Tandem affinity purification–mass spectrometry (TAP-MS) is a popular strategy for the identification of protein–protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression. FRT) recombination system, SH-based TAP-MS has been successfully applied to the in-depth analysis of human signaling networks  and K-562 and KCL-22 from DSMZ . HT-29 was kindly provided by P. Schneider (Lausanne). Cells were cultured in DMEM  or RPMI medium (Sigma-Aldrich) supplemented with 10% (v/v) FBS  and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) (Sigma-Aldrich). Ba/F3 was obtained from DMSZ and grown in RPMI supplemented with 10% (v/v) FBS (Gibco) and 1–3 ng/ml recombinant murine IL-3 . The reagents used were as follows: doxycycline , necrostatin-1 , necrosulfonamide , geldanamycin , MG132 , chloroquine , selumetinib , trametinib , and ponatinib .Antibodies used were HA , HA-7-HRP , MEK1/2 , phospho-MEK1/2 , ERK1/2 , phospho- ERK1/2 , STAT5 , phospho-STAT5A/B , phospho-p70 S6 kinase , p70 S6 kinase , RIPK3 , HSP90 , actin , and tubulin . The secondary antibodies used were goat anti-mouse HRP , goat anti-rabbit HRP , and Alexa Fluor 680 goat anti-mouse .Inducible retroviral expression vectors are derived from the pQCXIX self-inactivating retroviral vector backbone . pRSHIC vectors were assembled using standard cloning techniques and final expression constructs contain the following elements: pSIN-TREtight or TRE3G-HA-StrepII-Gateway cassette-IRES-mCherry-PGK-BlastR for N-terminal StrepHA tagging and pSIN-TREtight or TRE3G-Gateway cassette-StrepII-HA-IRES-mCherry-PGK-BlastR for C-terminal StrepHA tagging. Detailed cloning strategies, primers, and vector information are available upon request. NRAS coding sequence was PCR-amplified from K562 cDNA and cloned into the Gateway-compatible pDONR221 entry vector using BP recombination . The G12D mutant version of NRAS was generated by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit  using the following primers 5′-GTGGTGGTTGGAGCAGATGGTGTTGGGAAAAGC-3′ and 5′-GCTTTTCCCAACACCATCTGCTCCAACCACCAC-3′. Cloning of RIPK3, MLKL, and MLKL S358D has been described elsewhere . Followirpm, 45 min, room temperature). 24 h after infection the medium was exchanged and replaced with fresh medium. Another 24 h later, the medium was supplemented with 1–2 μg/ml puromycin (Sigma-Aldrich) to select for infected cells. Following puromycin selection, RIEP- or rtTA3-expressing cell lines were similarly transduced with retrovirus produced in HEK293T cells using the respective target gene-encoding pRSHIC vector, and pGAG-POL, pADVANTAGE, and pEcoEnv. Infected cells were selected by addition of 15–25 μg/ml blasticidin (InvivoGen). Target gene expression was induced by addition of 1–2 μg/ml doxycycline.Human cell lines were retrovirally transduced using vector pMSCV-rtTA3-IRES-EcoR-PGK-PuroR (pMSCV-RIEP) , and murm HEPES (pH 7.4), 250 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 10 mm NaF, 1 mm Na3VO4, one tablet of EDTA-free protease inhibitor  per 50 ml) or IP lysis buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 50 mm NaF, 1 mm Na3VO4, 1 mm PMSF, 5 μg/ml TPCK and protease inhibitor mixture) for 10 min on ice. Lysates were cleared by centrifugation . The proteins were quantified with BCA  or Bradford assay using γ-globin as a standard . Cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes Protran BA 85 . The membranes were immunoblotted with the indicated antibodies. Bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies using the ECL Western blotting system  or Odyssey Infrared Imager .Cells were lysed using Nonidet-40 lysis buffer (50 mm HEPES-KOH (pH 7.9), 150 mm NaCl, 20 mm Na2MoO4, 2 mm EDTA, 5% glycerol, 0.5% Triton X-100, one tablet of EDTA-free protease inhibitor (Roche) per 50 ml, 20 mm NaF, and 0.4 mm Na3VO4) for 10 min on ice. Lysates were cleared by centrifugation , quantified with BCA (Pierce), and precleared  on Sepharose6 beads (Sigma-Aldrich). Subsequently, lysates were incubated  with monoclonal anti-HA agarose antibody (Sigma-Aldrich). Beads were recovered by centrifugation and washed three times with lysis buffer before analysis by SDS-PAGE and immunoblotting.Cells were washed in PBS and lysed in ice-cold HENG buffer (50 mm HEPES (pH 8.0), 150 mm NaCl, 5 mm EDTA, 0.5% Nonidet P-40, 50 mm NaF, 1 mm Na3VO4, 1 mm PMSF, and protease inhibitor mixture. Cell lysates were cleared by centrifugation . Proteins were quantitated by Bradford assay using γ-globin as standard (Bio-Rad). 50 mg total lysate were incubated with StrepTactin Sepharose beads . Tagged proteins were eluted with d-biotin  followed by a second purification step using HA-agarose beads (Sigma-Aldrich). Protein complexes were eluted with 100 mm formic acid and immediately neutralized with triethylammonium bicarbonate buffer (Sigma-Aldrich). Samples were digested with trypsin , and the resultant peptides desalted and concentrated with customized reversed-phase tips . The threshold for switching from MS to MS2 was 2,000 counts. All samples were analyzed as technical, back-to-back replicates.Mass spectrometry was performed on a hybrid linear trap quadrupole Orbitrap Velos mass spectrometer  using the Xcalibur version 2.1.0 coupled to an Agilent 1200 HPLC nanoflow system  (Agilent) via a nanoelectrospray ion source using liquid junction . Solvents for liquid chromatography mass spectrometry separation of the digested samples were as follows: solvent A consisted of 0.4% formic acid in water and solvent B consisted of 0.4% formic acid in 70% methanol and 20% isopropanol. From a thermostatic microautosampler, 8 μl of the tryptic peptide mixture were automatically loaded onto a trap column  with a binary pump at a flow rate of 45 μl/min. 0.1% TFA was used for loading and washing the precolumn. After washing, the peptides were eluted by back-flushing onto a 16 cm fused silica analytical column with an inner diameter of 50 μm packed with C18 reversed phase material . The peptides were eluted from the analytical column with a 27 min gradient ranging from 3 to 30% solvent B, followed by a 25 min gradient from 30 to 70% solvent B, and, finally, a 7 min gradient from 70 to 100% solvent B at a constant flow rate of 100 nl/min. The analyses were performed in a data-dependent acquisition mode using a top 15 collision-induced dissociation method. Dynamic exclusion for selected ions was 60 s. A single lock mass at employed . The maxp value <10−3). The validated proteins retrieved by the two algorithms were merged and any spectral conflicts discarded and grouped according to shared peptides. By applying the same procedure against a reversed database, a false-positive detection rate of <1 and <0.1% (including the peptides exported with lower scores) was determined for proteins and peptides, respectively. The significance of the interactions from affinity purification-mass spectrometry (AP-MS) experiments was assessed using the SAINT software  and converted into Mascot generic format (mgf) files. The resultant peak lists were searched against either the human or mouse SwissProt database v2014.03_20140331 . Cell viability was determined using CellTiter Glo Luminescent Cell Viability Assay (Promega) according to the instructions provided by the manufacturer. Luminescence was recorded with a SpectraMax M5Multimode plate reader . Data were normalized to values of untreated controls.Samples were analyzed on an LSR Fortessa (BD Biosciences), and data analysis was performed using FlowJo software version 7.6.3 .To analyze the influence of inducible SH-tagged bait protein expression on cell proliferation and survival, pRSHIC-NRAS G12D (mCherry+) and pRSHIC-GFP (mCherry+/GFP+) transduced Ba/F3 rtTA3 cells were induced with 1 μg/ml doxycycline. After 24 h, cells were mixed in a 1:1 ratio and cultured in the presence of doxycycline with or without IL-3. The percentage of mCherry+ and mCherry+/GFP+ populations was monitored daily by flow cytometry, gating only viable cells (FSC/SCC).Microscopy images were taken at 10× with a Leica DFC310 FX on a Leica DM IL LED microscope  or at 20× on an Operetta automated confocal microscope  and analyzed with ImageJ 1.44p . The fluorophores used contained no overlapping spectrums and channels were imaged sequentially.n = 2) and analyzed by LC-MSMS as technical duplicates. Cell viability assay data were normalized to untreated control and are shown as mean value ± s.d. of at least two independent experiments (n ≥ 2) performed in triplicates. Flow-cytometry-based proliferation competition assay data are shown as mean value ± s.d. of at least two independent experiments (n ≥ 2). Flow cytometry and immunoblot results shown are representative of at least two independent experiments (n ≥ 2).Tandem affinity purifications were performed as biological replicates (A). The recombination efficiency of the gateway system allows high-throughput cloning, and thus, the vector is suitable for use with gateway-compatible cDNA and ORF libraries. Furthermore, we linked a fluorescent mCherry marker to the cDNA expression cassette via an internal ribosome entry site (IRES) sequence to enable tracing of bait protein-expressing cell populations by flow cytometry or microscopy. The doxycycline-controlled reverse tet transactivator protein 3 (rtTA3) . Transduced cells are selected using blasticidin, and transgene expression in the target cell lines can be assessed by flow cytometry or immunoblotting prior to TAP-MS and follow-up experiments.We assembled an inducible expression system in a self-inactivating retroviral vector containing a tetracycline response element tight (TREtight) promoter . For expFig. 