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Sample records for cellular receptor car

  1. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

    2001-08-01

    The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5 (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).

  2. [Prerequisite for hematopoietic cellular therapy programs to set up chimeric antigen receptor T-cell therapy (CAR T-cells): Guidelines from the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)].

    Yakoub-Agha, Ibrahim; Ferrand, Christophe; Chalandon, Yves; Ballot, Caroline; Castilla Llorente, Cristina; Deschamps, Marina; Gauthier, Jordan; Labalette, Myriam; Larghero, Jérôme; Maheux, Camille; Moreau, Anne-Sophie; Varlet, Pauline; Pétillon, Marie-Odile; Pinturaud, Marine; Rubio, Marie Thérèse; Chabannon, Christian

    2017-12-01

    CAR T-cells are autologous or allogeneic human lymphocytes that are genetically engineered to express a chimeric antigen receptor targeting an antigen expressed on tumor cells such as CD19. CAR T-cells represent a new class of medicinal products, and belong to the broad category of Advanced Therapy Medicinal Products (ATMPs), as defined by EC Regulation 2007-1394. Specifically, they are categorized as gene therapy medicinal products. Although CAR T-cells are cellular therapies, the organization for manufacturing and delivery is far different from the one used to deliver hematopoietic cell grafts, for different reasons including their classification as medicinal products. Currently available clinical observations were mostly produced in the context of trials conducted either in the USA or in China. They demonstrate remarkable efficacy for patients presenting advanced or poor-prognosis hematological malignancies, however with severe side effects in a significant proportion of patients. Toxicities can and must be anticipated and dealt with in the context of a full coordination between the clinical cell therapy ward in charge of the patient, and the neighboring intensive care unit. The present workshop aimed at identifying prerequisites to be met in order for French hospitals to get efficiently organized and fulfill sponsors' expectations before initiation of clinical trials designed to investigate CAR T-cells. Copyright © 2017 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

  3. Car Deceleration Considering Its Own Velocity in Cellular Automata Model

    Li Keping

    2006-01-01

    In this paper, we propose a new cellular automaton model, which is based on NaSch traffic model. In our method, when a car has a larger velocity, if the gap between the car and its leading car is not enough large, it will decrease. The aim is that the following car has a buffer space to decrease its velocity at the next time, and then avoid to decelerate too high. The simulation results show that using our model, the car deceleration is realistic, and is closer to the field measure than that of NaSch model.

  4. Mode of Action and Human Relevance Analysis for Nuclear Receptor-Mediated Liver Toxicity: A Case Study with Phenobarbital as a Model Constitutive Androstane Receptor (CAR) Activator

    The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are key nuclear receptors involved in the regulation of cellular responses. to exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non­ genotoxic i...

  5. Chimeric Antigen Receptor T Cell (Car T Cell Therapy In Hematology

    Pinar Ataca

    2015-12-01

    Full Text Available It is well demonstrated that immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation (HSCT. Adoptive T cell transfer has been improved to be more specific and potent and cause less off-target toxicities. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR and chimeric antigen receptor (CAR modified T cells. On July 1, 2014, the United States Food and Drug Administration granted ‘breakthrough therapy’ designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the beneficiaries of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical-clinical studies, effectiveness and drawbacks of this strategy.

  6. Androgen receptor drives cellular senescence.

    Yelena Mirochnik

    Full Text Available The accepted androgen receptor (AR role is to promote proliferation and survival of prostate epithelium and thus prostate cancer progression. While growth-inhibitory, tumor-suppressive AR effects have also been documented, the underlying mechanisms are poorly understood. Here, we for the first time link AR anti-cancer action with cell senescence in vitro and in vivo. First, AR-driven senescence was p53-independent. Instead, AR induced p21, which subsequently reduced ΔN isoform of p63. Second, AR activation increased reactive oxygen species (ROS and thereby suppressed Rb phosphorylation. Both pathways were critical for senescence as was proven by p21 and Rb knock-down and by quenching ROS with N-Acetyl cysteine and p63 silencing also mimicked AR-induced senescence. The two pathways engaged in a cross-talk, likely via PML tumor suppressor, whose localization to senescence-associated chromatin foci was increased by AR activation. All these pathways contributed to growth arrest, which resolved in senescence due to concomitant lack of p53 and high mTOR activity. This is the first demonstration of senescence response caused by a nuclear hormone receptor.

  7. A drive through cellular therapy for CLL in 2015: allogeneic cell transplantation and CARs.

    Mato, Anthony; Porter, David L

    2015-07-23

    Over the past decade the development of safer reduced-intensity conditioning regimens, expanded donor pools, advances in supportive care, and prevention/management of graft-versus-host disease have expanded stem cell transplantation (SCT) availability for chronic lymphocytic leukemia (CLL) patients. However, there are now increasingly active treatment options available for CLL patients with favorable toxicity profiles and convenient administration schedules. This raises the critical issue of whether or not attainment of cure remains a necessary goal. It is now less clear that treatment with curative intention and with significant toxicity is required for long-term survival in CLL. In addition, the demonstrated safety and activity of genetically modified chimeric antigen receptor (CAR) T cells present the opportunity of harnessing the power of the immune system to kill CLL cells without the need for SCT. We attempt to define the role of SCT in the era of targeted therapies and discuss questions that remain to be answered. Furthermore, we highlight the potential for exciting new cellular therapy using genetically modified anti-CD19 CAR T cells and discuss its potential to alter treatment paradigms for CLL. © 2015 by The American Society of Hematology.

  8. The cellular receptors of exogenous RNA

    Patryk Reniewicz

    2016-04-01

    Full Text Available One of the key determinants of survival for organisms is proper recognition of exogenous and endogenous nucleic acids. Therefore, high eukaryotes developed a number of receptors that allow for discrimination between friend or foe DNA and RNA. Appearance of exogenous RNA in cytoplasm provides a signal of danger and triggers cellular responses that facilitate eradication of a pathogen. Recognition of exogenous RNA is additionally complicated by fact that large amount of endogenous RNA is present in cytoplasm Thus, number of different receptors, found in eukaryotic cells, is able to recognize that nucleic acid. First group of those receptors consist endosomal Toll like receptors, namely TLR3, TLR7, TLR8 and TLR13. Those receptors recognize RNA released from pathogens that enter the cell by endocytosis. The second group includes cytoplasmic sensors like PKR and the family of RLRs comprised of RIG-I, MDA5 and LGP2. Cytoplasmic receptors recognize RNA from pathogens invading the cell by non-endocytic pathway. In both cases binding of RNA by its receptors results in activation of the signalling cascades that lead to the production of interferon and other cytokines.

  9. Boundary effects on car accidents in a cellular automaton model

    Yang Xianqing; Ma Yuqiang; Zhao Yuemin

    2004-01-01

    In this paper we numerically study the probability P ac of occurrence of car accidents in the Nagel-Schreckenberg (NS) model with open boundary condition. In the deterministic NS model, numerical results show that there exists a critical value of extinction rate β above which no car accidents occur, and below which the probability P ac is independent of the speed limit v max and the injection rate α, but only determined by the extinction rate β. In the non-deterministic NS model, the probability P ac is a non-monotonic function of β in the region of low β value, while it is independent of β in the region of high β value. The stochastic braking not only reduces the occurrence of car accidents, but splits degenerate effects of v max on the probability P ac . Theoretical analyses give an agreement with numerical results in the deterministic NS model and in the non-deterministic NS model with v max = 1 in the case of low β value region. Qualitative differences between open and periodic systems in the relations of P ac to the bulk density ρ imply that various correlations may exist between the two systems

  10. Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes

    Smetanina, Mariya A.; Pakharukova, Mariya Y.; Kurinna, Svitlana M.; Dong, Bingning; Hernandez, Juan P.; Moore, David D.; Merkulova, Tatyana I.

    2011-01-01

    2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car -/- ) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car -/- livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor. - Highlights: → The azo dye and mouse carcinogen OAT is a very effective mCAR activator. → OAT increases mCAR transactivation in a dose-dependent manner. → OAT CAR-dependently increases the expression of a specific subset of CAR target genes. → OAT induces an unexpectedly deferred, but CAR-dependent hepatocyte proliferation.

  11. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.

