O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38–54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2–6 sialylated N-acetyl-D-galactosamine  on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2.Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated  Podoplanin (PDPN), the endogenous ligand of C-type lectin-like receptor-2 (CLEC-2) ,2, is hiα2–6 linked sialic acid residue [O-glycan on Thr52 is required for the platelet aggregating activity of hPDPN [Interaction of human PDPN (hPDPN) with CLEC-2 mainly involves Glu47 and Asp48 in the platelet aggregation-stimulating domain-3 (PLAG3) and the  residue . The seq residue . CLEC-2  residue . In our of hPDPN . TherefoHere, we describe the development and characterization of a new anti-hPDPN mAb, LpMab-12, which specifically binds to glycosylated Thr52, and might serve as a novel modality to study hPDPN-CLEC-2 interaction.2 and 95% air. LEC was cultured in endothelial cell medium EGM-2MV supplemented with 5% FBS (Cambrex Corp.). Antibiotics including 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 25 μg/ml of amphotericin B  were added to all media.Chinese hamster ovary (CHO)-K1, LN229, HEK-293T, COS-7, and P3U1 were obtained from the American Type Culture Collection . Human lymphatic endothelial cell (LEC) was purchased from Cambrex . The human glioblastoma cell line, LN319, was donated by Dr. Kazuhiko Mishima . LN229 was transfected with human PDPN plasmids (LN229/hPDPN) using Lipofectamine 2000  according to the manufacturer’s instructions [Three female BALB/c mice (four-weeks old) were purchased from CLEA Japan . Animals were housed under pathogen-free conditions. "The Animal Care and Use Committee of Tohoku University" approved the animal experiments described herein. The use of one oral cancer tissue was reviewed and approved by "Tokyo Medical and Dental University Institutional Review Board" . Written8 LN229/hPDPN cells together with Imject Alum (Thermo Fisher Scientific Inc.), as previously described [Three BALB/c mice were immunized by intraperitoneal (i.p.) injection of 1 × 10Recombinant hPDPN or glycopeptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.) at a concentration of 1 μg/ml for 30 min. After blocking with 1% BSA in 0.05% Tween20/phosphate buffered saline , the plates were incubated with culture supernatant followed by 1:1000 diluted peroxidase-conjugated anti-mouse IgG or anti-rat IgG . The enzymatic reaction was conducted with a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories Inc.).Cell lysates (10 μg) were boiled in sodium dodecyl sulfate (SDS) sample buffer . The proteins were electrophoresed on 5–20% polyacrylamide gels  and were transferred onto a PVDF membrane . After blocking with 4% skim milk  in 0.05% Tween20/PBS, the membrane was incubated with 1 μg/ml of LpMab-12, LpMab-7, 1E6 , RcMab-3 (anti-IDH1) , or AC-1Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA . After washing with PBS, the cells were incubated with LpMab-12 (1 μg/ml) for 30 min at 4°C, followed by the incubation with Oregon Green 488 goat anti-mouse IgG (Thermo Fisher Scientific Inc.). Fluorescence data were collected using a Cell Analyzer EC800 .5 cells) and LEC (1 × 105 cells) were resuspended in 100 μl of serially diluted LpMab-12 (0.061–100 μg/ml) followed by Oregon Green 488 goat anti-mouse IgG (Thermo Fisher Scientific Inc.). Fluorescence data were collected using a cell analyzer . The apparent dissociation constants (KD) were obtained by fitting the binding isotherms using the built-in one-site binding models in GraphPad PRISM 6 .LN319 . Samples were blocked in 10% normal donkey serum  for 30 min at room temperature, incubated with 10 μg/ml of LpMab-12 overnight at 4°C, and then with Alexa Fluor 568-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific Inc.) for 1 h at 37°C. Subsequently, the sections were incubated with goat anti-human LYVE-1  and mouse anti-α-sarcomeric actin (α-SA)  for 2 h at 37°C, followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG and Alexa Fluor 647-conjugated donkey anti-mouse IgM  and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)  for 1 h at 37°C. The sections were then treated with 1% solution of Sudan Black B (Sigma-Aldrich Corp.) for 30 min at room temperature, and mounted in Vectashield medium . Images were acquired with Olympus FluoView FV100 laser scanning confocal microscope equipped with CCD camera (Bio-Rad Laboratories Inc.).Four-μm-thick histologic sections were deparaffinized in xylene and rehydrated. Without antigen retrieval procedure, sections were incubated with 1 μg/ml of LpMab-12 or LpMab-7 for 1 h at room temperature followed by treatment with Envision+ kit (Dako) for 30 min. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride , and then the sections were counterstained with hematoxylin .2 , and 250 μM CMP-Neu5Ac (Sigma-Aldrich Corp.) was used. A half volume of purified ST6GalNAcT-I enzyme was added to the reaction mixture and incubated at 37°C for 24 h. The recombinant ST6GalNAcT-I enzyme was bound to anti-FLAG M2 affinity gel (Sigma-Aldrich Corp.). After enzymatic reaction, the resin was removed by filtration using an Ultrafree-MC column (EMD-Millipore). Then, the glycopeptides were purified using a reversed-phase SPE cartridge . The other glycopeptides were produced sequentially as previously described [The hPDPN glycopeptide (hpp3854) with a GalNAc residue was purchased from Peptide Institute , and used as an acceptor substrate. For synthesis of the sialylated GalNAc on hpp3854, 25 mM HEPES (pH 7.0) containing 30 μM of acceptor substrate, 10 mM MnClInhibition assays were performed by ELISA. The recombinant proteins of hPDPN-Fc  and hCLE1, kappa) was developed. We immunized mice with hPDPN-expressing LN229 glioma cells (LN229/hPDPN), which possess cancer-type glycan patterns including highly sulfated polylactosamine and aberrant sialylation [As shown in −8 M for LN319 and 1.8 × 10−8 M for LEC, suggesting that the binding affinity of LpMab-12 is comparable with previously established anti-hPDPN mAbs [Using flow cytometry analysis, apparent dissociation constant of LpMab-12 was determined to be 1.2 × 10DPN mAbs  for hPDPTo confirm the utility of LpMab-12 for immunolabeling of tissues, we compared the reactivity of LpMab-12 with LpMab-7, the most sensitive anti-hPDPN mAb for this type of analysis . Both LpTo determine the critical epitope for the LpMab-12 interaction with hPDPN, we compared the mAb binding to the hPDPN carrying different point mutations. Using Western blot, we found that LpMab-12 did not detect protein sequences with the following amino acid substitutions: D49A, V51A, T52A, and P53A .O-glycan on Thr52 is critical for platelet aggregating activity of hPDPN [O-glycan on Thr52 in the Asp49-Pro53 sequence of hPDPN.In agreement, flow cytometry analysis demonstrated that LpMab-12 did not react with D49A, T52A, and P53A mutant proteins  Thus, ouof hPDPN . Therefo49-DVVTP-53 P-53 .1, kappa) with LpMab-12 [in vitro assay.Finally, we determined whether LpMab-12 inhibits hPDPN-CLEC-2 interaction using ELISA. Additional anti-hPDPN mAbs such as LpMab-2, LpMab-3, and LpMab-9 were employed as controls. Similarly to LpMab-12, LpMab-2 , LpMab-3LpMab-12 . As showLpMab-12  also modet al., also recognize PLAG1-3 domains, which are shown to be immunodominant antigenic sites of PDPN [O-glycosylation in hPDPN: LpMab-2 on Thr55/Ser56, LpMab-3 on Thr76, and LpMab-9 on Thr25. Although the glycosylation on Thr52 is the most critical for the binding of hPDPN to CLEC-2 and platelet aggregating-activity of hPDPN [As of today, almost all anti-hPDPN mAbs produced using conventional methods react with the non-glycosylated peptide spanning the PLAG1-3 domains ,12,31 or of PDPN . Recentl of PDPN ,29,36–38of hPDPN , no GpMa1, kappa), which specifically detects the glycosylation on Thr52 of hPDPN by flow cytometry Click here for additional data file.S1 File(DOCX)Click here for additional data file.


