In this study, we defined additional mechanisms involved in B cell antitumor immunity. Administration of IL-2 significantly augmented the therapeutic efficacy of adoptively transferred tumor-draining lymph node (TDLN) B cells which express IL- 2R. Culture supernatant of purified B splenocytes harvested from the mice that received adoptive transfer of 4T1 TDLN B cells plus IL-2 administration produced larger amounts of IgG which bound to 4T1, resulting in 4T1 lysis. Furthermore, we detected CXCR4 expression on 4T1 TDLN B cells, and 4T1 tumor cells produced its ligand CXCL12. Transwell experiments demonstrated the chemoattraction of CXCR4-expressing 4T1 TDLN B cells towards CXCL12- producing 4T1 cells. Blockade of CXCR4 using a CXCR4-specific inhibitor, AMD3100, significantly reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Blockade of FasL and CXCR4 concurrently inhibited B cell-mediated direct killing of tumor cells in an additive manner, indicating that both Fas/FasL and CXCL12/CXCR4 pathways are involved in the direct killing of 4T1 cells by 4T1 TDLN B cells. TDLN B cells produced perforin. Additional transwell experiments showed that effector B cells could directly kill tumor cells in cell-cell contact via the Fas/FasL and CXCR4/CXCL12 pathways as well as perforin, while without cell contact, perforin secreted by B cells led to tumor cell cytotoxicity. These findings underscore the diversity of function by which B cells can play an important role in the host immune response to tumor.We previously reported that antitumor B cells directly kill tumor cells  Furtherotherapy . In hostotherapy . This obotherapy . In a muInterleukin 2 (IL-2) is a pleiotropic cytokine that stimulates T-cell proliferation; enhances NK cytolytic activity, induces the differentiation of Tregs, and causes activation- induced cell death , 5. Howe6 cells/mouse) in conjunction with or without IL-2 administration. Fourteen days after 4T1 tumor cells were injected into the mammary fat pad, mice were administered with activated TDLN B cells alone or TDLN B cells plus IL-2. Fourteen days later, mice were euthanized to quantify pulmonary metastases. A suboptimal dose of B cells alone showed no efficacy, but B cells plus IL-2 administration i.p. significantly inhibited the induction of spontaneous pulmonary metastases  was expressed on activated B effector cells. We purified TDLN B cells for this purpose. Unsorted 4T1 TDLN cells before purification are composed of approximately 30% CD19% Figure . We then% Figure . Before % Figure .in vitro with anti-CD3/anti-CD28 or LPS/anti-CD40 respectively. Lastly, culture supernatants were collected for antibody detection. While we found no antibody in T cell cultures as expected, we found significantly (p<0.01) higher IgG production by B cells isolated from animals treated with B cells + IL-2 compared with B cell alone treatment groups  that IL-2 enhances the antitumor immunity of 4T1 TDLN B effector cells in adoptive therapy of 4T1-bearing animals, we collected host spleens at the end of the treatments. Splenic T and B cells were enriched and subsequently activated s Figure .To test the specificity of the antibody, we assessed the binding of the immune supernatants of cultured splenic B cells against 4T1 tumor cells. As shown in Figure Interaction of chemokine and chemokine receptors represents a key mechanism in the attraction and recruitment of immune cells to cancer cells , 15. We CXCR4 is the receptor for CXCL12 (SDF-1) which has been shown to mediate target cell chemotaxis by cells expressing CXCR4 . We deteTo ascertain the chemotactic effects of CXCL12 on TDLN B cell migration, transwell assays were performed. TDLN B cells were purified and used for the chemotaxis assays as described in the Methods section. Compared with the control group (complete medium supplemented with 10% FBS) at each time point, the addition of CXCL12 to the medium in the lower chambers resulted in significant (p < 0.01) migration of TDLN B cells from the upper chamber to the lower chamber with time Figure .Righi et al. have previously shown that AMD3100, a highly specific CXCR4 antagonist, blocks the binding of CXCR4 to CXCL12 . We addeWe have recently reported that TDLN B cells directly kill 4T1 tumor cells via the Fas/FasL pathway . To examCollectively, the above experiments demonstrated that B cells could directly kill tumor cells via cell-cell contact. It is well known that perforin, a small secreted pore-forming granule protein, can result in cell death after it is introduced into the target cell , 18. To −, e.g. 4T1 cells. More 4T1 cells expressed cleaved caspase-3  than those 4T1 cells in the transwell experiments as shown in Figure To better assess the cell death of 4T1 cells, we examined one of the cell death pathway molecules, cleaved caspase 3, by flow cytometry to verify the data assessed by trypan blue in Figure In Figure We previously reported that purified TDLN B effector cells could kill tumor cells directly involving the Fas/FasL pathway and are regulated by IL-10 . Like T in vitro, are effective in cancer immunotherapy and could kill tumor cells more effectively in vivo in combination with IL-2 administration [in vivo to enhance their antitumor immunity. In this study, we have shown that B cells express IL-2R (CD25). Our in vitro experiments demonstrated that IL-2 modulated expression of CD25 on B cells after activation /expansion (A/E) before adoptive transfer; enhanced CXCR4 expression on B cells; augmented CXCL12 production by 4T1 tumor cells, and increased production of perforin by B cells. In addition, we found significantly more binding to 4T1 tumor cells by IgG containing culture supernatants of splenic B cells collected from the hosts subjected to B cell therapy plus IL-2 administration vs. B cell therapy only. Collectively, these data indicate the direct effects of IL-2 on B cells. As a corollary, IL-2 administration in mice may stimulate CD4+ T cell proliferation in tumor-bearing mice, and it is well-known that the Th2 cells can provide help for B cell function [Our data revealed that IL-2 can enhance B cell killer/effector function. It has been well documented that T cells from tumor or tumor draining lymph nodes, after activation stration , 20, 21.function , 23. In We have previously reported that activated TDLN B cells mediate tumor cytotoxicity, the Fas/FasL pathway . In the Perforin is a cytolytic protein that binds to the tumor cell plasma membrane to form pores leading to its destruction , 27. PerIn summary, exogenous IL-2 administration significantly enhanced the antitumor reactivity of adoptively transferred effector B cells. Purified B effector cells kill tumor cells directly and such killing involves not only the Fas/FasL pathway, but also the CXCR4/CXCL12 pathway and perforin. These studies further identify the mechanisms by which effector B cells mediate antitumor reactivity which can be applied to future cancer immunotherapies.Female BALB/c mice were purchased from the Jackson Laboratories, Bar Harbor, ME. They were maintained in a pathogen-free environment and used at age 7 weeks or older. Principles of laboratory animal care  were followed. The animal protocols were approved by the University of Michigan Laboratory of Animal Medicine.in vitro in complete medium (CM).The 4T1 cell line is a mammary carcinoma syngeneic to BALB/c mice . Inoculating 4T1 cells into the mammary fat pad induces the development of spontaneous pulmonary metastases. 4T1 cells were maintained 6 4T1 tumor cells in 0.1 ml PBS were injected subcutaneously (s.c.) into the lower flanks of BALB/c mice. Nine days after 4T1 cell inoculation, the draining inguinal lymph nodes were collected. The TDLNs were processed as previously described [In order to induce TDLNs, 1 × 10escribed .+ B cells were purified from the TDLN cells or splenocytes using anti-CD19- coupled microbeads and the MACS separator . CD3+ T cells were purified from the TDLN or splenocytes using anti-CD3-coupled microbeads. B cells were activated with lipopolysaccharide  plus anti-CD40 (FGK45) mAb ascites in complete medium at 37°C with 5% CO2 for 3-4 days. T cells were activated with immobilized anti-CD3 and anti-CD28 mAbs in CM containing IL-2 . The culture supernatants were collected and stored at −20°C for future experiments.CD192. After fixation and permeabilization with Fixation/Perm Buffer , the cells were stained with PerCP rat anti-mouse CXCL12 mAb. In transwell assays, after 6 hours, 4T1 cells in the lower chamber were fixed and permeabilizated, followed by staining with anti-mouse cleaved caspase-3 mAb. In cell-cell contact assays, mixed B cells and 4T1 cells were harvested after incubation for 6 hours, surface stained with anti-CD19, and then fixed and permeabilizated by eBioscience Fixation and Permeabilization fixed kit, followed by staining with anti-mouse cleaved caspase-3 mAb. The 4T1 cells were gated from the CD19 negative population. Flow cytometry was performed on a LSRII flow cytometer (BD Biosciences). BD FACSDiva software (version 7.0) was used for all flow cytometry analysis.Cell surface expression of CD19, CD25 and CXCR4 and intracellular expression of CXCL12 and cleaved caspase-3 were analyzed by immunofluorescence assay. All fluorescein isothiocyanate (FITC)-or phycoerythrin (PE) -or peridinin chlorophyll protein (PerCP) - conjugated antibodies  and matched isotype controls were purchased from BD Biosciences . Alexa Fluor® 488-conjugated Rabbit anti-mouse/human cleaved caspase-3 antibody and matched control were purchased from R&D system . To measure intracellular CXCL12 expression, 1 million 4T1 cells were incubated with 2 μl/ml Leukocyte Activation Cocktail  and 0.67 μl/ml Golgistop  in 6- well plate for 4-6 hours at 37°C with 5% CO4 4T1 cells into the mammary fat pad to induce spontaneous pulmonary metastases. Fourteen days after tumor inoculation, the tumor- bearing mice were treated with tail vein injection of activated 4T1 TDLN B cells. Commencing on the day of the effector B cell transfer, intraperitoneal (i.p.) injections of IL-2   were administered in 0.5 ml of PBS and continued twice daily for 8 doses. About 2 weeks