1 (rtTA3)  in combi(in vivo . To gene(in vivo . Cell liA–2C). Target protein expression was confirmed by immunoblotting for SH-tagged GFP (D–2F). Additionally, we observed strong correlation between GFP and mCherry fluorescence , indicating that flow cytometry-based detection of the mCherry marker provides a reliable surrogate measure for efficient induction of transgene expression. The TREtight promoter exhibits low basal expression while promoting high-level transcription upon induction. Depending on the promoter used, the efficiency of inducible expression by Tet-regulated systems and the basal expression levels can vary between different cell types , which provides strongly reduced basal expression compared with earlier versions of the TRE promoter . As demonstrated in K-562 RIEP GFP cells, expression of bait proteins can be modulated by the addition of increasing concentrations of doxycycline . Furthermore, we monitored induction kinetics, indicating that GFP was induced within hours after doxycycline addition and continued to accumulate over 24 h . Removal of doxycycline led to a decline in GFP levels, illustrating the reversibility of bait expression . Altogether, these data establish pRSHIC as a versatile inducible vector system that enables scaling and reversible expression of SH-tagged bait proteins in multiple mammalian cell types.To characterize the properties of this novel expression system, we transduced human leukemia K-562 RIEP, KCL-22 RIEP and colorectal adenocarcinoma HT-29 RIEP cells with a vector construct encoding SH-tagged green fluorescent protein (GFP). Following selection using blasticidin, the cells were cultured in the presence of doxycycline for 24 h to induce GFP expression. In all three cell lines, >85% of the cell population efficiently induced gene expression as determined by the detection of the mCherry reporter using flow cytometry A–2C. Targged GFP D–2F. Addll types . For baipromoter  . To examine NRAS G12D-mediated growth factor independence, we performed flow cytometry-based proliferation-competition assays. While both cell populations showed equal growth in the presence of IL-3, NRAS G12D-expressing cells rapidly out-competed GFP-expressing control cells upon IL-3 withdrawal (A). Cytokine removal led to loss of signal transducer and activator of transcription 5 (STAT5) phosphorylation in both cell lines; however, NRAS G12D cells maintained elevated mitogen-activated protein kinase (MEK) 1/2 phosphorylation and hence activation of the mitogen-activated protein kinase pathway (B). Consequently, NRAS G12D-expressing cells showed marked sensitivity to the MEK 1/2 inhibitors trametinib (GSK1120212) (C) and selumetinib (AZD6244) (D) in the absence of IL-3, as increasing drug concentrations reduced mitogen-activated protein kinase pathway activation and ribosomal protein S6 kinase 1 (S6K1) phosphorylation . In order to map the interactome of NRAS G12D, we induced bait protein expression for 24 h with doxycycline in the presence of IL-3 and performed TAP coupled to one-dimensional gel-free liquid chromatography tandem mass spectrometry (TAP-LC-MSMS). Significance analysis of interactome (SAINT) analysis using GFP purifications as a control for nonspecific protein interactions identified Ras and Rab interactor 1 (RIN1) among the high-confidence interacting proteins of NRAS G12D . Indeed, RIN1 has been described as associating with harvey rat sarcoma viral oncogene homolog (HRAS) and to preferentially bind active, GTP-loaded RAS  C and selAZD6244) D in the aded RAS . RIN1 coaded RAS . Furtheraded RAS , 40 and . As we have shown previously  and microscopy . The MLKL inhibitor necrosulfonamide (NSA) . Conversely, the RIPK1 inhibitor necrostatin-1 (Nec-1)  and RIPK3 that in recent years has been the subject of very intense research efforts –28. Upon43Fig. 4eviously , exogenode (NSA)  inhibitede (NSA)  in a dos (Nec-1)  that bloD). Furthermore, heat-shock-related 70 kDa protein 2 (HSP72), HSP90A/B, and the kinase-adaptor cochaperone cell division cycle 37 (CDC37) . In order to investigate the functional relevance of HSP90 for MLKL S358D, we induced expression in HT-29 RIEP MLKL S358D cells by doxycycline addition for 3 h in the presence of geldanamycin, Nec-1, or NSA. Geldanamycin led to a strong decrease in MLKL S358D protein levels, whereas the other inhibitors had no effect (A). To exclude the possibility that geldanamycin interfered with the inducible expression system per se, we verified that the mCherry reporter was equally expressed in both control and geldanamycin-treated samples by flow cytometry . The rapid degradation of MLKL S358D upon HSP90 inhibition suggested that this protein constitutes a novel HSP90/CDC37 client. Indeed, the closely related mixed lineage kinase 3 (MLK3) has previously been shown to be stabilized by association with HSP90 and the cochaperone CDC37 (B), whereas blocking lysosomal protein degradation using chloroquine had no effect. This data suggested that MLKL S358D was subjected to proteasomal degradation in the absence of HSP90-mediated stabilization, similar to previously described HSP90 client proteins (C), further corroborating the requirement of HSP90 for MLKL S358D.To identify novel protein interaction partners of MLKL S358D, the cells were induced for 7 h with doxycycline before harvest and TAP-LC-MSMS analysis. The known interactor RIPK3  was signFig. 4 (CDC37)  were ide (CDC37) . These h (CDC37) . The com (CDC37)  assigned (CDC37)  has been (CDC37) . This in (CDC37) –58. Our o effect A. To excne CDC37 . The gelne CDC37 B, whereaproteins . Neitherproteins , 60, bloproteins C, furtheD). To confirm the interaction between HSP90 and wild-type MLKL as well as the MLKL S358D mutant, we performed coimmunoprecipitation experiments. MLKL copurified HSP90, similar to the previously described HSP90 client protein RIPK3 (E). As demonstrated by the identification and characterization of MLKL as a novel HSP90 client, pRSHIC is an efficient tool to perform phenotypic and TAP-MS analysis of toxicity-promoting proteins.Finally, we investigated the requirement of HSP90 function for the MLKL wild-type protein. Similar to the S358D mutant, geldanamycin induced destabilization of the wild-type MLKL protein and this degradation could be blocked by concomitant MG132 treatment D. To conin RIPK3 E. As demi.e. morphology, surface marker expression, drug resistance). Intracellular localization of the bait proteins can be assessed by probing for the N- or C-terminally fused SH-tag. Moreover, the inducibility of bait expression allows proteins that promote cell death to be studied and opens the opportunity to perform targeted chemical screens in the cell system of choice.We have established a retroviral-based expression system that expands the repertoire of cell lines amenable to SH-based TAP-MS experiments and thus enables interaction proteomic experiments in the physiologically relevant cellular background. The IRES-linked fluorescent reporter protein allows quick evaluation of bait protein induction by flow cytometry, fluorescence-activated cell sorting of specific cell populations and live tracing of bait-expressing cells to assess phenotypic changes (Here, we demonstrated efficiency and applicability of pRSHIC for TAP-MS-based interaction proteomics studies on the oncogenic NRAS G12D mutant protein  in murin


However, whether they counteract each other or synergize in regulating immune reactions remains controversial. To determine their interactions, we describe the results of experiments employing mouse models of intestinal inflammation by transferring antigen-specific Th cells  differentiated in vitro followed by the administration of the cognate antigen via enema. We show that cotransfer of induced Tregs (iTregs) suppressed Th1- and Th2-mediated colon inflammation. In contrast, colon inflammation induced by transfer of Th17 cells, was augmented by the cotransfer of iTregs. Furthermore, oral delivery of antigen potentiated Th17-mediated colon inflammation. Administration of a blocking antibody against cytotoxic T lymphocyte-associated antigen 4 (CTLA4) abrogated the effects of cotransfer of iTregs, while the injection of a soluble recombinant immunoglobulin (Ig) fusion protein, CTLA4-Ig substituted for the cotransfer of iTregs. These results suggest that antigen-specific activation of iTregs in a local environment stimulates the Th17-mediated inflammatory response in a CTLA4-dependent manner.Th17 cells and Foxp3 Ap. Ap+ T cl scores , was cal+CD11b+ mononuclear cells in the colon lamina propria (cLP) was more prominent in mice that received Th2 cells  –31. Moreantigens . Howeverm spleen . Howeveranalysis . Consistanalysis . Further+Gr-1+ cells in the cLP in addition to the induction of colon thickening. Th17 cells derived and differentiated from IL17A-deficient mice mediated these processes, although to a diminished but significant extent  phenotype in vivo to induce an antigen-specific IgA production [In contrast, recent reports show that Th17 cells acquired a follicular helper T cell  mice were obtained from The Jackson Laboratory . Il17A-KO mice were provided by Dr. Iwakura. Il10-KO mice and Il17A-KO mice were backcrossed with Balb/c genetic background more than 10 times in our facility to produce Il10-KO or Il17A-KO×DO11.10×Rag2-KO mice. Female mice 8 to 12 weeks older were used and bred in specific pathogen-free facilities at the Tokyo Metropolitan Institute of Medical Science. This study was carried out in strict accordance with the guidelines in the Proper Conduct of Animal Experiments, as defined by the Science Council of Japan and the Animal Care and Use Committees of the Tokyo Metropolitan Institute of Medical Science approved all experimental procedures (Permit Number:15035and 15036).Balb/c mice were purchased from Nihon SLC , DO11.10×The colon, starting from just below the cecum to above the anal, was excised. Connective tissues were removed, cut longitudinally, washed to remove fecal material, following which length and weight were measured to calculate the length-to-weight ratio. Independent experimental groups were used to obtain transverse sections of mid-to distal-colons. for histology. Specimens were formalin-fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.We purchased OVA from Sigma-Aldrich  and OVA peptide (323–339) from Scrum Inc. .eFluor 450-CD11b , APC-eFluor 780-Gr-1 (RB6-8C5), FITC- and Pacific Blue-IFN-gamma (XMG1.2), PE-Cy7-IL-4 (BVD6-24G2), Alexa Fluor 647- and Alexa Fluor 488-IL-17A (eBio17B7), PerCP-Cy5.5-FoxP3 (FJK-16s), PE-RORgammat (B2D), PE-IL-10 (JES5-16E3), APC-eFluor 780-CD4 (RM4-5), purified CD16/32 (93), and 7-AAD were purchased from eBioscience. We purchased fluorescein-CCR3 (#83101) from R&D Systems; PE-Cy5-CD45 (30-F11), PE-Siglec-F (E50-2440), PE-CD152 (UC10-4F10-11), and PE-DO11.10  from BD Biosciences; Alexa Fluor 488-CD25 (PC61) and Alexa Fluor 647-IL-17F (9D3.1C8) from BioLegend; and VioBlue-DO11.10 (KJ1-26) from Miltenyi Biotec. Purified anti-CD152(CTLA4) antibody (9H10) and isotype control antibody (SHG-1) used for in vivo studies was purchased from BioLegend, and anti-TGF-beta1,2,3 antibody (1D11) was R&D systems. The soluble fusion protein of CTLA4 and immunoglobulin (Ig) G1 Fc region  was purchased from Bristol-Myers Squibb.7 viable effector T cells were transferred intravenously with or without 1 × 107 viable