In the later phase of inoculation, PCV2 further activated p38 MAPK and ERK pathways to enhance IL-10 production by promoting Sp1 binding to the il10 promoter. For PCV2-induced IL-10 production in macrophages, PCV2 capsid protein Cap, but not the replicase Rep or ORF3, was the critical component. Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways to enhance IL-10 expression. In the whole process, gC1qR mediated PCV2-induced PI3K/Akt and p38 MAPK activation to enhance IL-10 induction by interaction with Cap. Depletion of gC1qR blocked PI3K/Akt and p38 MAPK activation, resulting in significant decrease in IL-10 production in PCV2-inoculated cells. Thus, gC1qR might be a critical functional receptor for PCV2-induced IL-10 production. Taken together, these data demonstrated that Cap protein binding with host gC1qR induction of PI3K/Akt and p38 MAPK signalings activation is a critical process in enhancing PCV2-induced IL-10 production in porcine alveolar macrophages.Porcine circovirus type 2 (PCV2) infection caused PCV2-associated diseases (PCVAD) is one of the major emerging immunosuppression diseases in pig industry. In this study, we investigated how PCV2 inoculation increases interleukin (IL)-10 expression in porcine alveolar macrophages (PAMs). PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1. Upon initial PCV2 inoculation, PI3K/Akt cooperated with NF-κB pathways to promote IL-10 transcription via p50, CREB and Ap1 transcription factors, whereas inhibition of PI3K/Akt activation blocked Ap1 and CREB binding to the  As a major immunosuppressive cytokine, IL-10 can inhibit the production of various pro-inflammatory cytokines, such as IL-12, IL-6 and TNF-α, promoter . In addipromoter . IL-10 hpromoter –8. Specipromoter , 8.Circoviridae which is considered to originate from a plant nanovirus [haemophilus parasuis, suggesting that PCV2 associated diseases (PCVAD) is actually an immunosuppressive disease. Previous studies have revealed that PCV2 could upregulate IL-10 production both in vivo and in vitro [PCV2 is a major pathogen to cause emerging viral infectious disease in world pig husbandry . PCV2 beanovirus . Besidesanovirus , 12. In anovirus , but no anovirus . PCV2 inin vitro , 15. Howc-myc, zinc finger protein 265 (ZNF265), thymine DNA glycosylase, E3 ubiquitin ligase family member MKRN1 and pPirh2, C1q receptor gC1qR, chaperonin Hsp40, and a porcine regulator of G protein signaling related to human RGS16, poRGS16 [PCV2 genome contains 11 putative open reading frames (ORFs), but only four of them are described to encode proteins. ORF1 encodes the replicase (Rep) of the virus, which is considered to be the essential protein for viral replication . ORF2 en poRGS16 , 24. HowIn the present study, we firstly investigated the activation characteristics of NF-κB, PI3K/Akt, ERK and p38 MAPK signaling pathways in PCV2-inoculated PAMs and the roles of these pathways in PCV2-induced IL-10 expression. Then, we identified which component of PCV2 and their binding cellular proteins played the central role in PCV2-induced IL-10 secretion in PAMs. Notably, in this study, we found PCV2 inoculation activated variant signalings in different characteristics to induce and enhance IL-10 transcription in PAMs, and gC1qR protein, a C1q binding protein, was firstly found to play a pivotal role in PCV2 induction of PI3K/Akt and p38 MAPK activation and IL-10 expression via binding with the Cap protein of PCV2.in vivo and in vitro studies have revealed that PCV2 infection induces porcine IL-10 expression in spleens, lymph nodes and thymus in infected pigs, and cultured monocytes and dendritic cells, whereas PCV1 was not reported to induce IL-10 production in vivo or in vitro studies [Previous  studies , 15. We il10 gene was induced to be transcripted for twice upon PCV2 inoculation, and that there might be some different regulation involved in twice induction.Next, we examined the kinetic of IL-10 production in PAMs response to different titers of PCV2 inoculation by ELISA assay. A detailed time-course showed that, after PCV2 inoculated PAMs at a multiplicity of infection (MOI) of 0.1, 1 or 10, IL-10 secretion greatly increased in the first 8 hours, and reach in highest levels in the second 8 hours, then declined in third 8 hours Figure . At 1 MONF-κB, PI3K/Akt and MAPKs signalings have been demonstrated to regulate IL-10 production in macrophages . To idenPI3K/Akt and MAPK signalings have been reported to be activated in PCV2 infected PK-15 cells -27. In PNext, we explored the roles of these signalings activation in PCV2-induced IL-10 production. As predicted, addition of NF-κB inhibitor (BAY11-7082), PI3K/Akt inhibitor (LY249002), p38 MAPK inhibitor (SB203580) or ERK inhibitor (PD98059) could significantly attenuate IL-10 accumulation in PCV2-inoculated PAMs Figure . Consistil10 promoter in situ within live cells by ChIP assays. Control (mock infection) cells virtually exhibited no binding of either Ap1, CREB or NF-κB1 p50 to the il10 promoter , rAd-ORF2 could significantly up-regulate p-Akt, p-p38 MAPK and p-ERK with no visible change of Akt, p38 MAPK and ERK Figure . Except gC1qR gene , receptor protein for the globular heads of complement component C1q (gC1qR), prostate apoptosis response-4 (Par-4), nucleosome assembly protein-1 (NAP1), nucleophosmin-1 (NPM1) and heat shock protein 40 (HSP40). To investigate which are involved in IL-10 induction, siRNAs specific for MKRN1, gC1qR, Par-4, NAP1, NPM1 and HSP40 were transfected into cells before PCV2 inoculation, respectively, and gene silencing was confirmed by western blotting . Down ree Figure , and thee Figure . The seqe Figure . In the e Figure .Next, PCV2 inoculated the gC1qR knockout and wild-type cells at 1 MOI for 24 h, and the secreted IL-10 were detected by ELISA. Results showed that the gC1qR knockout cells produced less IL-10 in the supernatants compared to the wild-type cells, whereas the cell clones (#1 and #3) that gC1qR were not knockout produced IL-10 as well as the wild-type cells Figure . ConsistThe data from the current study demonstrated that PCV2 Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways, but only PI3K/Akt signaling was involved IL-10 induction in the both early and late phase of PCV2 inoculation in macrophage, while PCV2 Cap protein binding with host gC1qR to induce IL-10 production at the both early and late phase of infection. Thus, we further assessed the roles of gC1qR in the activation of PI3K/Akt, p38 MAPK, and ERK signaling pathways induced by PCV2. Compared with gC1qR wild-type cells (gC1qR+/+), gC1qR knockout cells (gC1qR−/−) did not appear significant phosphorylation of Akt at 15 min and 12 hours post inoculation, while the phosphorylation of p38 MAPK was reduced significantly at 12 h p.i. Figure . HoweverTo eliminate the possibility of off-target effects for the CRISPR/Cas9 system sgRNA, we introduced two mutation sites into gRNA-c1qR-2 Figure  to test il10 promoter and increased the levels of NF-κB p50 . In further studies, we found that both empty recombinant adenoviruses (rAd-blank) and recombinant adenoviruses expressing Cap protein (rAd-ORF2) could activated NF-κB signaling, and Cap expression did not further increased the phosphorylation level of IκB (data not shown), suggesting Cap protein were not able to activate NF-κB signaling, NF-κB signaling were activated by adenoviruses as previous studies [PCV2 contains 11 predicted ORFs . ORF1, O studies . HoweverPrevious studies found that there were six possible Cap-binding proteins in PCV2-infected cells  . In thisIn summary, the present data in this study provide certain evidences for the roles of gC1qR, PI3K/Akt and p38 MAPK pathways in PCV2 induction of IL-10 production, and systematically demonstrate the regulation roles and patterns of different signaling pathways in PCV2-induced IL-10 expression in macrophage. These finding should help us to further understand why PCV2 can induce immune suppression and cause PMWS whereas PCV1 can not.All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Forth Military Medical University (permit number: 13024), and were performed according to the Animal Ethics Procedures and Guidelines of the People's Republic of China. No other specific permissions were required for these activities. This study did not involve endangered or protected species.PK-15 cells and porcine alveolar macrophages cell line 3D4/21 (CRL-2843) were purchased from ATCC. The primary porcine alveolar macrophages were obtained from the lungs of SPF piglets as previously described . PK-15 cPCV1 (AY193712) and PCV2 (EU366323) were both isolated and stocked in our laboratory . And thePAMs were adhered to 6 well plates followed by PCV infection at 1 MOI. The supernatants were harvested to detect IL-10 secretion by enzyme-linked immunosorbent assay (ELISA) kit (R&D) according to the manufacturer's instructions.The diluted plasmids containing PCV1 and PCV2 whole genomes were used as templates to draw standard curve. Virus DNA was isolated by proteinase K and SDS to determine the copy numbers of the harvested viruses. The primers were: PCV-F: AGTACCGGGAGTGGTAGGAG; PCV-R: GTTGAATTCTGGCCCTGCTC.Total cellular RNA was isolated by TRIZOL according to the manufactory's instructions. Reverse transcription was performed using M-MLV reverse transcriptase (Invitrogen) with Random primer. IL-10 mRNA level was analyzed by SYBR-green based Q-PCR using a Bio-Rad IQ5 Real-Time PCR System. IL-10 mRNA were normalized by comparing to β-actin and expressed relative to mock control. Primer sequences were: IL-10-F: AATCTGCTCCAAGGTTCCCG; IL-10-R: TGAACACCATAGGGCACACC; β-actin-F: GGACTTCGAGCAGGAGATGG; β-actin-R: AGGAAG GAGGGCTGGAAGAG.il10 promoter sequence was amplified and cloned into pGL3 basic vector (Promega) according to Rong Quan, et al. [Porcine , et al. . 3D4/21 For specific inhibitors treated cells, PAM cells were pre-incubated with DMSO, or NF-κB inhibitor BAY11-7082 (Merck), PI3K/Akt inhibitor LY294002 (Merck), ERK1/2 MAPK inhibitor PD98059 (Merck), p38 MAPK inhibitor SB203580 (Merck) and JNK inhibitor SP6000125 (Sigma) for 1 h. For siRNA treated cells, PAM cells were transfected with specific siRNAs  or negatXho I and Not I sites or recombinant shuttle vector pShuttle-CMV between Sal I and Not I. The confirmed pShuttle-ORFs were recombinant with the bone vector pAdeasy-1 in E. coli BJ5183, and the recombined plasmids were transfected into 293A cells after linearization to generate recombinant adenoviruses according to the manufactory's instruction. The recombinant adenoviruses infected 3D4/21 cells at 100 MOI for further analysis.Genes encoding viral proteins were amplified from PCV2 genomes and cloned into pCI-neo between The cells were lysed in RIPA with 1 mM PMSF and protease inhibitors (Sigma) for 15 min on ice. The cytosol and nuclear fraction were isolated according to the manufactory's instruction (Thermo). Similar amounts of protein from each extract were subjected to SDS-PAGE analysis and transferred to polyvinyl difluoride (PVDF) membranes (Millipore). After blocking for 1 h with blocking buffer (5% nonfat milk and 0.1% Tween-20 in PBS), the membranes were incubated with the following primary antibodies at 4°C overnight: anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-JNK, anti-JNK (Santa Cruz), anti-phospho-ERK, anti-ERK, anti-phospho-Akt, anti-Akt, anti-phospho-IκBα, anti-IκBα, anti-p50/p105, anti-p65, anti-Histone H3 , anti-gC1qR (Hycult biotech) and anti-β-actin (Tianjin Sungene Biotech) antibodies. HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Santa Cruz) were used as secondary antibodies. And ECL (Bio-Rad) was used for chemiluminescent detection according to the manufacturer's instructions.2O and analyze by PCR, and further quantified by Q-PCR. The specific primers for PCR and Q-PCR: AP1-F: CCAGCTGTGGAAGCTCACAA; AP1-R: GGAACAACGGGCCATGCTTA; NF-F: TTGGAGAGGTCTAGGGAAGGG; NF-R: AGAGCT GTGCCTTCTTCGTT; CREB-F: CAGCAAGGAGAA CCCTTGAGT; CREB-R: AGGTCACAATGACG TGGACA; SP1-F: ACACGTGAATGGAACCCACA; SP1-R: GAGGCTACCTCTCTCCCCTT; STAT3-F: TGCAAAGTTGGAGAGGTCTAGG; STAT3-R: AGTGAGGCCTCCCCATTCAT; NS-F: GTAAGCATGGCCCGTTGTTC; NS-R: GTTTTCTGTTCCAAGCCCGC.The ChIP assays were proceeded following the Cold Spring Harbor Protocols . BrieflyThe pCI-ORF2 plasmids were transfected into wild-type and gC1qR knockout cells. The cells were harvested and lysed by RIPA buffer on ice for 15 min. The supernatant were pre-cleared to reduce non-specific binding to the Protein A/G agarose beads. 1 μg rabbit anti PCV2 Cap polyclonal antibodies or mouse anti gC1qR monoclonal antibodies were added into the cell lysates and incubated with gentle rocking overnight at 4°C. Protein A/G agarose beads were added and incubated, then centrifuged, and the pellets were washed five times with cell lysis buffer. The pellets were resuspended with SDS sample buffer to perform western blotting analysis.Three pairs of oligos were designed based on the porcine gC1qR sequence (KM068128). The oligos were cloned into the BsmB I sites of lentiCRISPRv2 plasmid (Addgene). And the recombinant plasmids were transfected into 293T cells to obtain recombinant lentivirus. The recombinant lentiviruses were used to infect PAMs, respectively. 24 hours later, 5 μg/ml puromycin were added into the cell cultures to select the knockout cells. The selected cells were checked by western blotting and sequencing.All experiments were performed at least three times, and results are representative of three of independent experiments. Data were presented as means ± SEM (SD). Multiple group data were analyzed by ANOVA and Bonferroni post-hoc test, while comparisons between 2 groups were performed by unpaired Student's t test. Statistical significance was defined as P < 0.05.


Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application. ITGA2B or ITGB3 genes encoding the plasma membrane glycoproteins αIIb  and β3 , respectively. GT is characterized by quantitative and qualitative defects in the αIIbβ3 integrin (CD41/CD61 complex), the main receptor for fibrinogen ..28].2 in PBS), transferred onto irradiated C3H10T1/2 cells and differentiated with IMDM medium containing 10 mg/l insulin, 5.5 mg/l transferrin, 6.7 mg/ml selenium, 2 mmol/l L-glutamine, 15% fetal bovine serum , 0.45 μmol/l α-monothioglycerol (Sigma-Aldrich), 50 μg/ml ascorbic acid (Sigma-Aldrich) and 20 ng/ml recombinant human vascular endothelial growth factor (Invitrogen). On day 15, hiPSC-Sacs were disrupted with a cell scraper, crushed with a pipette and passed through a 40 μm cell strainer . The yielded cells were transferred onto irradiated C3H10T1/2 cells and cultured in the same medium without vascular endothelial growth factor containing 100 ng/ml human TPO (R&D), 50 ng/ml human SCF (R&D), and 25 U/ml heparin (Sigma-Aldrich). Medium was changed every 3 days. According to Takayama et al. . Fu. Fu31]. Subsequently, we analyzed platelet-like particles generated from hiPSC-derived MKs. The criteria used for platelet identification are defined in the Material and Methods section. The average platelet yield of 3.8 platelets per hiPSC-derived MK was comparable to published data , with noin vitro, hiPSC-derived and peripheral blood platelets were stimulated with adenosine diphosphate and thromboxane A2 (ADP/TXA2) or thrombin and stained with PAC-1, which specifically detects only the activated conformation of αIIbβ3. GT platelets from both sources showed diminished PAC-1 binding after both agonist treatments compared to corresponding CTR cells  protein but also presented a concurrent lack of CD61  surface expression. This finding suggests that mutated αIIb subunit negatively affects wild type β3, likely by impaired complex formation in the rough endoplasmatic reticulum and subsequent degradation of both proteins, as already reported [In our assay of platelet adhesion on fibrinogen, we measured an overall higher level of adhesion of freshly isolated blood platelets compared to hiPSC-derived cells. We can in part attribute this difference to a reduced viability of hiPSC-derived platelets in culture, as measured by shedding of CD42b in CD41/CD61+ or CD31+ positive platelets. In addition, the differences in platelet purity between the two sources might influence the adhesion rate: hiPSC-derived platelets isolated from cell culture supernatant cannot be completely separated from cell culture debris and have a higher degree of heterogeneity compared to thrombocytes in platelet rich plasma, leading to an underestimation of platelet adherence. Furthermore, it has been reported that hiPSC-derived cells are less mature than their native counterparts, which potentially could be another reason for lower platelet adhesion in the hiPSC-derived platelet group . Nonethein vitro. Our data shows that hiPSC-derived cells bear sufficient, but not complete resemblance to their native counterparts, a finding that highlights the importance of our strategy of side-by-side comparison. This approach could serve as an example for evaluating hiPSCs and their differentiated derivatives to reveal both validity and limitation of their disease modeling capabilities. Likewise, our results encourage the use of hiPSC-based disease models, especially in settings where the native target cell is not readily available [The wide application of hiPSCs in disease modeling, drug screening and therapy is limited by the difficulty in comparing diseased hiPSC-derived cells with their native counterparts . Taking vailable ,38.Despite a general bleeding disorder in GT patients, bleeding severity differs considerably between affected individuals, even within the same family or ethnic group . UnderstFurthermore, hiPSCs allow for the continuous production of diseased MKs and their derivatives in culture, creating new possibilities for studying the ontogeny of megakaryopoiesis in health and disease . In addiin vitro. Few reports have described higher platelets yields using non-patient-specific hiPSC lines; however, the numbers still remain too low for transfusion therapies. Advancements in dynamic three-dimensional culture conditions mimicking bone marrow and donor-specific immortalized megakaryocytic lines, which are expandable over longer periods, have improved the efficiency of in vitro platelet generation [in vitro platelet numbers to clinically significant amounts. However, it still needs to be proven that these methods are applicable to patient-specific lines and thus can be used for personalized transfusions therapies in patients with known genetic platelets disorders or life-threating bleedings. This is of special interest for platelets, since anucleated cells circumvent the concern of tumorigenesis in recipients of stem cell therapies.However, for a broader application of platelets and other cells derived from hiPSCs, the low efficiency and high variability of lineage-specific differentiation poses a substantial challenge . Increasneration ,45. ThisS1 Fig(A) Representative images of PBMCs on day 0 (i) and on day 5 (ii) of activation in the presence of anti-CD3 antibody and IL-2. Generated hiPSC lines show normal hESC morphology (iii) and typical alkaline phosphatase activity (iv). Scale bars represent 100 μm. (B) Immunostaining of hiPSC clones at passage 6 for Sendai viral antigen (red) and co-staining with DAPI (blue). Scale bar represents 50 μm. (C) Reverse transcription PCR analysis confirms presence (lane 3) of Sendai viral transgenes in infected T lymphocytes (ITL) and loss (lane 4 and 5) in CTR- (left panel) and GT-hiPSC lines (right panel). TL indicates primary T lymphocytes. (D) Immunostaining of hiPSC clone for the pluripotency markers NANOG (red) and TRA1-81 (green) and co-staining with DAPI (blue). Scale bar represents 25 μm. (E) Quantitative real-time PCR analysis confirms upregulation of endogenous pluripotency genes (expression levels relative to T-lymphocytes). All expression values are normalized to GAPDH. (A) and (D) Representative images of GT-hiPSC colonies (clone 2). (B) Representative images for each sample.(TIF)Click here for additional data file.S2 Fig(A) Quantitative real-time PCR analysis confirms the potential of generated hiPSCs to differentiate into cells of all three germ layers (expression levels in embryoid bodies relative to respective hiPSC clone). All expression values are normalized to GAPDH. (B) Immunostaining of whole EBs (day 26) for FOXA2 , cTNT  and P75  and co-staining with DAPI (blue). Scale bar represents 25 μm. Representative images of CTR-hiPSCs (clone 2) (C) Gene expression analysis with PluriTest of undifferentiated GT- (clone 1) and CTR-hiPSCs (clone 1).(TIF)Click here for additional data file.S3 Fig(A) Strategy used for differentiation of hiPSCs. (B) HiPSC-Sac formation at day 14–15 of hematopoietic differentiation (i) and higher magnification of the content of the hiPSC-Sac (ii) showing round cells resembling hematopoietic progenitors. Scale bars represent 200 μm. (C) and (D) Flow cytometry of CD34 (C) and CD41/CD61 (D) expression of hiPSC-derived cells within hiPSC-Sacs. HiPSC-derived cells within hiPSC-Sacs of GT and CTR were stained with anti-CD34 and anti-CD41/CD61 antibodies. The FSC/SSC log gate of large cells was applied. (E) Quantification of the percentage of CD34+ cells of total events  and their viability  measured as 7-AAD-negative cells by flow cytometry. P-values from unpaired t-tests. Representative images for each sample.(TIF)Click here for additional data file.S4 Fig2. Arrows indicate lamellipodia. Dashed lines mark cell body. (C) Rhodamine-conjugated phalloidin stain of GT- (right) and CTR- (left) hiPSC-derived MKs to selectively label actin filaments. Cells were stained after activation with ADP/TXA2. (D) Quantification of the percentage of MKs (CD42b and CD45 double positive cells) of all non-platelets  and their viability  measured as 7-AAD-negative cells by flow cytometry . (E) Distribution of nuclei number per MK from DAPI staining assessed by fluorescence microscopy . (F) Flow cytometry of CD42b and CD31 expression on hiPSC-derived MKs. Cells were stained with anti-CD45, anti-CD42b (x-axis) and anti-CD31 (y-axis) antibodies. For gating hierarchy see (A) Hemacolor stain of GT- (right) and CTR- (left) hiPSC-derived cells. Arrows indicate cells with typical MK-like appearance. Lymphocyte-like cells and monocyte-like cells are marked by asterisks. (B) DIC microscopy images of GT- (right) and CTR- (left) hiPSC-derived MKs after activation with ADP/TXA(TIF)Click here for additional data file.S5 FigCells were stained with anti-CD42b (red) and two different anti-CD41/CD61  antibodies and DAPI (blue) with (A) or without (B) Triton-X100 permeabilization. All scale bars represent 20 μm. Representative images for each sample.(TIF)Click here for additional data file.S6 FigPeripheral blood platelets of CTR and GT were stained with anti-CD42b (y-axis) and two different anti-CD41/CD61 antibodies  and DAPI (blue).(TIF)Click here for additional data file.S7 Fig(A) Quantification of hiPSC-derived platelet yield per MK from GT and CTR  by flow cytometry. Total number of platelets was divided by the number of MKs. P-value from unpaired t-test (B) Transmission electron microscopy images of hiPSC-derived platelets from GT and CTR show granules (asterisks) and the open canalicular system (arrows). Scale bar represents 2 μm. (C) Flow cytometry of CD3 and CD14 expression of hiPSC-derived platelets. HiPSC-derived cells of GT and CTR were stained with anti-CD42b, anti-CD41/CD61, anti-CD45, anti-CD3 and anti-CD14 antibodies. The FSC/SSC log gate of peripheral platelets was applied. For gating hierarchy see (TIF)Click here for additional data file.S8 Fig2. HiPSC-derived and peripheral platelets of CTR and GT were stained with anti-CD42b and PAC-1 antibodies and exposed to ADP/TXA2. The percentage of PAC-1+ of all CD42b+ platelets after ADP/TXA2 exposure is shown. P-values from unpaired t-tests.(A) HiPSC-derived and peripheral blood platelets were stained with PAC-1 and anti-CD42b antibodies in the absence (top) and presence (bottom) of thrombin. The FSC/SSC log gate of peripheral platelets was applied and further gated for CD42b positive cells. Representative images for each sample. For gating hierarchy see (TIF)Click here for additional data file.S9 Fig(A) HiPSC-derived platelets were stained with anti-CD42b (y-axis) and anti-CD41/CD61 or anti-CD31 (x-axis) antibodies. Continuous frames comprise either CD41/CD61+ or CD31+ platelets. Dashed frames comprise either CD42b and CD41/CD61 double positive or CD42b and CD31 double positive platelets. Representative images for each sample. For gating hierarchy see (TIF)Click here for additional data file.S10 FigFlow cytometry gating hierarchy of hiPSC-derived MKs and platelets. Doublets of events were excluded with the FSC TOF (far left) and SSC TOF (left) parameters of the Gallios cytometer (Beckman Coulter). 7-AAD negative events (right) were further gated according to forward and sideward scatter parameters (far right). The FSC/SSC log gate of peripheral blood platelets was applied for analyzing platelets. A FSC high/SSC high population was selected for non-platelets or large cells including hiPSC-derived MKs and HPCs.(TIF)Click here for additional data file.S1 MethodsSupporting Material and Methods and Supporting References.(DOCX)Click here for additional data file.S1 MovieSpreading of a CTR-hiPSC-derived platelet with lamellipodia formation.(AVI)Click here for additional data file.S2 MovieSpreading of a CTR-hiPSC-derived platelet with filopodia formation.(AVI)Click here for additional data file.S3 MovieSpreading of a CTR-hiPSC-derived platelet and megakaryocyte.(AVI)Click here for additional data file.S4 MovieSpreading of CTR peripheral blood platelets.(AVI)Click here for additional data file.S5 MovieSpreading of a GT-hiPSC-derived platelet.(AVI)Click here for additional data file.S6 MovieSpreading of GT-hiPSC-derived platelets and megakaryocyte.(AVI)Click here for additional data file.S7 MovieSpreading of GT peripheral blood platelets.(AVI)Click here for additional data file.S1 TableITGA2B gene.PCR-Primers used for sequencing of exons containing the mutations in the (DOCX)Click here for additional data file.S2 TableAntibodies/Flow Cytometry Dye.(DOCX)Click here for additional data file.


