The mean migration rates of cancer cells transfected with pRNAT-shRNA-GPC5-1 were reduced compared with the controls in A549 (P<0.001) and H3255 (P=0.001), while the migration rate of SPC-A1 with GPC5 overexpression was higher than that of the control (P=0.001). The downregulation of GPC5 impeded the transmigration of A549 and H3255 while the upregulation of GPC5 expression promoted the transmembrane invasion of SPC-A1. Furthermore, a panel of formalin-fixed paraffin-embedded NSCLC tissues from 127 patients undergoing curative resection  between January, 2003 and December, 2008 were obtained in order to investigate the correlation between GPC5 expression and clinicopathological factors using immunohistochemical methods. The results demonstrated that high GPC5 expression levels in NSCLC were associated with respiratory symptoms in lung cancer diagnosis, poor differentiation, vascular invasion, regional lymph node metastasis and a higher TNM stage. Using the Kaplan-Meier method, NSCLC patients with high levels of GPC5 expression demonstrated a significantly shorter overall survival time compared with those with low GPC5 expression levels . GPC5 expression was also identified as an independent prognostic factor by Cox regression analysis . This study suggested that increased levels of GPC5 expression are a poor prognostic marker for NSCLC.Although the correlation between glypican-5 (GPC5) and lung cancer is well known, the effect of GPC5 expression on non-small cell lung cancer (NSCLC) survival remains to be determined. In the present study, GPC5 expression in A549, H3255, and SPC-A1 NSCLC cell lines was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. GPC5 mRNA and protein expression levels were found to be higher in A549 and H3255 cells compared with SPC-A1 cells. The role of GPC5 in NSCLC cell migration was evaluated  Lung cancer is one of the leading causes of cancer mortality worldwide . Non-smaGlypican-5 (GPC5) is one of the six members of the glypican family that are bound to the external surface of the plasma membrane by glycosyl-phosphatidylinositol (GPI) linkage . The GPCThe aim of the present study was to investigate the expression pattern of GPC5 in NSCLC cell lines and tumor tissue samples. The effects of GPC5 expression on cancer cell migration were assessed. The correlation of GPC5 expression levels with NSCLC survival was also examined.2. Coding sequences of human GPC5 was cloned into pEGFP-N1 and designated as pEGFP/GPC5. GPC5 targeted three shRNAs, these were predicted to be 5′-GATCCGTTCGGAAACTTTTCCAGTTCAAGAGACTGGAAAAGTTTCCGAACTTTTTTTTCAA-3′, 5′-GATCCTTTGTAAACAGATTTTTTGTCAAGAGCAAAAAATCTGTTTACAAATTTTTTTTCAA-3′ and 5′-GATCCAAAGTTATACTCAGCGTGTTCAAGAGACACGCTGAGTATAACTTTTTTTTTTTCAA-3′. All three shRNAs targeting GPC5 were constructed into pRNAT-U6.1/Neo, as pRNAT-shRNA-GPC5-1,2,3. The cell lines A549, H3255 and SPC-A1 were transfected with pEGFP/GPC5, pEGFP-N1, pRNAT-shRNA-GPC5-1,2,3 or pRNAT-U6.1 using TurboFect  according to the manufacturer’s instructions. GPC5 expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis.The NSCLC cell lines A549, H3255 and SPC-A1 were cultured in RPMI-1640  supplemented with 10% fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in an atmosphere of 5% COTotal RNA was extracted using TRIzol reagent  and reverse transcribed into cDNA using a PrimeScript RT reagent kit  according to the manufacturer’s instructions. PCR was performed using the Mastercycler Gradient . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified as the endogenous control. The primer sequences used were: GPC5: forward, 5′-CCCTCGAGGGAGGATGGACGCACAGACC-3′ and reverse, 5′-CGGGATCCCGCCAGGCATATGCAGA GAGAGAG-3′; GAPDH: forward, 5′-CAATGACCCC TTCATTGACC-3′ and reverse, 5′-TGGAAGATGGTGAT GGGATT-3′.http://rsbweb.nih.gov/ij/).Protein lysates were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane . Subsequently, the membrane was incubated with rabbit monoclonal antibody against human GPC5  followed by horseradish peroxidase-labeled goat anti-rabbit IgG  and detected by chemiluminescence. GAPDH was used as a protein loading control. The intensity of protein fragments was quantified with ImageJ software /the distance prior to healing × 100. A Transwell  assay was used to further analyze cell migration according to the manufacturer’s instructions. Fifty thousand cells were placed in the upper chambers in serum-free media, and the lower chambers were filled with RPMI-1640 + 10% FBS. Following incubation for 24 h at 37°C, non-migrating cells on the top surface of the membrane were removed with a cotton swab. The membranes were fixed with methanol for 10 min and stained with 0.5% crystal violet for 5 min. The number of cells that migrated to the bottom of the filter were counted manually under an inverted microscope.Cells were seeded in 12-well plates and cultured overnight to form a confluent monolayer. After being scratched with a sterile pipette tip, the cells were rinsed gently with PBS to remove the detached cells and subsequently incubated with medium containing 1% FBS at 37°C in an atmosphere of 5% COA panel of formalin-fixed paraffin-embedded (FFPE) NSCLC tissues from patients undergoing curative resection between January, 2003 and December, 2008 were obtained from the The Affiliated Drum Tower Hospital of Nanjing University Medical School . Full medical record abstraction was performed in order to obtain the following patient variables: age, gender, smoking status, respiratory symptoms upon lung cancer diagnosis, cell type, tumor differentiation, vascular invasion, regional lymph node metastasis, pTNM stage, adjuvant treatment and other medical conditions. Smokers were defined as those having smoked at least 100 cigarettes in their lifetime. pTNM staging designations were made according to the postsurgical pathological staging system according to the 7th edition of the TNM classification of malignant tumors . Non-specific binding was blocked with 10% normal goat serum (Abcam) for 30 min at room temperature. The slides were subsequently incubated with a rabbit polyclonal antibody to GPC5  for a further 30 min at room temperature and incubated overnight at 4°C. The slides were then washed in Tris-buffered saline and incubated sequentially with anti-rabbit IgG . Color was developed by 15 min incubation with 3,3′-diaminobenzidine. Samples were counterstained with hematoxylin, followed by dehydration and mounting. Negative controls were included by replacing the primary antibody with PBS. Normal human brain tissue was detected as a positive control. The quality of IHC was confirmed using H&E staining. The evaluation of immunostaining of these samples was performed by two trained pathologists (J Yang and FQ Meng) who were unaware of the clinical background of the samples. The intensity and percentage of positive cells were considered. Five visual fields were randomly observed, and 100 cells in each field were counted . Positive cells from the 100 tumor cells in each field were counted. Tumor cells with a brown cell membrane were considered to be positive and were scored as: 3+, strong; 2+, moderate; 1+, weak; and 0, no staining. The average percentages of positively stained tumor cells were classified as: 0 for 0%; 1 for 1–33%; 2 for 34–66%; and 3 for 67–100%. The intensity and percentage scores were multiplied to yield a composite score of 1–9 for each sample. Composite scores of 1–3 were defined as indicating a low GPC5 expression, while scores of 4–9 were considered to indicate a high GPC5 expression.in vitro studies were expressed as the means ± SD and statistical significance was assessed by the Student’s t-test. Survival was assessed up to December 31, 2011. Overall survival (OS) was calculated as the period from the date of surgery for NSCLC until death. Patients who were alive at the last contact were censored. Associations between clinicopathological variables and GPC5 protein expression were examined using Pearson’s χ2 test for categorical variables (or Fisher’s exact test if any sample number was <5), and the Student’s t-test for continuous variables. Univariate Cox proportional hazard models were used to evaluate the prognostic impact on survival of all factors of interest. Factors were included in multivariate models if P<0.05 in the univariate analysis. Kaplan-Meier analysis was performed for survival curves and statistical significance was assessed using the log-rank test. All analyses were performed with SPSS software, version 16.0 . All tests were two-sided and performed at a significance level of 0.05.Data from GPC5 expression in three NSCLC cell lines  were analyzed. GPC5 was highly expressed in A549 and H3255 cells, compared with SPC-A1 cells . FollowiFollowing transfection with pRNAT-shRNA-GPC5-1, A549 and H3255 cells migrated at a markedly slower rate than parental cells transfected with control vectors . The meaTypical immunohistochemical staining patterns observed for GPC5 protein are shown in The median OS time was 14.0 months (95% CI: 9.0–19.0) in patients with high levels of GPC5 expression and 59.0 months (95% CI: 30.2–87.8) in those with low levels of GPC5 expression, which demonstrated a significant difference P=0.001, . For theIn the present study, we identified GPC5 expression in NSCLC tumor cells and these expression levels may have affected tumor cell migration. To the best of our knowledge, this is the first study to investigate the role of GPC5 on NSCLC survival. High expression levels of GPC5 were identified as negative prognostic markers in patients with resected NSCLC.The 8-exon GPC5 gene is located at chromosome 13q31.3, encoding a 572-amino acid product. The GPC5 protein belongs to the glypican gene family (GPC1-GPC6), the members of which have been reported to be overexpressed in several human malignancies . The funin vivo and in vitro(et al(These findings reveal the crucial role of GPC5 in NSCLC metastasis and survival. However, the mechanism responsible for GPC5-mediated effects on tumor metastasis remain to be clarified. Studies have highlighted potentially significant roles for the Wnt signaling pathway in the development of lung cancer in addition to being involved in mammalian limb development ,15. The  in vitro. As the  in vitro. A studytro(et al revealedA recent GWAS suggested that genetic variants at 13q31.3 modulate GPC5 expression that had been downregulated in the adenocarcinomas in patients who had never smoked. The GPC5 expression levels were reduced in the mammary tumors of breast cancer patients . AssociaIn conclusion, our findings suggest that the evaluation of GPC5 expression may be useful clinically in recognizing patients who are more likely to have a poor NSCLC outcome. Specifically, higher levels of GPC5 expression suggest a tendency for a shorter survival time. Furthermore, GPC5 may be involved in NSCLC metastasis through enhancing cancer cell migration.