iTregs.Antigen-specific effector T cells were prepared as described previously . ApproxiTwo hundred microliters of OVA solution (10 mg/mL dissolved in PBS) was injected intra-rectally with animal feeding needles , such that the tip was 4 cm proximal to the anus. This treatment was repeated five times daily at approximately 10 min intervals for each. To establish oral tolerance, mice were fed with drinking water supplemented with 1 mg/mL OVA for 7 days before adoptive transfer of cells.Rag2-KO) and OVA (50 mg/mL), and cytokines in the supernatants were measured using ELISA kits for IL-17F, IL-22, IL-21 and GM-CSF (eBioscience) or a multiplex bead array for IL-5, IL-13, IL-4, IL-10, IL-17A, and IL-2 (Millipore). Fecal extracts were obtained by adding weighed pellets to PBS  containing protease inhibitors . The samples were mixed and centrifuged, and the supernatants were collected for assay. OVA-specific IgA titers were determined using an ELISA with OVA as the capture antigen, and immune complexes were detected using horseradish peroxidase-conjugated anti-mouse IgA (Southern Biotech).Cells were activated using Dynabeads T-Activator CD3/CD28 (Life Technologies) or by culturing with antigen-presenting cells  and 1 mg/mL DNase I (Roche). Mononuclear cells were isolated using a discontinuous Percoll gradient (40% and 75%) and subjected to flow cytometry . Following stimulation with PMA (20 ng/mL) and ionomycin (1μM) in the presence of monensin for 4h, intracellular staining of the cells was performed using Foxp3 Fixation/Permeabilization Concentrate and Diluent (eBioscience) according to the manufacturer’s instructions.t test. A P-values of <0.05 was considered statistically significant.Data were analyzed using an unpaired two-tailed Student’s S1 Fig+T cells prepared from DO11.10+:Rag2-KO mice were polarized under conditions appropriate for each T cell lineage in the presence of APCs and OVA323-339 peptide (0.3microM), as described in Materials and Methods. (A) After 7 days, the cultured cells were restimulated with PMA (20 ng /mL) and ionomycin (1 μM) in the presence of monensin for 4 h. Cells were reacted with anti-CD4, anti-DO11.10 TCR, and anti-CD25 antibodies and treated with Fixable Viability Dye (FVD). The detection of intracellular expression of the indicated cytokines, transcription factors (FoxP3 and RORgammat), and CTLA4 is described in Materials and Methods. CD4+DO11.10 TCR+ FVD− cells were gated for the analysis. (B) Cells (1 × 106) were stimulated in the presence of APCs  and OVA (50 microg/mL) for 48 h and cytokine levels in culture supernatants were measured using multiplex bead assay.CD4(TIF)Click here for additional data file.S2 FigIl10-deficient Th2 cells and were not treated (none), or challenged with (OVA), or with (BSA) via enema.Wild-type mice were engrafted or not engrafted with (TIF)Click here for additional data file.S3 Fig+T cells (1 × 107) prepared from DO11.10+:Rag2-KO mice were transferred to wild-type BALB/c mice, and OVA protein was administered in a drinking water (1mg/mL) for a week (OVA-fed) or not (control). Mononuclear cells (MNCs) were isolated from the spleen (SPL) and colon lamina propria (cLP) of mice and subjected to the flow cytometric analysis. Frequencies of CD4+DO (KJ1.26)+ cells were shown and gated populations were analyzed for FoxP3 expression and ratio of FoxP3+ cells were shown in histograms. (B) Wild-type BALB/c mice were treated as described in (A). MNCs were isolated from the spleen (SPL) and subjected to the flow cytometric analysis as described in (A).(A) CD4(TIF)Click here for additional data file.S4 Fig+ T cells derived from Il10-deficient (Il10-KO:DO11.10+:Rag2-KO) or Il10-sufficient (Il10+/+:DO11.10+:Rag2-KO) mice. (A) Il10-sufficient (Il10-WT) and Il10-deficient (Il10-KO) Th2 cells were restimulated with 12-O-Tetradecanoylphorbol-13-acetate (PMA) (20 ng /mL) and ionomycin (1 μM) in the presence of monensin for 4 h. Cells were stained with mAbs against CD4, and the T cell receptor (DO11.10), and treated with Fixable Viability Dye, and subjected to flow cytometric analysis after incubation with antibodies against IL-4 and IL-10. The frequencies of cells expressing IL-4 or IL-10 were determined according to populations gated on CD4+DO11.10 TCR+ cells. (B) Il10-WT and Il10-KO Th2 cells were restimulated for 48 h in vitro using the anti-CD3epsilon-/anti-CD28-conjugated beads, and secreted cytokines were quantified as described in Materials and Methods. All experiments were reproducibly repeated at least twice, and a representative data set is shown. (C) Il10-WT Th2 cells or Il10-KO Th2 cells along with Il10-sufficient iTregs or Il10-deficient iTregs were transferred to wild-type BALB/c mice and mice. Mononuclear cells (MNCs) were isolated from the cLP of mice engrafted with each combination of cells as indicated and incubated with antibodies against CCR3 and Siglec-F. Representative flow cytometric profiles are shown with the frequencies of CCR3+Siglec-F+ cells in MNCs isolated from the cLP. (D) CTI values were calculated and are shown as mean and standard error (SE). (E) Il10-sufficient (Il10-WT) and Il10-deficient (Il10-KO) iTregs were restimulated as in (A) and stained with anti-CD4, anti-DO11.10 TCR, and anti-CD25 mAbs followed by Fixable Viability Dye staining. Cells were subjected to flow cytometry to determine the intracellular expression of IL-4, IL-10, CTLA4, and FoxP3. CD4+DO11.10 TCR+ FVD− cells were gated for the analysis.Th2 and iTregs were differentiated in vitro from CD4(TIF)Click here for additional data file.S5 Fig+ T cells were enriched using anti-CD4 magnetic beads (Miltenyi Biotech) and stained with indicated antibodies. Representative flow cytometric profiles are shown with the frequencies of CD3epsilon+DO+ cells and FoxP3+ cells gated on CD3epsilon+DO+ cells. (B) As a control for FoxP3 staining, Th17 cells were used and analyzed in the same way as described in (A).(A) MNCs were isolated from the spleen and cLP of mice engrafted with iTregs in the presence of anti-CTLA4 antibody (20 microg) or control antibody (20 microg) and treated as described in (TIF)Click here for additional data file.S6 Fig(A) Fecal extracts were prepared the day after the third challenge with OVA, and OVA-specific IgA was detected using ELISA to estimate relative endpoint titers as described in Materials and Methods. (B) Th17 cells with or without iTregs were transferred in the absence or presence of an anti-CTLA4 antibody (20 microg). After the third challenge with OVA, fecal samples were collected, and relative titers of OVA-specific IgA were determined.(TIF)Click here for additional data file.S7 Fig+CD4+ cells were shown and gated populations were analyzed for KJ1.26 staining and ratio of DO (KJ1.26)+ cells were shown in histograms.Effector T cells (Th2 or Th17) were intravenously transferred in the presence or absence of CTLA4-Ig (50microg), and mice were treated as described in (TIF)Click here for additional data file.S8 Fig+ T cells prepared from DO11.10+:Rag2-KO mice were stimulated under the condition for iTreg lineage, namely medium supplemented with IL-2, TGF-beta1 and retinoic acid (iTreg recipe), or medium containing IL-2 and TGF-beta1 but not retinoic acid (iTreg recipe without retinoic acid) in the absence (control) or presence of CTLA4-Ig . After 7 days, cells were subjected to the analysis as described in CD4(TIF)Click here for additional data file.


AtTORF-Ex  collection of plant lines overexpressing TF genes, we screened for those TFs which restore root growth on 9-HOT. Out of 6,000 lines, eight TFs were recovered at least three times and were therefore selected for detailed analysis. Overexpression of the basic leucine Zipper (bZIP) TF TGA5 and its target, the monoxygenase CYP81D11 reduced the effect of added 9-HOT, presumably due to activation of a detoxification pathway. The highly related ETHYLENE RESPONSE FACTORs ERF106 and ERF107 induce a broad detoxification response towards 9-LOX-oxylipins and xenobiotic compounds. From a set of 18 related group S-bZIP factors isolated in the screen, bZIP11 is known to participate in auxin-mediated root growth and may connect oxylipins to root meristem function. The TF candidates isolated in this screen provide starting points for further attempts to dissect putative signaling pathways involving 9-LOX-derived oxylipins.13-Lipoxygenase-derived oxylipins, such as jasmonates act as potent signaling molecules in plants. Although experimental evidence supports the impact of oxylipins generated by the 9-Lipoxygenase (9-LOX) pathway in root development and pathogen defense, their signaling function in plants remains largely elusive. Based on the root growth inhibiting properties of the 9-LOX-oxylipin 9-HOT , we established a screening approach aiming at identifying transcription factors (TFs) involved in signaling and/or metabolism of this oxylipin. Making use of the  Reactive Oxygen Species (ROS) is harmful to cell integrity and survival but is also a consequence of life in an oxygen-containing atmosphere. Lipid peroxidation yields a plethora of oxidized polyunsaturated fatty acids (FA), summarized as oxylipins [Reactive Electrophilic Species; RES), display toxic and/or mutagenic properties [Membrane oxidation by xylipins ,2. In anxylipins . Some oxoperties . On the operties ,3.Arabidopsis six LOX gene family members have been described, encoding 9-LOXes  and 13-LOXes  [In plants, oxylipins are formed non-enzymatically or by enzymatic activities ,4. Enzym3, 4, 6) ,8.Arabidopsis seedlings using various oxylipins with respect to root growth and their dependence on COI-1, at least three independent signaling pathways for oxylipins have been postulated [With respect to the plant pro-hormone Jasmonic Acid (JA), biosynthesis and signaling are well-described ,10. Percstulated . WhereasOxo-Phytodienoic Acid) or phytoprostane A1 (PPA1) have been shown to inhibit cell cycle progression and root growth [Arabidopsis bZIP factor genes (tga2 tga5 tga6) [In contrast to JA, our knowledge on the signaling of RES oxylipins in plants is limited. RES compounds such as the cyclopentenone OPDA 12-a5 tga6)  revealed-a5 tga6 a5 tga6) . One wela5 tga6) . Howeverlox1/lox5 double mutant display an increased number of lateral root primordia [Arabidopsis leaves upon Pseudomonas syringae infection [During the last years, evidence supporting a signaling function of 9-LOX-oxylipins in plant development and stress response has accumulated. When applied extracellularly, 9-HOT  and 9-KOT  reduce lateral root initiation. In contrast, genetic approaches using a 9-LOX specific rimordia . 