NKX2-1, encoding a homeobox transcription factor, is amplified in approximately 15% of non-small cell lung cancers (NSCLC), where it is thought to drive cancer cell proliferation and survival. However, its mechanism of action remains largely unknown. To identify relevant downstream transcriptional targets, here we carried out a combined NKX2-1 transcriptome (NKX2-1 knockdown followed by RNAseq) and cistrome (NKX2-1 binding sites by ChIPseq) analysis in four NKX2-1-amplified human NSCLC cell lines. While NKX2-1 regulated genes differed among the four cell lines assayed, cell proliferation emerged as a common theme. Moreover, in 3 of the 4 cell lines, epidermal growth factor receptor (EGFR) was among the top NKX2-1 upregulated targets, which we confirmed at the protein level by western blot. Interestingly, EGFR knockdown led to upregulation of NKX2-1, suggesting a negative feedback loop. Consistent with this finding, combined knockdown of NKX2-1 and EGFR in NCI-H1819 lung cancer cells reduced cell proliferation  more than knockdown of either alone. Likewise, NKX2-1 knockdown enhanced the growth-inhibitory effect of the EGFR-inhibitor erlotinib. Taken together, our findings implicate EGFR as a downstream effector of NKX2-1 in NKX2-1 amplified NSCLC, with possible clinical implications, and provide a rich dataset for investigating additional mediators of NKX2-1 driven oncogenesis. EGFR, KRAS, BRAF and ERBB2 (HER2), as well as rearrangements of ALK and ROS1 , 3915, 3EGFR is amplified and/or mutated in at least 10–15% of NSCLC (predominantly adenocarcimomas) [EGFR mutations tend to occur in lung adenocarcinomas from female non-smokers [KRAS mutations, despite ostensibly functioning just downstream of EGFR, tend to occur in adenocarcinomas with mucinous histology from male smokers [NKX2-1 amplification mediates its oncogenic effects at least in part through upregulation/activation of EGFR and its downstream signaling pathways (MAPK and PI3K).From our integrative -omic analysis, EGFR emerged as a top candidate with obvious biological and clinical interest. cimomas) , and thocimomas) . EGFR mu-smokers . In cont smokers , 41. OurEgfr transgene is not present at its native genome locus). In contrast, in Kras mutant mice Nkx2-1 loss was shown to accelerate lung cancer (mucinous adenocarcinoma) [NKX2-1 amplified human lung cancer.Notably, our findings are consonant with prior observations from genetically-engineered mouse models. In particular, Nkx2-1 was recently shown to be required for Egfr-driven murine lung cancer . As suggrcinoma)  and potercinoma) . NKX2-1 Our findings further suggest a feedback inhibition loop between NKX2-1 and EGFR, which regulates downstream signaling . Table N: Overlap of substantially regulated genes and binding-peak genes.(XLSX)Click here for additional data file.S2 FileFigure A: NKX2-1 isoform expression in NKX2-1-amplified NSCLC cell lines. (A) Schematic representation of the two NKX2-1 transcript variants and corresponding protein isoforms. (B) HCC1195 and HCC1833 cells predominantly express the short isoform of NKX2-1 protein, as determined by western blot co-migration with NKX2-1 short (and not long) isoform, exogenously expressed in 293T cells. Figure B: Independent siRNAs recapitulate findings from siRNA pools.  Compared to non-targeting control (NTC) siRNA, two independent siRNAs targeting NKX2-1 in H1819 cells result in (A) reduced NKX2-1 transcript levels by Q-RT-PCR , (B) reduced NKX2-1 protein levels by western blot , and (C) reduced cell proliferation by Wst-1 assay .  The two independent siRNAs targeting NKX2-1 also result in (D) reduced EGFR transcript levels, and (E) reduced EGFR protein levels.  Two independent siRNAs targeting EGFR result in (F) reduced EGFR transcript levels, and (G) reduced EGFR protein levels. (H) The two independent siRNAs targeting EGFR result in increased NKX2-1 protein levels. (I) EGFR knockdown does not alter NKX2-1 transcript levels, suggesting that the resultant increased NKX2-1 protein levels are the consequence of post-transcriptional regulation. Figure C: Supporting transcriptome data. (A) Gene numbers well-measured by RNAseq across all 4 NSCLC cell lines. (B) Gene numbers substantially (≥25%) downregulated (blue) or upregulated (red) in each of the 4 NSCLC cell lines. (C) Validation by Q-RT-PCR of select genes identified by RNAseq to be substantially downregulated following NKX2-1 knockdown. Q-RT-PCR transcript levels normalized to GAPDH; error bars indicate max/min values. Figure D: Supporting cistrome data. (A) Histogram showing distribution of NKX2-1 binding sites with respect to annotated genes (transcription start sites). (B) NKX2-1 binding peaks identified at the EGFR locus in H661, H1819, and HCC1195 cells. Called binding peaks are identified by blue triangles, and numbered. Below, the 100bp sequence of each NKX2-1 ChIPseq binding site is shown, highlighting NKX2-1 binding site motifs  EGFR protein levels in NKX2-1 amplified/dependent NSCLC cell lines. EGFR protein levels quantified by western blot; α-tubulin serves as a loading control. (B) Erlotinib dose-response curve in H1819 cells. Fifty percent growth inhibitory concentration (IC50) is indicated.(PDF)Click here for additional data file.


If) flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (IK1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro.Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current  with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of non-treated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs.The rabbit If inhibitor and beta-adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an  The establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life, lead breaks, infection, electromagnetic interference, appearance, and heart rate response during exercise.Cardiac pacemaker activity originates in the sinus node. Spontaneous diastolic depolarization in phase 4 of the action potential is initiated in the sinus node, and the electrical impulse is conducted through the atria to the atrioventricular node. The sinus node can generate impulses faster than those generated in other areas. Different kinds of ionic currents are involved in the formation of spontaneous diastolic depolarization of the sinus node –6.If) flowing through hyperpolarization-activated cyclic nucleotide-gated potassium (HCN) channel is robustly present in the sinus node [If currents [HCN4 found in a patient suffering from sick sinus syndrome (SSS) [HCN4 [Among them, the funny current  . Other gS) [HCN4 , 13. TheS) [HCN4 .+ current (IK1), which maintains resting membrane potential in working myocytes and antagonizes spontaneous activity, is negligibly small in sinus node cells [IK1 can give rise to pacemaker activity in ventricular myocytes [IK1, Purkinje fibers show If-dependent pacemaking, but the pacemaking is slow and not robust. Thus, one of the most important factors responsible for the rapid and robust pacemaking of the sinus node is the absence of IK1 [In contrast, the inward rectifier Kde cells , 15. Intmyocytes . Despite of IK1 .If flowing through HCN channels and attenuation of IK1 flowing through Kir channels are required for pacemaker cells to undergo spontaneous diastolic depolarization. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show weak IK1 currents to achieve two prerequisites for forming spontaneous diastolic depolarization, large If currents and small IK1 currents, and investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro.That is, enhancement of Hcn4 in mESCs due to the traceability. CAG promoter-IRES-EGFP construct in pCAGIG (Addgene #11159) and kanamycin/neomycin resistance gene (KanR/NeoR) in pIRES2-AcGFP1 vector (Clontech) were amplified via the polymerase chain reaction (PCR) method using PrimeSTAR GXL DNA polymerase (TaKaRa), purified using a QIAquick PCR Purification Kit (Qiagen), and ligated using an In-fusion HD enzyme (Clontech), i.e., pCAGIG- KanR/NeoR. Rabbit Hcn4 cDNA in the pCI vector (previously reported and kindly provided by Dr Takano) [KanR/NeoR, i.e., pCAGIG-rabbit Hcn4-KanR/NeoR. PCR primers are shown in Vertebrate HCN4 proteins are highly conserved  and we uAddgene #159 and k2.Mouse ESCs  were cultivated on 0.1% gelatin-coated plates in high-glucose Dulbecco’s Modified Eagle’s medium  supplemented with 20% fetal bovine serum (Sigma), 50 μM ß-mercaptoethanol (2-ME), MEM nonessential amino acids solution , 1000 units/mL leukemia inhibitory factor , and 100 μg/mL kanamycin (Sigma) in a humidified atmosphere containing 5% COCGR8 cells were harvested using 0.05% trypsin/EDTA. Two μg of a non-linearized vector was used for nucleofection  and HCN4-overexpressing ESCs were selected using a medium containing 400 μg/ml G418 (Roche Applied Science) for 7 days. Three stable clones that were resistant to G418 and were EGFP-positive were selected and expanded.If) was recorded at room temperature by using the perforated patch-clamp technique. Cells were superfused with a bath solution containing (in mM): 132 NaCl, 4.8 KCl, 2.0 CaCl2, 1.2 MgCl2, 1.0 BaCl2, 2.0 MnCl2, 5.0 D-glucose, and 10 Hepes; pH 7.4. Pipettes (2–4 MΩ resistances) were filled with a pipette solution containing (in mM): 110 K-aspartate, 5.0 K2-ATP, 11 EGTA, 1.0 CaCl2, 1 MgCl2, and 5 Hepes; pH 7.2. Then 0.3 mg/mL Amphotericin B was added to the pipette solution to achieve patch perforation . The If current was activated by a standard activation protocol. If currents through activated HCN4 channels were obtained during hyperpolarizing test pulses of 5 seconds between -45 and -125 mV in 20 mV increments from a holding potential of -35 mV.The funny current (5/cm2 cells were replated on Matrigel-coated cover glasses and incubated for 48 hours. AP recordings were performed on monolayer cardiomyocytes. APs were measured respectively by the perforated patch‐clamp technique using an Axopatch 200B amplifier (Molecular Devices). Data acquisition of APs were performed with pClamp10.2/Clampfit (Axon Instruments). APs were measured using a modified Tyrode’s solution containing (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.5 glucose, and 5.0 HEPES; pH 7.4 (NaOH). The pipette solution contained (in mM) 110 DL-aspartic acid, 30 KCl, 1 CaCl2, 5 ATP-Mg, 5 Creatine P-Na, 5 HEPES, and 10 EGTA; pH 7.25 (KOH). To achieve patch perforation (series resistance: 10–20 MΩ), amphotericin B (0.3 mg/mL)  was added to the pipette solution. Temperature was maintained at 35–36°C by a TC-344B dual channel heating system (Warner Instruments).Action potentials (APs) were also measured with the perforated patch‐clamp technique. mESC-CMs were dissociated using 0.25% Trypsin/0.02% EDTA for 5minutes and resuspended in high-glucose DMEM supplemented with 20% FBS, 50 μM 2-ME, and NEAA. Then 1 x 10Emryoid bodies (EBs) were formed by cultivating 500 mESCs with 0.5 mmol/L 2-O-alpha-D-glucopyranosyl-L-ascorbic acid  and without LIF in a hanging drop for 5 days (culture day 0 to day 5). On day On day 14, the medium was changed to no glucose DMEM (Gibco) with 4 mM L-sodium lactate (Sigma-Aldrich). Until dTo investigate pacing ability of HCN4-overexpressing mESC-CMs, we used a co-culture system with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).Human dermal fibroblasts (HDFs) were obtained from abdominal skin of 41-year-old healthy Japanese male, the corresponding author of this article, by punch biopsy. The skin biopsy sample was dissected into 10 evenly sized pieces. The dissected pieces were plated in 6-well plates containing DMEM supplemented with 10% FBS and 0.1 mg/mL kanamycin and placed in a 37°C incubator. The medium was changed every 3 days. After reaching confluence, the cells were dissociated with trypsin/EDTA and passaged.OCT4, SOX2, KLF4 and c-MYC cDNA  twice using retroviral vectors produced by Platinum Retrovirus Expression System, Pantropic . Two days after retrovirus transfection, 1 x 105 HDFs dissociated with 0.05% trypsin/0.02% EDTA were replated on 10 cm dishes with an SNL feeder layer  and maintained in DMEM/F12 (GIBCO) supplemented with 20% knockout serum replacement (GIBCO), 100 μmol/L 2-ME, NEAA, 10 ng/mL basic fibroblast growth factor , 0.05 mmol/L AA-2G and 100 μg/mL kanamycin (Sigma). Twenty-five days after replating, hiPSC colonies were selected. The hiPSC colonies were expanded on the SNL feeder layer. For feeder-free culture, hiPSCs were maintained on human embryonic stem cell-qualified Matrigel (Corning) in TeSRE8 medium (Stemcell Techonologies).hiPSCs were generated from HDFs as previously reported . HDFs we5/cm2 in TeSRE8 medium supplemented with 10 μM Y27632 (WAKO). Five days after plating, at day 0, cells were treated with 6 μM CHIR99021 (Cayman) and 50 μM AA-2G in RPMI medium (GIBCO) supplemented with B27-insulin supplement (GIBCO) and kanamycin. After 24 hours, at day 1, the medium was changed to RPMI supplemented with B27-insulin and 50 μM AA-2G. On day 3, 5 μM IWR-1-endo (WAKO) was added and was removed during the medium change on day 5. Cells were maintained in RPMI supplemented with B27 supplement (GIBCO) starting from day 7, with the medium was changed every 2 or 3 days. On day 28, cardiomyocyte purification was performed by the Percoll gradient procedure mentioned above.hiPSCs were dissociated into single cells with StemPro Accutase Cell Dissociation Reagent (GIBCO) and seeded on Matrigel-coated dishes at 1 x 10Total RNA from mESC-CMs was extracted using a Trizol Plus RNA Purificarion Kit (Invitrogen). Complementary DNA was synthesized from 1 μg of total RNA using a QunatiTect Reverse Transcription Kit (QIAGEN) as prescribed in the manual and subjected to PCR amplification.Taq DNA polymerase (Roche Applied Science) was used for RT-PCR and PCR products were subjected to electrophoresis in 2% agarose gels and stained with ethidium bromide. RT-PCR experiments were performed twice in each of the 3 cell lines. SYBR Green PCR Master Mix and Applied Biosystems 7300 Real-Time PCR Systems (Applied Biosystems) were used for quantitative PCR (q-PCR). The q-PCR data were processed with a standard curve method. PCR primers are shown in mESCs, mESC-CMs and hiPSC-CMs were plated on matrigel-coated cover glasses and were fixed in 4% paraformaldehyde. Cells were stained with primary antibodies against OCT4 , EGFP , α-Actinin , Troponin I , MLC-2v  and MLC-2a . Secondary antibodies were FITC-conjugated rabbit anti-goat IgG antibody, TRITC-conjugated rabbit anti-mouse IgG antibody, TRITC-conjugated swine anti-rabbit IgG , and Alexa Fluor 488 goat anti-mouse IgG . Nucleus staining was performed with Hoechst 33342 . F-actin staining was performed with Rhodamine Phalloidin .4 cells/well in high-glucose DMEM supplemented with 20% FBS, 1% NEAA, and 100 μM 2-ME and were incubated for 4 days.The purified mESC-CMs were plated on a laminin (Sigma Aldrich)-coated 96-well plate at 4 x 10We counted spontaneous beating frequencies and examined responses to ivabradine and isoproterenol.4 mESC-CMs were made by using the hanging drop method for 3 days. The purified hiPSC-CMs were plated on a Matrigel-coated 24-well plate at 2 x 104 cells/well and maintained in RPMI supplemented with B27. Three days after plating of hiPSC-CMs, the aggregate of mESC-CMs was plated on the well layered with hiPSC-CMs. After 7 days, synchronized beating rates of hiPSC-CMs away from aggregates were counted.Aggregates containing 1 x 10t test for unpaired data or one-way ANOVA with comparison of different groups by Dunnett’s post hoc test. Values of P < 0.05 were considered to be significant.All data are expressed as means ± SD. Statistical analysis was performed by student’s Hcn4-IRES-EGFP/SV40 promoter-neomycin resistance gene plasmid vector (HCN4/EGFP vector) (EGFP/SV40 promoter-neomycin resistance gene plasmid vector (EGFP vector) was transfected in mESCs, and colonies of cells that were resistant to G418 and were EGFP-positive were selected (Hcn4 (A CAG promoter-rabbit  vector)  or CAG pselected . We geneed  but not in non-overexpressing mESCs   .We generated 3 mESC lines in each group. In all mESCs, cardiac differentiation