The study investigated the accuracy of two-view full-field digital (2D) mammograms (FFDM) by comparing its performance with two-view digital breast tomosynthesis (3D) plus FFDM combination in a symptomatic setting.k values) was calculated and the area under curve (AUC) was compared between 2D imaging and 2D+3D imaging, regarding both readers and cases as random events.A multi-case multi-reader study was conducted involving four imagers of varying experience. A total of 109 lesions from 103 patients who attended symptomatic breast clinics during a 7-month period between March 2012 and September 2012 were retrospectively read. All patients who had subtle signs on 2D images were retrospectively double read in a free response study and the findings recorded. The performance quality of the methods was evaluated using receiver operating characteristic (ROC) curves. Reader agreement (k = 0.391) compared with 2D images alone (k = 0.153). In total, 85.19% of M3 mammograms on 2D imaging were changed to 11.01% M3 and 77.06% M2 on combination of FFDM and DBT. There was more correlation between the mammographic scores and the final result for combination mode compared with 2D images alone (P = 0.0001). ROC analysis revealed the AUC for the combination (0.898) was significantly greater than 2D mammograms alone .There was more inter-reader agreement between the two readers with 2D+3D combination (DBT increases diagnostic sensitivity in a symptomatic setting and reduces the number of M3 mammograms, when used as an adjuvant to 2D images. Thus DBT has the potential of increasing work-flow efficiency in a symptomatic setting by reducing benign biopsies.


A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 µM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 µM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants.Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008–2009. The NA of such variant contained additional NA changes  that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants  and characterized them  in vitro and in vivo. By contrast, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008–2009. This discrepancy could be attributed to permissive NA mutations  that were identified in most Bris07-like oseltamivir-resistant variants. To verify this hypothesis, we developed a reverse genetics system for a sensitive Bris07-like isolate (275H) whose NA protein contains the 3 permissive mutations . Using mutagenesis, we first introduced the H275Y then reverted codons at positions 222, 234 and 344. The resulting 5 recombinants  were compared with regard to NA enzyme properties, replicative capacities in vitro as well as infectivity and contact-transmissibility in ferrets. Among the studied permissive mutations, Q222R was associated with a significant reduction of both affinity and activity of the NA enzyme resulting in a virus with a reduced replicative capacity in vitro and decreased replication in lungs of ferrets. Thus, the R222Q mutation may have been the major permissive NA change that facilitated the emergence and spread of NAI-resistant Bris07 variants.The H275Y neuraminidase (NA) mutation conferring resistance to oseltamivir was shown to impair old influenza H1N1 strains both  Influenza viruses are respiratory pathogens associated with significant public health consequences. Each year, influenza epidemics can be responsible for significant morbidity in the general population and excess mortality in elderly patients and individuals with chronic underlying conditions. Influenza A viruses of the H1N1 subtype have been associated with seasonal influenza epidemics for many decades and, in presence of immunological pressure, such viruses continue to evolve through genetic variability which is mainly confined to virus segments encoding surface glycoproteins i.e., the hemagglutinin (HA) and neuraminidase (NA) The H275Y (H274Y in N2 numbering) NA mutation conferring resistance to oseltamivir and peramivir has been detected with increasing frequency in seasonal A/H1N1 viruses since 2007 to the extent that almost all characterized A/Brisbane/59/2007-like (Bris07) (H1N1) influenza strains that circulated worldwide during the 2008–09 season were H275Y variants To further investigate which secondary NA mutations may have facilitated the introduction of the H275Y mutation in contemporarily seasonal H1N1 viruses and allowed their dissemination, we developed a reverse genetics system using a clinical Bris07 (H1N1) isolate as genetic background and evaluated the impact of the H275Y oseltamivir resistance mutation as well as several potential compensatory NA mutations on enzyme activity, viral fitness and transmissibility.Vmax values), which reflects the total NA activity per virion, and Km values, which reflect the affinity for the substrate. As shown in Km of 40.31 vs 11.95 µM, P<0.001) and relative NA activity  compared to the WT . The H275Y/N344D mutant showed a significantly reduced affinity  with no change in NA activity compared to the WT. When comparing the double mutants to the single H275Y mutant, the Km values were significantly increased for the H275Y/Q222R and H275Y/N344D mutants (P<0.001) whereas only the double H275Y/Q222R mutant had a significantly lower relative NA activity (P<0.001).In the present study, five recombinant Bris07 influenza viruses were generated i.e., the WT virus (containing the putative permissive mutations) that briefly circulated during the 2007–08 season, the single H275Y oseltamivir-resistant variant and three double mutants containing the H275Y mutation as well as reversion of potential permissive mutations . NA enzymatic properties using equivalent titers of recombinants were first analyzed with determination of relative NA enzymatic activity (o the WT . The H27P<0.01), 9.72% (P<0.001), 32.07% (P<0.001) and 54.89% (P<0.01) for the H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D mutant proteins, respectively. When compared to the single H275Y mutant, H275Y/Q222R (P<0.001), H275Y/M234V (P<0.001) and H275Y/N344D (P<0.05) double mutants also had significantly reduced surface NA activities. The differences observed in total surface NA activity between the different recombinant NA proteins may be due to a decreased number of NA molecules that reached the cell surface or to less activity per enzyme.Using recombinant NA proteins expressed in 293T cells, we further investigated the impact of NA mutations on the amount of NA activity at the cell surface. As shown in 50 values) and peramivir (mean fold increase of 998) with no impact on zanamivir susceptibility . A similar trend was observed for oseltamivir (IC50 of 651.86 nM vs 1024.54 nM) although, in this case, the difference between IC50 values was not statistically significant.We next determined the phenotype of resistance to NAIs for the 5 recombinant viruses. As expected, the presence of the H275Y mutation was associated with resistance to oseltamivir  whereas the remaining recombinants generated plaques of comparable sizes (P<0.001). In replication kinetics experiments, the peak viral titers for all recombinants were obtained at 36 h post-infection (PI) with viral titers ranging from 5.6×106 PFU/ml (H275Y/Q222R) to 5.3×107 PFU/ml (WT) (P<0.001) and 48 h (P<0.05) PI compared to the WT (P<0.001). When compared to the single (H275Y) mutant, viral titers of the double H275Y/Q222R and H275Y/M234V mutants were significantly lower at 36 h (P<0.001).Viral fitness of recombinant A/Brisbane/59/2007-like viruses was assessed le sizes . Of note/ml (WT) . The WT,o the WT . There w5±2.9×104 PFU/ml for the WT and 2.6×105±8.7×104 PFU/ml for the H275Y mutant) whereas the H275Y/Q222R mutant had a reduced mean viral titer . Similarly, mean viral titers in nasal wash samples of ferrets infected with the H275Y/Q222R were significantly lower than those of the H275Y mutant (P<0.05) and WT virus (P<0.01) on day 4 PI . On the other hand, the three recombinants were associated with comparable mean viral titers on day 6 PI .Intranasal inoculation of ferrets with the WT and two mutant (H275Y and H275Y/Q222R) Bris07 recombinant viruses resulted in a febrile response that peaked on day 2 PI . The are2±1.2×102 vs 1.2×104±3.5×103 PFU/ml, P<0.01) and day 6 PI .All contact ferrets seroconverted for A/Brisbane/59/2007 when tested 14 days after contact, with geometrical mean hemagglutination inhibition (HAI) titers of 160±33, 145±119 and 95±55 for the WT, H275Y and H275Y/Q222R recombinant viruses, respectively. A febrile response could be observed on days 4 and 5 in the WT and the H275Y groups, respectively, but not in the H275Y/Q222R group . The AUCin vitro and ferret experiments that the R222Q NA mutation was the main but possibly not the only permissive mutation that allowed the widespread dissemination of the oseltamivir-resistant H275Y mutant during the 2007–09 influenza seasons. Although such mutant seems to have disappeared since the emergence of the pandemic H1N1 virus in April 2009, understanding the mechanisms leading to the transmission of this unique virus is of great importance and could have an impact on the future use of NAIs.In this study, we used recombinant viruses derived from a clinical WT Bris07 strain to demonstrate using both The influenza NA protein plays a major role during the viral replication cycle. Its sialidase activity promotes virion release by removing sialic residues from viral glycoproteins and infected cells In this study, we focused on a.a. changes that occurred in the NA protein during the evolution of recent seasonal influenza H1N1 viruses and that may have been involved in the development and dissemination of resistance to NAIs. These changes included the well-known framework H275Y mutation, responsible for the resistance phenotype to oseltamivir and peramivir, as well as other substitutions  that may have contributed to the emergence and dissemination of resistance by acting as permissive/compensatory mutations.Vmax) and affinity (Km) Km values) in both 275H and 275Y strains Km values using our Bris07 recombinants  ferrets may not completely capture the fitness of Bris07 in humans with pre-existing immunity. Alternatively, the Q222R mutation could affect airborne transmission which has not been evaluated in our study. Of note, possibly due to the lower affinity of Q222R for MUNANA, less NAIs were required for competitive inhibition of the H275Y/Q222R mutant compared to the H275Y mutant. Residue 222 is located in the vicinity of the catalytic site of the N1 enzyme based on 3-D structure analysis Phylogenetic analyses previously demonstrated that the V234M mutation was already present in oseltamivir-susceptible A/Solomon Islands/3/2006 (SI06) viruses mbinants . Besidesmbinants  and totambinants , in linein vitro. Nevertheless, a possible synergy between these mutations and Q222R cannot be completely excluded.In addition to the R222Q mutation, a permissive role was also suggested for V234M and D344N substitutions in vitro experiments. In addition, these recombinants had comparable infectivity and contact transmissibility in ferrets. Thus, the presence of the permissive mutations  in the NA protein of our WT strain was apparently not sufficient to alter the viral fitness to the level that a compensatory change, such as the H275Y mutation, would be necessary. Therefore, we believe that changes in the NA gene