    Levine, B L

    2015-03-01

    Performance enhancement of the immune system can now be generated through ex vivo gene modification of T cells in order to redirect native specificity to target tumor antigens. This approach combines the specificity of antibody therapy, the expanded response of cellular therapy and the memory activity of vaccine therapy. Recent clinical trials of chimeric antigen receptor (CAR) T cells directed toward CD19 as a stand-alone therapy have shown sustained complete responses in patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. As these drug products are individually derived from a patient's own cells, a different manufacturing approach is required for this kind of personalized therapy compared with conventional drugs. Key steps in the CAR T-cell manufacturing process include the selection and activation of isolated T cells, transduction of T cells to express CARs, ex vivo expansion of modified T cells and cryopreservation in infusible media. In this review, the steps involved in isolating, genetically modifying and scaling-out the CAR T cells for use in a clinical setting are described in the context of in-process and release testing and regulatory standards.

  12. Cysteinyl-Leukotriene Receptors and Cellular Signals

    G. Enrico Rovati

    2007-01-01

    Full Text Available Cysteinyl-leukotrienes (cysteinyl-LTs exert a range of proinflammatory effects, such as constriction of airways and vascular smooth muscle, increase of endothelial cell permeability leading to plasma exudation and edema, and enhanced mucus secretion. They have proved to be important mediators in asthma, allergic rhinitis, and other inflammatory conditions, including cardiovascular diseases, cancer, atopic dermatitis, and urticaria. The classification into subtypes of the cysteinyl-LT receptors (CysLTRs was based initially on binding and functional data, obtained using the natural agonists and a wide range of antagonists. CysLTRs have proved remarkably resistant to cloning. However, in 1999 and 2000, the CysLT1R and CysLT2R were successfully cloned and both shown to be members of the G-protein coupled receptors (GPCRs superfamily. Molecular cloning has confirmed most of the previous pharmacological characterization and identified distinct expression patterns only partially overlapping. Recombinant CysLTRs couple to the Gq/11 pathway that modulates inositol phospholipids hydrolysis and calcium mobilization, whereas in native systems, they often activate a pertussis toxin-insensitive Gi/o-protein, or are coupled promiscuously to both G-proteins. Interestingly, recent data provide evidence for the existence of an additional receptor subtype that seems to respond to both cysteinyl-LTs and uracil nucleosides, and of an intracellular pool of CysLTRs that may have roles different from those of plasma membrane receptors. Finally, a cross-talk between the cysteinyl-LT and the purine systems is being delineated. This review will summarize recent data derived from studies on the molecular and cellular pharmacology of CysLTRs.

  13. Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

    Takahashi, Tadanobu; Moriyama, Yusuke; Ikari, Akira; Sugatani, Junko; Suzuki, Takashi; Miwa, Masao

    2008-01-01

    Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism

  14. Clinical pharmacology of CAR-T cells: Linking cellular pharmacodynamics to pharmacokinetics and antitumor effects.

    Norelli, M; Casucci, M; Bonini, C; Bondanza, A

    2016-01-01

    Adoptive cell transfer of T cells genetically modified with tumor-reactive chimeric antigen receptors (CARs) is a rapidly emerging field in oncology, which in preliminary clinical trials has already shown striking antitumor efficacy. Despite these premises, there are still a number of open issues related to CAR-T cells, spanning from their exact mechanism of action (pharmacodynamics), to the factors associated with their in vivo persistence (pharmacokinetics), and, finally, to the relative contribution of each of the two in determining the antitumor effects and accompanying toxicities. In light of the unprecedented curative potential of CAR-T cells and of their predicted wide availability in the next few years, in this review we will summarize the current knowledge on the clinical pharmacology aspects of what is anticipated to be a brand new class of biopharmaceuticals to join the therapeutic armamentarium of cancer doctors. Copyright © 2015. Published by Elsevier B.V.

  15. Foreign or Domestic CARs: Receptor Ligands as Antigen-Binding Domains

    Donald R. Shaffer

    2014-01-01

    Full Text Available Chimeric antigen receptors (CARs are increasingly being used in clinical trials to treat a variety of malignant conditions and recent results with CD19-specific CARs showing complete tumor regressions has sparked the interest of researchers and the public alike. Traditional CARs have been generated using single-chain variable fragments (scFv, often derived from murine monoclonal antibodies, for antigen specificity. As the clinical experience with CAR T cells grows, so does the potential for unwanted immune responses against the foreign transgene. Strategies that may reduce the immunogenicity of CAR T cells are humanization of the scFv and the use of naturally occurring receptor ligands as antigen-binding domains. Herein, we review the experience with alternatively designed CARs that contain receptor ligands rather than scFv. While most of the experiences have been in the pre-clinical setting, clinical data is also emerging.

  16. The cellular receptors for infectious bursal disease virus | Zhu ...

    Virus receptors are simplistically defined as cell surface molecules that mediate binding (attachment, adsorption) and/or trigger membrane fusion or entry through other processes. Infectious bursal disease virus (IBDV) entry into host cells occurs by recognition of specific cellular receptor(s) with viral envelope glycoprotein, ...

  17. Safety and Efficacy of Intratumoral Injections of Chimeric Antigen Receptor (CAR) T Cells in Metastatic Breast Cancer.

    Tchou, Julia; Zhao, Yangbing; Levine, Bruce L; Zhang, Paul J; Davis, Megan M; Melenhorst, Jan Joseph; Kulikovskaya, Irina; Brennan, Andrea L; Liu, Xiaojun; Lacey, Simon F; Posey, Avery D; Williams, Austin D; So, Alycia; Conejo-Garcia, Jose R; Plesa, Gabriela; Young, Regina M; McGettigan, Shannon; Campbell, Jean; Pierce, Robert H; Matro, Jennifer M; DeMichele, Angela M; Clark, Amy S; Cooper, Laurence J; Schuchter, Lynn M; Vonderheide, Robert H; June, Carl H

    2017-12-01

    Chimeric antigen receptors (CAR) are synthetic molecules that provide new specificities to T cells. Although successful in treatment of hematologic malignancies, CAR T cells are ineffective for solid tumors to date. We found that the cell-surface molecule c-Met was expressed in ∼50% of breast tumors, prompting the construction of a CAR T cell specific for c-Met, which halted tumor growth in immune-incompetent mice with tumor xenografts. We then evaluated the safety and feasibility of treating metastatic breast cancer with intratumoral administration of mRNA-transfected c-Met-CAR T cells in a phase 0 clinical trial (NCT01837602). Introducing the CAR construct via mRNA ensured safety by limiting the nontumor cell effects (on-target/off-tumor) of targeting c-Met. Patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 × 10 7 or 3 × 10 8 cells. CAR T mRNA was detectable in peripheral blood and in the injected tumor tissues after intratumoral injection in 2 and 4 patients, respectively. mRNA c-Met-CAR T cell injections were well tolerated, as none of the patients had study drug-related adverse effects greater than grade 1. Tumors treated with intratumoral injected mRNA c-Met-CAR T cells were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, all surrounded by macrophages at the leading edges and within necrotic zones. We conclude that intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors. Cancer Immunol Res; 5(12); 1152-61. ©2017 AACR . ©2017 American Association for Cancer Research.

  18. Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    Bipulendu Jena

    Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  19. Similarities of cellular receptors for interferon and cortisol

    Filipic, B.; Schauer, P.; Likar, M.