was performed well via EB formation with or without HCN4 overexpression. Cardiomyocytes were purified by changing culture media to glucose-free and lactic acid-supplemented media. Most of the collected cells spontaneously beat and expressed cardiac sarcomere proteins as assessed by immunostaining: non-overexpressing mESC-CMs, 91.6 ± 6.5% α-actinin positive and 90.2 ± 6.7% troponin I-positive; HCN4 overexpressing mESC-CMs, 86.2 ± 1.3% α-actinin positive and 88.8 ± 6.9% troponin I-positive. Proportions of α-actinin, troponin I, myosin light chain (MLC)-2v and MLC-2a-positive cells were not significantly different between the non-overexpression and HCN4 overexpression groups .Oct4 and Nanog) had disappeared and that cardiac markers (Nkx2.5 and Tnnt2) were positive in all 3 mESC-CM lines in each group (HCN4/EGFP or EGFP-stably-transfected mESC-CM group)  . Expresshe cells .Hcn4 than did non-overexpressing mESC-CMs (EGFP mESC-CMs)  . Both HCentricle .If current than did non-overexpressing mESC-CMs (EGFP mESC-CMs)  showed a significantly larger ESC-CMs) .P < 0.0001)  .If currents, in a dose-dependent manner  (P < 0.0001)   and incr 0.0001) .We generated hiPSCs in which immunofluorescent staining showed nuclear accumulation of OCT4 and the expression pattern of cell surface markers: SSEA-1 negative, SSEA-4 positive, TRA-1-60 positive and TRA-1-81 positive . Then wein vitro and indicated that these cells could function as a biological pacemaker.Co-culture of mESC-CMs with aggregates composed of mESC-CMs  resultedIf currents and small IK1 currents. The cells have five specific abilities to become an appropriate biological pacemaker. (1) HCN4-overexpressing mESC-CMs expresses high levels of Hcn4 and Cacna1h genes and a low level of the Kcnj2 gene. (2) The cells show large If currents and rapid spontaneous beating, in other words, rapid impulse generation. (3) Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions and the Scn5a gene, which encodes sodium channels, are expressed in the cells. (4) Furthermore, the beating of the cells responds to an If inhibitor and beta-adrenergic receptor agonist. (5) HCN4-overexpressing mESC-CMs have pacing ability in an in vitro co-culture system with other excitable cells.We established HCN4-overexpressing mESC-CMs as a candidate for a biological pacemaker. We generated the cells in order to achieve two prerequisites for forming spontaneous diastolic depolarization: large Hcn1-ΔΔΔ, Hcn2 or Hcn4 gene [Hcn2- or Hcn4-overexpressing mesenchymal stem cells [Tbx18 gene transfer in mature ventricular cardiomyocytes increases HCN4 channel expression and yields pacemaker activity [If currents. Enhancement of If currents flowing through HCN channels is a core strategy for generating a biological pacemaker. Mouse ESC-derived myocytes expressed If currents [If current and a large amount of cells.Several investigators have reported overexpression of HCN channels as a strategy for generating a biological pacemaker. Injection of the cn4 gene –27 and tem cells , 29 haveactivity . We alsocurrents . Since Hcurrents , 32, thecurrents –35. AlthIf current through HCN channels but also attenuation of IK1 current and presence of other currents through T-type Ca2+ channels are required for diastolic depolarization [v1.5 channel is necessary as a pacemaker [Not only enhancement of rization , 37. Addacemaker , 39. OurIf inhibitor and reduces the firing rate of pacemaker cells [Ivabradine is an er cells , 41, ander cells . HCN4-ovA sufficient number of cells is necessary to engraft well and capture the surrounding myocardium . Since pin vivo. Further studies are needed to clarify this point.Recently, Inada et al reported that the spatial heterogeneous nature of the sinus node is important for its normal functioning and that the presence of Na channel and connexin 43 in the periphery may be essential for the node to be able to drive the atrial muscle , 39. Mouin vitro experiment, and we need to evaluate the efficacy of these cells in bradycardia model animals.Transplantation of human ESC-CMs into the ventricle of a complete atrioventricular block model animal has been reported . Our expin vitro. The results indicated that these cells could be applied to a biological pacemaker.We generated HCN4-overexpresssing mESC-CMs showing rapid spontaneous beating, responses to drugs and improved pacing ability S1 FigHcn4/EGFP-transfected mESCs observed by phase contrast microscopy (a to c) and fluorescence microscopy (d to f). Hcn4/EGFP-transfected mESCs were positive for EGFP (green) fluorescence (d to f). B. Representative living EGFP-transfected mESCs observed by phase contrast microscopy (a to c) and fluorescence microscopy (d to f). EGFP-transfected mESCs were positive for EGFP (green) fluorescence (d to f). C. Representative living control mESCs observed by phase contrast microscopy (a) and fluorescence microscopy (b). Control mESCs were negative for EGFP (b). Bar = 50 μm.A. Representative living (TIF)Click here for additional data file.S2 FigNkx 2.5, Tnnt2, connexin, Scn5a, Cacna1h, and mouse endogenous Hcn4 (lanes 7 to 12). Rabbit exogenous Hcn4 was expressed in HCN4/EGFP mESCs (lanes 4 to 6) and mESC-CMs (lane 10 to 12). B. Rabbit Hcn4 mRNA levels in 3 HCN4/EGFP mESC-CM lines assessed by q-PCR.A. In all cell lines with or without HCN4 overexpression, RT-PCR showed increases in mRNA expression for cardiac markers (TIF)Click here for additional data file.S3 FigNanog and Oct4 mRNA levels were not significantly different in all mESC lines with or without HCN4 overexpression.A. Immunofluorescent staining of OCT4 (a and b) and nuclear DNA staining by Hoecst (c and d) in HCN4/EGFP mESC-1 and 3 (immunofluorescent staining in HCN4/EGFP mESC-2 shown in (TIF)Click here for additional data file.S4 FigA. Generated hiPSCs observed by phase contrast microscopy (a). Immunofluorescent staining showed nuclear accumulation of OCT4 (red) (b to d) and cell surface antigen expression (green) pattern of human pluripotent stem cells . Bar = 50 μm. B. Differentiated cardiomyocytes from hiPSCs were positive for α-actinin (green) (a) and troponin I (green) (d). Counter staining with f-actin (red) (band e) and merge (c and f). Bar = 50 μm.(TIF)Click here for additional data file.


Background: Bevacizumab and temsirolimus are active agents in gynecologic tumors. Temsirolimus attenuates upregulation of HIF-1α levels, a resistance mechanism for antiangiogenics, and targets the PI3-kinase/AKT/mTOR axis, commonly aberrant in these tumorsPatients and Methods: We analyzed safety and responses in 41 patients with gynecologic cancers treated as part of a Phase I study of bevacizumab and temsirolimus.Results: Median age of the 41 women was 60 years ; median number of prior systemic therapies was 4 (1-11). Grade 3 or 4 treatment-related toxicities included: thrombocytopenia (10%), mucositis (2%), hypertension (2%), hypercholesterolemia (2%), fatigue (7%), elevated aspartate aminotransferase (2%), and neutropenia (2%). Twenty-nine patients (71%) experienced no treatment-related toxicity greater than grade 2. Full FDA-approved doses of both drugs (bevacizumab 15mg/kg IV Q3weeks and temsirolimus 25mg IV weekly) were administered without dose-limiting toxicity. Eight patients (20%) achieved stable disease (SD) ≥ 6 months and 7 patients (17%), a partial response (PR) [total = 15/41 patients (37%)]. Eight of 13 patients (62%) with high-grade serous histology  achieved SD ≥ 6 months/PR.Conclusion: Bevacizumab and temsirolimus was well tolerated. Thirty-seven percent of heavily-pretreated patients achieved SD ≥ 6 months/PR, suggesting that this combination warrants further study. Angiogenesis plays a key role in the pathogenesis of metastases, as new vessels provide growing tumors with nutrients, growth factors, oxygen, proteolytic enzymes, hemolytic factors, and hormones -3. The vDiverse receptors in interconnected signaling pathways communicate with each other through cross-talk , 10. Becn vitro studies with temsirolimus, as well as reduced levels of HIF-1α, HIF-2α and VEGF 532-554 of exon 9  and c1011-1062 of exon 20 (kinase domain)[PIK3CA proto-oncogene by Sanger sequencing following amplification of 276 bp and 198 bp amplicons, respectively. Codons 12, 13, and 61 were examined for KRAS and NRAS mutations and for BRAF, codons 468-474, codons 595-600, and mutations of exon 15 by pyro-sequencing were examined as previously described [e domain), which iescribed . PTEN loescribed -38. Form


The association between specific HLA alleles and risk of severe cutaneous drug hypersensitivity reactions is increasingly recognised. Vancomycin mediated pseudo-allergic 'red man syndrome' is well recognised, yet urticaria/anaphylaxis is uncommon and some clinical reactions are delayed. Although this may suggest the presence of vancomycin initiated T cell responses, no predisposing vancomycin HLA association has been identified. Therefore we set out to examine the role of T cell responses in vancomycin hypersensitivity reactions.ex-vivo IFN-/ IL-4 and proliferation above background.Of our tested cohort of 103 individuals who suffered delayed skin drug hypersensitivity reactions, vancomycin was deemed the culprit in 17, who developed drug exanthems (59%), DRESS (35%), or SJS/TEN (18%). We used ELISpot and [3H]-Thymidine assays to measure vancomycin-specific Individuals with drug hypersensitivity showed a mean circulating PBMC frequency of vancomycin-specific cells of 470.0 x10-4% (IFN-) / 33.5 x10-4% (IL-4) and SI 4.08 if tested within 30 days, whilst those tested after 30 days showed 316.0 x10-4% (IFN-) / 20.0 x10-4% (IL-4) and SI 3.43. Individuals never previously exposed to vancomycin (n=11) showed a lower mean circulating frequency: 2.1 x10-4% ) / 1.7 x10-4%  and SI 1.28. In controls who had been exposed to vancomycin  detectable frequencies were also lower than allergics: 11 x10-4% (IFN-) / 7.1 x10-4% (IL-4) and SI 0.98. We co-cultured non-allergic PBMC with vancomycin for two-weeks in vitro. In two lines, we saw expansion of vancomycin-specific T cells in ELISpot to an average frequency of 954 x10-4% (IFN-) and 1130 x10-4% (IL-4) which was confirmed by ELISpot and intracellular cytokine staining.These data confirm that robust Th1 responses target vancomycin in cutaneous hypersensitivity reactions, which supports the use of T cell inhibition such as steroids and ciclosporin. Vancomycin-specific responses appear reduced with time which advocates early diagnostic testing, but the clinical possibility of loss of sensitisation is intriguing. The finding that vancomycin-specific T cells can be artificially expanded from individuals without clinical reactivity, suggests that both immune predisposition and potentially adaptive regulation may be important in development of hypersensitivity responses to vancomycin.