after B cell transfer, all mice were sacrificed and lungs were harvested for enumeration of spontaneous pulmonary metastatic nodules. At the same time, spleens were collected for purification of splenic T and B cells as described above.Healthy Balb/c mice were inoculated with 5×10For 4T1 TDLN B cell cytotoxicity in the presence of anti-FasL antibody  and/or AMD3100 , the effector B cells were generated as described above and cultured with 4T1 cells with the admixture of 20 μg /ml anti-FasL and/or 2.5 μg /ml AMD3100 to block FasL and/or CXCR4; respectively.nd antibody: FITC-conjugated anti-mouse IgG. The concentration of total IgG was measured by ELISA quantitation kits . The binding of supernatant antibody to 4T1 cells was assessed using flow cytometry.4T1 cells were incubated with the immune supernatants collected from the cultured B cells with equal quantities of IgG followed by incubation with the 25 4T1 TDLN B cells in 200 μl were added to the upper chamber of a transwell . CXCL12  was added to the lower chamber in a volume of 900 μl. After 2, 4, 6, and 8h of incubation at 37°C, cells that migrated into the lower chamber were enumerated using a hemocytometer. The chemotactic index (%) represents the ratio of cells migrating in the presence of chemokines to the number of cells migrating in the absence of cytokines at each time point analyzed in triplicate.In transwell experiments, 5×10Cell cytotoxicity was assessed by measuring the release of cytoplasmic lactate dehydrogenase (LDH) into cell culture supernatants according to the manufacturer's protocol . For TDLN B cell cytotoxicity, effector B cells were generated from 4T1 TDLN B cells using LPS/anti-CD40 as described above. Target cells were plated in triplicates in a 96-well U-bottom tissue culture plate (5000 cells/well) and co-incubated with TDLN B cells at effector to target cell ratios of 1:1, 3:1, 10:1 and 30:1. After 12 hours of incubation, cells were centrifuged and 50 μl supernatant from each well was transferred to a fresh 96-well plate, 50 μl of the substrate mix was added and incubated at room temperature in the dark for 15 to 30 min. Before LDH measurement, 50 μl of stop solution was added to each well. Maximal release of LDH was performed by incubating the target cells with Lysis Solution . Target cells without effector cells were used as a spontaneous release control. Absorbance was measured at 490 nm using a 96-well plate reader.T cells and B cells purified from 4T1 TDLNs were activated as described above. Culture supernatants were then collected for perforin release detection using ELISA kits .5 cells/well, 1ml) and co-incubated with 4T1 TDLN B cells . In 0.4 μm transwell assay, 1×106 4T1 TDLN B cells were added to the upper chamber of a transwell, and 1×105 4T1 cells were added to the lower chamber. Anti-FasL(20μg/ml), anti-perforin (5μg/ml) and AMD3100 (2.5μg/ml) were added either alone or in combination as indicated in the experiments. After 12 hours of incubation, trypan blue staining was used to count the viable 4T1 cells either in the mixed co-culture or in the lower chamber of the transwell. The suspended B cells in the medium of the lower chambers were removed, and the well was washed by PBS for 3 times. B cells were distinguished from 4T1 cells by their significant different morphological characteristics under the microscope.In cell-cell contact assays, 4T1 cells were plated in triplicates in a 12-well tissue culture plate (1×10P<0.05 was considered statistically significant between the experimental groups. For comparison of more than 2 groups, statistical significance was determined using a one-way ANOVA followed by a Bonferroni multiple-group comparison test. Statistical analyses were performed with Graph- Pad Prism 6.0.Statistics were analyzed using the mean ± standard error. The significance of differences in numbers of metastatic nodules and cell lysis was determined using Student's t-test.


C. elegans zygote. Analysis of p150 mutants engineered by genome editing suggests that microtubule tip tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex and for generating robust cortical pulling forces. Instead, mutations in p150's CAP-Gly domain inhibit cytoplasmic pulling forces responsible for centration of centrosomes and attached pronuclei. The centration defects are mimicked by mutations of α-tubulin's C-terminal tyrosine, and both p150 CAP-Gly and tubulin tyrosine mutants decrease the frequency of early endosome transport from the cell periphery towards centrosomes during centration. Our results suggest that p150 GAP-Gly domain binding to tyrosinated microtubules promotes initiation of dynein-mediated organelle transport in the dividing one-cell embryo, and that this function of p150 is critical for generating cytoplasmic pulling forces for centrosome centration.The microtubule-based motor dynein generates pulling forces for centrosome centration and mitotic spindle positioning in animal cells. How the essential dynein activator dynactin regulates these functions of the motor is incompletely understood. Here, we dissect the role of dynactin's microtubule binding activity, located in the p150 CAP-Gly domain and an adjacent basic patch, in the  C. elegans embryos that dynactin, an essential dynein regulator, uses its microtubule binding activity to help dynein pull on microtubules for centrosome positioning during the first mitotic division. Our results with engineered dynactin and tubulin mutants suggest that microtubule binding by dynactin increases the efficiency with which dynein can initiate the transport of small organelles towards centrosomes. More organelles moving along microtubules through the viscous cytoplasm means that correspondingly larger pulling forces act on centrosomes. Thus, our work provides evidence for a novel functional link between dynactin's role in initiating transport of dynein cargo and the generation of cytoplasmic pulling forces critical for the positioning of centrosomes.Animal cells rely on molecular motor proteins to distribute intracellular components and organize their cytoplasmic content. The motor cytoplasmic dynein 1 (dynein) uses microtubule filaments as tracks to transport cargo from the cell periphery to the cell center, where the microtubule minus ends are embedded at the centrosome. Conversely, when dynein is anchored at the cell cortex or on organelles in the cytoplasm, the motor can pull on microtubules to position centrosomes within the cell. The intracellular location of centrosomes determines cell geometry and cell fate, and studying the underlying mechanisms will help us understand polarized cell behaviors such as cell migration or neurite outgrowth, and how cleavage plane orientation is established during cell division. Here, we show in  Cytoplasmic dynein 1 (dynein) is the major microtubule (MT) minus-end directed motor in animals and transports various cargo from the cell periphery to the cell interior. The motor also moves and positions intracellular structures such as nuclei and centrosomes by pulling on the MTs to which they are connected. To generate pulling force, dynein is either attached to anchor proteins fixed at the cell cortex  , or dyneDynactin is an essential multi-subunit activator of dynein that forms a tripartite complex with the motor and cargo-specific adaptors proteins –11, but in vitro demonstrated that the interaction between p150's CAP-Gly domain and tyrosinated MTs enhances the initiation of processive dynein motility . We. We22]. ogenesis . We muta (YA/YA) . Immunobd effect . Combinid effect . We thend effect . By conttba-1/2(YA/YA) mutant exhibited NCC centration/rotation defects reminiscent of those observed in p150dnc-1 CAP-Gly mutants (tba-1/2(YA/YA) mutant with the p150dnc-1(G45R) mutant did not significantly exacerbate the centration/rotation defects of the either mutant on its own, indicating that both mutants act in the same pathway  mutant  mutant .tba-1/2(YA/YA) mutant on GFP::p50DNC-2 localization and found that dynactin levels at MT tips were identical to controls  mutant also strongly reduced the number of observed tracks to 5.3 ± 1.1 tracks/min (tba-1/2(YA/YA) mutant had a less severe effect but still substantially reduced the number of tracks to 10.6 ± 2.2 per min. We also determined the maximal velocity in each track (determining the mean speed was complicated by frequent pausing of particles) and the total track displacement. This revealed only minor differences between controls and either mutant  ,63,64. Tacks/min . The tbar mutant . We concin vitro .C. elegans embryo, we tracked early endosomes after ebp-2(RNAi). The number of early endosome tracks was reduced from 16.3 ± 3.5 to 6.9 ± 1.7 per minute after EBP-2 depletion (ebp-2(RNAi) in the tba-1/2(YA/YA) mutant further reduced the number of early endosome tracks to 2.7 ± 1.0 per minute  and mammalian cells/filamentous fungi (dynein depends on dynactin). Surprisingly, similar to what was reported for the fungus U. maydis . Homozygous F1 progeny from balanced heterozygous mothers were identified by the absence of GFP fluorescence. None of the homozygous F1 p150dnc-1null progeny reached adulthood, and homozygous F2 p150dnc-1(G33S) progeny died during embryogenesis.Worm strains used in this study are listed in 2HPO4, 16.5 mM KH2PO4, 6.3 mM NaCl, 14.1 mM NH4Cl). dsRNAs were delivered by injecting L4 hermaphrodites, and animals were processed for live imaging after incubation at 20°C for 24 h or 48 h for partial and penetrant depletions, respectively.For production of double-stranded RNA (dsRNA), oligos with tails containing T3 and T7 promoters were used to amplify regions from N2 genomic DNA or cDNA. Primers used for dsRNA production are listed in DYCI-1 (residues 1–177) was generated as described previously  for 2 h at room temperature. After washing for 4 x 5 min in PBS, embryos were incubated with secondary antibodies conjugated with fluorescent dyes  for 1h at room temperature. Embryos were washed for 4 x 5 min in PBS and mounted in Prolong Gold with DAPI stain (Invitrogen).For immunofluorescence of Images were recorded on an inverted Zeiss Axio Observer microscope at 1 x 1 binning with