clinical, hormonal and metabolic indexes and plasma platelet-activating factor acetylhydrolase (PAF-AH) activity, and defined the association of apoC1 gene variants with apoE ε2/ε3/ε4 polymorphisms.Elevated serum levels of apolipoprotein (apo) C1 may be an early protein marker of metabolic abnormality in women with polycystic ovary syndrome (PCOS). It is not clear, however, whether there are any relationships between the apoC1 rs4420638A/G genotype was determined by a Taqman real-time PCR allelic discrimination assay. The apoC1–317H1/H2 and apoE ε2/ε3/ε4 genotypes were measured using PCR and restriction fragment length polymorphism analysis. The clinical, hormonal and metabolic parameters and PAF-AH activity were measured.This is a cross-sectional study of 877 women with PCOS and 761 controls. The apoC1 rs4420638A/G and -317H1/H2 genotypes and alleles were similar between PCOS and control groups (P > 0.05). However, the rs4420638 G allele was related to increased serum luteinizing hormone, cholesterol and apoB levels, and the ratio of apoB to apoA1 (P < 0.05), and the -317H1H1 genotype was associated with a higher acne grade score and a higher ratio of apoB-PAF-AH to H-PAF-AH activity (P < 0.05) in patients with PCOS. We also demonstrated that the apoC1 rs4420638A/G and -317H1/H2 gene variants existed in moderate to reasonably high linkage disequilibrium with apoE ε2/ε3/ε4 polymorphisms in Chinese women.The frequencies of apoC1 rs4420638A/G and -317H1/H2 gene variants might be involved in endocrine abnormalities of reproductive axis, metabolic abnormalities and chronic inflammation in PCOS, although no association was observed between the apoC1 genetic variants and the risk of PCOS in Chinese women.The  Polycystic ovary syndrome (PCOS) is a common heterogeneous female endocrine metabolic disorder affecting 4–18% of reproductive-aged women . In addiApolipoprotein (apo) C1 is a constituent of high-density lipoprotein (HDL) and triglyceride-rich lipoproteins and plays an important role in the regulation of lipoprotein metabolism and serum lipid levels. ApoC1 affects catabolism and clearance of apoB-containing lipoproteins by inhibiting lipoprotein lipase (LPL), liver-specific low density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein (LRP) –12. ThisapoE–apoC1–apoC4–apoC2 gene cluster is located on chromosome 19. Several genome-wide association studies have demonstrated that the rs4420638 A → G variant of the apoC1 gene, located in the non-coding region, is associated with increased serum total cholesterol (TC) and LDL-C levels  taking medication known to affect the metabolism of carbohydrates, lipids, or hormones within 3 months before the study; [ii] being pregnant or in the luteal phase; and [iii] smoking. Finally, 575 patients and 506 controls were included in the metabolism-related study. In addition, PAF-AH activity was measured in 281 patients and 277 controls of the metabolic analysis groups.Women with or without PCOS aged 17 to 40 years were recruited from 2006 to 2015 from the Outpatient Clinic of Reproductive Endocrinology at West China Second University Hospital. For the genetic association study of Each patient with PCOS met the revised 2003 Rotterdam ESHRE/ASRM consensus criteria . Oligo-oNone of the subjects had clinically evident acute or chronic diseases, such as infection, tumors, cardiovascular disease, thyroid dysfunction, endometriosis, hyperprolactinemia, hypogonadotropic hypogonadism or premature ovarian insufficiency.2), systolic and diastolic blood pressure (SBP and DBP), the degree of hirsutism and acne, and ultrasound ovarian volume [Clinical and anthropometrical parameters, including waist circumference, waist-to-hip ratio, body mass index  or mutant (G) DNA fragments of the apoC1 gene were used as positive controls. The primers, probes and positive controls were designed and synthesized by the Genecore Biotech Co. Ltd., Shanghai, China. For the -317H1/H2 genotype, the 221-bp  and 225-bp  fragments were amplified using the forward primer: 5’-TTTGAGCTCGGCTCTTGAGACAGGAA-3′ and the reverse primer 5’-GGTCCCGGGCACTTCCCTTAGCCCCA-3′ [-317H1/H2 PCR products were digested with HpaI (Thermo) and analyzed by electrophoresis on a 3.0% agarose gel and visualized by staining with Genecolour fluorescent dye. The enzyme digestion resulted in 159- and 66-bp fragments for the -317H2 allele and a non-digested 221-bp fragment for the -317H1 allele. ApoE ε2/ε3/ε4 polymorphisms (rs429358 and rs7412) were measured as described previously [Genomic DNA was isolated from peripheral blood leukocytes of the subjects , 28. ForCCCCA-3′ . The -31eviously . For qua2), TT, triglyceride (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), apoA1, apoB, plasma glucose and insulin concentrations, plasma PAF-AH, apoB-containing lipoprotein-associated PAF-AH (apoB-PAF-AH) and HDL-associated PAF-AH (H-PAF-AH) activity were measured as previously described [Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol  13.0 for Windows .Data were presented as the mean ± standard deviation (SD). Differences in variables were evaluated by an independent sample t-test between PCOS and control subjects. Variables with asymmetric distribution were evaluated by a Mann-Whitney U test. A chi-square analysis was used to determine allele or genotype frequencies and to test deviations in the genotype distribution from Hardy-Weinberg equilibrium between patients and controls. An analysis of covariance (ANCOVA) or a two way analysis of variance (ANOVA) was used to estimate the differences in clinical parameters, hormonal levels, metabolic profile and PAF-AH activity between the two groups or genotype subgroups after correction for differences in age and BMI. A http://analysis.bio-x.cn/myAnalysis.php.The analysis of linkage disequilibrium between different genetic polymorphic loci was performed by the SHEsis online software at Because mean age and BMI were different between the PCOS and the control groups Table , differeAs shown in Table TT and LH levels, the ratio of LH to FSH, fasting insulin concentration, HOMA index, TG, TC, LDL-C, non-HDL-C and apoB levels, AI, the ratio of apoB to apoA1, and the ratio of apoB-PAF-AH to H-PAF-AH were significantly increased, whereas FSH and HDL-C levels and H-PAF-AH activity were significantly reduced in the PCOS group compared with the control group Table .Table 2HapoC1 rs4420638A/G and -317H1/H2 were in Hardy-Weinberg equilibrium in the PCOS and control groups. No significant differences were observed in the frequencies of the apoC1 rs4420638A/G and -317H1/H2 genotypes and alleles between PCOS and control groups , apoC1–317H1/H2 loci and rs4420638A/G loci , and apoC1 rs4420638A/G loci and apoE ε2/ε3/ε4 loci .The degree of linkage disequilibrium (LD) among the rs4420638GG or -317H2H2 homozygotes were too small, we combined them into the heterozygous subgroups.Because the sample sizes of the AA homozygotes, G allele carriers (AG + GG) of the rs4420638 polymorphism had significantly higher apoB levels and a higher ratio of apoB to apoA1 in patients with PCOS or the controls (P < 0.05). Patients with the G allele had higher serum LH, TC, LDL-C and non-HDL-C levels (P < 0.05), and tended to have a reduced waist-to-hip ratio, DBP and fasting insulin levels (P < 0.100) and an increased ratio of LH to FSH (P = 0.074) compared with patients with the AA genotype. The controls with the G allele had higher fasting insulin and glucose concentrations, HOMA indexes, AI, plasma PAF-AH and apoB-PAF-AH activity (P < 0.05), and tended to have increased LDL-C and non-HDL-C levels (P < 0.080) and decreased HDL-C concentrations (P < 0.070) than the controls with the AA genotype.As shown in Table H1H1 genotype, patients with the H2 allele (H1H2 + H2H2) in -317H1/H2 polymorphism had lower acne grade score and the ratio of apoB-PAF-AH to H-PAF-AH (P < 0.05). The controls with the H2 allele had lower TC and LDL-C levels (P < 0.05), and tended to have increased waist-to-hip ratio and HOMA index (P < 0.09) and reduced non-HDL-C and apoB levels (P < 0.09) than the controls with the H1H1 genotype  were associated with apoC1 H2 alleles. Of the 319 apoE ε4 alleles, 303 (95%) were H2 alleles or rs4420638 G alleles and