    1977-01-01

    Cellular receptors are molecules located on the cell membrane. Their function is to bind different molecules to the cell surface. These molecules can penetrate into the cytoplasm and trigger cellular changes. One kind of such bound molecules are interferons and corticosteroids. Until very recently very little was known about interferon's receptors on the cell surface, mechanisms of interferon's binding to them or about kinetics of such binding. On the basis of results published elsewhere and on the basis of experimental results, the authors suggest: receptors for interferon and cortisol are glycoproteins located on the cell surface, in analogy with PHA receptors they are chemically sialoglycoproteins, binding kinetics of cortisol and interferon is similar, interferon and cortisol compete for cellular receptors, binding of cortisol or interferon is dependent on allosteric configuration of receptor molecules. (author)

  20. The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of car protein structure.

    Okegawa, T; Pong, R C; Li, Y; Bergelson, J M; Sagalowsky, A I; Hsieh, J T

    2001-09-01

    The coxsackie and adenovirus receptor (CAR) is identified as a high-affinity receptor for adenovirus type 5. We observed that invasive bladder cancer specimens had significantly reduced CAR mRNA levels compared with superficial bladder cancer specimens, which suggests that CAR may play a role in the progression of bladder cancer. Elevated CAR expression in the T24 cell line (CAR-negative cells) increased its sensitivity to adenovirus infection and significantly inhibited its in vitro growth, accompanied by p21 and hypophosphorylated retinoblastoma accumulation. Conversely, decreased CAR levels in both RT4 and 253J cell lines (CAR-positive cells) promoted their in vitro growth. To unveil the mechanism of action of CAR, we showed that the extracellular domain of CAR facilitated intercellular adhesion. Furthermore, interrupting intercellular adhesion of CAR by a specific antibody alleviates the growth-inhibitory effect of CAR. We also demonstrated that both the transmembrane and intracellular domains of CAR were critical for its growth-inhibitory activity. These data indicate that the cell-cell contact initiated by membrane-bound CAR can elicit a negative signal cascade to modulate cell cycle regulators inside the nucleus of bladder cancer cells. Therefore, the presence of CAR cannot only facilitate viral uptake of adenovirus but also inhibit cell growth. These results can be integrated to formulate a new strategy for bladder cancer therapy.

  1. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  2. Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation.

    Shizu, Ryota; Min, Jungki; Sobhany, Mack; Pedersen, Lars C; Mutoh, Shingo; Negishi, Masahiko

    2018-01-05

    The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

  3. Exploiting natural killer group 2D receptors for CAR T-cell therapy.

    Demoulin, Benjamin; Cook, W James; Murad, Joana; Graber, David J; Sentman, Marie-Louise; Lonez, Caroline; Gilham, David E; Sentman, Charles L; Agaugue, Sophie

    2017-08-01

    Chimeric antigen receptors (CARs) are genetically engineered proteins that combine an extracellular antigen-specific recognition domain with one or several intracellular T-cell signaling domains. When expressed in T cells, these CARs specifically trigger T-cell activation upon antigen recognition. While the clinical proof of principle of CAR T-cell therapy has been established in hematological cancers, CAR T cells are only at the early stages of being explored to tackle solid cancers. This special report discusses the concept of exploiting natural killer cell receptors as an approach that could broaden the specificity of CAR T cells and potentially enhance the efficacy of this therapy against solid tumors. New data demonstrating feasibility of this approach in humans and supporting the ongoing clinical trial are also presented.

  4. Cellular receptors for human enterovirus species A

    Yorihiro eNishimura

    2012-03-01

    Full Text Available Human enterovirus species A (HEV-A is one of the four species of HEV in the genus Enterovirus in the family Picornaviridae. Among HEV-A, coxsackievirus A16 (CVA16 and enterovirus 71 (EV71 are the major causative agents of hand, foot, and mouth disease (HFMD. Some other types of HEV-A are commonly associated with herpangina. Although HFMD and herpangina due to HEV-A are common febrile diseases among infants and children, EV71 can cause various neurological diseases, such as aseptic meningitis and fatal encephalitis.Recently, two human transmembrane proteins, P-selectin glycoprotein ligand-1 (PSGL-1 and scavenger receptor class B, member 2 (SCARB2, were identified as functional receptors for EV71 and CVA16. In in vitro infection experiments using the prototype HEV-A strains, PSGL-1 and SCARB2 could be responsible for the specific receptors for EV71 and CVA16. However, the involvement of both receptors in the in vitro and in vivo infections of clinical isolates of HEV-A has not been clarified yet. To elucidate a diverse array of the clinical outcome of HEV-A-associated diseases, the identification and characterization of HEV-A receptors may provide useful information in understanding the HEV-A pathogenesis at a molecular level.

  5. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    Kanno, Yuichiro; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-01-01

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR

  6. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

  7. Chimeric-antigen receptor T (CAR-T) cell therapy for solid tumors: challenges and opportunities.

    Xia, An-Liang; Wang, Xiao-Chen; Lu, Yi-Jun; Lu, Xiao-Jie; Sun, Beicheng

    2017-10-27

    Chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) have been shown to have unprecedented efficacy in B cell malignancies, most notably in B cell acute lymphoblastic leukemia (B-ALL) with up to a 90% complete remission rate using anti-CD19 CAR-T cells. However, CAR T-cell therapy for solid tumors currently is faced with numerous challenges such as physical barriers, the immunosuppressive tumor microenvironment and the specificity and safety. The clinical results in solid tumors have been much less encouraging, with multiple cases of toxicity and a lack of therapeutic response. In this review, we will discuss the current stats and challenges of CAR-T cell therapy for solid tumors, and propose possibl e solutions and future perspectives.

  8. Chimeric antigen receptor (CAR T cell therapy for malignant cancers: Summary and perspective

    Aaron J. Smith

    2016-11-01

    Full Text Available This paper will summarize the data obtained primarily from the last decade of chimeric antigen receptor (CAR T cell immunotherapy. It will do so in a manner that provides an overview needed to set the foundation for perspective on the state of research associated with CAR T cell therapy. The topics covered will include the construction of engineered CAR T cells from the standpoint of the different generations, the mode in which autologous T cells are transfected, the various biomarkers that have been used in CAR T cell immunotherapy, and setbacks associated with engineered T cells. Perspective on priorities of CAR T cell immunotherapy will also be addressed as they are related to safety and efficacy.

  9. Regional Delivery of Chimeric Antigen Receptor (CAR) T-Cells for Cancer Therapy.

    Sridhar, Praveen; Petrocca, Fabio

    2017-07-18

    Chimeric Antigen Receptor (CAR) T-cells are T-cells with recombinant receptors targeted to tumor antigens. CAR-T cell therapy has emerged as a mode of immunotherapy and is now being extensively explored in hematologic cancer. In contrast, CAR-T cell use in solid tumors has been hampered by multiple obstacles. Several approaches have been taken to circumvent these obstacles, including the regional delivery of CAR-T cells. Regional CAR-T cell delivery can theoretically compensate for poor T-cell trafficking and tumor antigen specificity while avoiding systemic toxicity associated with intravenous delivery. We reviewed completed clinical trials for the treatment of glioblastoma and metastatic colorectal cancer and examined the data in these studies for safety, efficacy, and potential advantages that regional delivery may confer over systemic delivery. Our appraisal of the available literature revealed that regional delivery of CAR-T cells in both glioblastoma and hepatic colorectal metastases was generally well tolerated and efficacious in select instances. We propose that the regional delivery of CAR-T cells is an area of potential growth in the solid tumor immunotherapy, and look towards future clinical trials in head and neck cancer, mesothelioma, and peritoneal carcinomatosis as the use of this technique expands.

  10. Regional Delivery of Chimeric Antigen Receptor (CAR T-Cells for Cancer Therapy

    Praveen Sridhar

    2017-07-01

    Full Text Available Chimeric Antigen Receptor (CAR T-cells are T-cells with recombinant receptors targeted to tumor antigens. CAR-T cell therapy has emerged as a mode of immunotherapy and is now being extensively explored in hematologic cancer. In contrast, CAR-T cell use in solid tumors has been hampered by multiple obstacles. Several approaches have been taken to circumvent these obstacles, including the regional delivery of CAR-T cells. Regional CAR-T cell delivery can theoretically compensate for poor T-cell trafficking and tumor antigen specificity while avoiding systemic toxicity associated with intravenous delivery. We reviewed completed clinical trials for the treatment of glioblastoma and metastatic colorectal cancer and examined the data in these studies for safety, efficacy, and potential advantages that regional delivery may confer over systemic delivery. Our appraisal of the available literature revealed that regional delivery of CAR-T cells in both glioblastoma and hepatic colorectal metastases was generally well tolerated and efficacious in select instances. We propose that the regional delivery of CAR-T cells is an area of potential growth in the solid tumor immunotherapy, and look towards future clinical trials in head and neck cancer, mesothelioma, and peritoneal carcinomatosis as the use of this technique expands.

  11. Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

    Vigl, Benjamin; Zgraggen, Claudia; Rehman, Nadia; Banziger-Tobler, Nadia E.; Detmar, Michael; Halin, Cornelia

    2009-01-01

    Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity

  12. Sexually dimorphic regulation and induction of P450s by the constitutive androstane receptor (CAR)

    Hernandez, J.P.; Mota, L.C.; Huang, W.; Moore, D.D.; Baldwin, W.S.