9-HOT hrimordia ,16–19. Infection . Recentlnfection .Arabidopsis mutants in the response to 9-HOT  [coi-1 mutant. Some noxy mutants are allelic to the ethylene signaling mutants ctr1 (CONSTITUTIVE ETHYLENE RESPONSE1) and eto1 (ETHYLENE-OVERPRODUCER1) and accordingly, 9-HOT and ethylene were found to act antagonistically [Supporting evidence that 9-LOX-oxylipins perform as signaling molecules comes from genetic approaches identifying YLIPINS) . The resstically . NeverthAtTORF-EX  collection holds seeds of transgenic TF overexpressing plants, which can be used for identifying regulators in development or stress response [® technology, a batch transformation procedure has been developed, which results in seed pools derived from transformants of 30–50 TF genes under control of the Cauliflower 35S promoter (35S). Currently, this screening collection enables analysis of more than 700 TFs and has successfully been applied for several screening purposes [In order to get an insight into putative 9-LOX-oxylipin signaling pathways in plants, we set-up a screen aiming at TFs involved in the transcriptional response to 9-HOT. The response ,23. Usinpurposes . Here, wArabidopsis Columbia (Col-0) wt and AtTORF-Ex seeds [Roots tool software (zysko@tlen.pl). To study gene induction by flooding, 2-week old seedlings were grown on MS medium and submerged with water for 1–6h. For chemically-induced gene expression, 10 μM Estradiol  was added.For root growth assays, Ex seeds ,23 were Ex seeds  under lotga2,5,6, tag2,5, tag6 [tga2,5,6 TGA5g [tga2,5,6 (35S:TGA2) and tga2,5,6 (35S:TGA5) [cyp81d11 [erf106 (SALK_097771) and erf107 (SALK_015182) were obtained from the Nottingham Arabidopsis Stock Centre and crossed for generating erf106/erf107 double mutants.The following mutant lines were used: ,5, tag6 , the com,6 TGA5g  and tga25S:TGA5) , 35S:CYPcyp81d11 . The Estcyp81d11 ,29. T-DNS)-hydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid), 9-KOT (9-keto-10(E),12(Z),15(Z)-octadecatrienoic acid) and OPDA (12-Oxo-Phytodienoic Acid) were prepared as described in [The following compounds were tested in root growth assays using the indicated concentrations. 9-HOT  and ERF107 (At5g61590) orfs were PCR-amplified using the primers provided in Agrobacterium tumefaciens strain GV3101.AtTORF-Ex lines, PCR-amplification was performed as described in [–ΔCT method [UBQ5 (At3g62250) gene as a reference. Immunoblots were performed as described in [To identify TF genes in the selected ribed in  using prribed in  using SYT method  using thribed in  using α-Bioinformatic and statistical analyses were performed with the Excel GraphPad Prism, Origin and XCELL software using the statistic tests indicated in the figure legend.Arabidopsis seedlings on MS-Agar supplemented with 25 μM 9-HOT. In contrast to [AtTORF-Ex resource [®-derived Arabidopsis TF overexpression lines employing the 35S promoter and scored plants showing wildtype (wt) root growth after 9-HOT application to the media. Screening of more than 6000 seedlings recovered 201 individuals (3%) which display tolerance to 9-HOT  is strongly impaired in primary root growth on medium containing 19μM 9-HOT  (TGA-bZIPs (group D)  have alrμM 9-HOT . Althoug mutants . Whereasle to wt . Moreove (TGA5g)  or 35S-d (TGA5g)  could pa (TGA5g) . Taken tCYP81D11 is transcriptionally induced by lipid peroxidation products like phytoprostanes, OPDA and JA as well as electrophilic xenobiotics  [cyp81d11 mutant on 9-HOT is slightly but not significantly reduced. Most likely, other CYP450 proteins may share redundant functions. Further studies are needed to define the substrate specificity of CYP81D11 with respect to xenobiotics and endogenous compounds.TGAs have been proposed to be involved in controlling detoxification pathways with respect to oxylipins or xenobiotics. The cytochrome P450 monoxygenase gene ic acid) ,15. AlonAtTORF-Ex screen, bZIP11 and bZIP2 lines were most frequently found . This finding is in contrast to previous studies, demonstrating that overexpression of these bZIPs leads to severely reduced plant growth [AtTORF-Ex lines isolated showed reduced bZIP transcript levels  [AtbZIP41, AtbZIP16), which have been shown to heterodimerize with S and C bZIPs [i.e., precisely how these bZIPs control root apical growth, is currently not resolved. Particularly, 9-LOX-oxylipins have been implicated in controlling overall root growth and lateral root formation [Among the group S bZIP members identified in the t growth . We conft levels . These f2,11,44) . These t2,11,44) . bZIP11-2,11,44) –45. More2,11,44) . Moreove C bZIPs  were alsormation . Here, wAtTORF-Ex screen  family members from various subgroups have been identified in the x screen . ERF106 x screen . Along tx screen . As seveERF106 and ERF107 can be induced by hypoxia or flooding. RT-qPCR studies confirmed that both lines are knock-outs  Screening for restoration of root growth during treatment with an inhibiting compound has been found to be an easy-to-use approach. However, root growth as the screening output is pleiotropic and may be caused by numerous factors, which are not specific for the oxylipin compound used. (2) It is conceivable, that oxylipin signaling is closely linked to general plant stress, detoxification or developmental processes and hence, that their responses are channeled via general transcriptional regulators.Whereas the signaling properties of 13-LOX-oxylipins, such as JA is well-defined, the function of 9-LOX-derived oxylipins as signaling compounds is still a matter of debate. In particular, perception mechanisms are yet not elucidated. Screening approaches for components of signaling pathways are therefore straightforward . Here, win vivo. In animal systems, pathways leading to detoxification of xenobiotics are well-described and TF regulators are known [Here, we identified two sets of TGA and ERF factors, which both induces mechanisms of detoxification/metabolism related to a broader spectrum of oxylipin and xenobiotic compounds. It will be interesting to determine, whether independent detoxification pathways are used and which endogenous substrates are detoxified re known . In contre known . MoreoveS1 Figtga triple  double  and single tga6 mutants and the triple mutant complemented with a genomic TGA5 fragment (gTGA5) [t-test: *p ≤ 0.05, **p≤0.01, ***p ≤ 0.001.The  (gTGA5)  were ass(TIFF)Click here for additional data file.S2 FigAtTORF-Ex lines (A-C) revealed presence of the bZIP11 transgene, suggesting overexpression of this TF. However, recent publications demonstrate a severe dwarf phenotype resulting from bZIP11 ectopic expression41. As the AtTORF-Ex collections are build or primary transformants, the T2 generation of the three selected bZIP11-AtTORF-Ex lines segregates. RT-qPCR analysis of 5 offspring lines each (1–5) show no overexpression, but partially reduced bZIP11 transcript levels, presumably due to co-suppression. Hence, these data suggest, that the observed phenotype is due to lower bZIP11 expression.Combined PCR and sequencing analysis of three selected (TIFF)Click here for additional data file.S3 FigERF transcripts in the erf106, erf 107 and double mutants. Low background transcription (black) was enhanced by 4h flooding (white). Given are mean values + SD (n≥3); t-test: *p≤0.05, **p≤0.01.(A) Schematic view of the T-DNA insertions and primer attachment sites used for genotyping. (B) Homozygous mutants were identified by PCR genotyping using the primers given in (TIF)Click here for additional data file.S4 FigArabidopsis Col-0 seedlings were grown on MS-Agar supplemented with 25μM 9-HOT for 1 to 6h. ERF106 (blue) and ERF107 (green) transcript abundance relative to the uninduced situation was analyzed by RT-qPCR in shoot (A) and root (B). Given are mean values +SD (n≥3).2-week old (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The fully human monoclonal antibodies nivolumab (Nivo) and ipilimumab (Ipi) block the interaction between the immune checkpoint receptors programmed death-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4), respectively, and their cognate ligands, restoring antitumor immune response. Phase 1 studies of Nivo monotherapy  or Nivo+Ipi combination therapy  demonstrated durable clinical activity and objective response rates (ORRs) of 31% to 40%, respectively, in patients with advanced melanoma (MEL). Evaluation of PD-L1 expression using immunohistochemistry (IHC) suggested a correlation between pretreatment tumor PD-L1 expression and clinical response to Nivo monotherapy [MEL patients received Nivo monotherapy (n=107) or Nivo+Ipi combination therapy . Tumor and tumor infiltrating lymphocyte (TIL) surface programmed death ligand 1 (PD-L1) expression in formalin-fixed, paraffin-embedded (FFPE) tumor tissue was evaluated by IHC with an automated assay (Dako) using the 28-8 Ab. Tumor PD-L1 positivity (PD-L1+) was defined as ≥5% cell membrane staining of any intensity; any expression on TILs was considered positive. Absolute lymphocyte counts (ALC) were measured in serial peripheral blood samples and lymphocyte subsets were evaluated using flow cytometry.P< 0.05).Tumor PD-L1-positive expression was observed in 45% and 37% of samples from the 003 and 004 studies, respectively. In 003, inclusion of any immune cell staining increased PD-L1 positivity to 92%. A numerically higher ORR was observed in MEL patients with PD-L1+ tumors with Nivo monotherapy or with sequential but not concurrent combination therapy. Neither study demonstrated an obvious change in ALC; however, phenotypic changes in T-cell subsets, including increases in the percentage of CD4 and CD8 expressing HLA-DR, ICOS and/or Ki67, were seen with combination therapy. In both studies, responses were observed irrespective of tumor PD-L1 or ALC status. In an exploratory analysis low pretreatment myeloid derived suppressor cells (MDSC) correlated with higher ORR with combination therapy (PD-L1 positivity is associated with tumor response with Nivo monotherapy; however, some responses were observed independent of PD-L1 or ALC status. No correlation between response and PD-L1 or ALC status was seen with combination therapy. MDSC levels may correlate with response to combination therapy. Future phase 3 randomized studies will explore these markers and other phenotypic changes in immune cell populations that might predict activity of Nivo in patients with MEL and other advanced cancers.