alone may not provide a complete explanation for the emergence and spread of the oseltamivir-resistant H275Y Bris07 variant. Other changes in the genome might have been involved in this event. For instance, Yang and colleagues recently demonstrated that the dominant H275Y variant that emerged in Taiwan in 2007–2008 was a result of intra-subtypic reassortments between HA, NA, PB2 and PA genes from one clade (clade 2B) and the remaining 4 genes from another one (clade 1) Our study revealed that the H275Y NA mutation was not deleterious to fitness in the Bris07 genetic context in contrast to older H1N1 strains. However, this mutant did not have a replicative advantage compared to the WT as suggested by epidemiological studies. Indeed, the recombinant WT virus and its H275Y variant demonstrated similar replication kinetics during in vitro characterization, assessment of viral fitness and contact transmission in ferrets as well as NA enzyme properties of recombinant mutants. In particular, our investigation clearly demonstrated the positive impact of one specific NA substitution (i.e. R222Q) in conjunction with the oseltamivir resistance H275Y mutation on enzymatic properties and viral fitness of the Bris07 H1N1 strain. Noteworthy, our results suggest that total NA activity was more likely predictive of in vitro and in vivo viral fitness than the enzyme affinity (Km) parameter. Whether the Q222R mutation is also deleterious in the absence of H275Y was not investigated here; however, in a previous work, influenza A/Paris/497/2007 (222Q/275H) and A/Solomon Islands/3/2006 (222R/275H) seasonal H1N1 isolates grew to comparable titers in in vitro kinetics experiments Despite the fact that the secondary mutations described here were not investigated individually but in conjunction with H275Y, our study provides a comprehensive analysis of relevant permissive NA mutations in the contemporarily seasonal H1N1 background. This included All procedures were approved by the Institutional Animal Care Committee at Laval University according to the guidelines of the Canadian Council on Animal Care.275Y mutant plasmid was then used for reverting potential compensatory mutations  as described above. All recombinant plasmids were sequenced to confirm the absence of undesired mutations. The eight bidirectional plasmids were cotransfected into 293T human embryonic kidney cells using the LipofectamineTM 2000 reagent  as previously described Reverse transcription-PCR using universal influenza primers 6 plaque forming-units (PFU)/ml and incubated at 37°C in 50-µl reactions with different concentrations of MUNANA. The final concentration of the substrate ranged from 0 to 3000 µM. Fluorescence was monitored every 90 s for 53 min (35 measures). The Michaelis-Menten constant (mK) and the relative NA activity (maxV) were calculated with the Prism software , by fitting the data to the Michaelis-Menten equation using nonlinear regression A fluorometric based assay using MUNANA (Methylumbelliferyl-N-acetylneuraminic acid)  as substrate was performed to determine total NA enzymatic activity per infectious virus Recombinant NA plasmids and pCAGGS-PA, -PB1, -PB2 and -NP plasmids were used to co-transfect 293T cells in order to express recombinant NA enzymes 50) was determined from the dose-response curve.The drug resistance phenotype was determined by NA inhibition assays using the MUNANA substrate as previously described Replicative capacities of the recombinant viruses were evaluated by infecting ST6Gal1-MDCK cells with a multiplicity of infection (MOI) of 0.001 plaque-forming units (PFUs)/cell. Supernatants were collected every 12 h until 60 h PI and titrated by plaque assays. The mean viral plaque area of recombinant viruses was determined from a minimum of 16 plaques obtained after 60 h of incubation under agarose overlay using the ImageJ software (version 1.41), developed by Wayne Rasband of the National Institutes of Health as previously described 5 PFUs of the recombinant Bris07-like WT, H275Y or H275Y/Q222R variants. Temperature of ferrets was measured by rectal thermometers every day until day 10 PI. Ferrets were weighed daily and nasal wash samples were collected from animals on days 2, 4 and 6 PI. Virus titers from nasal wash samples were determined by plaque assays using ST6Gal1-MDCK cells. Serum samples were collected from each ferret before intranasal infection and on day 14 PI to evaluate specific antibody levels against the seasonal Bris07 strain using standard HAI assays. To evaluate contact-transmissibility, inoculated-contact animal pairs were established by placing a naïve ferret into each cage 24 h after inoculation of the index ferret Groups of 4 seronegative (900–1500 g) male ferrets  were lightly anesthetised by isoflurane and received an intranasal instillation of 1.25×10mK and maxV values), NAI IC50 values and viral titers in vitro and in nasal washes of ferrets were compared by one-way ANOVA analysis of variance, with the Tukey's multiple comparison post test. The amount of NA activity on the cell surface and plaque sizes of the recombinants were compared to those of the WT virus and/or the H275Y mutant by the use of unpaired two-tailed t tests.NA kinetic parameters (


MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17∼92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17∼92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17∼92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment.Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include  ALK and NF1Neuroblastoma is an embryonal tumor arising from sympathetic neuronal progenitor cells and is responsible for 15% of all pediatric cancer deaths. This tumor exhibits a remarkably diverse clinical behavior ranging from spontaneous regression to aggressive disease, often refractory to multi-modal therapies miR-17∼92 cluster as an important mediator of MYCN signaling in neuroblastoma with several components of this cluster down regulating expression of target genes such as DKK3 and members of the TGFβ pathway that contribute to the MYCN driven tumor phenotype miR-17∼92 polycistron in a neuroblastoma cell line. Further analyses of a large series of neuroblastoma tumors and cell lines (n = 223) revealed enrichment for known MYCN up regulated genes in focal gains and amplicons. Moreover, by applying an integrated genomics approach including mRNA and miRNA gene expression data and subsequent validation using MYCN and miR-17∼92 inducible cellular model systems, we identified several genes, within the regions of focal loss, that are putatively under the control of MYCN activated miRNAs. Of these, RGS5 and RNF11 were identified as two new MYCN regulated therapeutically relevant genes, which are implicated in vascular remodeling We recently identified the miR-17∼92 locus (13q31.3) in the MYCN amplified cell line NLF  losses in a large series of primary neuroblastoma tumor samples and cell lines.High resolution array CGH analysis revealed, amongst others, a 5 kb focal gain of the line NLF . FISH anll lines , in keepfication . TherefoKIF1B (1p36.22), CDKN2A/CDKN2B (9p21.3) and NF1 (17q11.2) as well as previously described focal heterozygous losses harboring genes such as APAF1 (12q23), ELAVL4 (1p34) and PTPRD (9p23-p24.3), while several amplifications were observed targeting well established oncogenes such as MYCN (2p24.1), ALK (2p23) and MDM2 (12q14.3-q15) CCND1, CDKN1C, CDK4), “cellular apoptosis”  and “protein kinase” (including e.g. INSR and NF1). Given the implication of genes involved in activated tyrosine kinase pathways in neuroblastoma, NF1 , INSR and ITK  were selected for mutation analysis in a panel of neuroblastoma cell lines. Loss-of-function mutations in the NF1 gene were detected in 23.5% of tested cell lines  MYCN amplified samples )<1.0E-4, p(FE (2B vs 2A))<1.0E-4). Focal (homozygous) deletions are also observed more frequently in MYCN amplified samples, however this difference is not significant with subtype 2A ) = 3.6E-2; p(FE (2B vs 2A)) = 0.13) ((FE (2B vs 1))<1.0E-4; p((FE (2B vs 2A)) = 1.9E-3).A set of 33 neuroblastoma cell lines and 190 primary neuroblastoma tumors, representative of all clinical and genetic subtypes, was analyzed for focal DNA copy number aberrations . In totafor INSR . Further = 0.13) . When comiR-17∼92, several known bona fide direct MYCN targets were affected by either gains or high level amplifications. A first example is the TERT gene TERT copies per haploid genome equivalent. Increased TERT expression could be confirmed in one amplified sample for which expression data were available, as compared to the tumors without TERT amplification (data not shown). Other examples of direct MYCN targets located within amplified regions in both our data set and previously reported array CGH data sets include CDK4 (in one MYCN amplified and one MYCN non-amplified tumor and two MYCN amplified cell lines) and MDM2 (in two MYCN amplified tumors and two MYCN amplified cell lines) MYCN and harbor direct MYCN target genes  MYCN as a separate amplicon (in 4/43 MYCN amplified tumors (9.3%) and 2/24 MYCN amplified cell lines (8.3%)) contains the bona fide oncogene ODC1 (2p25.1), which was recently described as a critical determinant of MYCN oncogenesis Further analysis showed that in addition to http://medgen.ugent.be/MYCNot). A separate analysis was performed (1) for the up regulated target genes with a validated direct interaction with MYCN (referred to as directly up regulated MYCN target genes) and (2) for all up regulated genes in the MYCNot database, meaning all indirect MYCN targets, putative direct MYCN targets  as well as the above mentioned validated direct MYCN target genes.In a next step, we tested the regions of focal gain and amplification for enrichment of up regulated MYCN target genes, as available in the MYCNot database (TERT) and 12q13.2-q14.1 (CDK4) are also gained or amplified in subtype 2B tumors .Investigation of focal gains and amplicons in our sample panel revealed significant enrichment for directly up regulated MYCN target genes in high risk MYCN non-amplified (subtype 2A) and MYCN amplified (subtype 2B) tumors as well as in the cell lines. When performing this analysis for all up regulated MYCN target genes listed in the MYCNot database, significant enrichment could be seen for all tumor subtypes and cell lines . FurtherDKK3 and ESR1 as well as several TGFβ pathway components were down regulated as a result of increased levels of miRNAs encoded by the miR-17∼92 cluster in neuroblastoma Using the MYCNot database, no enrichment of MYCN down regulated target genes could be observed in the focal deletions. This was not unexpected, since so far only a limited number of down regulated MYCN target genes have been carefully assessed and validated. Recent studies revealed that MYCN-induced miRNA activation acts as an important mechanism for indirect down regulation of gene expression following MYCN overexpression MYCN status and expression levels of MYCN activated miRNAs .First, we investigated a cohort of 101 neuroblastoma samples for RGS5 and RNF11, were also down regulated after MYCN induction. Down regulation was confirmed for both genes by qPCR , which is in line with the observed association of both genes with MYCN expression. RNF11 was recently shown to be regulated by the miR-19b component of the miR-17∼92 