only 16 (5%) were H1 alleles or rs4420638 A alleles. In contrast, the apoE ε3 allele was strongly associated with the apoC1 H1 or rs4420638 A alleles. Of the 2684 apoε3 alleles, 2604 (97%) were H1 alleles and 2594 (96.6%) were rs4420638 A alleles; however, only 80 (3%) were H2 alleles and 90 (3.4%) were rs4420638 G alleles. Nevertheless, these differences were not statistically significant for patients with PCOS versus the controls  of the rs4420638 polymorphism had significantly higher LH, TC, LDL-C, non-HDL-C and apoB levels, and a higher ratio of apoB to apoA1, and tended to have an increased ratio of LH to FSH. This suggests that this polymorphism may potentially be linked to endocrine abnormalities of the reproductive axis and dyslipidemia in the patients. In addition, we found a higher acne grade score and an increased ratio of apoB-PAF-AH to H-PAF-AH activity in patients carrying the H1H1 genotype of the -317H1/H2 polymorphism compared with patients carrying the H2 allele. This suggests that the -317H1/H2 genetic polymorphism is associated with systemic chronic inflammation in these patients. Furthermore, we demonstrated that the rs4420638A/G or -317H1/H2 polymorphisms of the apoC1 gene exist in moderate to reasonably high linkage disequilibrium with apoE ε2/ε3/ε4 polymorphisms.In this study, we show that rs4420638A/G polymorphism in the apoC1 gene is associated with serum lipid levels, chronic inflammation and disease risk. The G allele of rs4420638 is associated with a higher level of TC, LDL-C, TG and PAF-AH activity, abdominal obesity, and increased risk of type 2 diabetes and coronary heart disease [G allele is associated with lower serum C-reactive protein (CRP) levels [G allele carriers were associated with not only increased serum LDL-C levels but also elevated serum non-HDL-C and apoB levels, and the ratio of apoB to apoA1 than the AA homozygotes in patients with PCOS or the controls. Because apoB and the apoB/apoA-I ratio have been reported to be better predictors of cardiovascular diseases than traditional lipid measurements [A → G variant of rs4420638 in the apoC1 gene is associated with cardiovascular disease risk. We also demonstrated that the controls with the G allele had higher fasting insulin and glucose concentrations, HOMA indexes, plasma PAF-AH and apoB-PAF-AH activity than the AA homozygotic controls. Additionally, patients carrying the G allele tended to have a reduced waist-to-hip ratio than the AA homozygotic patients. Consistent with our findings, Avery et al. demonstrated that the rs4420638A/G polymorphism of the apoC1 gene was associated with elevated plasma glucose, atherogenic dyslipidemia, vascular inflammation, and central obesity in individuals of European descent [A → G variant of rs4420638 was associated with increased plasma PAF-AH activity [apoC1 rs4420638 G allele exist in strong linkage disequilibrium with the apoE ε4 allele, which may partially explain the effect of the rs4420638 G allele on lipoprotein metabolism and disease risk. The apoE ε4 allele was associated not only with higher serum TC and LDL-C levels but also with higher oxidative stress, a more pro-inflammatory state and increased risk of cardiovascular disease and late-onset Alzheimer’s disease [G allele had higher LH levels and tended to have increased ratio of LH to FSH than in patients with the AA genotype, suggesting that the A → G variant of rs4420638 may potentially link to endocrine abnormalities of the reproductive axis in the patients. However, the effect of this gene variant on the reproductive axis must be investigated further.Several studies have found that the  disease –17. On t) levels . In thisurements , our res descent . Graller disease , 35. For-317H1/H2 polymorphism of the apoC1 gene, which is localized to the transcription initiation site in the promoter region, has been identified to exist in linkage disequilibrium with the ε2/ε3/ε4 polymorphisms of the apoE gene and influences lipoprotein metabolism [H2 allele of the apoC1 gene has been reported to cause a significant 1.5-fold increase in apoC1 gene transcription in a reporter-gene