The combined ALT-803+TA99 therapy significantly exceeded either monotherapy in inhibiting melanoma tumor growth (p < 0.001) and prolonging survival (p < 0.01) of B16F10 tumor-bearing mice. Through immune cell depletion studies and immunophenotyping of peripheral cells as well as tumor-infiltrating leukocyte subsets, we found that ALT-803 enhances TA99-mediated antitumor immunity through activation of NK cells and expansion of the CD8+CD44high memory T cell arm. In contrast, CD4+ T cells were shown to play more of a suppressive role in the therapeutic effect of ALT-803+TA99, possibly through involvement of regulatory T cells or ALT-803-mediated induction of PD-L1 on CD4+ T cells in the periphery and tumor microenvironment. Addition of anti-PD-L1 mAb to ALT-803+TA99 therapy resulted in a further increase in antitumor activity against subcutaneous B16F10 tumors. Furthermore, tumor-bearing mice that survived due to ALT-803+TA99 combination therapy exhibited long term antitumor memory against B16F10 tumor cell rechallenge. Immune-depletion studies revealed that this antitumor memory was associated with CD4+ T cells, CD8+ T cells and NK cells. Our findings suggest a therapeutic opportunity for ALT-803 in combination with tumor-targeting antibodies to simultaneously augment targeted antitumor activities of therapeutic antibodies and induce a long-term vaccinal effect which will provide durable responses in the treated host.Cytokine-based and antibody-targeted immunotherapies have both been important approaches in the treatment of malignant cancers. However, combinational therapies of cytokines and tumor-targeting antibodies remain to be further explored, especially in advanced solid tumors. In this study, C57BL/6 mice bearing established subcutaneous B16F10 melanoma were treated with mouse melanoma targeting anti-gp75 monoclonal antibody (mAb), TA99, combined with interleukin (IL)-15 based superagonist ALT-803. This soluble protein complex consists of an IL-15 superagonist mutant (IL-15N72D) associated with an IL-15 receptor α Sushi domain - human IgG1 Fc fusion protein. Compared to native IL-15, ALT-803 possesses superior


NF-κB activation depends on the IKK complex consisting of the catalytically active IKK1 and 2 subunits and the scaffold protein NEMO. Hitherto, IKK2 activation has always been associated with IκBα degradation, NF-κB activation, and cytokine production. In contrast, we found that in SCF-stimulated primary bone marrow-derived mast cells (BMMCs), IKK2 is alternatively activated. Mechanistically, activated TAK1 mediates the association between c-Kit and IKK2 and therefore facilitates the Lyn-dependent IKK2 activation which suffices to mediate mitogenic signaling but, surprisingly, does not result in NF-κB activation. Moreover, the c-Kit-mediated and Lyn-dependent IKK2 activation is targeted by MyD88-dependent pathways leading to enhanced IKK2 activation and therefore to potentiated effector functions. In neoplastic cells, expressing constitutively active c-Kit mutants, activated TAK1 and IKKs do also not induce NF-κB activation but mediate uncontrolled proliferation, resistance to apoptosis and enables IL-33 to mediate c-Kit-dependent signaling. Together, we identified the formation of the c-Kit-Lyn-TAK1 signalosome which mediates IKK2 activation. Unexpectedly, this IKK activation is uncoupled from the NF-κB-machinery but is critical to modulate functional cell responses in primary-, and mediates uncontrolled proliferation and survival of tumor-mast cells. Therefore, targeting TAK1 and IKKs might be a novel approach to treat c-Kit-driven diseases. The IKK complex consists of IKK1/2 and NEMO. IKK1 and 2 are the catalytically active components whereas NEMO is the regulatory scaffold protein of the IKK complex . StimulaThe RTK, c-Kit is critical for differentiation of cells of the haematopoietic system –12. MastThe IKK complex and NF-κB are important to mediate the effector functions of mature mast cells stimulated with ligands of the TIR superfamily  or by cWe show that stimulation of BMMCs with SCF, the overexpression of wt c-Kit or the expression of constitutively active c-Kit mutants mediates activation of IKK2. Surprisingly, the c-Kit-mediated IKK2 activation does not lead to NF-κB activation but is critical for differentiation, proliferation and survival of primary- and tumor-mast cells. Therefore, the c-Kit-mediated IKK2 activation regulates mast cell effector functions and might be a novel approach to treat c-Kit-driven diseases.Δikk2 mice . Membranes were washed in 0.1% Tween/TBS and incubated with HRP-conjugated secondary antibodies: anti-rabbit-Ig, anti-goat-Ig (Santa Cruz) and anti-mouse-Ig (Thermo-scientific). Detection was performed using ECL reagent (Pierce).Samples were separated in 10% sodium dodecyl sulfate (SDS)-Laemmli gels and transferred by electroblotting onto nitrocellulose membranes (biostep). Membranes were blocked with dry milk and incubated (overnight) with antibodies detecting phosphorylated/ non-phosphorylated proteins or Hemagglutinin (HA)-tagged TAK1. We used the anti-HA antibody , anti-pY719-c-Kit/c-Kit, anti-pY705-STAT3/STAT3, anti-pY694-STAT5/STAT5, anti-pS184/pS187-TAK1/TAK1, anti-pS176(IKK1)/pS-177(IKK2) or anti-pY199-IKK2 and anti-IKK1/2, anti-pS32-IκBNF-κB-EGFP- MC/9 cells (106 cells/ml) were IL-3-starved (1 h) and stimulated with SCF or IL-33 (8 h). After harvesting and washing with PBA-buffer, cells were analyzed for EGFP production by using an LSR II flow cytometer (BD) and FlowJo 9.8.0 .For detection of dead HMC-1.1 or HMC-1.2 cells, samples were washed with PBA . Cells were left untreated or were treated with propidium iodid (PI). For detection of mast cell-specific surface marker, cells from cell cultures or from the peritoneal cavity were washed with PBA. Non-specific binding was blocked with anti-CD16/CD32 (clone 2.4G2) and rat-IgG (Jackson). Cells were stained with biotinylated IL-33R antibody  and PE-5 cells/ml) were treated with DMSO or the IKK-inhibitor VII. BMMCs (106 cells/ml) were IL-3-starved (1 h) and pre-incubated (30 min) with vehicle (DMSO) or U-73122 (PLCγ inhibitor), BAPTA-AM (Ca2+ chelator), U0126 (MEK inhibitor), SP600125 (JNK inhibitor), the p38 inhibitor, SU6656 (SFK inhibitor), AG490 (Jak2 inhibitor), the IKK-inhibitor VII, the STAT3 inhibitor S3I-201  or Glivec® (imatinib mesylat) . BMMCs were stimulated with SCF, IL-33 or both. BaF/3 cells  were IL-3-starved (1 h) and treated with DMSO or the IKK-inhibitor VII. For proliferation assays cells were cultured for 54 h. [3H]-thymidine (1 μCi) in 25 μl complete IMDM (PAA) (without IL-3) was added to each well for additional 18 h. Incorporated radioactivity was measured by using a β-scintillation counter . For ELISA experiments supernatants were collected (after 8 h) and analyzed for cytokine production using matched pair antibodies (ebioscience).HMC-1.1 and HMC-1.2 cells (10HMC-1.1 and HMC-1.2 cells were treated with the IKK-inhibitor VII (48 h), harvested, washed (PBA-buffer) and treated with propidium iodide (PI). Cells were analyzed with the LSR II flow cytometer (BD) and FlowJo 8.1.1 (Treestar Inc.).5 BMMCs per well were resuspended in 80 μl Phenol Red-free DMEM (Life Technologies), were mixed with 20 μl Ringer's solution  and an equal volume of loading dye. Prior to stimulation with SCF cells were pre-treated with inhibitors (30 min).For calcium assays, the FLIPR Calcium 4 Assay Kit and the FlexStation3 microplate reader  were used. All recordings were done on PLL coated 96 well plates (Greiner). 1 × 10Samples of back skin or ear pinna were fixed in 4% (v/v) buffered formalin and embedded in paraffin ensuring a cross-sectional orientation. For detection of mast cells, 5 μm sections were metachromatically stained (Giemsa). Mast cell numbers were counted per ear or back skin section over a length of 1 cm. Mean of 3 sections per mouse was calculated ± SD.T-test. Statistical significance was accepted for p < 0,05 .All experiments were performed at least three times. Proliferation assays, and ELISAs were performed at least in a 6-fold determination. Cytokine concentration, stimulation indices (SI) and mast cell numbers per peritoneum are indicated as the mean of measurements ± standard error of the mean (SEM). The statistical analysis was performed with IBM SPSS Statistics version 21.0 . Statistical significance was assessed by students


All cause mortality increases with lower levels of estimated glomerular filtration rates. As part of a comorbidity focus, we assessed the eGFR in CSF-flow disorder patients guided by NICE recommendations, current literature recognising a relative hazard ratio per 15mls/min decrease in eGFR of 1.04[182 CSf-flow disorder patients who had eGFR measurement were grouped into the respective subtypes; IIH (n=41), NPH (n=72), CSF hypovolaemia (n=7) and other causes (n=45). Others included those investigated for NPH but found not to be suitable for shunt.Estimated GFR was categorised as per NICE guidelines; (ml/min/1.73m) in line with current clinical laboratory facilities;>90 normal high, 60-89 mild reduction related to normal range for a young adult, 45-59 mild-moderate reduction, 30-44 moderate-severe reduction, 15-29 severe-reduction, <15 kidney failure.The eGFR's were recorded and the mean calculated for each subgroup.IIH cohort; mean eGFR 77.73;26 patients >90 normal high, 14 patients in 60-80 mild reduction, and 1 patient 45-59 mild-moderate category.Primary NPH; mean eGFR 73.32mls/min; 18 patients >90 normal high, 35 patients 60-89 mild reduction, 17 patients 45-59 mild-moderate and 2 patients 30-44 moderate-severe category.Secondary NPH; mean eGFR 81.94mls/min; 16 patients 60-89 mild reduction range and 1 patient 45-59 mild-moderate category.Orthostatic HA; mean eGFR 87.25mls/min; 5 patients >90 normal high and 2 patients 60-89 mild reduction.Other cohort; mean eGFR 76.96mls/min; 22 patients >90, 21 patients 60-89 mild reduction and 2 patients 45-59 in the moderate-severe reduction.Of the 182 patients, 71 patients had high normal eGFRs >90mls/min representing 39% of patients. 48% fell into the 60-89 mild reduction range, 11.5% into the 45-59 mild-moderate reduction range and 1% into the moderate-severe reduction range.A reduced estimated GFR is commonly seen in the CSF-flow disorders patient cohorts. Given the emphasis towards comorbidity factors influencing surgical outcomes, this acts as a marker of comorbidity and physiological reserve for surgical tolerance and post-operative performance and should therefore be taken into consideration.The altered eGFR could represent the comorbid state of the patient or it could form an element of the underlying pathophysiology of CSF-flow disorders or indeed both. Larger studies are required with more translational medical research models.