Alum adjuvant significantly improved protection as evidenced by effective viral clearance compared to unadjuvanted FI-RSV. However, in contrast to unadjuvanted FI-RSV, alum-adjuvanted FI-RSV (FI-RSV-A) induced severe vaccine-enhanced RSV disease including weight loss, eosinophilia, and lung histopathology. Alum adjuvant in the FI-RSV-A was found to be mainly responsible for inducing high levels of RSV-specific IFN-γ-IL4+, IFN-γ-TNF-α+ CD4+ T cells, and proinflammatory cytokines IL-6 and IL-4 as well as B220+ plasmacytoid and CD4+ dendritic cells, and inhibiting the induction of IFN-γ+CD8 T cells. This study suggests that alum adjuvant in FI-RSV vaccines increases immunogenicity and viral clearance but also induces atypical T helper CD4+ T cells and multiple inflammatory dendritic cell subsets responsible for vaccine-enhanced severe RSV disease.Respiratory syncytial virus (RSV) is well-known for inducing vaccine-enhanced respiratory disease after vaccination of young children with formalin-inactivated RSV (FI-RSV) in alum formulation. Here, we investigated alum adjuvant effects on protection and disease after FI-RSV immunization with or without alum in comparison with live RSV reinfections. Despite viral clearance, live RSV reinfections caused weight loss and substantial pulmonary inflammation probably due to high levels of RSV specific IFN-γ Brie6) per well were cultured with peptide F92-106 , F85-93 (KYKNAVTEL), G183-195 (WAICKRIPNKKPG), and M282-90 (SYIGSINNI) as previously described [Splenocytes, lung, and bone marrow cells were isolated from tissue samples at 5 d.p.c.. RSV F-specific antibody secreting cells were determined on Multi-screen 96 well plates  pre-coated with purified recombinant F protein 400ng/ml) by enzyme-linked immunospot (ELISpot) assay as previously described . The spo0ng/ml byescribed , 44. Aft92-106, F85-93, G183-195, and M282-90 peptide. Intracellular FACS staining was performed using a cytofix/cytoperm kit (BD Biosciences) according to manufacturer’s instructions. Cells were stained with antibodies for intracellular cytokines and surface markers including IFN-γ, IL-4 (eBioscience), TNF-α (BioLegend), CD45, CD3, CD4, CD8, Siglec F (BD Biosciences). To determine memory B cells, plasma cells, and DC phenotypes, mediastenial lymph node (MLN), spleen, and bone marrow cells were isolated from mice at 5 d.p.c. and 7 d.p.i.. Cells were stained with CD19, IgD, F4/80, CD11b, CD11c, and PNA or GL7 (eBioscience) as well as with CD45R/B220, CD138, and CD103 (BioLegend). The PNA+ or GL7+ germinal center B cells and CD138+ plasma cells were analyzed from pre-gated mature B cells (CD19+IgD-B220+/CD4-CD8- and CD19+IgD-B220int/CD4-CD8-) in spleens and MLN. The DC populations of B220+, CD103+ and CD11b+ in MLN and lungs were determined from pre-gated CD11c+F4/80- cells. Stained cells were acquired on a FACSCanto flow cytometer (BD), and analyzed using Flow Jo software .BAL fluids were obtained by infusing 1 mL of PBS into the lungs via the trachea using a 25-gauge catheter  at 5 d.p.c. and 7 days post immunization (d.p.i) before challenge infection. Lung cells were isolated by homogenization of tissue through 70 μm cell strainers and Percoll gradient (44% and 67%) centrifugation. For detection of intracellular cytokine-secreting cells, lung cells were stimulated with FThe lungs collected at 5 d.p.c. and 7 d.p.i. were fixed with 10% formalin in PBS, embedded in paraffin blocks. Lung tissue sections were stained with hematoxylin and eosin (H&E) and periodic acid-schiff stain (PAS) to assess histologic changes and airway mucin expression respectively as described . The secp value less than 0.05 was considered significant.The data obtained using the mean and the standard error of means (SEM) value from two independent experiments and 5 mice per group. The statistical analyzes were performed by either a one-way ANOVA with Tukey multiple comparison test or two-way ANOVA in GraphPad Prism version 5 . A The underlying mechanisms by which FI-RSV-A immunization induces vaccine-enhanced RSV disease have not been well understood. RSV F and G proteins are the major targets for neutralizing antibodies. We investigated the effects of alum adjuvant on the immunogenicity of FI-RSV after intranasal immunization of mice with alum-adjuvanted FI-RSV (FI-RSV-A) in comparison with unadjuvanted FI-RSV (FI-RSV) and live RSV reinfections (live RSV). First, we evaluated RSV F and G-specific antibody responses . Live RSWe further analyzed the levels of RSV-specific antibody responses in immune sera using whole live RSV or FI-RSV as an ELISA coating antigen . As showp<0.05, Neutralizing antibody is an important parameter in protective efficacy against RSV infection. We determined neutralizing activity against RSV in boost immune sera using a plaque reduction assay . The neuMice were challenged with RSV at 12 weeks after boost immunization and weight changes of mice were monitored . Mice wiMock (PBS) treated naïve mice or FI-RSV immune mice were found to have highest viral loads in the lungs among the groups. The FI-RSV-A group showed significantly lower levels of RSV lung titers than those in the FI-RSV group  B cells was observed among groups before challenge  B cell responses, the bone marrow, lymph nodes, and spleen cells were collected 7 days post boost immunization for flow cytometry and ELISpot analysis . The livhallenge .+B220intCD19+IgD- phenotypic plasma cells (p<0.001) and GC phenotypic B cells (p<0.001) were observed in spleens of live RSV and FI-RSV-A immune mice compared to those in FI-RSV immune mice , PAS positive area was analyzed to determine mucus production . In continophils  as deterinophils . Interesinophils . Next, winophils . Interesinophils . Howeverinophils . Therefo183-195 or M282-90 peptide   .in vitro stimulation with peptide F92-106 or G183-195  to better understand the effector T cell responses  in the lung  in MLN than those in FI-RSV alone immune mice  were observed at higher levels in the lung and MLN from FI-RSV immune mice regardless of alum adjuvant compared to those in live RSV reinfection or naïve mice (+ (CD103+CD11c+F4/80-CD45+) DCs were recruited into the lungs from live RSV, FI-RSV-A, FI-RSV immune mice compared to those in PBS control mice with RSV infection  immune mice, which were not observed in mice with inactivated RSV (FI-RSV) alone immune mice. These results in this study are consistent with previous studies reporting that Th2 cytokines and chemokines were associated with pulmonary eosinophilia and RSV vaccine-enhanced disease , 51, 52.in vitro stimulation with F92-106, F51-66, F85-93, and G183-195 peptides. In other words, F92-106-stimulated IFN-γ-IL4+ and IFN-γ-TNF-α+ Th2 CD4 T cells as well as G183-195-specific IFN-γ-IL-4+ Th2 CD4 T cells were observed at significantly higher levels in the lungs of alum-adjuvanted FI-RSV immune mice than those in FI-RSV alone immune mice without alum adjuvant. It is interesting to note that high levels of IFN-γ-IL-4+ and IFN-γ-TNF-α+ CD4 T cells in response to CD8 T cell epitope F92-106 peptide stimulation were observed in the FI-RSV-A group as determined by intracellular cytokine staining  or FI-RSV alone immune mice are likely to induce RSV F and G specific CD8 T cells expressing IFN-γ+ but not IL-4+ CD4 T cells. Experimental evidence in this study suggests that alum adjuvant in FI-RSV suppresses the induction of RSV specific IFN-γ+ CD8 T cells. RSV specific effector T cell responses are differentially induced depending on primary RSV infection of naïve mice (PBS mock control), RSV re-infection, alum-adjuvanted FI-RSV, or inactivated RSV immunization.Mice with RSV re-infection induced high levels of IFN-γBoth FI-RSV with and without alum groups were significantly lower in the levels of IFNγ producing CD4 and CD8 T cells, in particular F and M2 specific IFN-γ CD8 T cells . Alum ad+ pDC populations (B220+CD11cintF4/80-CD45+) in the lung and MLN compared to those in inactivated RSV alone or live RSV immune mice. Therefore, recruitment of high levels of pDCs in both lungs and MLN is likely to be associated with inflammatory pulmonary RSV disease in addition to IL-4 and IL-6 proinflammatory cytokines as well as mostly CD4 T cells secreting IL-4 or TNF-α. Consistent with possible roles of pDCs in RSV disease in FI-RSV alum immune mice, pDCs were shown to increase mortality during influenza virus infections by inhibiting CD8 T cell responses [+ DCs in MLN from FI-RSV alum immune mice. In support of possible roles of CD4+ DCs in inducing IL-4 cytokine and IL-4+ CD4 T cells in FI-RSV alum immune mice, CD4+ DCs were reported to enhance Th2 differentiation by inhibiting IL-12 and IFN-γ production [in vitro  cytokines in alum-adjuvanted FI-RSV immune mice.It is considered that mobilization of DCs would provide an important immune regulator for modulating innate microenvironment and adaptive immune T cell responses as well as for pulmonary histopathology. In particular, different subsets of DCs were suggested to play a critical role in inducing RSV-specific adaptive immune responses . Howeveresponses . Also, hesponses –60. Detaoduction , 61. To + pDCs, and CD4+ DCs, and inhibition of effector CD8 T cells. Results in this study suggest that induction of a balanced immune response with appropriate CD4 and CD8 T cells, and appropriately regulated innate pulmonary DC subsets in addition to RSV neutralizing activity would be required for a safe and effective RSV vaccine. Further studies are required to delineate possible roles of respiratory DC subsets in the RSV protection and pulmonary histopathology with different RSV vaccine platforms.In summary, live RSV re-infection, alum-adjuvanted FI-RSV, and FI-RSV without alum significantly influence the induction of Th1 and Th2 cytokines by differential CD4 and CD8 T cell responses as well as the mobilization of multiple DC subsets and eosinophils. Both innate and adaptive host immune components play significant roles in RSV protection and disease in mice. In particular, alum adjuvant in FI-RSV vaccine formulations has considerable impacts on increasing immunogenicity and virus clearance as well as Th2 type CD4 T cells, proinflammatory cytokines, eosinophilia, mucus production, B220S1 File(DOCX)Click here for additional data file.