cluster In a next step, we sought further evidence for MYCN driven regulation of these candidate genes using the SHEP-TET21-N-MYC inducible model system  by qPCR . SurvivaRGS5 is homozygously deleted on chromosome 1q23 in cell line NLF. Using ultra-high density (1M) oligo array CGH breakpoints of this 27 kb deletion were further delineated and complete loss of RGS5 expression was confirmed by qPCR (RGS5 3′UTR and significant negative correlation between RGS5 expression and seven of these miRNAs (RGS5 expression. We selected four of the seven MYCN driven miRNAs (p<5.0E-2): miR-9, miR-20a, miR-92a and miR-181a and tested binding to the 3′UTR of RGS5. To this purpose, HEK-293T cells were co-transfected with a 3′UTR luciferase reporter plasmid, containing the full length 5074 bp RGS5 3′UTR, and the selected pre-miRs. This showed significant decrease in luciferase activity for miR-20a, miR-92a and miR-181a indicating a direct interaction between these miRNAs and the 3′UTR of RGS5 . by qPCR . In a nee miRNAs , we eval of RGS5 . No evid of RGS5 . A rescuIn addition to posttranscriptional down regulation, we also investigated the possibility that RGS5 might be targeted by loss-of-function mutations but sequencing of a panel of cell lines revealed no mutations, in keeping with low mutation rate in neuroblastoma miR-17∼92, an important oncogenic locus under the direct transcriptional control of MYCN. In this cell line, MYCN is amplified but with moderate increased copy number levels. In keeping with this observation, MYCN mRNA expression is clearly elevated but within the lower range of the cohort of MYCN amplified cell lines. We therefore hypothesize that miR-17∼92 copy number gain may have been selected to reinforce the MYCN-driven oncogenic phenotype in this cell line as this miRNA cluster is a well-established direct oncogenic target of MYCN and MYC and is consistently up regulated in MYCN/MYC amplified neuroblastoma as well as in other tumor entities bona fide up regulated MYCN target genes such as ODC1, TERT, CDK4 and MDM2  whereas a higher frequency of large segmental aberrations was observed in subtype 2A tumors in keeping with previously published data bona fide oncogenes such as ODC1  CDK4  MDM2  ALK  CDKN2A  NF1  PTPRD  APAF1  IKIP  WWOX  FAF1 (n = 4) MYCN mRNA, was amplified in rare neuroblastoma cases and more recently transgenic mice overexpressing LIN28B in the developing sympathetic nervous tissue were shown to develop neuroblastoma The present study investigates gene content of focal gains, amplicons and deletions in neuroblastoma. Interestingly, these focal aberrations occur more frequently in http://www.ncbi.nlm.nih.gov/geo/ accession number: GSE32664).A series of 190 primary neuroblastoma tumor samples was collected prior to therapy  for which a written informed consent was obtained and approved by the commision for medical ethics of the respective institutions . In addition, 33 neuroblastoma cell lines were included in this study. Patient and cell line information (including cell line references) are summarized in http://medgen.ugent.be/arraycghbase) <−2.0), (b) amplification >2.0), (c) focal recurrent loss and (d) focal recurrent gain. We defined focal losses and gains for (c) and (d) as alterations <2 Mb involving at least 2 non-overlapping contiguously altered oligos <−0.3 and >1.0, respectively). Since we aimed to find genes important in neuroblastoma biology we looked for recurrent alterations defined by their occurrence in at least 2 samples. Chromosomal regions that are homozygously deleted or amplified (>8 haploid copies) are typically small and very likely to contain tumor suppressor genes and oncogenes, respectively. Therefore, all (and not only recurrent) homozygously deleted regions or amplicons were taken into account for further analysis. Throughout the text we will refer to (a) and (c) as focal copy number losses and to (b) and (d) as focal copy number gains. The Y chromosome was excluded from the analysis. Copy number variants characteristic for control DNAs were excluded. Aberrations that were present in more than 5 cases in a separate in-house screened clinical genetics cohort of 1000 samples were considered as possible copy number variants and excluded from further analysis (http://medgen.ugent.be/arraycghbase). All reported results were confirmed by manual inspection of the array profiles. All homozygous deletions, amplifications and focal recurrent losses and gains that met the aforementioned criteria are provided in http://medgen.ugent.be/arraycghbase .Samples were profiled on a custom designed 44K (156 samples), 60K (66 samples) or 180K (1 sample) array (Agilent Technologies) enriched for critical regions in neuroblastoma , cancer gene census genes http://medgen.ugent.be/MYCNot). Most of the genes listed in the database have been identified as putative MYCN target genes through one or more differential expression screens without any other evidence suggesting that they are directly regulated by MYCN alone or by both MYCN and other transcription factors that work in a co-ordinated manner. Currently, 1041 up and down regulated MYCN mRNA targets are included in this database.To summarize the current knowledge on MYCN downstream cascades, a thorough literature search was performed to track down all hitherto identified MYCN downstream genes. We grouped all the information and assembled and annotated the individual genes into the ‘MYCN oncogene targets’ (MYCNot) database (BMCA (ID 14) and SDC4 (ID 15). mRNA expression levels were measured and normalized to at least 2 internal reference genes, TBP (ID 8098), YWHAZ (ID 8100), B2M (ID 2) and UBC (ID 8099). Primer sequences are available in RTPrimerDB (http://www.rtprimerdb.org) hsa-miR26a-2 (ID 8243), hsa-miR-766 (ID 8250), RGS5 (ID 8244), TCF4 (ID 8245), ITK (ID 8246), INSR (ID 8247), TERT (ID 8248), SEPT6 (ID 8249) CDKN2A (ID 8251), NF1 (ID 8252), DIAPH2 (ID 8254), IGSF6 (ID 8255), THRAP3 (ID 8257), A2BP1 (ID 8258), MYCN (ID 11), RGS5 (ID 8261-expression) and RNF11 (ID 8592-expression). Copy number and mRNA data analysis as well as error propagation was done using qbasePLUS software 1.5 (http://www.biogazelle.com) Real-Time qPCR was performed for validation of DNA CNAs and for expression evaluation. DNA and RNA concentration was measured using the Nanodrop (Thermo Scientific). First-strand cDNA was synthesized from total RNA using an iScript cDNA synthesis kit (Bio-Rad). qPCR reactions were performed with SYBR Green I detection chemistry, using the LC480 real-time PCR detection system (Roche). qPCR reactions were performed in a total volume of 5 µl consisting of 2.5 µl of SYBR Green I qPCR master mix (2×) , 0.25 µl of forward and reverse primer (5 µM), 2 µl of 2.5 ng/µl DNA or cDNA . Cycling conditions were as follows: 10 minutes at 95°C followed by 45 cycles of 10 seconds at 95°C, 45 seconds at 60°C and 1 second at 72°C. DNA copy number levels were measured relative to human genomic DNA (Roche Diagnostics) and normalized to the internal reference genes, 17∼92 expression level were determined for 27 neuroblastoma cell lines for which gene expression profiling was performed using Sureprint G3 human gene expression microarrays (Agilent Technologies) according to the manufacturer's instructions as well as miRNA profiling using a stemloop RT-qPCR platform as described previously SHEP-TET21N-MYC cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal calf serum and treated with 1 µg/ml tetracycline (Sigma-Aldrich) to switch off MYCN expression  test. For the prioritization of genes in focal losses, selection of the candidate genes was performed by combining data of spearman correlation analysis between miRNA and mRNA expression data and paired t-test. Statistical significance was defined as p<5.0E-2.RGS5 and the tyrosine kinase domain of ITK was performed on genomic DNA. NF1 . Electropherograms were analyzed using Seqscape v2.5 software (Applied Biosystems). Primer info is summarized in Sequence analysis of the coding exons and flanking intronic sequences of RGS5 gene was cloned in a vector downstream of the firefly luciferase open reading frame . Seed regions were mutated using the QuickChange II mutagenesis kit (Stratagene). Specifically, the complementary sites of miR-20a (2), miR-92a and miR-181a, CACTTTTTGTTTCTTTCAGGTAGACCCACACATACACACACA and CACTTTTCACCAAATAGGTTCCTGTTTGTGTTAAACA, TGCAATATCCAAATGTTTAAAAATTCTATTCACATTATT and TGAATGTGGGTTAAGTAATAGGACTATGATATCAA were changed into AAAGGTTTGTTTCTTTCAGGTAGACCCACACATACACACACA and AAAGGTTCACCAAATAGGTTCCTGTTTGTGTTAAACA, GGACCTATCCAAATGTTTAAAAATTCTATTCACATTATT and GGCCGGTGGGTTAAGTAATAGGACTATGATATCAA, respectively. HEK-293T cells were plated at 10,000 cells per 96-well in RPMI medium containing 10% FBS 24 hrs prior to transfection. Transfection mixes contained RPMI medium, 50 nM pre-miR (Applied Biosystems), 0.4% DharmaFect Duo (Dharmacon) and 100 ng oligo-psiCHECK vector. The ratios between Firefly and Renilla luciferase were measured 48 hr post-transfection using the dual-glo luciferase kit (Promega) according to the manufacturer's protocol.The 3′UTR of the human Figure S1MYCN copy number and gene expression for neuroblastoma cell lines.MYCN copy number data (log2) and MYCN expression data were plotted for a panel of MYCN amplified and MYCN non-amplified cell lines. MYCN expression levels were significantly higher in the MYCN amplified cell lines <0.001). A linear relation was observed between MYCN copy number and expression within the group of MYCN amplified cell lines (p<0.005).(TIF)Click here for additional data file.Figure S2Distribution of focal aberrations. A. Distribution of recurrent focal gains and amplifications and B. recurrent focal losses and homozygous deletions in the neuroblastoma tumors according to subtype. Amplification of the MYCN locus was excluded for this analysis as MYCN amplification is characteristic of subgroup 2B. ***, p(FE)<0.0001; *, p(FE)<0.05(TIF)Click here for additional data file.Figure S3Confirmation of RGS5 and RNF11 down regulation. A. Confirmation of RGS5 and RNF11 down regulation using qPCR after induction of MYCN in the SHEP-TET21-N-MYC and B. miR-17∼92 in the SHEP-TR-miR-17∼92 inducible model systems.(TIF)Click here for additional data file.Figure S4Confirmation of RGS5 homozygous deletion in NLF. A. Confirmation of RGS5 homozygous deletion by 1M high-resolution array CGH. B. RGS5 mRNA expression by qPCR in NLF versus average expression in NB cell lines.(TIF)Click here for additional data file.Table S1Overview of patient and cell line information.(XLSX)Click here for additional data file.Table S2Overview of focal aberrations in tumors and cell lines.(XLSX)Click here for additional data file.Table S3Detail of enrichment analyses.(XLSX)Click here for additional data file.Table S4Correlation of MYCN regulated miRNAs to RGS5 and RNF11 expression and miRNA seed information.(XLSX)Click here for additional data file.Table S5Fold change data from the tetracycline treated and untreated SHEP-TR-miR-17∼92 inducible model using Affymetrix GeneChip Human Gene 1.0 ST arrays .(XLSX)Click here for additional data file.Table S6MYCN amplification status and expression and miR-17∼92 expression in a panel of neuroblastoma cell lines.(XLSX)Click here for additional data file.Table S7List of sequencing primers.(XLSX)Click here for additional data file.