These cells reside within the B cell follicles, are mostly IgM+IgD+, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils.Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19 B lymphocytes are key players in adaptive immune response due to their ability to differentiate into cells producing antigen-specific antibodies following encounter with micro-organisms or vaccination. B cells have been classified into various sub-populations including memory, germinal center and follicular B cells, each identified by particular phenotypic arrays of surface markers. Together, these populations constitute conventional B cells or B-2 B cells) which react adaptively to antigen challenges with antibody responses after differentiation in plasma cells by affinity maturation . In rece B cells +IgM+CD5+CD43+ and the ability to produce granulocyte–macrophage colony-stimulating factor (GM-CSF). These murine cells represent a transitional B-1a-derived population, reside in peritoneal and pleural cavities during the steady state, respond quickly after infection, and expand in the spleen during sepsis (or LPS stimulation) [A new subpopulation of B lymphocytes, called Innate Response Activator (IRA) B cells, has been described in mice. They can be identified by the expression of CD19ulation)  and atheulation) , and in ulation) .The production of GM-CSF by IRA B cells may exert different effects, depending on the pathology and on the compartments where they reside. During the onset of intestinal sepsis, IRA B cells may participate in neutrophil-dependent bacterial clearance , while iWe were interested in (i) evaluating whether IRA B cells could be identified in human palatine tonsils that, as strategic secondary lymphoid organs, represent a first line of defense against invasive microorganisms in the upper respiratory tract; (ii) characterizing them phenotypically, and (iii) investigating their potential function.We recruited patients undergoing tonsillectomy at the Otorhinolaryngology Unit of the University Hospital of Siena . Eligible tonsillectomized patients were clinically stable children (aged ≤ 16 years) with recurrent tonsillitis. Enrolment criteria were: ≥ 7 well-documented, clinically important, adequately treated episodes of throat infection in the preceding year, or ≥ 5 such episodes in each of the two preceding years, or ≥ 3 such episodes in each of the three preceding years. Written informed consent for each single patient was obtained from the next of kin, caretakers, or guardians of the minors involved in the study, which was approved by the ethical committee of Siena University.Tonsil tissues were maintained in cold HBSS medium in a plastic container until processing (within 1 to 3 h after surgery). Using a sterile scalpel, the tissue was fragmented in a Petri dish with 2 ml HBSS + 700 U/ml collagenase (Gibco) + 20 μg/ml DNase (Sigma). Samples were incubated for 40 min at 37°C. After enzymatic digestion, tonsil-derived cell suspensions were filtered twice through 70-μm diameter cell-strainers, washed, and suspended in 20 ml HBSS. Mononuclear cells were stratified on Ficoll-Paque PREMIUM , and centrifuged for 30 min at 1000×g at room temperature. The interphase ring containing mononuclear cells was transferred in 10 ml of HBSS; after 2 washes, mononuclear cells were counted with a Vi-cell XR counter (Beckman Coulter).The following anti-human antibodies were used for flow cytometry analysis: anti-CD19-BV605 SJ25C1, anti-CD20-APC-H7 2H7, anti-CD5-PECy5 UCHT2, anti-CD11b-V450 ICRF44, anti-CD27 PECy7 M-T271, anti-CD43-FITC 1G10, anti-CD183-PECy5 1C6, anti-CD185(CXCR5)-A488 RF8B2, anti-CD184-PECy5 12G5 (BD Pharmingen); anti-IgM-PE-CF594 G20-127, anti-IgG-V450 G18-145 (BD Horizon); anti-CD20-PerCP-Cy5.5 L27 (BD Biosciences); anti-CD93-PE R3, anti-IgD-PerCP-eFluor710 IA6-2, anti-CD11c-APC-eFluor780 BU15, anti-GM-CSF-eFluor660 GM2F3, anti-CD40-eFluor450 5C3 (eBioscience); anti-IgA-FITC IS11-8E10 (Miltenyi Biotec)anti-CD86-FITC 2331 (FUN-1) (BD Pharmingen); anti-CD284-AlexaFluor488 HTA125 (eBioscience). The following anti-human isotype controls were used: anti-IgG1-FITC, anti-IgG1-PECy5, anti-IgG1-PECy7 MOPC-21, anti-IgG2b-A488 A95-1, anti-IgG2a-PECy5 G155-178 (BD Pharmingen); anti-IgG1-V450 MOPC-21, anti-IgG1-PE-CF594 X40 (BD Horizon); anti-IgG1-eFluor450, anti-IgG1-eF660, anti-IgG1-APC-eFluor780 P3.6.2.8.1, anti-IgG1-PE P3, anti-IgG2a-PerCP-eFluor710 eBM2a, (eBioscience); anti-IgG1-FITC IS5-21F5 (Miltenyi Biotec); anti-IgG1-AlexaFluor488 (eBioscience). Acqua Live/Dead Stain (Life Tecnhnologies) was used to select live cells. For intra-cytoplasmic staining, cells were permeabilized and fixed using the Cytofix/Cytoperm Plus kit (BD Biosciences). Data were acquired on either LSRII special order or Fortessa flow cytometers (BD Biosciences), and analyzed with FlowJo .5/200 μl) were plated in medium alone , or in medium plus 10 μg/ml CpG (Primm), 10 μg/ml goat F(ab')2 anti-human IgM-UNLB (SouthernBiotech), or Staphylococcus aureus (S. aureus) Cowan I  at the final dilution of 1:20000. The incubation was performed in the presence of 1000 U/ml IL-2 (Peprotech) for 2 days at 37°C with 5% CO2. Brefeldin-A (Sigma) was added at 5 μg/ml 3 h before fixation and staining. Alternatively, 1.5 x 106 cells/200μl RPMI supplemented with 10% fetal calf serum were plated in a 96-well plate and immediately stained using the antibodies listed above.Tonsil-derived cells (5x105 (300 μl/well) tonsil-derived cells were seeded on a poly-L-lysine-coated coverslip in a 24-well plate. Cells were incubated for 2 h at 37°C or 4°C with or without heat-inactivated S. aureus or PhRodo Green S. aureus Bioparticles at a MOI of 10:1 (bacteria:cells). After incubation, cells were fixed with 2% formaldehyde for 30 min at RT, washed with PBS and permeabilized with 0.1% Triton X-100. Coverslips were then washed again with PBS and blocked with 3% BSA—10% goat serum (Invitrogen) for 20 min at RT. Finally, coverslips were washed with PBS and incubated for 30 min at RT with antibody mix containing human anti-CD19-V450 HIB19 (BD Pharmingen), anti-GM-CSF-eFluor660 GM2F3, anti-MPO-PE 455-8E6 (eBioscience), all diluted in 0.1% BSA + 1% goat serum. After incubation, coverslips were washed and mounted on a slide with Gold Anti-Fade reagent (Life Technologies) for analysis by confocal microscopy.5 x 10A portion of each tonsil was collected in dry conditions, immediately frozen in liquid nitrogen, and stored at -80°C until processing. Tonsil cryosections (8-μm thick) were obtained with the cryo-microtome CM1950 (Leica), fixed with 4% formaldehyde for 10 min at RT and washed three times with PBS. Sections were blocked with 1 mg/ml of human immunoglobulins (Gentaur) for 30 min at RT, washed with PBS and stained with an antibody mix containing anti-human CD19-V450 HIB19 (BD Pharmingen), anti-CD3-PE SK7 (BD bioscience), anti-GM-CSF-eFluor660 GM2F3 (eBioscience), anti-IgD-BrillantViolet421 IA6-2 (Biolegend), all diluted in 1 mg/ml human immunoglobulins for 1 h at RT. After incubation, sections were washed again with PBS and mounted with a coverslip using Gold anti-Fade reagent (Life Technologies) for analysis by confocal microscopy.Images from human tonsil samples were acquired by Axio Observer LSM700 confocal microscope (Zeiss) at 20°C, using Plan-Apochromat 40X or 100X objective lenses with 1.3 numerical aperture. The 40X and 100X objective lens was used with the Zeiss Immersol 518F imaging medium. Images were processed with Zen 2008 software (Zeiss).Statistical differences between groups were evaluated using the non-parametric Mann–Whitney test. P-values ≤ 0.05 were considered statistically significant.+CD20+GM-CSF+ cells, the gating strategy showed in in vitro, probably because of the chronically inflamed environment of the tonsils at the time of tonsillectomy. To confirm this hypothesis, we incubated tonsil-derived cells for 2 days with polyclonal stimuli  or Toll-like receptor (TLR) agonist (CpG). Even under these conditions, we did not observe a significant increase in the frequencies of GM-CSF+ hIRA B cells  never localized hIRA B cells out of the follicles, suggesting that the localization of these cells is restricted to the B cell areas. In addition, hIRA B cells were detected predominantly within the mantle zone of B cell follicles (IgD+ area) and not in GCs (IgD- area)  .et al. [+CD20+GM-CSF+) from conventional B lymphocytes (defined as CD19+CD20+GM-CSF-). Typical mature B cell markers CD19 and CD20 were clearly expressed by hIRA B cells; expression of CD40 and the chemokine receptor CXCR4 was also detected in all the samples analyzed , naïve B cells (CD38dimIgD+CD27-), germinal center B cells (CD38+IgD-), memory B cells (CD38dimCD27+IgG+) and plasma cells (CD38hiCD27+CD43+), data reported in our work suggest that the majority of hIRA B cells seem to be part of the small fraction of CD19+CD38-IgD+CD27- B cells and appear to be excluded from GC-dependent B cell maturation processes [+IgD+), allow us to conclude that the cell population characterized for the first time in human tonsils very likely represents the human counterpart of murine IRA B cells.The phenotypic properties of tonsil hIRA B cells were analyzed by flow cytometry evaluating the expression of a panel of surface markers modified by Rauch et al.  , and confocal microscopy analyses were performed after staining with anti-CD19, anti-GM-CSF, and anti-myeloperoxidase (MPO)  ,6, and pse (MPO) . InteresS. aureus bacteria are actively internalized by GM-CSF+CD19+ hIRA B cells within an acidic MPO+ compartment, and that this activity was exclusively restricted to hIRA B cells. The uptake of bacteria by tonsil hIRA B cells required a metabolically active phagocytic process, as the uptake was not observed when the experiment was carried out at 4°C. These findings suggest that hIRA B cells may have innate effector functions by entrapping invading pathogens in acidic compartments.Previous studies in mice reported that MPO is expressed by B-1a peritoneal B lymphocytes . In the In mice, antigens can be captured in the periphery by DCs, macrophages and neutrophils, and subsequently presented to MZ B cells secreting IgM by a T-independent pathway. In these conditions, secretion of GM-CSF by IRA B cells was suggested to enhance the survival and activation of antigen-presenting cells in the splenic red pulp . GM-CSF i) are present in human tonsils; (ii) exclusively reside within the follicular areas; and (iii) can actively phagocyte bacterial microorganisms. Further work is required to clarify a possible relationship between hIRA and follicular B cells, the role of hIRA in the adaptive immune response, and the potential cellular targets of GM-CSF within the tonsils. Additionally, somatic hypermutation (SHM) analysis of Ig genes would be required to clearly define the nature (naïve or memory) of hIRA B cells.In conclusion, the data presented in this work show for the first time that hIRA B cells Click here for additional data file.S2 FigExpression of CD3 (T cells), CD14 (monocytes) and CD38 (plasmacells) was analyzed in hIRA B cells. Flow cytometry plots showed that hIRA B cells are single positive for CD19.(TIF)Click here for additional data file.S3 Fig+GMCSF+ cells or CD19+GMCSF- cells. In panel B, we reported only the graph for CD27 and IgM, since the expression of CD86,CD284 (TLR4) was very low. Means and standard deviations are shown. P value was calculated using the non-parametric Mann–Whitney test (n = 4).Flow cytometry plots of CD27, IgM, IgD, CD284 (TLR4),CD86, CD43, CD5 and CD11c were shown as percentage (panel A) or as GeoMFI (panel B) on total CD19(TIF)Click here for additional data file.


Whether concomitant HIV antiretroviral therapy (ART) affects the safety and efficacy of interferon-free HCV therapies or whether HCV treatment may negatively affect HIV control is unclear. We assessed the 3 direct-acting antiviral (3D) regimen of ombitasvir, ABT-450  and dasabuvir with ribavirin (RBV) in HCV/HIV-1 co-infected patients with and without cirrhosis, including HCV treatment-experienced, receiving atazanavir (ATV)- or raltegravir (RAL)-based ART therapy.3 or CD4 + % ≥14%, and plasma HIV-1 RNA suppressed on stable ART received open-label 3D + RBV for 12 or 24 weeks. Rates of HCV-sustained virologic response at post-treatment weeks 4 and 12  and bilirubin-related adverse events (AEs) are reported from post-hoc analyses for subgroups defined by treatment duration and ART regimen.HCV genotype 1-positive treatment-naïve or pegIFN/RBV-experienced patients, with or without Child-Pugh A cirrhosis, CD4+ count ≥200 cells/mmThe SVR12 rate for patients receiving 12 weeks of 3D + RBV was 93.5% with comparable rates in patients receiving either ATV (93.8%) or RAL therapy (93.3%) . The SVRIn this first study to evaluate an IFN-free regimen in HCV genotype 1-positive treatment-naïve and experienced patients with HIV-1 co-infection, including those with cirrhosis, high rates of SVR were comparable to those with HCV monoinfection. Indirect hyperbilirubinemia was consistent with the known ABT-450 inhibition of the OATP1B1 bilirubin transporter, RBV-related haemolytic anaemia and inhibitory effect of ATV on bilirubin conjugation. The laboratory abnormalities and AEs observed did not negatively affect treatment response or lead to treatment discontinuation.