a 100x NA 1.46 Plan-Apochromat objective and an Orca Flash 4.0 camera (Hamamatsu). Image files were imported into Fiji for further processing.For each condition, 100 worms were collected into 1 mL M9 buffer and washed 3 x with M9 buffer and once with M9 / 0.05% Triton X-100. To 100 μL of worm suspension, 33 μL 4x SDS-PAGE sample buffer  and ~20 μL of glass beads were added. Samples were incubated for 3 min at 95°C and vortexed for 2 x 5 min. After centrifugation at 20000 x g for 1 min at room temperature, supernatants were collected. Proteins were resolved by 7.5% or 10% SDS-PAGE and transferred to 0.2 μm nitrocellulose membranes . Membranes were rinsed 3 x with TBS , blocked with 5% non-fat dry milk in TBST (TBS / 0.1% Tween 20) and probed at 4°C overnight with the following primary antibodies: mouse monoclonal anti-FLAG M2 , mouse monoclonal anti-α-tubulin B512 , rat monoclonal anti-tyrosinated α-tubulin YL1/2 , rabbit polyclonal anti-DYCI-1 , rabbit polyclonal anti-DNC-1 , and rabbit polyclonal anti-DNC-2 . Membranes were washed 5 x with TBST, incubated with goat secondary antibodies coupled to HRP  for 1 hour at room temperature, and washed again 3 x with TBST. Proteins were detected by chemiluminescence using Pierce ECL Western Blotting Substrate (Thermo Scientific) and X-ray film (Fuji).p150dnc-1 exon 3 (GAATGTCACCTGCTGCTT); forward oligo on p150dnc-1 exon 4 (AAAGCGGTCTACAACTCC); reverse oligo on p150dnc-1 exon 5 (GATTGCGATAAGTTGGAGA); reverse oligo on p150dnc-1 exon 6 (AGTAGTCGTGGACGCTTT). For the SL1 PCR shown in p150dnc-1 exon 6 (TCCAGTATCATCAATCTTCTT).Total RNA was isolated from adult hermaphrodites using the TRIzol Plus RNA Purification Kit (Invitrogen). After 3 washes with M9, pelleted worms were homogenized in 200 μL of TRIzol reagent with a pellet pestle homogenizer and incubated at room temperature for 5 min. After addition of 40 μL chloroform, samples were shaken vigorously by hand, incubated at room temperature for 3 min, and centrifuged at 12000 x g for 15 min at 4°C. The upper phase containing the RNA was transferred to an RNase-free tube and an equal volume of 70% ethanol was added. Further RNA purification steps were performed according to the manufacturer's instructions. Purified RNA was treated with DNase I (Thermo Scientific), and cDNA was synthesized with the iScript Select cDNA Synthesis Kit (Bio-Rad Laboratories). The following oligos were used for the PCR reactions in Embryonic viability tests were performed at 20°C. L4 hermaphrodites were grown on NGM plates with OP50 bacteria for 40 h at 20°C, then singled-out to mating plates . After 8 h, mothers were removed and the number of hatched and unhatched embryos on each plate was determined 16 h later.2, 3.4 mM CaCl2, 5 mM HEPES, pH 7.4), and embryos were mounted onto a fresh 2% agarose pad. Imaging was performed in rooms kept at 20°C. Embryos co-expressing GFP::histone H2B and GFP::γ-tubulin were imaged on an Axio Observer microscope (Zeiss) equipped with an Orca Flash 4.0 camera (Hamamatsu), a Colibri.2 light source, and controlled by ZEN software (Zeiss). Embryos expressing GFP::p50DNC-2, dynein heavy chainDHC-1::GFP, EBP-2::mKate2, and mCherry::RAB-5 were imaged on a Nikon Eclipse Ti microscope coupled to an Andor Revolution XD spinning disk confocal system composed of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner , and a CSU-X1 confocal scanner (Yokogawa Electric Corporation), controlled by Andor IQ3 software (Andor Technology).Gravid hermaphrodite worms were dissected in a watch glass filled with Egg Salts medium .Time-lapse sequences of GFP::histone H2B and GFP::γ-tubulin, consisting of 7 x 1 μm z-stacks for GFP fluorescence and one central differential interference contrast (DIC) image captured every 10 s, were recorded at 2 x 2 binning with a 63x oil immersion objective from the start of pronuclear migration until the onset of cytokinesis. Embryo length was defined as the distance between the outermost points of the egg shell visible in DIC. After maximum intensity projection of GFP z-stacks, the x and y coordinates of pronuclei and centrosomes were recorded over time using the MTrackJ plugin by manually clicking in the center of the centrosome or nucleus. The position of centrosomes and pronuclei along the anterior-posterior axis was then calculated relative to embryo length, with the anterior reference point set to 0%. Tracks from individual embryos were aligned relative to pronuclear meeting or NEBD.Time-lapse sequences of GFP::γ-tubulin, consisting of 12 x 1 μm z-stacks captured every 2 s, were recorded at 2 x 2 binning with a 63x oil immersion objective from the beginning of metaphase until the end of anaphase. After maximum intensity projection of z-stacks, the x and y coordinates for centrosomes were determined over time with MTrackJ and the transversal distance of each centrosome to a line bisecting the embryo along the anterior-posterior axis was calculated.Time-lapse sequences, consisting of 8 x 1 μm z-stacks captured every 10 s, were recorded at 1 x 1 binning with a 60× NA 1.4 oil immersion objective from 40–50 s prior to pronuclear meeting until the onset of cytokinesis. Nuclear envelope (NE) signal was quantified 3 frames prior to pronuclear meeting using a maximum intensity projection of the 3 z-sections representing the best in-focus images of the NE. A 2 pixel-wide line was drawn on top of the NE along its entire circumference, and a similar line was drawn next to the NE on the cytoplasmic side around the nucleus. The mean fluorescence signal of the cytoplasmic line was then subtracted from the mean fluorescence signal of the NE line.Kinetochore signal was measured 7–8 frames before the onset of sister chromatid separation using a maximum intensity projection of the z-stack. The top 10 local maxima intensities on kinetochores were identified using the "Find Maxima" function, and the 10 values were averaged. The mean fluorescence intensity of the spindle background close to the kinetochore region was measured and subtracted from the kinetochore signal.Mitotic spindle signal was measured 2 frames after the onset of chromosome segregation using a maximum intensity projection of the z-stack. The mean intensity in 3 separate 10 x 10 pixel squares on the spindle was determined and the three values were averaged. The mean intensity of three equivalent squares in the cytoplasm adjacent to the spindle served as background signal and was subtracted from the spindle signal.DNC-2 signal at MT plus ends after ebp-2(RNAi)  and the integrated fluorescence intensity was measured in the GFP channel. The circle was then expanded by increasing the radius by 2 pixels, and the integrated intensity of this larger circle was measured. Background was defined as the difference in integrated intensities between the larger and the smaller circle. The background value was scaled in proportion to the smaller circle and then subtracted from the integrated intensity of the smaller circle to obtain a final value for the GFP signal. For presentation in graphs, all values were normalized to the mean value of the control. For quantification of the GFP::p50-2(RNAi) , embryosTime-lapse sequences, consisting of a single cortical confocal section captured every 200 ms, were recorded at 1 x 1 binning with a 100× NA 1.4 oil immersion objective for 1 min beginning at metaphase. Image sequences were analyzed using the LoG Detector (estimated blob diameter 10 pixels) and the Simple LAP Tracker  in the TrackMate plugin. The values for track duration were considered the cortical residency times for GFP and mKate2 puncta.Time-lapse sequences, consisting of 8 x 1 μm z-stacks captured every 30 s, were recorded at 1 x 1 binning with 60× NA 1.4 oil immersion objective. Four-cell embryos were imaged from the beginning of nuclear envelope breakdown in AB cells until cytokinesis of the P2 cell. The signal at the EMS-P2 cell border was measured at the time of EMS spindle rotation in maximum intensity projections of the z-stacks. The top 10 local maxima intensities on the EMS-P2 cell border were identified using the "Find Maxima" function, and the 10 values were averaged. The mean fluorescence intensity of an adjacent area (20 x 20 pixels) in the EMS cytoplasm served as background and was subtracted from the EMS-P2 cell border signal.Time-lapse sequences, consisting of a single confocal section captured every 400 ms, were recorded at 1 x 1 binning with a 60× NA 1.4 oil immersion objective for 6 min beginning at pronuclear meeting. Image sequences were analyzed using the LoG Detector (estimated blob diameter 6 pixels) and the Simple LAP Tracker  in the TrackMate plugin. All tracks whose particles showed directed movement towards centrosomes and had a track displacement of at least 0.9 μm (5 pixels) were considered.P values below 0.05.Values in figures and text are reported as mean ± SEM with a 95% confidence interval. Statistical analyses was performed with GraphPad Prism 7.0 software. The type of statistical analysis performed is indicated in the figure legends. Differences were considered significant at S1 Fig(A), (B) Correlation plots of GFP::p50DNC-2 versus EBP-2::mKate2 intensity (A) and dynein heavy chainDHC-1::GFP versus EBP-2::mKate2 intensity (B), measured at the cortex of metaphase one-cell embryos. Pearson correlation coefficient (r) and P-value indicating statistical significance are on top right. The best-fit line of a linear regression with 95% confidence bands is also shown.(C), (D) Residency times of EBP-2::mKate2 (C) and GFP::p50DNC-2(D) puncta at the cortex of metaphase one-cell embryos. The total number (n) of MT plus ends scored is indicated, derived from at least 8 embryos.(TIF)Click here for additional data file.S2 Fig(A) Cortical confocal section of one-cell embryos in metaphase co-expressing endogenous dynein heavy chainDHC-1::GFP and EBP-2::mKate2, showing that depletion of p150DNC-1 delocalizes dynein from MT tips. Images are maximum intensity projections over time (12 images acquired every 5 s). Scale bar, 5 μm; insets, 2 μm.