It has been shown that Sema3A competitively antagonizes VEGF165 signaling. This study investigated whether Sema3A is expressed in synovial tissues, and is associated with disease activity and the histological features of synovial tissues from RA patients.Rheumatoid arthritis (RA) is an autoimmune disease of which the pathogenetic mechanisms are not fully understood. Semaphorin3A (Sema3A) has an immune regulatory role. Neuropilin1 (NRP1), the primary receptor for Sema3A, is also a receptor for vascular endothelial growth factor 165 .Human synovial tissues samples were obtained from RA and osteoarthritis (OA) patients. Disease activity of RA patients was calculated using the 28-joint Disease Activity Score based on C-reactive protein (DAS28-CRP). The histological features of RA synovial tissues were evaluated using Rooney’s inflammation scoring system. The localization of Sema3A, VEGF165 and NRP1 proteins were expressed in the synovial lining and inflammatory cells beneath the lining. Immunohistochemistry revealed the protein expression of Sema3A in synovial lining cells was decreased in RA tissues compared with OA samples. qPCR analysis demonstrated a significant reduction of Sema3A mRNA levels in RA synovial tissue samples than in OA and a significant correlation of the ratio of Sema3A/VEGF-A mRNA expression levels with DAS28-CRP . Sema3A mRNA levels also correlated with Rooney’s inflammation score, especially in perivascular infiltrates of lymphocytes , focal aggregates of lymphocytes  and diffuse infiltrates of lymphocytes .In OA specimens, Sema3A, VEGFReduction of Sema3A expression in RA synovial tissues may contribute to pathogenesis of RA. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by progressive joint destruction that accompanies the proliferation of synovial cells and blood vessels as well as invasion of inflammatory cells -3. AlthoSemaphorins are a large family of proteins that function as guidance cues for axonal/dendritic projections. Class 3 semaphorins are vertebrate secreted proteins and include seven members, semaphorin3A (Sema3A) to Semaphorin3G (Sema3G) . Sema3A The action of Sema3A is not limited to the nervous system as NRP1 is expressed on endothelial cells, keratinocytes, T cells, and tumor cells in breast and prostate cancer. Sema3A inhibits angiogenesis, migration of keratinocytes, proliferation of T cells, and migration of tumor cells -11. In aNRP1 mediates signal transduction through PlexinA co-receptors , which a165), a spliced isoform of VEGF-A [165 [165 is attributed to Sema3A expression. Indeed, the imbalance between Sema3A and VEGF expression levels is also associated with disease activity in several tumors [Vascular endothelial growth factor165  samples. The Sema3A expression level was also significantly associated with the RA pathological score and disease activity score.These facts prompted us to investigate the possibility that Sema3A expression and/or the balance of Sema3A and VEGFSynovial tissue samples were obtained from RA n = 30) and OA (n = 23) patients during total knee arthroplasty. The diagnosis of patients with RA and OA was based on the revised 1987 American Rheumatism Association Criteria for RA [ and OA  were stained with HE. The histological features of RA synovial tissues were evaluated using Rooney’s inflammation scoring system . These h165, CD3 and CD20, the tissue sections were subjected to antigen retrieval by autoclaving in 10 mM citrate buffer (pH 6.0) for 15 min at 121°C. Slides were then treated with 0.3% H2O2 for 30 min to block endogenous peroxidases. Sections for Sema3A, NRP1 and VEGF165 staining were blocked with 10% normal goat serum. All sections were incubated at 4°C overnight with rabbit anti-Sema3A polyclonal antibody , mouse anti-CD3 monoclonal antibody , mouse anti-CD20 monoclonal antibody (DAKO), rabbit anti-NRP1 polyclonal antibody  or rabbit anti-VEGF165 polyclonal antibody . This was followed by incubation with Envision™, Rabbit/HRP (DAKO) for Sema3A, NRP1 and VEGF165 or Envision™/HRP (DAKO) for CD3 and CD20. Immunoreactivity was visualized using 3,3’-diaminobenzidine plus  for Sema3A, NRP1 and VEGF165, or 3,3’-diaminobenzidine tetrahydrochloride  for CD3 and CD20. Finally, the sections were counterstained with hematoxylin and mounted. Controls for Sema3A immunohistochemistry included preabsorption and co-incubation of the antibody with the antigen peptide .For immunostaining of Sema3A, antigen retrieval was performed by incubating at 95°C for 30 min in DAKO Target Retrieval Solution . For immunostaining of NRP1, VEGFTissue sections immunostained with anti-Sema3A antibody were analyzed for 12 patients with OA and 12 patients with RA. The total immunostaining intensity in the lining layer was measured using a BZ-9000 microscope  equipped with Dynamic Cell count software BZ-H1C (Keyence). Immunostaining intensity per unit was calculated as described previously .Sema3A , VEGF-A , NRP1  and β-actin , 5 μl Taqman Fast Advanced Master Mix 2X, 2 μl cDNA template and 2.5 μl RNase-free water. The default ABI 7900HT amplification conditions were 20 sec at 95°C, followed by 1 sec at 95°C and 20 sec at 60°C for 40 cycles. A standard curve, derived from known serial dilutions of RA synovial tissue, was constructed to calculate arbitrary values of mRNA levels and to correct for differences in primer efficiencies. The obtained data were standardized using the reference gene, β-actin.Total RNA was extracted from synovial tissues using an Illustra RNA spin Mini Kit  according to the manufacturer’s instructions. RNA was reversed transcribed into cDNA using a PrimeScript RT Reagent Kit . The cDNA synthesized from 1 μg of total RNA was used as the template in each reaction. qPCR analysis was performed using an Applied Biosystems 7900HT Fast Real-time PCR system (Applied Biosystems LLC) based on the TaqMan® PCR manufacturer’s protocol. The assay was performed in triplicate in optical 96-well reaction plates covered with optical adhesive cover in a volume of 10 μl containing 0.5 μl Taqman Gene Expression Assay 20X for human Synovial tissue specimens embedded in Optimum Cutting Temperature compound  were sectioned (5 μm thick). The sections were then fixed in cold acetone for 5 min at 4°C and rinsed in phosphate buffered saline (PBS). To eliminate nonspecific protein binding, the samples were incubated with 10% normal goat serum for 30 min at room temperature. The samples were incubated with rabbit anti-NRP1 polyclonal antibody  and mouse anti-CD20 monoclonal antibody (DAKO) overnight at 4°C. This was followed by incubation with Alexa 488-labeled goat anti-rabbit antibody  and Alexa 594-labeled goat anti-mouse antibody (Applied Biosystems LLC) for 40 min at 37°C. Finally, the sections were mounted with aqueous mounting medium. The distributions were analyzed by confocal microscopy using a Zeiss LSM510 confocal laser microscope .U-test and Spearman’s rank correlation coefficient were used to test the differences. A p value < 0.05 was considered significant. We calculated a posterior power of this study using G*Power . All statistical powers in this study were greater than 80%.Statistical analyses were performed using SPSS 11.0 for Windows . The Mann–Whitney 165 and NRP1 expression in serial synovial serial sections from RA and OA patients. HE staining demonstrated that synovial tissues from OA patients contained two layers, the lining and sublining layers  were sporadically localized in the sublining layer of OA specimens, the number of T cells was greater in RA synovial tissues  than in OA  and B cells (CD20). NRP1 was abundantly expressed in the inflammatory cells of lymphoid follicles  and OA patients (n = 23), whereas Catalano used relatively small numbers of RA (n = 10), OA (n = 10) patients and healthy controls (n = 5). For qPCR, we used the standard curve method for relative quantification, whereas Catalano used a comparative Ct method.Catalano reported synovial tissues derived from healthy controls, OA and RA patients exhibited no significant differences although the relative Sema3A expression was lowest in RA samples . This diet al. recently suggested that Sema3A can modulate the autoimmune properties of B cells in SLE [et al. also reported Sema3A serum levels were significantly lower in SLE patients (p < 0.0001) and RA patients (p = 0.047) compared with healthy controls [Sequential immunohistochemical staining revealed that B cells were observed mainly in the lymphoid follicles, consistent with a previous report . Here wes in SLE . Thus, Scontrols . Since Scontrols ,42, loweet al. reported that the presence of calcium and histamine could modulate Sema3A expression levels in human keratinocytes