assay [H2 allele disrupts the binding of a transcription suppressor to produce a positive effect on transcription [H1H1 homozygotes, the H2 allele was associated with lower ratio of apoB-PAF-AH to H-PAF-AH and acne grade score in patients with PCOS. Furthermore, the H2 allele decreased or tended to decrease TC, LDL-C, non-HDL-C and apoB levels, but tended to increase waist-to-hip ratio and HOMA index in the controls. We also determined that the apoC1 H2 allele has complete linkage disequilibrium with the apoE ε2 allele, and to a lesser extent, the apoE ε4 allele in Chinese women. Consistent with our findings, Xu et al. reported that the H2 allele was associated with a decrease or tending decrease in apoB and LDL-C levels compared with H1H1 homozygotes in African-Americans with the apoE ε3/ε3 genotype but not the apoE ε3/ε4 genotype [apoE ε2 and apoE ε3 contexts; however, higher apoC1 levels may increase plasma lipid levels via decreased remnant clearance [apoC1–317H1/H2 polymorphism are different between patients and controls. Plasma PAF-AH specifically hydrolyzes and inactivates PAF and PAF-like oxidized phospholipids that are potent pro-inflammatory mediators [H1 allele might relate to increased systemic chronic inflammation in patients with PCOS.The tabolism . The H2 ne assay . The H2 cription . This alcription  and famicription . In thisgenotype . Similarlearance . Expresslearance . This maediators . Given tediators , 36, 37.A → G variant and the CGTT insertion (H2) allele of apoC1 appeared to have a greater impact on metabolic profile in the controls relative to in patients with PCOS (Tables apoC1 genetic variations on metabolic profile may be disturbed or weakened by these factors in patients. Further research is needed to clarify this issue.In addition, our study also showed that the S Tables  and 5. Isertion H allele oS Tables  and 5, iapoC1 and apoE genetic variants differ among ethnic groups, and these variants exhibit an ethnically distinct linkage disequilibrium pattern [apoC1 H2 allele combined with apoE ε2, ε3 and ε4 is 0.95, 0.02 and 0.85, respectively, in European-Americans; 1.00, 0.08 and 0.55, respectively, in African-Americans [apoC1 and apoE genetic polymorphisms and lipoprotein levels or diseases may differ among ethnic groups.In particular, the allele frequencies for  pattern , 20, 30.mericans ; and 1.0rs4420638GG and -317H2H2, we could not analyze them in the form of subgroups. A larger sample size of patients and controls are needed to properly evaluate dose-dependent genotype characteristics. Second, due to insufficient sampling, we did not measure plasma and lipoprotein-associated PAF-AH activity in some subjects, which might influence the statistical power of these parameters. Third, we could not determine plasma apoC1 concentrations due to plasma or serum sample inadequacy. Further study to detect apoC1 levels may help provide clues to the mechanisms responsible for the genetic association with PCOS.We should point out that this study has some limitations. Firstly, given the low frequency of homozygosity of minor alleles apoC1 rs4420638A/G and -317H1/H2 genetic polymorphisms are not associated with the risk of PCOS in Chinese women. However, the apoC1 rs4420638A → G variation may potentially be linked to elevated serum LH levels, a relatively high ratio of LH to FSH, and an adverse lipid metabolic profile; and the -317 H1H1 genotype is associated with increased the ratio of apoB-PAF-AH to H-PAF-AH activity and the acne grade score in the patients with PCOS. Additionally, this study also indicates that apoC1 rs4420638A/G and -317H1/H2 genetic variations are associated with an adverse glucose and lipid metabolic profile and the rs4420638G allele increases plasma PAF-AH and apoB-PAF-AH activity in the control women. These results suggest that apoC1 rs4420638A/G and -317H1/H2 genetic variants are related to metabolic abnormalities and chronic inflammation in patients and controls, while the rs4420638A/G polymorphism might be involved in endocrine abnormalities of reproductive axis in patients.The present study demonstrates that