(B) Quantification of dynein heavy chainDHC-1::GFP levels at MT plus ends using fluorescence intensity measurements at the cortex. Error bars represent the SEM with a 95% confidence interval, and n indicates the total number of measurements from 7–8 embryos per condition. The t-test was used to determine statistical significance. ****P < 0.0001.(C) Central confocal section of metaphase one-cell embryos expressing transgene-encoded mKate2::EBP-1, demonstrating the efficiency of ebp-1/3(RNAi). Images are maximum intensity projections over time (10 images acquired every 300 ms). Note that for reasons that are not clear, mKate2::EBP-1 does not localize to MT plus ends. Scale bar, 5 μm.(D) Schematic of the clip-1 locus. Mutations introduced by CRISPR-Cas9-based genome editing to generate a null allele (Δclip-1) are indicated in black font.(TIF)Click here for additional data file.S3 Fig(A) Schematic of the p150dnc-1 locus with engineered modifications. By deleting and/or fusing exons, p150dnc-1 expression was restricted to single N-terminal splice isoforms , as indicated on the right. Introduction of a frameshift mutation after the p150dnc-1 start codon generated a null allele.(B) Results of reverse transcription PCRs using RNA isolated from adult worms and primer pairs that allow detection of the four splice isoforms. Primer locations and predicted sizes of PCR products for the different isoforms are indicated. Crosses (x) indicate that the PCR will not amplify any product. All four N-terminal splice isoforms are detected in wild-type and p150dnc-1 null mutant adults, whereas only one isoform is detected in each p150dnc-1 isoform mutant. Asterisks next to gel bands denote unspecific PCR products. M, DNA size marker.(C) Embryonic viability assay for p150dnc-1 isoform mutants. Error bars represent the SEM with a 95% confidence interval, and n indicates the number of hermaphrodite mothers whose progeny was counted . Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ns = not significant, P > 0.05.(D) Result of a reverse transcription PCR using RNA isolated from adult wild-type worms with one of the primers recognizing the spliced leader sequence 1 (SL1) and the other located in exon 6 of p150dnc-1. In C. elegans, about 70% of mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders, SL1 or SL2, which replace the 5' ends of pre-mRNAs. One major product was amplified in the SL1 PCR and identified as the Δexon 4 isoform by sequencing. A PCR reaction with a primer recognizing SL2 did not amplify any product. M, DNA size marker.(E) Immunoblot of wild-type or p150dnc-1::3xflag adult worms with an antibody against the 3xFLAG tag, showing that there is no detectable p150DNC-1 isoform corresponding to human p135. α-Tubulin was used as the loading control. Molecular mass is indicated in kilodaltons.(TIF)Click here for additional data file.S4 Fig(A) Embryonic viability assay for p150dnc-1 CAP-Gly mutants. Error bars represent the SEM with a 95% confidence interval, and n indicates the number of hermaphrodite mothers whose progeny was counted . Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001; ns = not significant, P > 0.05.(B) Quantification of dynactin levels at the nuclear envelope, kinetochores, and the mitotic spindle for the p150dnc-1 mutants G45R and F26L, using fluorescence intensity measurements of GFP::p50DNC-2. Circles represent measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001; ns = not significant, P > 0.05.(TIF)Click here for additional data file.S5 Fig(A) Stills from time-lapse sequences in 2-cell embryos expressing dynein heavy chainDHC-1::GFP, demonstrating that dynein is delocalized from MT plus ends in the p150dnc-1(G45R) mutant. Scale bar, 5 μm.(B) Quantification of dynein levels at MT plus ends using fluorescence intensity measurements of dynein heavy chainDHC-1::GFP at the cortex of metaphase one-cell embryos. Error bars represent the SEM with a 95% confidence interval, and n indicates the total number of individual measurements from 6–7 embryos per condition. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001.(C) Stills from time-lapse sequences in one-cell embryos expressing dynein heavy chainDHC-1::GFP, showing that the p150dnc-1(G45R) mutant reduces dynein levels on the mitotic spindle and at centrosomes, but not at the nuclear envelope and kinetochores. Scale bar, 5 μm.(D) Quantification of dynein levels at the nuclear envelope, kinetochores, and the mitotic spindle for the p150dnc-1 mutants G45R and F26L, using fluorescence intensity measurements for dynein heavy chainDHC-1::GFP in images as shown in (C). Circles represent measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ***P < 0.001; ns = not significant, P > 0.05.(TIF)Click here for additional data file.S6 Fig(A) Migration kinetics of the male pronucleus in one-cell embryos expressing single isoforms of p150DNC-1. The position of the male pronucleus, marked by GFP::histone H2B, was determined along the anterior-posterior axis in images captured every 10 s. Individual traces were normalized to embryo length, time-aligned relative to pronuclear meeting, averaged for the indicated number (n) of embryos, and plotted against time. Error bars represent the SEM with a 95% confidence interval.(B) Positioning of centrosomes, marked by GFP::γ-tubulin, measured in time-lapse sequences as described for (A) and plotted relative to nuclear envelope breakdown. Solid lines indicate the midpoint between the two centrosomes (spindle position). Anaphase begins at 200 s. Error bars represent the SEM with a 95% confidence interval.(C) Angle between the centrosome-centrosome (C-C) axis and the anterior-posterior (A-P) axis in one-cell embryos at nuclear envelope breakdown (NEBD) and anaphase onset. Circles correspond to measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. **P < 0.01; ns = not significant, P > 0.05.(TIF)Click here for additional data file.S7 Fig(A) Selected frames from time-lapse sequences in one-cell embryos co-expressing GFP::histone H2B and GFP::γ-tubulin, showing that congression of chromosomes is delayed in the p150dnc-1(F26L) mutant. Time is relative to nuclear envelope breakdown. Scale bar, 5 μm.(B) Interval duration for nuclear envelope breakdown (NEBD) to full alignment of chromosomes and full alignment of chromosomes to anaphase (onset of sister chromatid separation). Error bars represent the SEM with a 95% confidence interval, and n indicates the number of embryos analyzed. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ***P < 0.001.(C) Embryonic viability assay for p150dnc-1 CAP-Gly mutants with and without depletion of the spindle assembly checkpoint component Mad1MDF-1. Error bars represent the SEM with a 95% confidence interval, and n indicates the number of hermaphrodite mothers whose progeny was counted . Statistical significance was determined by two-way ANOVA followed by Bonferroni's multiple comparison test. ***P < 0.001; *P < 0.05; ####P < 0.0001; ###P < 0.001.(TIF)Click here for additional data file.S8 Fig(A) Migration kinetics of the male pronucleus, marked by GFP::histone H2B, along the anterior-posterior axis. Nuclear position was determined in images captured every 10 s, individual traces were normalized to embryo length, time-aligned relative to pronuclear meeting, averaged for the indicated number (n) of embryos, and plotted against time. Error bars represent the SEM with a 95% confidence interval.(B) Angle between the centrosome-centrosome (C-C) axis and the anterior-posterior (A-P) axis in one-cell embryos at nuclear envelope breakdown (NEBD) and anaphase onset. Circles correspond to measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. The t-test was used to determine statistical significance. ns = not significant, P > 0.05.(TIF)Click here for additional data file.S1 MovieDNC-2 and EBP-2::mKate2. The anterior side is to the left. A single confocal section in the embryo center was acquired every 0.4 s. Playback speed is 30 frames per second. Scale bar, 10 μm.One-cell embryo in metaphase co-expressing GFP::p50(AVI)Click here for additional data file.S2 MovieDHC-1::GFP. The anterior side is to the left. A single confocal section near the cortex was acquired every 0.2 s. Playback speed is 30 frames per second. Scale bar, 10 μm.Two-cell embryo expressing dynein heavy chain(AVI)Click here for additional data file.S3 MovieDNC-2. The anterior side is to the left. A single confocal section in the embryo center was acquired every 0.2 s. Playback speed is 30 frames per second. Scale bar, 10 μm.One-cell embryos in metaphase expressing GFP::p50(AVI)Click here for additional data file.S4 MovieDNC-2. The anterior side is to the left. A single confocal section at the cortex was acquired every 0.2 s. Playback speed is 30 frames per second. Scale bar, 10 μm.One-cell embryos in metaphase expressing GFP::p50(AVI)Click here for additional data file.S5 MovieDNC-2. The anterior side of the embryo is to the left. A single confocal section at the cortex was acquired every 0.2 s. Playback speed is 30 frames per second. Scale bar, 10 μm.One-cell embryos in metaphase expressing GFP::p50(AVI)Click here for additional data file.S6 MovieOne-cell embryos expressing GFP-labelled histone H2B and γ-tubulin to mark chromosomes and centrosomes, respectively. The anterior side is to the left. Time lapse is 10 s and playback speed is 6 frames per second. Scale bar, 10 μm.(AVI)Click here for additional data file.S7 MovieOne-cell embryos expressing GFP-labelled histone H2B and γ-tubulin to mark chromosomes and centrosomes, respectively. The anterior side is to the left. Time lapse is 2 s and playback speed is 12 frames per second. Scale bar, 10 μm.(AVI)Click here for additional data file.S8 MovieOne-cell embryos expressing GFP-labelled histone H2B and γ-tubulin to mark chromosomes and centrosomes, respectively. The anterior side is to the left. Time lapse is 10 s and playback speed is 6 frames per second. Scale bar, 10 μm.(AVI)Click here for additional data file.S9 MovieOne-cell embryos during the centration phase. Early endosomes are marked by mCherry::RAB-5. The anterior side is to the left. Time lapse is 0.4 s and playback speed is 30 frames per second. Scale bar, 10 μm.(AVI)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.