    2009-01-01

    The constitutive androstane receptor (CAR) is a xenosensing nuclear receptor and regulator of cytochrome P450s (CYPs). However, the role of CAR as a basal regulator of CYP expression nor its role in sexually dimorphic responses have been thoroughly studied. We investigated basal regulation and sexually dimorphic regulation and induction by the potent CAR activator TCPOBOP and the moderate CAR activator Nonylphenol (NP). NP is an environmental estrogen and one of the most commonly found environmental toxicants in Europe and the United States. Previous studies have demonstrated that NP induces several CYPs in a sexually dimorphic manner, however the role of CAR in regulating NP-mediated sexually dimorphic P450 expression and induction has not been elucidated. Therefore, wild-type and CAR-null male and female mice were treated with honey as a carrier, NP, or TCPOBOP and CYP expression monitored by QPCR and Western blotting. CAR basally regulates the expression of Cyp2c29, Cyp2b13, and potentially Cyp2b10 as demonstrated by QPCR. Furthermore, we observed a shift in the testosterone 6α/15α-hydroxylase ratio in untreated CAR-null female mice to the male pattern, which indicates an alteration in androgen status and suggests a role for androgens as CAR inverse agonists. Xenobiotic-treatments with NP and TCPOBOP induced Cyp2b10, Cyp2c29, and Cyp3a11 in a CAR-mediated fashion; however NP only induced these CYPs in females and TCPOBOP induced these CYPs in both males and females. Interestingly, Cyp2a4, was only induced in wild-type male mice by TCPOBOP suggesting Cyp2a4 induction is not sensitive to CAR-mediated induction in females. Overall, TCPOBOP and NP show similar CYP induction profiles in females, but widely different profiles in males potentially related to lower sensitivity of males to either indirect or moderate CAR activators such as NP. In summary, CAR regulates the basal and chemically inducible expression of several sexually dimorphic xenobiotic metabolizing P

  13. Distribution of cellular HSV-1 receptor expression in human brain.

    Lathe, Richard; Haas, Juergen G

    2017-06-01

    Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

  14. Polyvalent cation receptor proteins (CaRs) are salinity sensors in fish.

    Nearing, J; Betka, M; Quinn, S; Hentschel, H; Elger, M; Baum, M; Bai, M; Chattopadyhay, N; Brown, E M; Hebert, S C; Harris, H W

    2002-07-09

    To determine whether calcium polyvalent cation-sensing receptors (CaRs) are salinity sensors in fish, we used a homology-based cloning strategy to isolate a 4.1-kb cDNA encoding a 1,027-aa dogfish shark (Squalus acanthias) kidney CaR. Expression studies in human embryonic kidney cells reveal that shark kidney senses combinations of Ca(2+), Mg(2+), and Na(+) ions at concentrations present in seawater and kidney tubules. Shark kidney is expressed in multiple shark osmoregulatory organs, including specific tubules of the kidney, rectal gland, stomach, intestine, olfactory lamellae, gill, and brain. Reverse transcriptase-PCR amplification using specific primers in two teleost fish, winter flounder (Pleuronectes americanus) and Atlantic salmon (Salmo salar), reveals a similar pattern of CaR tissue expression. Exposure of the lumen of winter flounder urinary bladder to the CaR agonists, Gd(3+) and neomycin, reversibly inhibit volume transport, which is important for euryhaline teleost survival in seawater. Within 24-72 hr after transfer of freshwater-adapted Atlantic salmon to seawater, there are increases in their plasma Ca(2+), Mg(2+), and Na(+) that likely serve as a signal for internal CaRs, i.e., brain, to sense alterations in salinity in the surrounding water. We conclude that CaRs act as salinity sensors in both teleost and elasmobranch fish. Their tissue expression patterns in fish provide insights into CaR functions in terrestrial animals including humans.

  15. Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization.

    Ozawa, Akihiko; Brunori, Gloria; Mercatelli, Daniela; Wu, Jinhua; Cippitelli, Andrea; Zou, Bende; Xie, Xinmin Simon; Williams, Melissa; Zaveri, Nurulain T; Low, Sarah; Scherrer, Grégory; Kieffer, Brigitte L; Toll, Lawrence

    2015-08-19

    The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [(35)S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with μ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These

  16. Receptor Oligomerization as a Process Modulating Cellular Semiotics

    Giorgi, Franco; Bruni, Luis Emilio; Maggio, Roberto

    2010-01-01

    be another level of quality control that may help maintaining GPCRs rather stable throughout evolution. We propose here receptor oligomerization to be a basic molecular mechanism controlling GPCRs redundancy in many different cell types, and the plasma membrane as the first hierarchical cell structure...... at which selective categorical sensing may occur. Categorical sensing can be seen as the cellular capacity for identifying and ordering complex patterns of mixed signals out of a contextual matrix, i.e., the recognition of meaningful patterns out of ubiquitous signals. In this context, redundancy...

  17. Cars, Cars, Cars

    McIntosh, Phyllis

    2013-01-01

    Cars are the focus of this feature article, which explores such topics as the history of cars in the United States, the national highway system, safety and pollution concerns, mobility and freedom for women, classic car shows, and the road trip in American literature and film. Also included are links to the websites of Automobile in American Life…

  18. Incorporation of Immune Checkpoint Blockade into Chimeric Antigen Receptor T Cells (CAR-Ts): Combination or Built-In CAR-T.

    Yoon, Dok Hyun; Osborn, Mark J; Tolar, Jakub; Kim, Chong Jai

    2018-01-24

    Chimeric antigen receptor (CAR) T cell therapy represents the first U.S. Food and Drug Administration approved gene therapy and these engineered cells function with unprecedented efficacy in the treatment of refractory CD19 positive hematologic malignancies. CAR translation to solid tumors is also being actively investigated; however, efficacy to date has been variable due to tumor-evolved mechanisms that inhibit local immune cell activity. To bolster the potency of CAR-T cells, modulation of the immunosuppressive tumor microenvironment with immune-checkpoint blockade is a promising strategy. The impact of this approach on hematological malignancies is in its infancy, and in this review we discuss CAR-T cells and their synergy with immune-checkpoint blockade.

  19. Incorporation of Immune Checkpoint Blockade into Chimeric Antigen Receptor T Cells (CAR-Ts: Combination or Built-In CAR-T

    Dok Hyun Yoon

    2018-01-01

    Full Text Available Chimeric antigen receptor (CAR T cell therapy represents the first U.S. Food and Drug Administration approved gene therapy and these engineered cells function with unprecedented efficacy in the treatment of refractory CD19 positive hematologic malignancies. CAR translation to solid tumors is also being actively investigated; however, efficacy to date has been variable due to tumor-evolved mechanisms that inhibit local immune cell activity. To bolster the potency of CAR-T cells, modulation of the immunosuppressive tumor microenvironment with immune-checkpoint blockade is a promising strategy. The impact of this approach on hematological malignancies is in its infancy, and in this review we discuss CAR-T cells and their synergy with immune-checkpoint blockade.

  20. Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV.

    Liu, Dongfang; Tian, Shuo; Zhang, Kai; Xiong, Wei; Lubaki, Ndongala Michel; Chen, Zhiying; Han, Weidong

    2017-12-01

    Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells contribute to the body's immune defenses. Current chimeric antigen receptor (CAR)-modified T cell immunotherapy shows strong promise for treating various cancers and infectious diseases. Although CAR-modified NK cell immunotherapy is rapidly gaining attention, its clinical applications are mainly focused on preclinical investigations using the NK92 cell line. Despite recent advances in CAR-modified T cell immunotherapy, cost and severe toxicity have hindered its widespread use. To alleviate these disadvantages of CAR-modified T cell immunotherapy, additional cytotoxic cell-mediated immunotherapies are urgently needed. The unique biology of NK cells allows them to serve as a safe, effective, alternative immunotherapeutic strategy to CAR-modified T cells in the clinic. While the fundamental mechanisms underlying the cytotoxicity and side effects of CAR-modified T and NK cell immunotherapies remain poorly understood, the formation of the immunological synapse (IS) between CAR-modified T or NK cells and their susceptible target cells is known to be essential. The role of the IS in CAR T and NK cell immunotherapies will allow scientists to harness the power of CAR-modified T and NK cells to treat cancer and infectious diseases. In this review, we highlight the potential applications of CAR-modified NK cells to treat cancer and human immunodeficiency virus (HIV), and discuss the challenges and possible future directions of CAR-modified NK cell immunotherapy, as well as the importance of understanding the molecular mechanisms of CAR-modified T cell- or NK cell-mediated cytotoxicity and side effects, with a focus on the CAR-modified NK cell IS.