and fibroblasts [It is unclear which factors regulate Sema3A expression in synovial tissues. However, Fukamachi roblasts . Since hroblasts ,45, it mVEGF-A was not significantly altered in OA and RA synovial tissues. Hashimoto et al. reported staining intensity for VEGF expression did not differ between RA and OA synovial lining [et al. reported that VEGF165 expression did not differ in the chronically inflamed tissue of RA patients and OA patients [et al. observed significantly higher levels of VEGF protein in RA compared with OA synovial fluid and serum [et al. observed significant correlations of serum VEGF levels with DAS28-CRP scores [VEGF-A expression levels and DAS28-CRP scores  but not in OA samples (8 patients) using reverse transcription-PCR [NRP1 was up-regulated in RA synovial tissues. However, we did not observe significant alterations of VEGF-A , we cannot rule out the possibility of altered Sema3A expression by anti-rheumatoid drugs, because each group sample size is small in this study.Several limitations of our study should be addressed. First, this study population may be influenced by environmental factors as well as genetic factors. Second, patients enrolled in our study were in the advanced stages of RA disease (mean duration 16.3 years). Thus, our data do not reflect the pathogenesis of RA during the early stages. Third, anti-rheumatoid drugs may alter the expression level of Sema3A and other molecules. In this study 12 patients (40%) received methotrexate (MTX) (mean prednisolone daily dose 2.9 mg), 4 patients (13.3%) received TNF-α blockade , 11 patients received Disease Modifying Antirheumatic Drugs (DMARDs) without MTX (mean prednisolone daily dose 1.6 mg), and 3 patients received prednisolone alone (daily dose 6.2 mg). Although there was no statistically significant difference in This study demonstrated that Sema3A expression levels and the disease activity score in patients with RA negatively correlated with histological parameters in RA synovial tissues. This suggests that Sema3A may have a suppressive effect on the pathological severity of RA. Further elucidation of the role of Sema3A in joints may elucidate the biological and pathological functions of Sema3A and allow the development of new strategies for the treatment of RA.165: Vascular endothelial growth factor 165.DAS28-CRP: 28-joint disease activity score based on c-reactive protein; HE: Hematoxylin and eosin; IL: Interleukin; NRP1: Neuropilin1; NRP2: Neuropilin2; OA: osteoarthritis; qPCR: Quantitative real-time polymerase chain reaction; RA: Rheumatoid arthritis; Sema3A: Semaphorin3A; Sema3C: Semaphorin3C; Sema3F: Semaphorin3F; Sema3G: Semaphorin3G; SD: Standard deviation; SLE: Systemic lupus erythematosus; TNF-α: Tumor necrosis factor-α; VEGFThe authors declare that they have no competing interests. This work was supported by supported by Grants-in-Aid for Scientific Research nos. 30170517 (to T.S) and nos. 23590429 (to Y.N) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and by Grant nos. 07085023 (to Y.G.) from the National Project on Protein Structural and Functional Analyses of MEXT.TS participated in the design of the study, performed the experiments and statistical analysis, drafted and revised the manuscript. NF designed the study, analyzed data, drafted and revised the manuscript critically. KK helped with collection and acquisition of the data and the synovium and revised the manuscript critically. NY assisted in scoring histological parameters and revised the manuscript critically. GY designed the study and revised the manuscript critically. ST helped with collection of the clinical materials and revised the manuscript critically. All authors read and approved the final manuscript for publication.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/40/prepubExpression of VEGF165 mRNA in synovial tissues: preliminary study. Expression levels of VEGF165 mRNA were measured by real-time PCR. The mRNA levels were normalized to the expression of β-actin. The box plots demonstrate the 10th and 90th percentile (whiskers), the 25th and 75th percentile, and the median. VEGF165 mRNA levels were not significantly altered in RA (n = 25) synovial tissues compared with OA (n = 17) (A). VEGF165 expression levels did not significantly correlate with DAS28-CRP. (B)Click here for file


Hepatocellular carcinoma is one of the most common malignant neoplasms in the world and is the main cause of death in patients with liver cirrhosis. Surgical intervention is not suitable for majority of hepatocellular carcinoma. Investigation of the effective targeting to the tumor cells is essential for both primary tumors and metastases. Tumor specific cytotoxic T lymphocytes (CTL) have been considered to be the attractive vehicles for delivering therapeutic agents toward various tumor diseases. This study was to explore the distribution pattern of CTL carrying the lentiviral vectors with the characteristic of adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand promoter (Lenti/hCD40L/E1AB). Following transduction with adenoviral vectors containing chimeric type 5 and type 35 fiber proteins (Ad5/35-TRAIL), these CTLs produced infectious virus when exposed to HepG2 cells. We assessed the therapeutic ability of CTLs using MTT, Western blot and colony formation assay. The novel CTL harboring Lenti/hCD40L/E1AB and Ad5/35-TRAIL caused proliferation inhibition and significant apoptosis in hepatocellular carcinoma cell lines. Thus, the novel CTL may be useful for the development of gene therapy approaches to hepatocellular carcinoma. Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countriesTumor antigen–specific cytotoxic T lymphocytes (CTLs) have been shown in a number of preclinical and clinical models to be highly effective at infiltrating tumor sites in a multiplicity of organs + T cells for less than 24 hours + dendritic cell and B lymphocytes. The CD40L promoter is tightly regulated by the AT hook transcription factor AKNA, which is expressed only transiently following antigen-mediated T-cell activation CD40 and CD40 ligand (CD40L) are members of the TNF family, and their interaction provides a potent signal for DC activation. CD40L expression is tightly regulated, being transiently expressed on the surface of activated CD4In the development of gene therapy, Ad5 vectors transduce predominantly hepatocytes after intravenous injection, and because tumor cells do not often express the Ad5 receptor , these vectors are unsuitable for tumor targeting therapy. Because commonly used species C Ad serotype 5-based vectors do not efficiently transduce HCC, the chimeric Ad5 vectors carry fibers from species B Ad serotype 35 (Ad5/35) In this study, we constructed a novel CTL harboring LV-CD40Lpr and Ad5/35-TRAIL which caused proliferation inhibition and significant apoptosis in hepatocellular carcinoma cell lines. We wonder if this kind of CTL could be applied in the HCC treatment.The study protocol and consent forms conform to the Declaration of Helsinki and were approved by the Ethical Review Board (ERB) Committee  and informed consent was obtained from all subjects. All the participants provided their written informed consent to participate in this study under the inspection of ERB committee.2.The human embryonic kidney cell line (HEK293), HEK293T, human hepatocellular carcinoma cell line (HepG2) were obtained from the American Type Culture Collection , and cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% FBS and 100 IU/ml penicillin/streptomycin in a 37°C humidified incubator with 5% COAAGCTTAAGAAAGCAGGTGCTAACTATATAG-3′5′- CCC and downstream: GGATCCGCTGTGTTAAAGTTGAAATGGTATC-3′5′- CG) and subcloned into the lentiviral shuttle vector pLenti, named pLenti/hCD40L. The E1 gene  was polymerase chain reaction amplified and subcloned into the downstream of pLenti/hCD40L vector, pLenti/hCD40L/E1AB. Lentiviral particles were produced by performing transient co-transfection involving a three-plasmid expression system in HEK293T cells according to user's manual, and concentrated through ultracentrifugation.The CD40L promoter was cloned from human genome using the specific primers , precoated with RetroNectin  and anti-CD3 antibody  at 1×106 cells per well, and incubated for 48 hours for optimal activation before transduction. The stimulated CTL lines were resuspended at 1×106 cells/mL in complete medium supplemented with IL-2 (300 U/ml), INF-γ (400 U/ml), phytohaemagg lutinin (10 U/ml), rhIL-4 (100 U/ml), GM-CSF (10 U/ml) and rhIL-2 (100 IU/mL), and then incubated for 36 hours at 37°C and 5% CO2. CD3-FITC, CD4-PE, CD8-PE, CD56-PE, CD226-FITC, CD11-PE and CD305-FITC monoclonal antibodies (MAbs) with the