Introduction: Tamoxifen is one of the most common treatment opportunities for hormonal positive breast cancer. Despite its good tolerability, patients demonstrate decreasing adherence over years impacting on therapeutic success. PBPK modeling was applied to demonstrate the impact of drug holidays on plasma levels of tamoxifen and its active metabolite endoxifen for different CYP2D6 genotypes.Materials and Methods: A virtual study with 24,000 patients was conducted in order to investigate the development of tamoxifen steady-state kinetics in patient groups of different CYP2D6 genotypes. The impact of drug holidays on steady-state kinetics was investigated assuming changing drug holiday scenarios.Results: Drug holidays in CYP2D6 extensive and intermediate metabolizers  exceeding 1 month lead to a decrease of endoxifen steady-state trough levels below the 5th percentile of the control group. Assuming drug holidays of 1, 2, or 3 months and administering a fixed-dose combination of 20 mg tamoxifen and 3 mg endoxifen EMs demonstrated re-established endoxifen steady-state trough levels after 5, 8, and 9 days. IMs receiving the same fixed-dose combination demonstrated re-established endoxifen steady-state trough levels after 7, 10, and 11 days.Discussion: The PBPK model impressively demonstrates the impact of drug holidays in different CYP2D6 genotypes on PK. Population simulation results indicate that drug holidays of more than 2 weeks cause a tremendous decrease of plasma levels despite the long half-life of tamoxifen. To improve therapeutic success, PBPK modeling allows identifying genotype-specific differences in PK following drug holidays and adequate treatment with loading doses. Tamoxifen treatment proved to be highly successful against hormone receptor positive breast cancer , http://www.cypalleles.ki.se/cyp2d6.htm). Patients with the extensive metabolizer (EM) genotype show significantly higher endoxifen steady-state levels than patients with an intermediate metabolizer (IM) genotype than patients with a poor metabolizer (PM) genotype, each undergoing tamoxifen treatment with the standard dose of 20 mg daily   could prove useful in accelerating the establishment of the steady-state metabolic pattern, too Figure  Group A.The presented work shows how genotype-specific PBPK modeling can be used to address the relevant question of patient adherence undergoing long-term therapy and the subsequent impact on PK. Moreover, PBPK modeling opens the door for minimizing the risk of decreased treatment benefit due to lack of adherence when combined with therapeutic drug monitoring as it enables for the calculation of individualized doses to re-establish efficacious metabolic patterns in all patients, irrespective of their genotype.in silico . PK-Sim® also calculated the PK-parameters from the simulation results. Population simulations were conducted using the MoBi® Toolbox for MATLAB® 2.2/2.3 (www.mathworks.com/products/matlab). This tool was also used for the evaluation of simulation results in terms of plasma concentration-time profiles, goodness-of-fit plots, box-whisker-plots, as well as calculation of median plasma concentrations of the simulated compounds.The presented coupled PBPK model was developed by means of the computational systems biology software platform including PK-Sim® 4.2.4 and MoBi® 2.3.5 (tss of CYP2D6 EMs was exceeded (cases) or the day when endoxifen Ctss of CYP2D6 EMs was again achieved (controls). Results were compared to CYP2D6 EMs and IMs under the fixed-dose combination of 20 mg/d tamoxifen and 3 mg/d endoxifen. Thus, in group A, controls consisted of CYP2D6 EMs and IMs each receiving either standard tamoxifen (CYP2D6 EMs) or the CYP2D6 IM-specific fixed-dose combination once daily (CYP2D6 IMs) to ensure equalized endoxifen exposure in both genotypes. Cases consisted of CYP2D6 EMs and IMs receiving the CYP2D6 fixed-dose combination of 20 mg tamoxifen and 3 mg endoxifen once daily. Population simulations were conducted in each the four groups .A virtual study with two groups was conducted. In group A the time to development of endoxifen steady-state kinetics in CYP2D6 EMs under standard therapy (20 mg/d) and CYP2D6 IMs under fixed-dose combination  was investigated. Time to steady-state was the day before endoxifen CN = 1000, respectively).In group B the impact of drug holidays in CYP2D6 EMs and IMs after 6 months of tamoxifen was investigated. To ensure equalized endoxifen exposure in both genotype groups CYP2D6 EMs received standard tamoxifen and IMs received a fixed-dose combination . After 2, 4, 8, or 12 weeks of no drug intake administration of standard tamoxifen  or 20 mg/d tamoxifen and 1 mg/d endoxifen  were compared to the intake of 20 mg/d tamoxifen and 3 mg/d endoxifen in CYP2D6 EMs and IMs (cases). Thus, in group B, controls consisted of CYP2D6 EMs and IMs each receiving standard tamoxifen once daily (CYP2D6 EMs) or the CYP2D6 IM-specific fixed-dose combination once daily (CYP2D6 IMs) for 6 months. Subsequently, drug holidays of different lengths was simulated, namely for 2, 4, 8, and 12 weeks. Consequently, drug intake was re-established in terms of CYP2D6 EMs with standard tamoxifen once daily and in terms of CYP2D6 IMs with the CYP2D6 IM fixed-dose combination once daily until 12 months of intake were completed. Cases were made up of CYP2D6 EMs and IMs receiving standard tamoxifen once daily (CYP2D6 EMs) or the CYP2D6 IM-specific fixed-dose combination once daily (CYP2D6 IMs) for 6 months. Subsequently, drug holidays of different lengths was simulated, namely drug holidays taken for 2, 4, 8, and 12 weeks. Consequently, drug intake was re-established with CYP2D6 PM-specific fixed-dose combination in CYP2D6 EMs and IMs. Population simulations were conducted in each group (tss) observed in CYP2D6 EMs receiving standard tamoxifen assuming full adherence. Time to steady-state (either achievement or re-establishment) was the day before endoxifen Ctss of CYP2D6 EMs was exceeded (cases) or the day when endoxifen Ctss of CYP2D6 EMs was again achieved (controls). Overall, 20 different administration protocols in two study groups were investigated in 20,000 virtual female patients. The administration protocols investigated are illustrated in detail in Table The goal of the clinical trial simulation is to analyze the time to target concentration of END either at therapy onset or following drug holidays for CYP2D6 EMs and IMs. To match END exposure in both groups, IMs received their fixed dose combination of 20 mg tamoxifen and 1 mg endoxifen whereas EMs remained under standard therapy. Target concentration range in each study arm was the endoxifen steady-state trough concentration  to a range around the 25th percentile of the median Ctss of endoxifen in CYP2D6 EMs under standard tamoxifen with full adherence (Ct after 2 weeks drug holidays: 14.9 μg/L vs. Ctss 25th percentile with full adherence: 13.8 μg/L)  funded by the German Federal Ministry of Education and Research (BMBF). This work was financially supported by a research grant from Bayer Technology Services GmbH as part of a Ph.D. thesis of KD. The research grant was received by GH (between 2010 and 2013).KD and MB are employed by Bayer AG. SW is employed by Bayer Pharma AG. A patent for fixed-dose combinations of tamoxifen and endoxifen has been filed by KD and SW. GH received research grants from Bayer Technology Services GmbH, now Bayer AG that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We conducted a study on a continuous series of 249 confirmed influenza infections. Incidence was three fold greater than in the previous three years. Most isolates were A(H3N2) viruses (78%) and clustered in subgroups 3C.2a (57%) and 3C.3b (43%). We identified 23 non-synonymous mutations that had already been identified during previous seasons at low frequencies, except mutation Q197H, present in 26% of 3C.3b isolates. We identified lung disorder, tobacco smoking and A(H1N1)pdm09 infection as risk factor of severe influenza disease. In contrast, young age (< 5 years), A(H3N2) infection and initial admission to an emergency department were associated with a better outcome of influenza infection.Current influenza vaccination strategy is based on limited analyses of circulating strains and has some drawbacks, as illustrated during the 2014–2015 season with the circulation of A(H3N2) viruses belonging to divergent genetic subgroups. We reasoned that these strains, poorly neutralized  Influenza viruses are characterized by high genetic diversity resulting from mutations or genetic re-assortment with other influenza viruses. Influenza viruses mutate frequently, resulting in continuous antigenic drift. This permanent evolution necessitates annual updates of vaccine composition, relying on the selection of strains that antigenically match currently circulating influenza viruses . This strategy has some drawbacks, as demonstrated during the recent 2014–2015 influenza season. The northern hemisphere influenza vaccine composition for that season was decided upon in February 2014 by the WHO , based on currently circulating strains. It included the A/California/07/2009 (H1N1)pdm09, A/Texas/50/2012 H3N2, 3C.1), and B/Massachusetts/2/2012 (Yamagata) strains. However, the circulating strains of influenza A(H3N2) evolved rapidly in 2014, with the emergence of the divergent genetic subgroups 3C.3a (reference strain A/Switzerland/9715293/2013) and 3C.2a (reference strain A/Hong Kong/5738/2014) [N2, 3C.1,in vitro, may have been associated with vaccination failure and more cases of severe disease. We addressed this issue by conducting a large retrospective study on a continuous series of laboratory-confirmed influenza infections. The aim was to obtain a representative overview of influenza infections in the Centre-Val de Loire region . We determined the clinical profiles of patients and the genetic diversity of circulating influenza viruses and then identified the clinical and viral factors associated with severe influenza disease and factors associated with vaccination among patients with influenza A infection.In France, the 2014–2015 influenza season lasted nine weeks and was associated with 2.9 million consultations, 1,558 cases of severe influenza necessitating intensive care, and 18,300 excess deaths [™ II RV16 & RB5 PCR assays (Seegene) or the Filmarray® respiratory panel (Biomerieux) for emergency cases [A continuous series of patients (N = 1323) were hospitalized at the Bretonneau hospital  between December 24, 2014 (week 52) and April 1, 2015 (week 14) with symptoms of respiratory infection , with or without headache, myalgia, or arthralgia. They had diagnostic procedures