  1. Opportunistic activation of TRP receptors by endogenous lipids: exploiting lipidomics to understand TRP receptor cellular communication.

    Bradshaw, Heather B; Raboune, Siham; Hollis, Jennifer L

    2013-03-19

    Transient receptor potential channels (TRPs) form a large family of ubiquitous non-selective cation channels that function as cellular sensors and in many cases regulate intracellular calcium. Identification of the endogenous ligands that activate these TRP receptors is still under intense investigation with the majority of these channels still remaining "orphans." That these channels respond to a variety of external stimuli (e.g. plant-derived lipids, changes in temperature, and changes in pH) provides a framework for their abilities as cellular sensors, however, the mechanism of direct activation is still under much debate and research. In the cases where endogenous ligands (predominately lipids) have shown direct activation of a channel, multiple ligands have been shown to activate the same channel suggesting that these receptors are "promiscuous" in nature. Lipidomics of a growing class of endogenous lipids, N-acyl amides, the most famous of which is N-arachidonoyl ethanolamine (the endogenous cannabinoid, Anandamide) is providing a novel set of ligands that have been shown to activate some members of the TRP family and have the potential to deorphanize many more. Here it is argued that activation of TRPV receptors, a subset of the larger family of TRPs, by multiple endogenous lipids that are structurally analogous is a model system to drive our understanding that many TRP receptors are not promiscuous, but are more characteristically "opportunistic" in nature; exploiting the structural similarity and biosynthesis of a narrow range of analogous endogenous lipids. In addition, this manuscript will compare the activation properties of TRPC5 to the activity profile of an "orphan" lipid, N-palmitoyl glycine; further demonstrating that lipidomics aimed at expanding our knowledge of the family of N-acyl amides has the potential to provide novel avenues of research for TRP receptors. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Development of second generation peptides modulating cellular adiponectin receptor responses

    Laszlo eOtvos

    2014-10-01

    Full Text Available The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC. In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399. The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM - low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400 was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400 at similar concentrations will be an important target validation tool to study adiponectin functions.

  3. Development of second generation peptides modulating cellular adiponectin receptor responses

    Otvos, Laszlo; Knappe, Daniel; Hoffmann, Ralf; Kovalszky, Ilona; Olah, Julia; Hewitson, Tim; Stawikowska, Roma; Stawikowski, Maciej; Cudic, Predrag; Lin, Feng; Wade, John; Surmacz, Eva; Lovas, Sandor

    2014-10-01

    The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM - low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

  4. Quantum dot multiplexing for the profiling of cellular receptors

    Lee-Montiel, Felipe T.; Li, Peter; Imoukhuede, P. I.

    2015-11-01

    The profiling of cellular heterogeneity has wide-reaching importance for our understanding of how cells function and react to their environments in healthy and diseased states. Our ability to interpret and model cell behavior has been limited by the difficulties of measuring cell differences, for example, comparing tumor and non-tumor cells, particularly at the individual cell level. This demonstrates a clear need for a generalizable approach to profile fluorophore sites on cells or molecular assemblies on beads. Here, a multiplex immunoassay for simultaneous detection of five different angiogenic markers was developed. We targeted angiogenic receptors in the vascular endothelial growth factor family (VEGFR1, VEGFR2 and VEGFR3) and Neuropilin (NRP) family (NRP1 and NRP2), using multicolor quantum dots (Qdots). Copper-free click based chemistry was used to conjugate the monoclonal antibodies with 525, 565, 605, 655 and 705 nm CdSe/ZnS Qdots. We tested and performed colocalization analysis of our nanoprobes using the Pearson correlation coefficient statistical analysis. Human umbilical vein endothelial cells (HUVEC) were tested. The ability to easily monitor the molecular indicators of angiogenesis that are a precursor to cancer in a fast and cost effective system is an important step towards personalized nanomedicine.The profiling of cellular heterogeneity has wide-reaching importance for our understanding of how cells function and react to their environments in healthy and diseased states. Our ability to interpret and model cell behavior has been limited by the difficulties of measuring cell differences, for example, comparing tumor and non-tumor cells, particularly at the individual cell level. This demonstrates a clear need for a generalizable approach to profile fluorophore sites on cells or molecular assemblies on beads. Here, a multiplex immunoassay for simultaneous detection of five different angiogenic markers was developed. We targeted angiogenic receptors

  5. Potential cellular receptors involved in hepatitis C virus entry into cells

    Muellhaupt Beat

    2005-04-01

    Full Text Available Abstract Hepatitis C virus (HCV infects hepatocytes and leads to permanent, severe liver damage. Since the genomic sequence of HCV was determined, progress has been made towards understanding the functions of the HCV-encoded proteins and identifying the cellular receptor(s responsible for adsorption and penetration of the virus particle into the target cells. Several cellular receptors for HCV have been proposed, all of which are associated with lipid and lipoprotein metabolism. This article reviews the cellular receptors for HCV and suggests a general model for HCV entry into cells, in which lipoproteins play a crucial role.

  6. Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

    Gaëlle Gonzalez

    Full Text Available Cell microparticles (MPs released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5, and serotype 35 (HAdV35, respectively. We found that MPs derived from CHO cells (MP-donor cells constitutively expressing CAR (MP-CAR or CD46 (MP-CD46 were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

  7. Chimeric Antigen Receptor (CAR) T Cells: Lessons Learned from Targeting of CD19 in B-Cell Malignancies.

    Hay, Kevin A; Turtle, Cameron J

    2017-03-01

    Adoptive immunotherapy with chimeric antigen receptor-modified (CAR)-T cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T cell therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers.

  8. Chimeric antigen receptor T cell (CAR-T) immunotherapy for solid tumors: lessons learned and strategies for moving forward.

    Li, Jian; Li, Wenwen; Huang, Kejia; Zhang, Yang; Kupfer, Gary; Zhao, Qi

    2018-02-13

    Recently, the US Food and Drug Administration (FDA) approved the first chimeric antigen receptor T cell (CAR-T) therapy for the treatment CD19-positive B cell acute lymphoblastic leukemia. While CAR-T has achieved remarkable success in the treatment of hematopoietic malignancies, whether it can benefit solid tumor patients to the same extent is still uncertain. Even though hundreds of clinical trials are undergoing exploring a variety of tumor-associated antigens (TAA), no such antigen with comparable properties like CD19 has yet been identified regarding solid tumors CAR-T immunotherapy. Inefficient T cell trafficking, immunosuppressive tumor microenvironment, suboptimal antigen recognition specificity, and lack of safety control are currently considered as the main obstacles in solid tumor CAR-T therapy. Here, we reviewed the solid tumor CAR-T clinical trials, emphasizing the studies with published results. We further discussed the challenges that CAR-T is facing for solid tumor treatment and proposed potential strategies to improve the efficacy of CAR-T as promising immunotherapy.

  9. Chimeric Antigen Receptor (CAR) T cells: Lessons Learned from Targeting of CD19 in B cell malignancies

    Hay, Kevin A; Turtle, Cameron J

    2017-01-01

    Adoptive immunotherapy with chimeric antigen receptor-modified T (CAR-T) cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers. PMID:28110394

  10. Cellular targets of inhalational anaesthetic- and opioid receptor ...

    Secondly, the cardioprotective effects occur independently of the ... cardioprotection take place and highlights the cellular ... Activation of sarcolemmal KATP channels hyperpolarizes cells, ..... respiration and its supramolecular organization.

  11. Targeting cellular adhesion molecules, chemokines and chemokine receptors in rheumatoid arthritis

    Haringman, Jasper J.; Oostendorp, Roos L.; Tak, Paul P.