Experimental evidence indicates that prostate apoptosis response-4  is a key regulator of cancer cell survival and may be a target for cancer-selective targeted therapeutics. Par-4 was first identified in prostate cancer cells undergoing apoptosis. Both intracellular and extracellular Par-4 have been implicated in apoptosis. Relatively little is known about the role of Par-4 in breast cancer cell apoptosis. In this study, we sought to investigate the effects of Par-4 expression on cell proliferation, apoptosis and drug sensitivity in breast cancer cells. MCF-7 cells were stably transfected with expression vectors for Par-4, or transiently transfected with siRNA for Par-4 knockdown. Cell proliferation assays were performed using MTT and apoptosis was evaluated using acridine orange staining, fluorescence microscopy and flow cytometry. Par-4 overexpression reduced MCF-7 proliferation rates. Conversely, Par-4 knockdown led to increased MCF-7 proliferation. Par-4 downregulation also led to increased BCL-2 and reduced BID expression. Par-4 overexpression did not affect the cell cycle profile. However, MCF-7 cells with increased Par-4 expression showed reduced ERK phosphorylation, suggesting that the inhibition of cell proliferation promoted by Par-4 may be mediated by the MAPK/ERK1/2 pathway. MCF-7 cells with increased Par-4 expression showed a marginal increase in early apoptotic cells. Importantly, we found that Par-4 expression modulates apoptosis in response to docetaxel in MCF7 breast cancer cells. Par-4 exerts growth inhibitory effects on breast cancer cells and chemosensitizes them to docetaxel. Breast cancer is the most frequently diagnosed cancer in the industrialized world . Like otProstate apoptosis response-4 (Par-4), also known as PAWR, was first identified as upregulated in rat prostate cells undergoing apoptosis . Par-4 eExperimental evidence indicates that Par-4 plays an important role in tumor cell survival and may be considered a candidate for tumor cell selective therapy . This sePar-4 acts in both intrinsic and extrinsic apoptotic pathways. The mechanisms of Par-4 apoptosis induction involve activation of the Fas-FADD-caspase-8 apoptotic pathway, including the translocation of Fas/FasL to the plasma membrane and inhibition of the NF-κB pro-survival pathway ,17. Studμg/ml streptomycin, 0.25 μg/ml fungizon and 10% fetal bovine serum (FBS)  in a humidified atmosphere of 5% CO2 at 37°C. For assays involving drug treatment, cells were grown in medium supplemented with 5% FBS until they reached 60–70% confluence and the medium was replaced with RPMI-1640 medium without phenol red and supplemented with 5% FBS containing 5 or 100 nM docetaxel  and cells were cultured for an additional 24 h.Human MCF-7 breast cancer cells were purchased from the American Type Culture Collection (ATCC) and maintained in RPMI-1640 medium  supplemented with 100 U/ml penicillin, 100 μg/ml . The selected clones were maintained in 800 μg/ml geneticin and screened for Par-4 mRNA expression by real-time PCR, or for Par-4 protein by western blotting.MCF-7 cells were stably transfected with plasmid pcCMV6-XL6-Par-4 expressing a full length Par-4 cDNA, or empty vector (pcCMV6-XL6-Neo) acquired from OriGene . Transfection assays were performed in 6-well plates using TurboFectin 8.0 transfection reagent according to a standard protocol (OriGene). Twenty-four hours after transfection, MCF-7 cells were selected with geneticin 3000 ® . In brief, 1×105 cells were seeded and cultured in 6-well plates until they reached 30–50% confluence and transiently transfected with the specific Par-4 siRNA or scrambled oligonucleotides using TurboFectin 8.0 according to the manufacturer’s instructions (OriGene). Cells were harvested 24–120 h after transfection and screened for Par-4 mRNA by real-time PCR, or for Par-4 protein content by western blotting. Twenty-four hours after transfection, cells were harvested and plated in 96-well plates for cell proliferation assays using MTT, or seeded in 6-well plates for flow cytometric apoptosis assays.The duplex RNA oligonucleotides for Par-4 and silencer-negative control (scramble) were purchased from IDTPAR-4, forward 5′-CCAGAGAAGGGCAAGAGCTCGG-3′; reverse 5′-ATTGCATCTTCTCGTTTCCGC-3′; GADPH, forward 5′-CCTCCAAAATCAAGTGGGGCG-3′; reverse 5′-GGGGCAGAGATGATGACCCTT-3′; BCL-2, forward 5′-CGACTTCGCCGAGATGTCCAG-3′; reverse 5′-CCGTCC CTGAAGAGCTCCTCC-3′; BAX, forward 5′-GGCCGG GTTGTCGCCCTTTTC-3′; reverse, 5′-GTCCAGCCCATG ATGGTTCTG-3′; BID, forward 5′-AACTCCTGTGACCAC AACATG-3′; reverse 5′-CAGGAAAGCATCTGGTAA GAA-3′; BECLIN forward, 5′-ACTTTCCAGAGCTACAAC ATG-3′; reverse 5′-GTCCATGGGGTTAAGAATCAA-3′. Relative expression was calculated by 2−ΔΔCT ; ΔΔCT = ΔCT of the target sample − ΔCT of the reference sample). The average value of the control cells served as the reference sample. The results were expressed as n-fold differences in mRNA expression relative to expression of GAPDH and the reference sample.For quantitative RT-PCR analysis, cells were harvested and total RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform method. cDNA synthesis was performed using the High Capacity cDNA Archive kit . Real-time PCR (qPCR) was performed using the Power SYBR Green kit  following the manufacturer’s recommendations. qPCR reactions were processed in the GeneAmp 7500 Sequence Detection System (Applied Biosystems) under the following conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 55°C for 1 min. The PCR primers used were: for 3VO4), followed by centrifugation at 13,000 rpm for 15 min at 4°C. Thirty micrograms of protein lysate were separated on a 10% SDS-PAGE gel and blotted onto nitrocellulose membranes . Blots were incubated with anti-Par-4 monoclonal antibody , anti-p-ERK, anti-ERK  and anti-β-actin mouse monoclonal antibody  for 2 h at room temperature. Membranes were washed in TBS-T  and incubated with appropriate peroxidase-conjugated secondary IgG antibody for 2 h at room temperature. Incubations were performed in 5% skim milk diluted in TBS-T. Specific proteins were detected using an enhanced chemiluminescence system  and exposed to Hyperfilm ECL film .Cells were harvested and total cell lysates were prepared in lysis buffer  and maintained in RPMI-1640 medium without phenol red supplemented with 5 or 0.5% FBS. Cell growth was assessed at 0, 48, 72, 96 and 144 h. At the end of incubation, the absorbance was measured at 570 nm using the Biotrak II Plate reader .Cell proliferation and viability were measured using a 3--2,5-diphenyltetrazolium bromide  assay following the manufacturer’s instructions. Cells were seeded in 96-well plates  for 48 h before treatment. For immunocytochemistry, cells were fixed using 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Next, cells were incubated with primary mouse monoclonal anti-Par-4 antibody 1:50  and rabbit polyclonal anti-tubulin antibody 1:300 (OriGene), followed by the appropriate secondary antibodies conjugated with Alexa Fluor 546 and Alexa Fluor 488 1:300 . Nuclei were counterstained with Hoechst 33342 1:3000 (Invitrogen). Cells were visualized with Carl Zeiss LSM 510 Meta  laser scanning confocal microscopy. All immunofluorescence images were recorded at magnification ×20 and ×63.μg/ml RNase A, 0.1% Triton X-100 and 20 μg/ ml propidium iodide (PI) and immediately analyzed by fluorescence-activated cell sorting using the Becton-Dickinson FACSort flow cytometer . The cell cycle analysis program CellQuest (BD Biosciences) was used to determine the percentage of cells at different stages of the cycle  and the percentage of cell death using sub-G1 cells.Floating and adherent cells were collected, washed once with PBS, fixed with 70% ice-cold ethanol and stored at −20°C until analysis by flow cytometry. The fixed cells were washed twice with PBS, resuspended in PBS containing 200 ® Acoustic Focusing Cytometer . Apoptosis was also analyzed using a double-fluorescence staining technique with Hoechst 33342 and acridine orange (AO). Briefly, transfected MCF-7 cells treated with docetaxel (5 or 100 nM) for 24 h were co-stained with AO (8.5 μg/ml) and with nuclear dye Hoechst 33342 (5 μg/ml) for 10 min in the dark and examined under magnification ×20 and ×63 using a fluorescence microscope. Viewed by fluorescence microscopy, viable cells appear to have an intact and bright green nucleus, while apoptotic cells exhibit a bright green nucleus in which chromatin condensation is observed as dense green areas. Apoptotic cells were calculated as the ratio of apoptotic cells (with characteristic apoptotic morphology) to total cells. At least 10 fields were counted for each treatment, using the ImageJ Launcher software .Apoptosis was evaluated in MCF-7-pcPar-4 and MCF-7-pcNeo cells before and after treatment with 5 or 100 nM docetaxel for 24 h using the Annexin V-FITC Apoptosis Detection Kit I , following the manufacturer’s instructions. Cytometric analyses were performed using the AttuneStatistical analyses were performed by Student’s and Mann-Whitney tests using Statistical Package for the Social Sciences 20.0 . P-values were considered statistically significant at P<0.05. All data are presented as the mean ± SD of three or more replicate experiments.To evaluate the possible effects of Par-4 expression on cell proliferation, MCF-7 cells were transfected with expression vector pCMV6-XL6-Par-4 (MCF-7-pcPar-4) or with the empty vector pCMV6-XL6-Neo (MCF-7-pcNeo). Clones were selected with geneticin and were characterized by qPCR and western blotting. MCF-7-pcPar-4 tranfectants exhibiting increased Par-4 protein expression compared with MCF-7-pcNeo transfectant cells showed no significant morphological differences. Cell proliferation was significantly inhibited in MCF-7 cells expressing increased Par-4 compared with control MCF-7-pcNeo transfectants . BecauseWe also examined whether increased Par-4 expression affected the expression profile of genes involved in apoptosis  and autophagy (BECLIN). MCF-7-pcPar-4 cells exhibited slightly reduced BAX expression and marginally increased BID expression relative to the control cells . No detePar-4 overexpression did not affect the cell cycle profile . HoweverTo better characterize the effects of Par-4 on breast cancer cell proliferation, MCF-7 cells were transiently transfected with specific siRNA duplexes. Three different siRNA duplexes synthesized by TriFECTA were tested for Par-4 knockdown efficiency. Two Par-4 siRNAs (named siSeq1 and siSeq3) efficiently reduced Par-4 protein and mRNA expression in MCF-7 breast cancer cells compared with the control scramble siRNAs .The following experiments were performed with MCF-7 cells transfected with a mixture of siSeq1 and siSeq3 siRNAs and control cells transfected with control siRNA. Western blot analysis revealed that reduced Par-4 expression leads to increased ERK phosphorylation , which iWe also evaluated the effect of Par-4 knockdown on expression of the pro-apoptotic genes BAX and BID, the anti-apoptotic gene BCL-2 and the pro-autophagic gene BECLIN. Compared with MCF-7 cells transfected with scrambled siRNA, we observed significantly decreased BID mRNA expression  and significantly increased BCL-2 protein expression (4.76-fold) in MCF-7 cells transfected with Par-4-specific siRNAs . MCF-7 cPar-4 overexpression has been associated with increased sensitivity to apoptotic stimuli in different cell types . To evalA semi-quantitative analysis of the number of apoptotic cells was also performed by AO/Hoechst double labeling. MCF-7-pcPar-4 cells displayed a higher proportion of AO/ Hoechst double-stained cells exhibiting condensed chromatin, nuclear fragmentation, nucleolar disappearance, increased nuclear fluorescence and the appearance of granular apoptotic bodies compared with control cells (MCF-7-pcNeo) with vehicle  and in low and high concentrations of docetaxel  .The present study provides evidence that Par-4 plays an important role in cell proliferation and survival in breast cancer cells. Our findings indicate that elevated levels of intracellular Par-4 can sensitize MCF-7 cells to apoptosis and growth inhibition by docetaxel, which is a fundamental and effective chemotherapeutic agent against primary and advanced breast cancer, suggesting that intracellular Par-4 are involved in the pro-apoptotic and growth inhibitory action of docetaxel in breast cancer cells.et al  protein expression. Conversely, MCF-7 cells with increased Par-4 expression exhibited slower proliferation and decreased p-ERK protein expression compared with control cells. ERK kinase participates in the Ras/Raf/MEK/ERK signaling pathway, which is activated by mitogens and growth factors . ERK canc. Thus, Bid acts by amplifying the apoptotic signal by also activating the mitochondrial pathway  and other taxanes, such as paclitaxel, are anti-microtubule drugs used as a therapeutic regimen for patients with breast, lung and ovarian cancer . TreatmeIn colon cancer cells, Par-4 overexpression led to increased apoptosis in the presence of 5-fluorouracil chemotherapy by inhibiting NF-κB activity . The incTaken together, these findings suggest that Par-4 expression increases the sensitivity of breast cancer cells to chemotherapeutic agents and co-parallel elevation of Par-4 may be an effective strategy to increase the efficacy of docetaxel treatment in breast cancer.Our findings demonstrate for the first time that Par-4 modulates cell proliferation and survival in MCF-7 breast cancer cells. We show that PAR-4 inhibits MCF-7 cell growth, possibly through the inhibition of ERK-driven proliferation. Our results also show that Par-4 expression increases the rate of early apoptosis and increases the sensitivity of MCF-7 cells to docetaxel treatment. Our findings have translational relevance as they suggest that co-parallel elevation of Par-4 may increase the efficacy of breast cancer therapy.