for viral respiratory tract infections. All patients with at least one respiratory sample  positive for influenza virus were included in our study. Laboratory diagnoses were performed using multiplex Anyplexcy cases .Patient clinical history was analyzed by reviewing the clinical chart of their medical records. Young children (< 5 years) and elderly patients (> 65 years) are known to be at high risk of complications  and werehttps://www.fludb.org). Genetic subgroups were determined according to available CDC recommendations [http://www.cbs.dtu.dk/services/NetNGlyc/), setting 0.5 as the threshold value of the average potential to predict glycosylation sites. Phylogenetic analyses were performed to confirm identification of genetic subgroups. Multiple alignments of HA gene nucleotide sequences were performed using the Bioedit program and MUSCLE algorithm. A neighbor-joining tree was constructed with Mega 7 software, using the Kimura 2-parameter model [Influenza A virus subtyping was performed by qPCR as recommended by the WHO . Sanger ndations . Potentier model . The inft-test or Mann-Whitney test were performed for continuous variables. All tests were two-sided at the 0.05 significance level. Factors associated with severe influenza infection or vaccination were identified using multivariate logistic regression adjusted for age as a potential confounding factor. Results are expressed as the odds-ratio (OR) with their 95% confidence interval (95% CI).Results are expressed as the mean and standard deviation (SD) or median and interquartile range (IQR) for continuous variables, and percentage for categorical variables. Statistical analyses were performed using Epi Info 7 (CDC). The Chi squared test or Fisher’s exact test were performed for categorical variables. Student The 2014–2015 influenza season started on week 52 of 2014 and ended on week 14 of 2015, with a peak incidence at weeks 7 and 8 . Rate ofSubtyping of the 249 influenza A positive samples identified 250 influenza A viruses (because one patient was co-infected with H1N1 and H3N2 viruses). These 250 influenza A viruses were mostly A(H3N2) viruses , including three viruses with H3N2-like subtyping results. The latter were H3 positive but N2 negative, probably because of poor performance of the N2-specific RT-qPCR. Other subtypes were A(H1N1)pdm09  and two atypical profiles (1%): one H1-/N1+ and one H3-/N2+, that we attributed to probable HA mutations at primer binding sites. Fourteen viruses were not typed, either due to an insufficient amount of biological material  or because all subtyping RT-PCRs were negative .The medical records of all patients tested positive for influenza A (N = 249) were retrospectively analyzed. Demographic data, comorbidities, and details on influenza infections were extracted . Young cIn contrast, elderly patients were at higher risk of comorbidities  but at lower risk of atypical infections  and they were more frequently vaccinated  in comparison to patients aged 5–65 years. Also, elderly patients were at higher risk of infection with A(H3N2)  and at lower risk of infection with A(H1N1)pdm09  in comparison to patients aged 5–65 years.The clinical presentation was atypical for 36 patients (14%), which had no symptoms of respiratory infection, but presented with asthma (n = 5), asthenia (n = 4) or seizures (n = 4).We sequenced the HA gene of 174 (90%) out of 194 A(H3N2) isolates to construct a phylogenetic tree . We obseA subset of 141 (57%) patients had a poor outcome of influenza infection . Among tAmong the 249 patient with influenza A infection, 10% (n = 26) had been vaccinated earlier in the current season. Most of these patients were > 65 years old , male , had at least one comorbidity  and were initially admitted to a clinical department other than emergency or ICU . Twenty-three (88%) were infected with an A(H3N2) strain (11 with 3C.2a strains and 8 with 3.C3b strains) and one with an A(H1N1)pdm09 strain (two samples were missing).Univariate analysis performed on these 249 influenza cases identified a higher prevalence of elderly patients (> 65 years) in the vaccinated group than in the non-vaccinated group  . TherefoWe accurately described both clinical and virological features of the 2014–2015 influenza epidemic, at a regional scale, on a continuous series of 249 hospitalized patients with influenza A infection. In comparison with previous years, rate of influenza detection was three-fold higher, confirming the increased incidence this year . Most paWhen A(H1N1)pdm09 emerged in 2009, mortality caused by this strain was similar to that of seasonal influenza with a mean hospitalization fatality rate (HFR) of 3%, but elderly patients (> 65 years) were at a lower risk of infection than adults and children –20. We cThe 174 HA sequences obtained in this study and their associated clinical features contribute to a better understanding of the genetic diversity AH3N2) virus in France and the northern hemisphere. Our regional results are in agreement with European data, with 3C.2a and 3C.3b being the most prevalent subgroups N2 virus . These 1The 2014–2015 influenza season in France started with a high risk of vaccination failure because of the predominant circulation of variant strains . Our stuAmong patients with influenza disease, this study identified that elderly patients  (OR = 3.The multisite US Flu Vaccine Effectiveness Network estimated the 2014–2015 influenza vaccine effectiveness (VE) to be 23% (95% CI = 8–36%). This is far below the 50% estimated for the previous seasons. This suggests that, apart from clinical factors, several viral factors could be responsible for this low VE ,26.The most important viral factor is probably the predominant circulation of 3C.2a viruses , poorly Strains belonging to 3C.3a subgroup, poorly neutralized in HI assay , could aA third potential factor to explain vaccination failure is HA mutation Q197H/R, located in epitope B and identified as a critical position for recognition by neutralizing-antibodies . MutatioIn addition to these viral factors, impaired immune response to influenza vaccine probably contributed to vaccination failure. Screening methods for evaluating new influenza vaccines relies mostly on antibody responses . Yet, anOverall, the circulation of antigenic drift variant 3C.2a strains remains the most likely explanation for vaccination failure in the northern hemisphere during the 2014–2015 influenza season .With three to five million cases of severe influenza-related illness every year and low VE (23–50%), there is an evident need to focus our efforts on more effective surveillance and prevention strategies . The cur


MYCC amplification. This is in contrast to patients from the Intermediate 3/4 Group who showed the best survival rate (100%). Overall and progression free survival were better for this group than for Group 3  and Group 4 . Our work supports the view that within the non-WNT/non-SHH tumors different risk groups exist and that the current two groups classifier may be not sufficient for proper clinical categorization of individual patients.Medulloblastoma, the most common malignant pediatric brain tumor, is a heterogeneous disease, with the existence of at least four molecular types: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 and Group 4 tumors. The latter two groups, which can be identified by an application of multi-gene expression or methylation profiling, show sometimes ambiguous categorization and are still classified for diagnostic reason as non-SHH/non-WNT medulloblastomas in updated WHO 2016 classification. In order to better characterize non-SHH/non-WNT tumors, we applied the method based on the Nanostring nCounter Technology, using the 26 genes codeset in 68 uniformly treated medulloblastoma patients. This allowed for identification of tumors, which shared common Group 3 and Group 4 gene signatures. We recognized three transcriptional groups within non-WNT/non-SHH tumors: Group 3, Group 4 and the Intermediate 3/4 Group. Group 3, in line with previously published results, showed poor prognosis with survival rate < 40%, frequent metastases, large cell/anaplastic pathology and presence of tumors with  Medulloblastoma is the most common malignant pediatric brain tumor. Recent studies revealed molecular heterogeneity of the disease, with the existence of at least four subtypes, which depend on distinctive profiles of gene expression and DNA alterations: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 and Group 4 medulloblastomas –5.These four molecular subtypes are now recognized as distinct biological entities and are acknowledged in the updated World Health Organization (WHO) 2016 classification of tumors of the central nervous system [In addition to microarray analysis, Group 3 and Group 4 tumors may be identified by an application of Nanostring nCounter Technology, which allows for transcriptional profiling of tumors from formalin-fixed paraffin-embedded (FFPE) tissue samples . This isTwo recently published studies based on NanoString method and 22-genes classifier have been employed for detection of four molecular groups in medulloblastoma and the results showed that a subset of tumors displayed expression of genes from both Group 3 and Group 4 signatures. Remarkably, these tumors were classified as Group 3 in a series by Li et al.  but as GTherefore, in this study, in order to better characterize non-SHH/non-WNT tumors, we applied the NanoString method using an increased 26 genes codeset, which includes four additional genes expressed in Group 3 or/and Group 4. The molecular results were correlated with histological and clinical features, including survival time of uniformly treated patients.Sixty-eight patients diagnosed with medulloblastoma between the years 2003 and 2013 in The Children’s Memorial Health Institute (CMHI) in Warsaw, Poland, were included in the analysis. Only patients who were treated according to the uniform protocol of the Polish Pediatric Neurooncology Group (PPNG) for children above 3 years of age were included in survival analyses. Since patients with Group 4 tumors are characterized by later relapses our analyzed cohort had at least 4 years of observation time.Presence of metastases at diagnosis was assessed according to Chang et al. .Informed consent was obtained to use tumor material according to the procedures outlined by the CMHI’s Ethical Committee.Analysis was performed on FFPE and frozen tumor samples obtained at diagnosis. All tumors were independently reviewed by two experienced pathologists and histologically defined according to the recent WHO 2016 morphological criteria of medulloblastoma [For identification of the