    2005-01-01

    The development of specific targeted therapies, such as anti-TNF-alpha treatment, for chronic inflammatory disorders such as rheumatoid arthritis, has significantly improved treatment, although not all patients respond. Targeting cellular adhesion molecules and chemokines/chemokine receptors as

  12. A Global Genomic Screening Strategy Reveals Diverse Activators of Constitutive Activated Receptor (CAR)

    A comprehensive survey of conditions that activate CAR in the mouse liver has not been carried out but would be useful in understanding their impact on CAR-dependent liver tumor induction. A gene signature dependent on CAR activation was identified by comparing the transcript pr...

  13. Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

    Freimuth, Paul I.

    2010-04-06

    The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

  14. Seatbelts in CAR therapy: How Safe Are CARS?

    Kentaro Minagawa

    2015-05-01

    Full Text Available T-cells genetically redirected with a chimeric antigen receptor (CAR to recognize tumor antigens and kill tumor cells have been infused in several phase 1 clinical trials with success. Due to safety concerns related to on-target/off-tumor effects or cytokine release syndrome, however, strategies to prevent or abate serious adverse events are required. Pharmacologic therapies; suicide genes; or novel strategies to limit the cytotoxic effect only to malignant cells are under active investigations. In this review, we summarize results and toxicities of investigations employing CAR redirected T-cells, with a focus on published strategies to grant safety of this promising cellular application.

  15. Osteochondral Biopsy Analysis Demonstrates That BST-CarGel Treatment Improves Structural and Cellular Characteristics of Cartilage Repair Tissue Compared With Microfracture

    Méthot, Stéphane; Changoor, Adele; Tran-Khanh, Nicolas; Hoemann, Caroline D.; Stanish, William D.; Restrepo, Alberto; Shive, Matthew S.; Buschmann, Michael D.

    2016-01-01

    Objective The efficacy and safety of BST-CarGel, a chitosan-based medical device for cartilage repair, was compared with microfracture alone at 1 year during a multicenter randomized controlled trial (RCT) in the knee. The quality of repair tissue of osteochondral biopsies collected from a subset of patients was compared using blinded histological assessments. Methods The international RCT evaluated repair tissue quantity and quality by 3-dimensional quantitative magnetic resonance imaging as co-primary endpoints at 12 months. At an average of 13 months posttreatment, 21/41 BST-CarGel and 17/39 microfracture patients underwent elective second look arthroscopies as a tertiary endpoint, during which ICRS (International Cartilage Repair Society) macroscopic scoring was carried out, and osteochondral biopsies were collected. Stained histological sections were evaluated by blinded readers using ICRS I and II histological scoring systems. Collagen organization was evaluated using a polarized light microscopy score. Results BST-CarGel treatment resulted in significantly better ICRS macroscopic scores (P = 0.0002) compared with microfracture alone, indicating better filling, integration, and tissue appearance. Histologically, BST-CarGel resulted in a significant improvement of structural parameters—Surface Architecture (P = 0.007) and Surface/Superficial Assessment (P = 0.042)—as well as cellular parameters—Cell Viability (P = 0.006) and Cell Distribution (P = 0.032). No histological parameters were significantly better for the microfracture group. BST-CarGel treatment also resulted in a more organized repair tissue with collagen stratification more similar to native hyaline cartilage, as measured by polarized light microscopy scoring (P = 0.0003). Conclusion Multiple and independent analyses in this biopsy substudy demonstrated that BST-CarGel treatment results in improved structural and cellular characteristics of repair tissue at 1 year posttreatment compared with

  16. INTERACTION OF RECOMBINANT DIPHTHERIA TOXOIDS WITH CELLULAR RECEPTORS in vitro

    K. Yu. Manoilov

    2016-06-01

    Full Text Available The aim of the research was to compare in vitro characteristics of reception of the natural diphtheria toxin — DT and its nontoxic recombinant analogs — toxoids. For assessing ligand-receptor interaction the method of immunoenzyme analysis and ELISA was used, where the bonding layer recombinant analogues of diphtheria toxin cell receptor HB-EGF from sensitive and resistant to the toxin of the organisms were served. According to the results of ELISA the natural diphtheria toxin, in contrast to recombinant toxoids — CRM197, and B subunit, interacted with mouse HB-EGF with a very low affinity. While human HB-EGF with an equally high affinity connected as toxoids as native diphtheria toxin. Therefore, the analyzed recombinant analogs of toxin obtained in E. coli cells did not reproduce in full measure the receptor specificity of the natural toxin, which should be considered in the case of using these proteins as biotech products.

  17. A Dual-Sensing Receptor Confers Robust Cellular Homeostasis

    Hannah Schramke

    2016-06-01

    Full Text Available Cells have evolved diverse mechanisms that maintain intracellular homeostasis in fluctuating environments. In bacteria, control is often exerted by bifunctional receptors acting as both kinase and phosphatase to regulate gene expression, a design known to provide robustness against noise. Yet how such antagonistic enzymatic activities are balanced as a function of environmental change remains poorly understood. We find that the bifunctional receptor that regulates K+ uptake in Escherichia coli is a dual sensor, which modulates its autokinase and phosphatase activities in response to both extracellular and intracellular K+ concentration. Using mathematical modeling, we show that dual sensing is a superior strategy for ensuring homeostasis when both the supply of and demand for a limiting resource fluctuate. By engineering standards, this molecular control system displays a strikingly high degree of functional integration, providing a reference for the vast numbers of receptors for which the sensing strategy remains elusive.

  18. Functional Proteomics Defines the Molecular Switch Underlying FGF Receptor Trafficking and Cellular Outputs

    Francavilla, Chiara; Rigbolt, Kristoffer T.G.; Emdal, Kristina B

    2013-01-01

    The stimulation of fibroblast growth factor receptors (FGFRs) with distinct FGF ligands generates specific cellular responses. However, the mechanisms underlying this paradigm have remained elusive. Here, we show that FGF-7 stimulation leads to FGFR2b degradation and, ultimately, cell proliferation...

  19. Cellular imaging and folate receptor targeting delivery of gum kondagogu capped gold nanoparticles in cancer cells.

    Kumar, Sathish Sundar Dhilip; Mahesh, Ayyavu; Antoniraj, M Gover; Rathore, Hanumant Singh; Houreld, N N; Kandasamy, Ruckmani

    2018-04-01

    In this study, the green synthesis of gum kondagogu capped gold nanoparticles (GK-GNPs) was prepared using a naturally available polysaccharide. The anionic gum capped GK-GNPs enabled the successful coupling of folic acid (FA) and fluorescein isothiocyanate (FITC) to produce a fluorescently labelled GNP (F2-GNP). F2-GNPs were further characterized using different physicochemical methods Cellular viability, cellular imaging, and targeted delivery of F2-GNPs were further evaluated in both folate receptor positive (MCF-7) and folate receptor negative (A549) cancer cells. Physicochemical characterization revealed a nanoparticle with a small size (37 nm), smooth surface (surface charge of -23.7 mV), crystallinity of gold nanoparticles and existence of gum kondagogu in the F2-GNPs. Cellular uptake of F2-GNPs indicated a greater affinity towards folate receptor positive cells. This study shows that the F2-GNPs is as an effective nanocarrier for targeted drug delivery and cellular imaging via folate receptors. Copyright © 2017. Published by Elsevier B.V.

  20. Organization of cellular receptors into a nanoscale junction during HIV-1 adhesion.

    Terrence M Dobrowsky

    2010-07-01

    Full Text Available The fusion of the human immunodeficiency virus type 1 (HIV-1 with its host cell is the target for new antiretroviral therapies. Viral particles interact with the flexible plasma membrane via viral surface protein gp120 which binds its primary cellular receptor CD4 and subsequently the coreceptor CCR5. However, whether and how these receptors become organized at the adhesive junction between cell and virion are unknown. Here, stochastic modeling predicts that, regarding binding to gp120, cellular receptors CD4 and CCR5 form an organized, ring-like, nanoscale structure beneath the virion, which locally deforms the plasma membrane. This organized adhesive junction between cell and virion, which we name the viral junction, is reminiscent of the well-characterized immunological synapse, albeit at much smaller length scales. The formation of an organized viral junction under multiple physiopathologically relevant conditions may represent a novel intermediate step in productive infection.