The valve wasrecaptured and repositioned during deployment (1C-E). Immediate (1F), one (1G) andthree-month (1H) transthoracic echocardiography confirmed significant reduction intransaortic gradients. The patient remained in functional class I.A 41-year-old man with history of surgical replacement of the aortic valve with a21mm-Mitroflow bioprosthesis (1A), presented with functional class IV heart failure.Transesophageal echocardiography confirmed severe bioprosthesis obstruction (1B). Weimplanted a 23mm-CoreValveOur experience in repositioning the valve during a valve-in-valve procedure demonstratesthe usefulness of this resource in such challenging procedures.


Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively.Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute ( Orbivirus, family Reoviridae (–Bluetongue virus (BTV) is the type species of the genus oviridae , 2, thatoviridae , 4. The oviridae –9.BTV strains from different continents have evolved separately, acquiring multiple point mutations, developing characteristic regional variants/“topotypes” of each genome segment. Full-genome sequence data are available for BTV-2 from Taiwan , belongiCulicoides throughout the year), together with susceptible and high-density sheep populations, all help to maintain the high incidence of BT. Until 1981, clinical BT in India was confined to exotic sheep breeds. However, severe outbreaks subsequently occurred in native Indian breeds  in native sheep breeds in Karnataka, Maharashtra, and Andhra Pradesh states, suggesting introduction of exotic BTV strain(s)  . Nine BTtrain(s) , with BTtrain(s) , Tamil ntrain(s) .http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01) at the Pirbright Laboratory, was grown in BHK-21 cells. Extracted genomic dsRNA was used to synthesize full-length cDNAs by reverse transcription (RT)-PCR, followed by sequencing on a 3730 ABI capillary sequencer using segment-specific primers  to South African isolates (w) .Similarly, Seg-9/VP6 shows 99% nt identity with the BTV-10(w) isolate USA/10O80Z [U55781]. Although Seg-2/VP2(OC1) of IND1994/01 shows up to 99% nt identity with other eastern BTV-2 strains, confirming its serotype. Seg-6/VP5(OC2) is identical to Indian BTV-1 strains (IND2003/05), demonstrating reassortment between its two outer-capsid protein genes. It would be useful to investigate the serological characteristics of this virus. Full-genome sequence data for this reassortant BTV-2(r) strain will facilitate investigations of phylogenetic and serological relationships between Indian BTVs.KP268774 to KP268783.The complete genome sequence of BTV isolate IND1994/01 was deposited in GenBank under the accession numbers


Akt1 deficiency abolished the miR-17/20-mediated apoptosis. These results demonstrated a novel pathway through which miR17/20 regulate p53 and Akt controlling breast cancer cell apoptosis.The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Herein, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse, demonstrates a requirement for Akt1 in miRNA-mediated cellular apoptosis. The miR-17/20 cluster is known to inhibit breast cancer cellular proliferation through G1/S cell cycle arrest via binding to the cyclin D1 3'UTR. Here we show that miR-17/20 overexpression sensitizes cells to apoptosis induced by either Doxorubicin or UV irradiation in MCF-7 cells via Akt1. miR-17/20 mediates apoptosis via increased p53 expression which promotes Akt degradation. Akt1  Akt1, also known as protein kinase B (PKB), is frequently hyperactivated in human cancers. Akt1 is recruited to the plasma membrane in the presence of phosphoinositide triphosphate . Akt1 plays a central role in the ability of external signals to promote cell survival by preventing cytochrome c release from mitochondria . Substrate specificity of the Akt1 isoforms is similar, although Akt1 is the predominant isoform expressed in most tissues. Constitutive activation of Akt1 kinase occurs in human cancer through deletion and mutation of the tumor suppressor gene PTEN, the phosphatase that negatively regulates Akt1, through amplification of the Akt1 genes, or through amplification of the catalytic subunit of PI3 kinase .Akt1 kinase activity . The expression of p53 and p27ent Fig. . Doxorubent Fig. . Consistent Fig. .The effect of miR-17/20 was next assessed in the NAFA cell line which was derived from an ErbB2-induced mouse mammary tumor. miR-17/20 transduction increased the sensitivity of NAFA cells to doxorubicin-induced apoptosis as assessed by TUNEL staining Fig. . The indThe anti-estrogen tamoxifen is the most commonly used treatment for patients with estrogen-receptor α (ER)-positive breast cancer. Tamoxifen resistance occurs in ERα+ breast cancer cells including MCF-7. miR-17/20 transduced MCF-7 cells and control were treated with 15 uM tamoxifen up to 36 hours. The relative cell survival was determined using the MTT assay. As shown in Figure 50 of MCF-7 cells to doxorubicin (48h treatment). The MCF-7 cell growth curve with 500 nM doxorubicin treatment demonstrated significantly enhanced sensitivity to doxorubicin to doxorubicin after 24h of treatment  of doxorubicin for 48h and 72h, followed by quantitative analysis of cell survival. Compared to control cells, miR-17/20 transduced MCF-7 showed increased sensitivity to 400 nM and 500 nM doxorubicin. The reduction in cell survival of miR-17/20 transduced cells was most pronounced (~20% vs. ~35%) after 72h of treatment Fig. . miR-17/ent Fig. .Akt and p53 both are important regulators of stress signal-induced cellular apoptosis . Using In the current studies, miR-17/20 enhanced tamoxifen-induced apoptosis. Addition of tamoxifen induced apoptosis with a 4-fold increase in cellular apoptosis at 24 hours. In prior studies, tamoxifen-induced apoptosis was shown to involve the estrogen receptor ERα and additional signaling kinases, including protein kinase C, calmodulin TGF-α, map kinases including JNK and p38 map kinase, and oxidative stress involving the mitochondrial permeability transition ). 10 uM cyclin D1 gene expression via a 3' UTR binding site. The cyclin D1 3' UTR is spliced in the normal population  on the 1,220 predicted target genes revealed a cell death network  and DNA-dependent Protein Kinases (DNA-PK) which sequentially implement the DNA damage response. In resting cells the levels of p53 protein are low due to the activity of E3 ubiquitin ligases . Akt2 was required for irradiation-induced p53 stabilization  were isolated from MMTV-ErbB2 mammary gland tumors generated in bi-transgenic mice, and maintained as previously described , RNAiMax (Invitrogen) was used following the manufacturer's instructions.miR-17/20Retroviral production and infection methods were described in detail before . The ecoWhole-cell lysates (50 μg) were separated by 10% SDS-PAGE, and the proteins were transferred to nitrocellulose membrane. The following antibodies were used for Western blotting: anti-cyclin D1 from Neomarker ; anti-Bcl-Xs from Calbiochem ; anti-β-Tubulin (sc-9104), anti-β-actin (sc-47778), anti-p53 (sc-6243G), anti-p57 (sc-1040), anti-p27 (sc-528), anti-Cytochrome C (SC-7159), anti-Bax (sc-7480), anti-BCL-2 (sc-7382), anti PARP (sc-7150), anti-Caspase 9 (sc-8355), anti-Caspase 3 (sc-7148) were from Santa Cruz Biotechnology ; anti-GDI from RTG Solutions ; anti-Akt1 (#2967 ), anti-Akt2 ( #5239 ), anti-Akt3 (#4059 ), anti-p-y-H2A ( #9718s ) were from Cell Signaling.puro-miR-17/20 cluster or the pMSCVpuro empty vector. After puromycin selection, 4x104 cells were seeded into each well of a 6-well plate in triplicate and cell number was counted daily for 3 to 4 days under a microscope using a hemocytometer. For the 3--2,5-diphenyltetrazolium(MTT) assay, 4x103 cells/well were seeded into a 96-well plate in triplicate and cell growth was measured after 24 hours' culture.MCF-7 Cells were infected with the pMSCVIn Situ Cell Death Detection Kit, TMR red  following the manufacturer's instructions.miR-17/20 transduced cells and control were cultured in medium containing doxorubicin (0.05μM). After 24-48 hours cells were plated into 96-well plate in triplicate. Apoptosis assays were performed using the +/+Akt1 and −/−Akt1 murine ErbB2 breast tumor cells were incubated with fluorochrome-conjugated Annexin V followed by Propidium Iodide staining solution treatment. Flow cytometry analysis was applied to measure apoptotic cells.http://www.targetscan.org/). The predicted target genes were further analyzed for pathway analysis and network analysis using Ingenuity Pathway Analysis (IPA).The target gene prediction of hsa-miR-17 and hsa-miR-20 was performed using TargetScan Human 6.2 version (released June 2012) (Data are presented as mean + SEM. The standard two-tailed student's t-test was used for analysis and P<0.05 was considered significant.