molecular groups NanoString nCounter system analysis  was applied in a series of 68 medulloblastoma tumors. Total RNA was extracted from FFPE or frozen tumor samples using RNeasy kits (Qiagen). RNA integrity was assessed using an Agilent 2100 Bioanalyzer.ACTB, GAPDH and LDHA). The marker genes included 22 genes as described by Northcott et al. [SNCAIP, since it is duplicated and expressed in Group 4 [MYCC, since it is amplified and highly expressed in some tumors from Group 3 [RCVRN (recoverin) and (4) PDC (phosducin), since photoreceptor signature genes were expressed in the subset of non-WNT/non-SHH type of tumors [For group assignment a custom NanoString CodeSet was applied, which consisted of 26 marker genes and three housekeeping genes  MYCf tumors , 3.The probes were designed to target the following regions of the genes:SNCAIP (RefSeq:NM_001242935.1), region of exon 4 and 5 TATTCATTACGCAGGTTGCTATGGCCAGGAAAAGATTCTTCTGTGGCTTCTTCAGTTTATGCAAGAACAGGGCATCTCGTTGGATGAAGTAGACCAGGATfor MYCC (RefSeq:NM_002467.3), region of exon 3 CACCGAGGAGAATGTCAAGAGGCGAACACACAACGTCTTGGAGCGCCAGAGGAGGAACGAGCTAAAACGGAGCTTTTTTGCCCTGCGTGACCAGATCCCGfor RCVRN (RefSeq:NM_002903.2), region of exon 1 and 2 CCCTCTACGACGTGGACGGTAACGGGACCATCAGCAAGAATGAAGTGCTGGAGATCGTCATGGCTATTTTCAAAATGATCACTCCCGAGGACGTGAAGCTfor PDC (RefSeq:NM_002597.4), region of exon 4 ACTGCCTTCGTAAATACCGTAGACAGTGTATGCAGGATATGCACCAGAAGCTGAGTTTTGGGCCTAGATATGGGTTTGTGTATGAGCTGGAAACTGGAAA,for Hybridization of the probes was performed in NanoString Technologies, Seattle, USA for an initial set of 44 tumors. An additional set of 24 tumors was analyzed in the Clinical Research Centre, Medical University of Białystok, Poland, according to NanoString Technologies procedures for hybridization, detection and scanning. Raw counts for each gene underwent technical and biological normalization using nSolver 2.5 software. Clustering of the samples was performed with Pearson, Spearman and Eucledian distance metrics and average settings.PDC gene at the protein level  was detected using anti-PHOS antibody . Antigen retrieval was performed using Target Retrieval Solution Citrate pH 6 (DAKO), for 20 min at 99 °C. Whole preparations were scanned in Hamamatsu NanoZoomer 2.0 RS scanner at an original magnification of x40. Positive reaction was considered where areas with > 10% positive cells were encountered within examined tumor samples.Expression of the CTNNB1 and detection of MYCC amplification by FISH are described elsewhere [Procedures for detection of mutations in exon 3 of lsewhere .Detection of isochromosome 17q or 17q gain was carried out using MLPA (Multiplex Ligation-dependent Probe Amplification) analysis of genomic DNA extracted from the frozen tumor tissues. The analysis was performed using the SALSA MLPA kit P301-A2  according to the manufacturer’s protocol. Probe amplification products were run on ABI Prism 3130 Genetic Analyzer . The peak plots were visualized and normalized, and the dosage ratios were calculated using GeneMarker software v 2.2.0 .Statistical analyses were performed using T-test and Fisher Exact test. Overall survival (OS) and progression free survival (PFS) were calculated using Kaplan–Meier estimation and group comparisons were made using the log-rank test. All patients had at least 4 years of the follow-up period.The average age of 68 pediatric patients at diagnosis was 9.2 years, range 0.5–17 years. 43 patients were males, 25 patients were females. The tumors were histologically diagnosed as classic medulloblastoma in 48 cases, large cell/anaplastic (LCA) in 12 cases, desmoplastic/nodular (DN) in 7 cases, and not otherwise specified (NOS) in 1 case.CTNNB1 to ensure the presence of the WNT group in the analyzed series.An initial set of 44 tumors was analyzed by the NanoString method for assignment into one out of four molecular groups. Our set of tumors included 6 patients with mutation in exon 3 of Unsupervised hierarchical clustering using Pearson correlation and average settings identified four clusters of tumors: 10 tumors from the WNT subtype, 8 tumors from the SHH subtype and two clusters from the non-SHH/non-WNT subtype: cluster 3 with 12 tumors (Group 3) and cluster 4 with 14 tumors (Group 4). However, the analysis revealed that Group 3 parent cluster consisted of two sub-clusters, one with 5 tumors and the second with 7 tumors. The latter sub-cluster was characterized by expression of genes which belong both to Group 3 and Group 4 signatures. Therefore, we named this sub-cluster as the Intermediate 3/4 Group , , Fig.  a, b. ThPDC gene was present in Group 3 and Intermediate 3/4 Group, but showed low expression in all Group 4 tumors  level, we performed immunohistochemical analysis using antibody anti-PHOS ab77523) in a series of FFPE preparations. Intense immunoreactivity for PHOS, mainly cytoplasmic and membrane, was present in all 8 analyzed tumors from Group 3 and in all 10 analyzed tumors from the Intermediate 3/4 Group  study Northcott et al. , 7 and fClinical and genetic features present in the analyzed patients are shown in Table MYCC expression and FISH analysis identified high amplification (> 10 copy number) of MYCC gene in those cases.Two tumors from Group 3 (No 12 and 18) displayed a high level of Isochromosome 17q or 17q gain, as detected by MLPA, was present in 88% of analyzed cases and the frequency was not significantly different between the groups.5 year OS and PFS were significantly better for Intermediate 3/4 Group , when compared with Group 3  and better when compared to Group 4 ,  and also significantly better when compared with Group 4 .The results from several previous studies clearly identified the WNT and the SHH medulloblastomas, regardless of the method applied. By contrast, the remaining non-WNT/non-SHH tumors warrant further characterization in order to establish clinically more effective sub-grouping of these tumors. Although they are provisionally categorized into Group 3 and Group 4 in the recent WHO 2016 classification [Earlier studies based on expression microarrays using Affymetrix HG U133 Plus 2.0 Array identified 3–4 groups within non-WNT/non-SHH tumors, with several tumors displaying an expression of mixed signatures, e.g. group D in Kool et al. study [k-means consensus clustering. Still, when the samples were compared with the gene expression sub-grouping, several incompatible cases involved switches between Group 3 and Group 4, but not between the WNT and SHH groups [By contrast, two groups within non-WNT/non-SHH tumors were recognized by subsequent analyses based on Affymetrix Human Exon 1.0 ST Array and unsupervised hierarchical clustering of 1450 high–standard deviation genes. However, principal component analysis (PCA) showed overlapping of samples within these groups [H groups . FinallyH groups . TherefoAs the result of an ambiguous classification, proportion of tumors belonging to Group 3 or Group 4 may vary, also in the large analyzed series. For example, ratio of Group 3 tumors to Group 4 tumors was 1:1.35 for exon array data, 1:1.56 for methylation profiling data and 1:2 for NanoString data . This isFrom the clinical point of view, the most important issue is related to the survival rate of patients. Patients with Group 3 tumors showed consistently worse prognosis than patients with Group 4 but the difference varied from significant to not significant, depending on the study , 13, 14.MYCC amplification. Importantly, survival rate was < 40% and significantly lower than in the remaining groups. This was not contributed to the presence of two MYCC amplified cases, since one of the patient has been a long time survivor. Patients with Group 4 medulloblastoma had an intermediate survival rate what is expected from the previous studies. The most noteworthy result is related to the Intermediate 3/4 Group where patients > 3 years of age showed an excellent survival rate. This was the only statistically significant feature which could distinguish this group from the other two in our series. Although we did not provide further extensive molecular characteristics of tumors, it seems that tumors with transitional features, as detected in this study, may constitute a clinically relevant cohort. They may be compatible with group c4 which showed > 90% OS and > 75% EFS rates [In our study, we recognized three transcriptional groups within non-WNT/non-SHH tumors. Although the results were obtained on the limited number of cases and marker genes, the identified Group 3 showed all features of poor prognosis, including frequent metastases, LCA pathology and presence of tumors with Recently, four risk groups based on DNA methylation profiling were proposed for non-WNT/non-SHH tumors where Group 3 and Group 4 were subdivided further into high risk (HR) and low risk (LR) tumors . InteresSNCAIP gene was significantly higher in Group 4 than in the Intermediate 3/4 Group, therefore we assume that our Group 4 tumors may include cases recognized as group 4α by Northcott et al. [In our study, we applied the NanoString method as a potentially useful diagnostic tool for analysis of FFPE material. The increased number of genes, including two genes regulating phototransduction cascade, allowed for identification of tumors, which shared common Group 3 and Group 4 signatures and the existence of these “middle ground” tumors allowed for the separation of “genuine” Group 3 from Group 4. Expression of t et al. .From the diagnostic point of view, it seems that the NanoString method requires further exploration in terms of the establishment of the most clinically relevant genes signature and algorithms used for the tissue samples clustering. The fact that several tumors could be originally classified as either Group 3 or Group 4 medulloblastoma in our study, depending on the algorithm applied, is not acceptable for diagnostic purposes. Therefore, we suggest that more effort should be undertaken to adjust nonetheless a very promising NanoString method for a better classification of non-WNT/non-SHH tumors.Also, since there are currently no reliable immunohistochemical markers for individual groups detection, we suggest that expression of PDC protein present in Group 3 and Intermediate 3/4 Group may be one of the useful biological markers for the future diagnostic panel.In summary, our work supports the view that within the non-WNT/non-SHH tumors different risk groups exist. We confirmed the presence of the poor prognostic group (Group 3) and proved that also favorable group, conventionally named as Intermediate 3/4 Group, exists. This altogether suggests, that the current two groups classifier is not sufficient for proper clinical categorization of individual patients.