  1. Development of a Systems Computational Model to Investigate Early Biological Events in Hepatic Activation of Constitutive Androstane Receptor (CAR) by Phenobarbital

    Activation of the nuclear receptor CAR (constitutive active/androstane receptor) is implicated in the control several key biological events such as metabolic pathways. Here, we combined data from literature with information obtained from in vitro assays in the US EPA ToxCast dat...

  2. A mollusk VDR/PXR/CAR-like (NR1J) nuclear receptor provides insight into ancient detoxification mechanisms

    Cruzeiro, Catarina, E-mail: catarinarcruzeiro@hotmail.com [ICBAS - Institute of Biomedical Sciences Abel Salazar, U. Porto - University of Porto (Portugal); CIIMAR/CIMAR - Interdisciplinary Center of Marine and Environmental Research, U. Porto (Portugal); Lopes-Marques, Mónica, E-mail: monicaslm@hotmail.com [ICBAS - Institute of Biomedical Sciences Abel Salazar, U. Porto - University of Porto (Portugal); CIIMAR/CIMAR - Interdisciplinary Center of Marine and Environmental Research, U. Porto (Portugal); Ruivo, Raquel, E-mail: ruivo.raquel@gmail.com [CIIMAR/CIMAR - Interdisciplinary Center of Marine and Environmental Research, U. Porto (Portugal); Rodrigues-Oliveira, Nádia, E-mail: nadia.oliveira@ciimar.up.pt [CIIMAR/CIMAR - Interdisciplinary Center of Marine and Environmental Research, U. Porto (Portugal); Santos, Miguel M., E-mail: santos@ciimar.up.pt [CIIMAR/CIMAR - Interdisciplinary Center of Marine and Environmental Research, U. Porto (Portugal); FCUP - Faculty of Sciences, Department of Biology, U. Porto (Portugal); Rocha, Maria João, E-mail: mjsrocha@netcabo.pt [ICBAS - Institute of Biomedical Sciences Abel Salazar, U. Porto - University of Porto (Portugal); CIIMAR/CIMAR - Interdisciplinary Center of Marine and Environmental Research, U. Porto (Portugal); Rocha, Eduardo, E-mail: erocha@icbas.up.pt [ICBAS - Institute of Biomedical Sciences Abel Salazar, U. Porto - University of Porto (Portugal); CIIMAR/CIMAR - Interdisciplinary Center of Marine and Environmental Research, U. Porto (Portugal); Castro, L. Filipe C., E-mail: filipe.castro@ciimar.up.pt [CIIMAR/CIMAR - Interdisciplinary Center of Marine and Environmental Research, U. Porto (Portugal); FCUP - Faculty of Sciences, Department of Biology, U. Porto (Portugal)

    2016-05-15

    Highlights: • A nuclear receptor orthologue of the NR1J group is isolated from a mollusc. • The molluscan NR1J transactivates gene expression upon exposure to okadaic acid but not a pesticide, esfenvarelate and triclosan. • Lineage specific gene duplications and gene loss have occurred in the NR1J of protostomes with likely impacts on detoxification mechanisms. - Abstract: The origin and diversification of the metazoan endocrine systems represents a fundamental research issue in biology. Nuclear receptors are critical components of these systems. A particular group named VDR/PXR/CAR (NR1I/J) is central in the mediation of detoxification responses. While orthologues have been thoroughly characterized in vertebrates, a sparse representation is currently available for invertebrates. Here, we provide the first isolation and characterization of a lophotrochozoan protostome VDR/PXR/CAR nuclear receptor (NR1J), in the estuarine bivalve the peppery furrow shell (Scrobicularia plana). Using a reporter gene assay, we evaluated the xenobiotic receptor plasticity comparing the human PXR with the S. plana NR1Jβ. Our results show that the molluscan receptor responds to a natural toxin (okadaic acid) in a similar fashion to that reported for other invertebrates. In contrast, the pesticide esfenvalerate displayed a unique response, since it down regulated transactivation at higher concentrations, while for triclosan no response was observed. Additionally, we uncovered lineage specific gene duplications and gene loss in the gene group encoding NRs in protostomes with likely impacts on the complexity of detoxification mechanisms across different phyla. Our findings pave the way for the development of multi-specific sensor tools to screen xenobiotic compounds acting via the NR1I/J group.

  3. A mollusk VDR/PXR/CAR-like (NR1J) nuclear receptor provides insight into ancient detoxification mechanisms

    Cruzeiro, Catarina; Lopes-Marques, Mónica; Ruivo, Raquel; Rodrigues-Oliveira, Nádia; Santos, Miguel M.; Rocha, Maria João; Rocha, Eduardo; Castro, L. Filipe C.

    2016-01-01

    Highlights: • A nuclear receptor orthologue of the NR1J group is isolated from a mollusc. • The molluscan NR1J transactivates gene expression upon exposure to okadaic acid but not a pesticide, esfenvarelate and triclosan. • Lineage specific gene duplications and gene loss have occurred in the NR1J of protostomes with likely impacts on detoxification mechanisms. - Abstract: The origin and diversification of the metazoan endocrine systems represents a fundamental research issue in biology. Nuclear receptors are critical components of these systems. A particular group named VDR/PXR/CAR (NR1I/J) is central in the mediation of detoxification responses. While orthologues have been thoroughly characterized in vertebrates, a sparse representation is currently available for invertebrates. Here, we provide the first isolation and characterization of a lophotrochozoan protostome VDR/PXR/CAR nuclear receptor (NR1J), in the estuarine bivalve the peppery furrow shell (Scrobicularia plana). Using a reporter gene assay, we evaluated the xenobiotic receptor plasticity comparing the human PXR with the S. plana NR1Jβ. Our results show that the molluscan receptor responds to a natural toxin (okadaic acid) in a similar fashion to that reported for other invertebrates. In contrast, the pesticide esfenvalerate displayed a unique response, since it down regulated transactivation at higher concentrations, while for triclosan no response was observed. Additionally, we uncovered lineage specific gene duplications and gene loss in the gene group encoding NRs in protostomes with likely impacts on the complexity of detoxification mechanisms across different phyla. Our findings pave the way for the development of multi-specific sensor tools to screen xenobiotic compounds acting via the NR1I/J group.

  4. Nuclear receptor CAR (NR1I3) is essential for DDC-induced liver injury and oval cell proliferation in mouse liver.

    Yamazaki, Yuichi; Moore, Rick; Negishi, Masahiko

    2011-11-01

    The liver is endowed with the ability to regenerate hepatocytes in response to injury. When this regeneration ability is impaired during liver injury, oval cells, which are considered to be postnatal hepatic progenitors, proliferate and differentiate into hepatocytes. Here we have demonstrated that 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) activates the nuclear receptor constitutive active/androstane receptor (CAR), resulting in proliferation of oval cells in mouse liver. Activation of CAR by DDC was shown by hepatic nuclear CAR accumulation and cytochrome P450 (CYP)2B10 mRNA induction after feeding a 0.1% DDC-containing diet to Car(+/+) mice. After being fed the DDC diet, Car(+/+), but not Car(-/-) mice, developed severe liver injury and an A6 antibody-stained ductular reaction in an area around the portal tract. Oval cell proliferation was confirmed by laser capture microdissection and real-time PCR; mRNAs for the two oval cell markers epithelial cell adhesion molecule and TROP2 were specifically induced in the periportal region of DDC diet-fed Car(+/+), but not Car(-/-) mice. Although rates of both hepatocyte growth and death were initially enhanced only in DDC diet-fed Car(+/+) mice, growth was attenuated when oval cells proliferated, whereas death continued unabated. DDC-induced liver injury, which differs from other CAR activators such as phenobarbital, occurred in the periportal region where cells developed hypertrophy, accumulated porphyrin crystals and inflammation developed, all in association with the proliferation of oval cells. Thus, CAR provides an excellent experimental model for further investigations into its roles in liver regeneration, as well as the development of diseases such as hepatocellular carcinoma.

  5. In vivo effects of chronic contamination with depleted uranium on CYP3A and associated nuclear receptors PXR and CAR in the rat

    Souidi, M.; Gueguen, Y.; Linard, C.; Dudoignon, N.; Grison, S.; Baudelin, C.; Marquette, C.; Gourmelon, P.; Aigueperse, J.; Dublineau, I.