We next investigated whether age-related diminished ARC POMC activity therefore impacts the potency of 5-HT obesity pharmacotherapies, lorcaserin, d-fenfluramine, and sibutramine and report that all compounds reduced food intake to a comparable extent in both chow-fed young lean (3–5 months old) and middle-aged obese (12–14 months old) male and female mice. We provide a mechanism through which 5-HT anorectic potency is maintained with age, via preserved 5-HT–POMC appetitive anatomical machinery. Specifically, the abundance and signaling of the primary 5-HT receptor influencing appetite via POMC activation, the 5-HT2CR, is not perturbed with age. These data reveal that although 5-HT obesity medications require ARC POMC neurons to achieve appetitive effects, the anorectic efficacy is maintained with aging, findings of clinical significance to the global aging obese population.The phenomenon commonly described as the middle-age spread is the result of elevated adiposity accumulation throughout adulthood until late middle-age. It is a clinical imperative to gain a greater understanding of the underpinnings of age-dependent obesity and, in turn, how these mechanisms may impact the efficacy of obesity treatments. In particular, both obesity and aging are associated with rewiring of a principal brain pathway modulating energy homeostasis, promoting reduced activity of satiety pro-opiomelanocortin (POMC) neurons within the arcuate nucleus of the hypothalamus (ARC). Using a selective ARC-deficient POMC mouse line, here we report that former obesity medications augmenting endogenous 5-hydroxytryptamine (5-HT) activity  Adiposity commonly accumulates with age from early adulthood (20 years old in humans) through late middle-age (65 years old in humans). This increase in adiposity has significant health implications; age-related obesity represents the primary cause of metabolic syndromes . Understd-fenfluramine, which stimulates 5-HT release/blocks 5-HT reuptake, and sibutramine, which blocks 5-HT/noradrenaline (NA) reuptake. However, both compounds have been withdrawn from clinical use due to off-target effects  and its release in 2013.To date, medications targeting 5-hydroxytryptamine  pathways have been among the most clinically successful obesity treatments . These i effects , 4. Tran5-HT2CRs . The the2CR activation, achieves effects on appetite and body weight has been revealed. Specifically, d-fenfluramine and 5-HT2CR agonists chiefly elicit appetitive effects through activation of pro-opiomelanocortin (POMC) neurons , ARC POMC neurons display an age-dependent upregulation of mammalian target of rapamycin (mTOR) signaling, which functions to reduce POMC neuronal activity, an effect reversed by mTOR inhibitor rapamycin  (am) with ad libitum access to chow diet and water unless otherwise stated. All experiments were in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986.Mice on a C57BL/6 background expressing enhanced green fluorescent protein (EGFP) under the control of POMC regulatory elements (POMC-EGFP mice) , 17, ARCeo/fneo) , and wilBody composition was compared in young adult  and middle-aged adult  male C57BL/6 mice  using dual-energy x-ray absorptiometry Lunar PIXImus2 mouse densitometer .fneo/fneo and wild-type littermates  and POMC-EGFP young adult , and middle-aged adult  mice were individually housed for 1 week before experimentation and received a habituation ip injection of vehicle 1 day before experimentation. Mice were then treated with vehicle, lorcaserin (8 mg/kg), d-fenfluramine (3 mg/kg), or sibutramine (5 mg/kg) 45 minutes before the onset of the dark cycle (lights on at 7:00 am and off at 7:00 pm) and food was removed. At the onset of the dark cycle, chow pellets were replaced and 3- to 4-hour food intake measured. These studies were performed using a within-subjects experimental design, with a minimum of a 3-day treatment-free period.POMCht2cr (5-HT2CR) and Pomc (ht2cr) or SP6 (Pomc) polymerase, according to the manufacturer's protocol (Promega and Ambion Inc) in the presence of 35S-labeled UTP. cRNA riboprobes were diluted to 2 × 107 cpm/mL in hybridization solution. Sections were mounted onto microscope slides, air-dried, and exposed to Biomax MR film (Kodak). Density of ht2cr mRNA and Pomc mRNA was examined in adjacent sections of brain tissue using densitometry analysis by computing the integrated density  using ImageJ . This was performed at 3 ARC levels  and the average integrated density was calculated.Young adult  and middle-aged adult  C57BL/6 mice were anesthetized with pentobarbitone (50 mg/kg ip) and transcardially perfused with diethylpyrocarbonate-treated PBS followed by 10% neutral buffered formalin (Sigma). Brains were extracted, immersed in the same fixative overnight, cryoprotected in 20% sucrose for 24 to 48 hours and then sectioned coronally on a freezing sliding microtome at 25 μm. Tissue was collected in 5 equal series. In situ hybridization histochemistry was conducted using antisense radiolabeled riboprobes specific to the mRNA sequences of and Pomc , 19. Rib3, 1.2 NaH2PO4, 10 glucose, 2 Na pyruvate, 1.2 MgCl2, 2.4 CaCl2 (pH 7.3), gassed with 95% O2 and 5% CO2. Slices were loaded with fura-2 AM . Imaging was performed at 34°C–36°C with an Olympus FluoView 1000MPE 2-photon laser-scanning microscope, equipped with a MaiTai DeepSee laser (Spectra-Physics) tuned to 790 nm and via an LUMPlanFI/IR ×40 (NA 0.8) objective. Images were acquired at a frame rate of 5.4 seconds, and spatial averages of somatic fluorescence were monitored as agonists were applied to the bath perfusion. Fluorescence values (F) were corrected for linear bleaching, background subtracted, and expressed as ΔF/F baseline.Coronal brain slices 180 μm) containing the ARC (−1.58 to −1.94 mm bregma) from male POMC-EGFP young adult  and middle-aged  mice were prepared as previously described (0 μm contd-fenfluramine (Tocris Biosciences), 5-HT and NA reuptake inhibitor sibutramine (Tocris Biosciences), and 5-HT2CR agonists WAY-161,503 -one; Tocris Biosciences) and lorcaserin (LGM Pharma) were used. Drugs were dissolved in 0.9% sterile saline and administered in vivo in a volume of 10 mL/kg body weight (ip).5-HT reuptake inhibitor and releaser t test, χ2, or repeated-measures ANOVA followed by Tukey's post hoc tests. For all analyses, significance was assigned at P < .05. Data are presented as mean ± SEM.Data were analyzed with paired or independent d-fenfluramine and 5-HT2CR agonists require melanocortin 4 receptors to produce effects on appetite , sibutramine (5 mg/kg), and lorcaserin (8 mg/kg) suppressed 3-hour dark cycle food intake to a comparable extent in young adult and middle-aged male , sibutramine  or lorcaserin . These data indicate that diminished ARC POMC activity in middle-age does not impair 5-HT obesity therapy efficacy.Given that ARC POMC neuron function is diminished with age, we next sought to determine whether 5-HT compound anorectic potency is thereby compromised with aging. As expected, middle-aged (12–14 months old) male mice displayed significantly elevated body weight A, percen2CR and POMC neuroanatomical appetitive machinery. We therefore compared Pomc mRNA and ht2cr mRNA abundance in young adult (3–5 months old) and middle-aged (12–15 months old) male and female mice. Despite observing age-associated obesity, ARC Pomc mRNA  activated a similar proportion of ARC POMC neurons in both young adult and middle-aged mice . Although the biological factors driving increased fat accumulation with age are not well understood, recent reports reveal age-related melanocortin system remodeling and link these functional changes with age-associated obesity , 16. Thid-fenfluramine and sibutramine to ARC POMC neurons. The mechanistic specificity of the hypophagic effect achieved via this small population of POMC neurons is remarkable given the wholesale effect on 5-HT and 5-HT/NA bioavailability produced by d-fenfluramine and sibutramine, respectively. Our findings are consistent with earlier reports demonstrating that 5-HT depolarizes and stimulates expression of ARC POMC neurons and that functional melanocortin 4 receptors (the primary appetitive receptor target of POMC) are required for d-fenfluramine and 5-HT2CR agonists to suppress food intake (2CRs expressed in global POMC neurons has emphasized the importance of this population of 5-HT2CRs in 5-HT hypophagia. Specifically, d-fenfluramine and 5-HT2CR appetite suppression is attenuated in 5-HT2CR–null mice but fully restored in mice with 5-HT2CRs exclusively re-expressed in POMC cells (2CRs in POMC neurons abolishes d-fenfluramine and 5-HT2CR agonist hypophagia (2CRs, POMC neurons specifically within the ARC are a critical component of the neural circuit through which obesity medications d-fenfluramine and sibutramine influence appetite.Here we distill a critical and necessary mechanism responsible for communicating the anorectic effect of obesity medications d intake , 20, 21.pophagia . Our resGiven that functional POMC is mechanistically required for 5-HT pharmacotherapy appetite suppression, recent reports revealing perturbed POMC function and melanocortin system remodeling in middle-age presented the possibility that 5-HT compound anorectic efficacy may be diminished with age. Specifically, middle-age is associated with an upregulation in mTOR signaling in ARC POMC cells, coupled with an age-dependent increase in inhibitory agouti-related protein inputs, which promotes lower resting membrane potential and reduction in POMC neuronal activity , 16.2CR–POMC appetitive machinery is not compromised with middle-age; both ARC ht2cr and Pomc abundance is preserved, as is 5-HT2CR signaling in POMC cells. Specifically, plasma membrane Gq-coupled 5-HT2CRs activate phospholipase C, which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate into the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, promoting calcium mobilization (2CR agonist was equally effective in stimulating intracellular calcium mobilization in young and middle-aged mice, which suggests that there is no inherent defect in components of the intracellular signaling cascade lying downstream of the 5-HT2CR. Thereby, these findings provide a mechanism through which 5-HT medication anorectic potency is maintained with age and suggest that pharmacological activation of the 5-HT2CR-POMC system is sufficient to overcome any age-related reduction in endogenous POMC neuronal activity.Nevertheless, in the present study, we demonstrate that 5-HT compounds suppress feeding with equal potency in young adult and middle-aged mice. Furthermore, we report that the 5-HTlization . Using 22CR densities in the young adult and middle-aged brain. These data indicate that 5-HT compounds are capable of overcoming components of age-related changes in physiology to activate POMC neurons and suppress feeding. These data reveal that although former and current 5-HT obesity medications require ARC POMC to achieve appetitive effects, a pathway that is remodeled with aging, the anatomical 5-HT2CR-POMC machinery is preserved and the anorectic potency is maintained, findings of clinical significance to the global obese population.In summary, we report that 5-HT compound anorectic potency is maintained with aging in association with equivalent 5-HT medication activation of POMC neurons at comparable 5-HT


Some individuals remain persistently HIV-seronegative despite multiple high-risk exposures to the virus (HIV-exposed seronegatives or ESN). Different mechanisms may influence host resistance to HIV infection. HIV-specific CTL has been suggested to play a role in HIV-1 protection.Ten HIV-1 ESN in HIV-discordant couples were enrolled at the Fann University hospital, Dakar, Senegal. Thirty HIV-1 infected patients (10 HIV-1 non transmitted partners in discordant coupes and 20 HIV-1 infected partners in HIV-concordant couples) and 10 HIV-negative unexposed subjects were included as controls. Levels of CD107a and b, and CD107a/b+IFN-γ+ in CD8+ T cells sub-set were measured in fresh PBMC by flow cytometry, in the presence or absence of stimulation with SEB.HIV-negative subjects (10 ESN subjects and 10 HIV-negative controls) showed significantly lower percentages of CD107a/b+ expression on CD8+ T-cells than did HIV-1 infected patients . Similar conclusions were reached when expression of CD107a/b+IFN-γ+ were analyzed. Interestingly, HIV-1 ESN subjects showed higher frequency of CD8+ T cells expressing CD107a and b compared with unexposed HIV-negative controls , concordant with production of intracellular IFN-γ.Taken together, our data suggest that resistance to HIV-1 infection among ESN partners in HIV-discordant couples may be associated with HIV-specific CTL responses.