Between September 2017 and February 2018, influenza A(H1N1)pdm09, A(H3N2) and B viruses  co-circulated in Europe. Interim results from five European studies indicate that, in all age groups, 2017/18 influenza vaccine effectiveness was 25 to 52% against any influenza, 55 to 68% against influenza A(H1N1)pdm09, −42 to 7% against influenza A(H3N2) and 36 to 54% against influenza B. 2017/18 influenza vaccine should be promoted where influenza still circulates.  Most countries in the European Union (EU) recommend and fund seasonal influenza vaccine for elderly people and individuals at increased risk of severe influenza . The UniThe trivalent influenza vaccines for the 2017/18 northern hemisphere influenza season include an A/Michigan/45/2015 (H1N1)pdm09-like virus, an A/Hong Kong/4801/2014 H3N2)-like virus and a B/Brisbane/60/2008-like virus (B/Victoria lineage). The quadrivalent vaccines also contain a B/Phuket/3073/2013-like virus (B/Yamagata lineage) [N2-like vThe early phase of the 2017/18 influenza season in Europe was characterised by the co-circulation of influenza A(H1N1)pdm09, influenza AH3N2) and influenza B, with different patterns of dominant type or subtype observed between countries . Up to FN2 and inHere we present the interim 2017/18 season influenza vaccine effectiveness (VE) estimates from three single-country studies  and Spain (ES)) and two multi-country studies (primary care (EU-PC) and hospital (EU-H) European Influenza - Monitoring Vaccine Effectiveness (I-MOVE/I-MOVE+) networks) .The methods of these five studies have been described in detail elsewhere -9. StudyAll five studies used a test-negative case control design (TND) . In shorThe ES, UK and EU-PC studies included patients at primary care level , the EU-H study included patients at hospital level , and the DK study included results from primary care and hospital level pooled together. Patients’ inclusion was foreseen to be systematic (or exhaustive) in the ES, EU-PC and EU-H studies and ad hoc in the DK and UK studies. In Spain, 268 of the 833 physicians included in the ES study were also included in the EU-PC study.The study population included all age groups in all studies except for EU-H, which was confined to individuals 65 years and older.In all studies, we defined patients as vaccinated with the 2017/18 influenza vaccine if they had been vaccinated at least 14 days (UK) or 15 days  before symptom onset. Patients were excluded if they were vaccinated less than 14 (UK) or 15 days  before symptom onset or if the date of vaccination was unknown.In seven EU-PC countries , the UK, ES and DK, all or a random sample of positive influenza specimens were selected for genetic sequencing.VE was computed by comparing the odds of vaccination between cases and controls (VE = (1 − OR) × 100). All studies used logistic regression to adjust VE for measured confounding variables, excluding patients with missing data for covariates in the model  . We compIf the number of events per parameter was lower than 10, we conducted a sensitivity analysis using penalised logistic regression to assess small sample bias .Between September 2017 and February 2018, the number of patients included in the VE analysis by study was 2,399  in the ES, 1,331 (421 cases) in the UK, 4,652  in the EU-PC, 11,907  in the DK and 931 (385 cases) in the EU-H study. Overall, more than two thirds of cases were positive for influenza B viruses in all studies except UK, where influenza A and B viruses were detected in similar proportions (51% (214/423) and 49% (209/423), respectively) . Where sAmong all ages, VE against any medically attended influenza ranged between 25% : −10 to 48) in the UK study and 52% (95% CI: 29 to 67) in the ES study. In UK, VE of the LAIV4 was 53% (95% CI: −56 to 86) in children and VE of the inactivated vaccine was 18% (95% CI: −23 to 45) in adults . Among tAll 76 influenza A(H1N1)pdm09 viruses sequenced belonged to clade 6B.1 (A/Michigan/45/2015) . VE against influenza A(H1N1)pdm09 was 68% (95% CI: 42 to 83) and 55% (95% CI: 23 to 74) among all ages in the EU-PC and DK studies, respectively. Among 18–64 year-olds, it was 63% (95% CI: 12 to 84) and 60% (95% CI: 13 to 82) in the EU-PC and DK studies, respectively. Among those aged 65 years and older, it was 37% (95% CI: −40 to 72) in the DK study .Of the 204 influenza A(H3N2) viruses sequenced, 63% (n = 129) belonged to genetic clade 3C.2a, 35% (n = 72) to 3C.2a1 and 1% (n = 3) to 3C.3a . Among all ages, VE against influenza A(H3N2) ranged from −42% (95% CI: −116 to 7) in the DK and 7% (95% CI: −74 to 51) in the ES study. VE against hospitalisation for influenza A(H3N2) in patients aged 65 years and older was −1% (95% CI: −93 to 47) in EU-H .Of the 596 influenza B viruses sequenced, 94% (n = 559) were B/Yamagata  and 6% (n = 37) were influenza B/Victoria  and 12 belonging to clade 1A) .Among all ages, VE against influenza B ranged between 36% (95% CI: 27 to 44) in the DK and 54% (95% CI: 24 to 72) in the UK study. Age group-specific VE was lowest at 15% (95% CI: −114 to 66) among those aged 65 years and older and highest at 83% (95%CI: 54 to 94) in the 0–14-year age group in the ES study. VE was 34% (95% CI: 8 to 52) against hospitalised influenza B in EU-H and 28% (95% CI: 14 to 39) against medically attended and hospitalised influenza B in the DK study among those aged 65 years and older . VE agaiFor all of the above analyses, sensitivity analyses for small sample size gave similar results (absolute difference ranging between 1% and 6%).Interim results from five established influenza VE studies across Europe indicate that 2017/18 VE against all influenza ranged between 25 and 52% among all ages and between 36 and 40% in the targeted groups. VE was moderate to good against influenza A(H1N1)pdm09 among all ages (55 to 68%), poor against influenza A(H3N2) with all point estimates below 8% for all ages, and moderate against influenza B, with point estimates between 39 and 52% for all ages.The good VE against medically attended influenza A(H1N1)pdm09 is consistent with historical data . HoweverThe influenza A(H3N2) component included in the 2017/18 northern hemisphere vaccine was the same as in the 2016/17 northern hemisphere vaccine . As antiThe interim VE against medically attended influenza B was moderate in the studies included here , similar to recently published estimates from northern hemisphere countries ,21,22. IThe UK study was the only one to provide VE estimates for the quadrivalent vaccines. Vaccine effectiveness against any influenza among children was similar in the UK study (53%), where children receive LAIV4, and in the EU-PC study (59%), where most vaccinated children received TIV. However, it is difficult to compare these estimates against any influenza since the relative proportion of circulating (sub)types was different in the UK, where there was a higher proportion of circulating influenza A(H3N2) viruses, compared with most countries participating in the EU-PC study. In past seasons where circulating and vaccine lineages were different, contradictory results were observed ,23,24. PEnd-of-season analyses are needed to verify the conclusions from these interim season results. A larger sample size should allow more precise estimates, especially in stratified analyses. Recent publications suggest a potentially strong (boosting or lowering) effect of previous vaccination on VE estimates ,30 and eThese early VE results from five studies were included in the Global Influenza VE (GIVE) report to help inform the World Health Organization vaccine strain selection committee meeting on 22 February 2018. For the 2018/19 northern hemisphere trivalent vaccine, this selection committee recommended to include the same influenza A(H1N1) component as in the 2017/18 northern hemisphere vaccine, an A/Singapore/INFIMH-16–0019/2016 (H3N2)-like virus and a B/Colorado/06/2017-like virus (B/Victoria/2/87 lineage) [In the context of an influenza season with co-circulation of influenza A(H3N2), influenza A(H1N1)pdm09 and influenza B viruses mismatched with the trivalent vaccine, results from these five EU studies indicate a moderate VE against all influenza. Vaccination continues to be the most effective preventive measure against influenza and uptake of the 2017/18 trivalent or quadrivalent influenza vaccines should still be promoted in countries with ongoing virus circulation. In particular in settings with evidence of influenza A(H3N2) virus circulation, prophylactic use of antiviral drugs, administered according to country-specific guidelines, could help prevent severe outcomes . Based o