appropriate fluorescein isothiocyanate (FITC)– or R-phycoerythrin (PE)–conjugated were purchased from BD Bioscience . Cells were washed and stained with the appropriate antibodies for 20 minutes at 4°C in the dark in phosphate-buffered saline (PBS) supplemented with 0.1% bovine serum albumin (BSA). Control cells were unstained. After incubation, the cells were washed twice and resuspended in PBS.Lymphocytes was isolated from human blood using the human lymphocyte separation medium, then transferred to a 25 cm6 cells/mL in complete medium, and then incubated with different volumes  of freshly generated Lenti/hCD40L/E1AB for 36 hours at 37°C and 5% CO2. To increase the efficiency of transduction, 2 to 3 rounds of transduction were performed. The transduced CTLs were subsequently maintained in Yssel medium  supplemented with 10% serum. Except where stated, cells were transduced at 37°C with 103 vp per cell in Opti-MEM medium. At 6 hours after transduction, the cells were washed in 4 mL PBS and resuspended in fresh medium supplemented with 10% FCS.The CTL lines were resuspended at 1×10CTLs were transduced with the Lenti/hCD40L/E1AB as described above, and then added into HepG2 cells for 48 h; the total cells were lysed using cold radioimmunoprecipitation assay buffer . After sonication, the lysates were centrifuged at 10,000 g for 10 minutes. Protein concentrations were determined by commercial assay . Proteins were separated on a 12% SDS-PAGE under denaturing conditions and transferred to a supported PVDF membrane . The membrane was blocked for 1 h at room temperature in TBS-T  buffer containing 5% nonfat milk. Membranes were then incubated overnight at 4°C or 1 h at room temperature with the respective primary antibodies: Adenovirus E1A (1∶500) and GAPDH (1∶500). Anti-mouse secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) was used to visualize the stained bands with an enhanced chemiluminescence visualization kit . The signal was detected by incubation with chemiluminescent substrate and developed on FX7 .3 cells/well and then treated with Lenti/hCD40L/E1AB and Ad5/35-TRAIL at the suitable MOIs in the growth medium for 24, 48 and 72 hours. The following day, the medium was removed, and 100 µL of fresh medium containing 0.5 mg/mL MTT  was added to each well. The cells were incubated at 37°C in humidified 5% CO2 atmosphere for 4 hours, followed by the addition of 150 µL of solubilization solution (0.01 mol/L HCl in 100 g/L sodium dodecyl [SDS]) to each well, and the incubation of cells for a further 10 minutes at 37°C with gentle shaking. The optical density of the plates was measured using the spectrophotometrical absorbance at 570 nm in the Microplate Reader Model 550 .The cytotoxic activity of Lenti/hCD40L/E1AB and Ad5/35-TRAIL on the CTLs was determined by an MTT assay. Briefly, cells were seeded in 96-well tissue culture plates at a density of 5×10To evaluate anti-angiogenic effects mediated by new CTLs, we used the new CTLs in subsequent experiments. To obtain the new CTLs, CTLs cells were seeded on 100- mm dishes containing medium with 10% FBS. After 24 hours, the medium was added into Lenti/hCD40L/E1AB and Ad5/35-TRAIL with the suitable MOIs, then, the cells was collected after 24 hours. Then, HUVEC were cultured in M199 medium in each treatment and their tube formation were evaluated in the microscope.3 cells in 2 mL of 0.375% (w/v) Noble agar in 10% NBCS DMEM were added above the polymerized base solution. All solutions were kept at 40°C before pouring to prevent premature agar polymerization and to ensure cell survival. Plates were incubated  under standard conditions for 10 days before colony number and diameter were quantified microscopically.To assess anchorage-independent growth, soft agar clonogenic assays were done. Each well of a 6-well plate contained 2 mL of 0.5% (w/v) Noble agar (Difco) in DMEM with 10% NBCS. HepG2 cells and the indicated CTLs were mixed equally, and 3×10All data are presented as means ±SD of multiple replicate experiments. Intergroup differences were analyzed using paired t testing. P values of 0.05 and below were taken as significant.In order to identify the CTL cells, we examined the expression of CD3, CD8, CD11a, CD56, CD226 and CD305 expression on the cell surface of CTL cells. After isolation and incubation, the cells were stained with antibodies to T cell markers (CD3-FITC and CD8-PE) and a mixture of FITC- or PE-labeled antibodies to CD56, CD226, CD11a and CD305 . CD3, CD11a, CD56 and CD226 were expressed in the CTL cells surface, and CD8 and CD305 were negative . These rThe schema for generating CTLs capable of producing adenovectors following the lentiviruses (Lenti/hCD40L/E1AB) was described in material and methods. We first transduced the CTLs with a Lenti/hCD40L/E1AB containing the adenovirus E1 gene driven by the CD40L promoter (named CD40L-CTLs). To determine the suitable MOIs of CTL transduction by the Lenti/hCD40L/E1AB, the cell lines were analyzed by western blot using a monoclonal antibody specific to adenoviral E1a. After infected with the Lenti/hCD40L/E1AB at different MOIs, the CD40L-CTLs expressed the E1a adenovirus protein within 48 hours was confirmed by western blot analysis of cell lysates .To ensure that adenovirus-transduced CD40L-CTLs continued to amplification, we tested their proliferative activity by MTT assay. CD40L-CTLs were infected with Ad5/35-GFP or Ad5/35-TRAIL in the 96 well plate, and cultured in minimum media (Yssel plus 10% FCS) alone. The cell counts revealed that the proliferation rate was the same for both cultures for 24, 48 and 72 h . The CD4In vitro angiogenesis assay showed that HUVEC formed vessel-like structures (tubes) when plated on ECM wells . HEK293 We also tested the effects of Ad-CD40L-CTL on colony formation of HepG2 cells in soft agar . Ad-CD40Hepatocellular carcinoma is one of the most common malignant neoplasms in the world and is the main cause of death in patients with liver cirrhosis. Surgical treatments including hepatic resection and liver transplantation are considered as the most effective treatment of HCC, however for various reasons, the vast majority of HCC patients are not suitable for surgery.Inducible gene expression from a non-integrating, non-toxic Ad5/35 vector in HCC has a series of potential applications in cancer gene therapy. We focused our attempts on incorporating the Lenti/hCD40L/E1AB and Ad5/35 expression systems into CTL system. The availability of a chimeric adenoviral vector (Ad5/35) that can infect human T cells and of an activation-dependent promoter (CD40L) controlling a critical early adenoviral replicative gene E1 can made it possible to generate cytotoxic T cells that produce adenoviral vectors when they encounter their target antigen. These antigen-specific T cells retain their specificity and function after exposure to lentiviral vectors encoding the E1 antigen driven by a CD40L promoter and to E1-deleted Ad5/35 vectors encoding eGFP or a gene of TRAIL. Upon antigen-induced activation, the T cells produce infectious virus that can enter proximate target cells. If the adenoviral vector contains a tumor therapy gene TRAIL, the approach can be used to produce a higher level of target cell killing than could be achieved by the CTL alone.It has been well established that cytotoxic T lymphocytes have the potential to directly kill malignant cells The stimulation of the CTLs transduced with both E1 lentivirus and E1-deficient Ad5/35 adenoviral vectors could produce substantial quantities of infectious adenoviral vector when these cells were cultivated with CTL target cells. Transduction of target cells was substantially lower when cultivation occurred with CTL targets, consistent with a lower degree of T-cell activation through an CTL-restricted receptor pathway. The adenoviral vector produced was capable of infecting a high proportion of CTL-positive target cells. If the vector encoded a potentially oncolytic gene such as TRAIL, then it was possible to subsequently kill the target cells. The substitution of Ad-CD40L-CTL was capable of producing substantially greater tumor cell death than the use of cytotoxic T cells alone.In summary, we present the Ad5/35 vector and Lenti/hCD40L/E1AB expression systems that allow for tightly regulated CTLs expression in human HCC. The function and specificity of these vector producer T cells appears unimpaired, and incorporation of an vector encoding a potentially oncolytic gene, such as TRAIL, can increase target killing compared with CTLs alone. It will be of interest to discover whether targeted delivery of adenoviral vectors by cytotoxic T cells will have wider applicability in cancer therapy.


Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies . The confirmation of key epitopes with development of effective mAb is urgently needed.in vivo.We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes. The results provide a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.In this study, we selected three neutralizing mAbs, which recognize different non-overlapping epitopes of BoNT/B, and compare the neutralizing effects among different antibody combinations. We found that 8E10, response to ganglioside receptor binding site, could collaborate with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination between two mAbs, 5G10 and 2F4, blocking protein receptor binding sites, did not achieve synergistical effects. We also found that the binding epitope of 8E10 was conserved among BoNT A, B, E and F. It could cross-protect the challenge of different serotypes of BoNTs E.coli BL21 (DE3) in soluble form and purified for preparation of a toxoid immunogen. Sixteen hybridomas were obtained and screened for the production of anti-BoNT/B Hc mAbs, using ELISA with purified BoNT/B Hc  was optimized and synthesized. The reconstructed Hc proteins were expressed in oNT/B Hc . Cell cuoNT/B Hc . The res (PDB 1F31) and with SytII peptide (PDB 2NM1). The crystal structure reveals two distinguished domains which bind to GT1b and SytII respectively. Therefore, it is possible to compare the binding site of protein and ganglioside receptor to BoNT/B Hc with the localization of the mAb 8E10, 5G10 and 2F4 epitope identified in this study. The illustration of structure of BoNT/B Hc is showed in in vitro, we used SytII transfected and ganglioside treated PC12 cells, a neuroendocrine cell line of which wild type is lack of functional toxin receptors In order to determine the toxin neutralization by IgG In vivo toxin neutralization was studied using a standard mouse protection assay. We incubated 100 µg mAbs with BoNT/B for 1 hour prior to intraperitoneal injection into BALB/c mice and the groups of mice with 6 animals for each group were used to test dose level. Mice were observed for morbidity and mortality over 30 days. Complete protection was observed with doses up to 20 LD50s for 8E10, and 40 LD50s for 5G10 and 2F4. Partial protection, as indicated by increased survival compared with antibody-free control mice, was afforded with higher doses  and 5G10 or 2F4 (recognizing protein receptor binding domain) could result in a more than 30–60 folds increase in potency compared with that of any the single mAb. However, for the combination of 5G10 and 2F4, which both blocked protein receptor binding sites by non-overlapping epitopes, no synergistic effect was observed. This indicates that double blocking the ganglioside and protein receptor binding domain simultaneously with two mAbs is helpful for the synergistic effects of the two antibodies' combination against BoNT/B.For the last two decades, the relationship between the structure and function of BoNT has been studied intensively. With the complex structure of BoNT/B and its receptors, protein receptor Syt-II CC  overlap the putative sialoganglioside binding site and cover a large portion of HCC. The other mAb C25 bound a conformational epitope that consisted of the sequence from the N- and C-terminal subdomains of BoNT/A Hc, which overlapped with a putative inositol phosphate binding site that may be important for attachment to the lipid membrane. This function domain is essential for the binding to anionic lipid in the environment of lipid raft, which is important for the translocation processing of the toxin. The combinations of antibodies, which recognize nonoverlapping epitopes, synergistically cooperate in neutralization potency have been reported previously N. This is a functionally important epitope for intoxication, as mAb binding leads to potent BoNT neutralization. In this study, a neutralizing mAb 8E10 binding the conservative domain on Hc was reported. The structure of Hc from BoNT/B (PDB 2NM1) in vivo. Although its synergistic effect with the mAbs blocking protein receptor binding domain in BoNT A, E or F has not been detected, we can predict that 8E10 could act synergistically with the mAbs recognizing protein receptor binding domain against BoNT A, E or F. This study provides a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.Neutralizing mAbs binding multiple serotypes of botulinum neurotoxin are rare have been reported previously Mice were purchased from Animal Center of Chinese Academy of Sciences and maintained under pathogen-free conditions. All animal experimental procedures were carried out in strict accordance with the guidelines of the Animal Experiment Committee of the International Joint Cancer Institute, and were approved by the Animal Experiment Committee of the International Joint Cancer Institute.BoNT/A, BoNT/B, BoNT/E and BoNT/F were provided by Lanzhou Institute of Biological Products . DNA encoding BoNT/A Hc (residues 868 through 1296), BoNT/B Hc (residues 853 through 1291), BoNT/E Hc (817 through 1255), BoNT/F Hc (847 through 1280), and Syt II-LD fragment (residue 37 through 86) were synthesized and subcloned into a pET expression vector, expressed in E. coli BL21(DE3). The recombinant histidine tagged BoNT/A, B, E, F Hc and Syt II-LD fragment were isolated and purified by nickel affinity gel column chromatography and their molecular weight and purity were verified by gel electrophoresis Peptides used in this study, P1 (YFCISKWYLKEV), P2 (PISDSDEFYNTI), P3 (IKLKKDSPVGEI), P4 (THPHLPRALMRS), were synthesized by Yeli Bio-Scientific Inc. .6- to 8-week-old female BALB/c mice were purchased from Animal Center of Chinese Academy of Sciences and maintained under pathogen-free conditions. PC12  was purchased from Cell Bank of Chinese Academy of Science . To generate cells that express syt II (syt II), full-length mouse syt II  was subcloned into pCDNA3.1  and transfected into PC12 cells via electroporation. Transfected cells were selected with 1 mg/ml G418, and several independent monoclonal cell lines were established. For experiment in which cells were preloaded with gangliosides , cells were grown to 80% confluence followed by incubation in serum-free media plus 250 µg/ml gangliosides. 24 h later, the serum-free/ganglioside media was replaced with complete media, and the cells were incubated with FITC labeled BoNT/B Hc.6 hybridoma cells were injected intraperitoneally into BALB/c mice, which had been primed with 0.5 ml pristane . Ascites fluid was collected, and the mAbs purified, using HiTrap Protein G coupled to Sepharose 4B . mAb isotypes were determined by the Mouse Typer Isotyping Panel Kit . The binding specificities of the mAbs were determined by western blotting. The binding strength of neutralizing mAbs to the BoNT/B Hc was analyzed by surface plasmon resonance technology using a Biacore T100 instrument  6-week-old female BALB/c mice were subcutaneously immunized twice at 3-week intervals with 10 ìg of BoNT/B Hc emulsified in Freund's complete or incomplete adjuvant . Three days after a final immunization with BoNT/B Hc antigen alone, spleen cells from the mice and mouse myeloma NS1 cells  were fused and maintained according to the standard procedure 0.1 µg of purified BoNT/B Hc were mixed with 2× SDS sample buffer , boiled and loaded onto 10% polyacrylamide gels. After electrophoresis, the samples were transferred on nitrocellulose sheets, probed with all of these 16 positive binding mAbs respectively (1∶1000), and stained with a goat anti-mouse IgG coupled to horseradish peroxidase (HRP)  and developed with ECL Plus.To determine the cross-reactivity of 8E10, Easy Wash 96-well plates  were coated at 4°C overnight with 100 µl/well purified BoNT A/B/E/F Hc (at 5 µg/ml) respectively (BSA was set as negative control). After blocked with blocking buffer (5% non fat dry milk in PBS) for 2 hours at room temperature, 1 ng of 8E10 in 100 ul PBS was added and incubated for 2 hour at 37°C. The wells were washed and incubated at 37°C for 1 h with HRP-conjugated goat anti-mouse IgG at a 1∶3000 dilution. After extensive washing, 3, 3, 5, 5-tetramethylbenzidine (TMB) was added (50 ul/well) and incubated for 10 min at room temperature in the dark and the reaction was stopped by the addition of 2 M H2SO4 (50 ul/well). Absorbance of the samples in the plates was read in an automated ELISA microplate reader at 450 nm. For western blot, purified BoNT A/B/E/F Hc were subjected to SDS PAGE, transfer to nitrocellulose membrane and probed with 8E10 as described above.BIAcore measurements were performed with the Biacore T200 instrument  at 25°C in running buffer . CM5 chips were coated with BoNT/B Hc. 7 neutralizing mAbs were passed over the chip surface at concentrations ranging from 4.4 nM to 22.2 nM for 240 seconds at a flow rate of 30 ul/min and dissociation was recorded during 60 minutes. The chip was regenerated with 20 µl of 35 mM EDTA at 50 µl/min. Binding kinetics were evaluated using the BiaEvaluation software package  using a Langmuir model 1∶1.To determine the binding abilities of mAb 8E10 and BoNT/A/B/E or F Hc, Protein A was cross-linked to the dextran surface of a CM5 sensor chip. Protein A was immobilized using amine coupling with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride and N-hydrosuccinimide to a density of 1000–2000 response units (RU). mAb 8E10 was captured to approximately 100 RU. The analyte BoNT/A/B/E or F Hc was passed over the chip surface at concentrations ranging from 4.4 nM to 22.2 nM as indicated for 240 seconds at a flow rate of 30 µl/min and dissociation was recorded during 60 minutes. Binding kinetics were evaluated as described above.To identify the binding domain which was blocked by the neutralizing mAbs, BoNT/B Hc was conjugated with horseradish peroxidase (HRP) using HRP Plus Activated Conjugation Kit  according to the instruction of the kit. Easy Wash 96-well plates  were coated at 4°C overnight with 100 µl/well recombinant syt II-LD (at 5 µg/ml) or GT1b (at 10 µg/ml) in PBS, washed with PBS/0.05% Tween-20  and then blocked for 1 hour at 37°C with PBS/0.05% Tween-20/5% bovine calf serum/3% goat serum . HRP-conjugated BoNT/B Hc was pre-incubated with neutralizing mAbs at molar ratio of 1∶100 for 1 hour at 37°C before added into coated plate. 3, 3, 5, 5-tetramethylbenzidine (TMB) was used as a substrate  to detect bound BoNT/B Hc.To determine the binding epitope of the mAbs were overlapping or not, competition was tested by the competition-ELISA 11 pfus) from Ph.D.-12 Phage Display Peptide Library  were pre-absorbed by immobilized mouse IgG. The phages, diluted in Tris-buffered saline, were incubated at 4°C for 1 h with immobilized mAbs. The wells were washed for five times with TBST . Then, the phages that bound with mAbs were amplified by direct infection with E. coli ER2738. The amplified phages were purified by precipitation with 20% PEG8000, 2.5 M NaCl and used in the next cycle. Three rounds of selection were routinely performed.Anti-BoNT/B antibodies  were separately immobilized on 96-well plate. Phages  was incubated with control medium, monoclonal antibody (100 µg) or pairs of mAbs (50 µg of each Ab) for one hour at room temperature. Sub-confluent Ganglioside-treated SytIIFor the initial screening of the positive hybridoma that produced the neutralizing antibody, 0.5 ml of culture supernatant from hybridomas grown in a 24-well tissue culture flask for 3–5 days, was pre-incubated with 4 LD50 of BoNT/B for 1 h at room temperature, and the reaction mixtures intraperitoneally (i.p.) injected to each of six mice. More than four survivals in four days were considered as positive. In order to define the activity of the positive neutralizing mAbs and synergistic effect of pairs of mAbs, 100 µg of the positive neutralizing mAbs were added to the indicated number of mouse LD50 of BoNT/B neurotoxin in a total volume of 0.5 ml of gelatin phosphate buffer and incubated at RT for 60 min. For pairs of mAbs, 50 µg of each mAb was added. The mixture was then injected i.p. into female BALB/c mice (16–22 g). Mice were studied in groups of six and were observed at least daily. The morbidity and mortality of mice were determined over 30 days. For the cross-protection assays, 100 µg of 8E10 was pre-incubated with 20 LD50s of BoNT/A, B, E, F for 1 hour respectively. The mixtures were injected into mice, and the final death tally was determined 4 days after injection.Figure S1The effect of BoNT/B-specific antibodies on binding of BoNT/B Hc with ganglioside-treated Syt II+PC12 cells. Ganglioside-treated Syt II+PC12 cells were cultured with FITC-labeled BoNT/B Hc (green), with or without mAbs as indicated and visualized by confocal microscopy.(DOC)Click here for additional data file.Figure S2Binding affinity of mAb 8E10 and BoNT A/B/E/F Hc. The 8E10 was immobilized on the surface of a Protein A cross-linked CM5 chip. Purified BoNT/A/B/E/F Hc at concentrations of 22.2 nM, 14.8 nM, 9.9 nM, 6.6 nM, 4.4 nM were directly injected over the target and the reference surface. The signal obtained from the reference surface was subtracted to avoid the binding of non-specificity.(DOC)Click here for additional data file.