    2005-01-01

    In addition to its natural presence at high concentrations in some areas, uranium has several civilian and military applications that could cause contamination of human populations, mainly through chronic ingestion. Reports describe the accumulation of this radionuclide in some organs (including the bone, kidney, and liver) after acute or chronic contamination and show that it produces chemical or radiological toxicity or both. The literature is essentially devoid of information about uranium-related cellular and molecular effects on metabolic functions such as xenobiotic detoxification. The present study thus evaluated rats chronically exposed to depleted uranium in their drinking water (1 mg/(rat day)) for 9 months. Our specific aim was to evaluate the hepatic and extrahepatic mRNA expression of CYP3A1/A2, CYP2B1, and CYP1A1 as well as of the nuclear receptors PXR, CAR, and RXR in these rats. CYP3A1 mRNA expression was significantly higher in the brain (200%), liver (300%), and kidneys (900%) of exposed rats compared with control rats, while CYP3A2 mRNA levels were higher in the lungs (300%) and liver (200%), and CYP2B1 mRNA expression in the kidneys (300%). Expression of CYP1A1 mRNA did not change significantly during this study. PXR mRNA levels increased in the brain (200%), liver (150%), and kidneys (200%). Uranium caused CAR mRNA expression in the lungs to double. Expression of RXR mRNA did not change significantly in the course of this study, nor did the hepatic activity of CYP2C, CYP3A, CYP2A, or CYP2B. Uranium probably affects the expression of drug-metabolizing CYP enzymes through the PXR and CAR nuclear receptors. These results suggest that the stimulating effect of uranium on these enzymes might lead to hepatic or extrahepatic toxicity (or both) during drug treatment and then affect the entire organism

  6. Modeling mechanical restriction differences between car and heavy truck in two-lane cellular automata traffic flow model

    Li, Xin; Li, Xingang; Xiao, Yao; Jia, Bin

    2016-06-01

    Real traffic is heterogeneous with car and truck. Due to mechanical restrictions, the car and the truck have different limited deceleration capabilities, which are important factors in safety driving. This paper extends the single lane safety driving (SD) model with limited deceleration capability to two-lane SD model, in which car-truck heterogeneous traffic is considered. A car has a larger limited deceleration capability while a heavy truck has a smaller limited deceleration capability as a result of loaded goods. Then the safety driving conditions are different as the types of the following and the leading vehicles vary. In order to eliminate the well-known plug in heterogeneous two-lane traffic, it is assumed that heavy truck has active deceleration behavior when the heavy truck perceives the forming plug. The lane-changing decisions are also determined by the safety driving conditions. The fundamental diagram, spatiotemporal diagram, and lane-changing frequency were investigated to show the effect of mechanical restriction on heterogeneous traffic flow. It was shown that there would be still three traffic phases in heterogeneous traffic condition; the active deceleration of the heavy truck could well eliminate the plug; the lane-changing frequency was low in synchronized flow; the flow and velocity would decrease as the proportion of heavy truck grows or the limited deceleration capability of heavy truck drops; and the flow could be improved with lane control measures.

  7. An investigation of merging and diverging cars on a multi-lane road using a cellular automation model

    Jetto, K.; Ez-Zahraouy, H.; Benyoussef, A.

    2012-11-01

    In this paper, we have investigated two observed situations in a multi-lane road. The first one concerns a fast merging vehicle. The second situation is related to the case of a fast vehicle leaving the fastest lane back into the slowest lane and targeting a specific way out. We are interested in the relaxation time τ, i.e., which is the time that the merging (diverging) vehicle spends before reaching the desired lane. Using analytical treatment and numerical simulations for the NaSch model, we have found two states, namely, the free state in which the merging (diverging) vehicle reaches the desired lane, and the trapped state in which τ diverges. We have established phase diagrams for several values of the braking probability. In the second situation, we have shown that diverging from the fast lane targeting a specific way out is not a simple task. Even if the diverging vehicle is in the free phase, two different states can be distinguished. One is the critical state, in which the diverging car can probably reach the desired way out. The other is the safe state, in which the diverging car can surely reach the desired way out. In order to be in the safe state, we have found that the driver of the diverging car must know the critical distance (below which the way out will be out of his reach) in each lane. Furthermore, this critical distance depends on the density of cars, and it follows an exponential law.

  8. Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

    Hinrich eAbken

    2013-11-01

    Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

  9. Aberrant localization of lamin B receptor (LBR) in cellular senescence in human cells

    Arai, Rumi; En, Atsuki; Ukekawa, Ryo [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Miki, Kensuke [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Ichiban Life Corporation, 1-1-7 Horai-cho, Naka-ku, Yokohama 231-0048 (Japan); Fujii, Michihiko, E-mail: mifuji@yokohama-cu.ac.jp [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Ayusawa, Dai [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Ichiban Life Corporation, 1-1-7 Horai-cho, Naka-ku, Yokohama 231-0048 (Japan)

    2016-05-13

    5-Bromodeoxyuridine (BrdU), a thymidine analogue, induces cellular senescence in mammalian cells. BrdU induces cellular senescence probably through the regulation of chromatin because BrdU destabilizes or disrupts nucleosome positioning and decondenses heterochromatin. Since heterochromatin is tethered to the nuclear periphery through the interaction with the nuclear envelope proteins, we examined the localization of the several nuclear envelope proteins such as lamins, lamin-interacting proteins, nuclear pore complex proteins, and nuclear transport proteins in senescent cells. We have shown here that lamin B receptor (LBR) showed a change in localization in both BrdU-induced and replicative senescent cells.

  10. Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

    Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

    2011-12-01

    Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

  11. Safety, tumor trafficking and immunogenicity of chimeric antigen receptor (CAR)-T cells specific for TAG-72 in colorectal cancer.

    Hege, Kristen M; Bergsland, Emily K; Fisher, George A; Nemunaitis, John J; Warren, Robert S; McArthur, James G; Lin, Andy A; Schlom, Jeffrey; June, Carl H; Sherwin, Stephen A

    2017-01-01

    T cells engineered to express chimeric antigen receptors (CARs) have established efficacy in the treatment of B-cell malignancies, but their relevance in solid tumors remains undefined. Here we report results of the first human trials of CAR-T cells in the treatment of solid tumors performed in the 1990s. Patients with metastatic colorectal cancer (CRC) were treated in two phase 1 trials with first-generation retroviral transduced CAR-T cells targeting tumor-associated glycoprotein (TAG)-72 and including a CD3-zeta intracellular signaling domain (CART72 cells). In trial C-9701 and C-9702, CART72 cells were administered in escalating doses up to 10 10 total cells; in trial C-9701 CART72 cells were administered by intravenous infusion. In trial C-9702, CART72 cells were administered via direct hepatic artery infusion in patients with colorectal liver metastases. In both trials, a brief course of interferon-alpha (IFN-α) was given with each CART72 infusion to upregulate expression of TAG-72. Fourteen patients were enrolled in C-9701 and nine in C-9702. CART72 manufacturing success rate was 100% with an average transduction efficiency of 38%. Ten patients were treated in CC-9701 and 6 in CC-9702. Symptoms consistent with low-grade, cytokine release syndrome were observed in both trials without clear evidence of on target/off tumor toxicity. Detectable, but mostly short-term (≤14 weeks), persistence of CART72 cells was observed in blood; one patient had CART72 cells detectable at 48 weeks. Trafficking to tumor tissues was confirmed in a tumor biopsy from one of three patients. A subset of patients had 111 Indium-labeled CART72 cells injected, and trafficking could be detected to liver, but T cells appeared largely excluded from large metastatic deposits. Tumor biomarkers carcinoembryonic antigen (CEA) and TAG-72 were measured in serum; there was a precipitous decline of TAG-72, but not CEA, in some patients due to induction of an interfering antibody to the TAG-72

  12. Development of CINPA1 analogs as novel and potent inverse agonists of constitutive androstane receptor

    Lin, Wenwei; Yang, Lei; Chai, Sergio C.; Lu, Yan; Chen, Taosheng

    2015-01-01

    Constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) are master regulators of endobiotic and xenobiotic metabolism and disposition. Because CAR is constitutively active in certain cellular contexts, inhibiting CAR might reduce drug-induced hepatotoxicity and resensitize drug-resistant cancer cells to chemotherapeutic drugs. We recently reported a novel CAR inhibitor/inverse agonist CINPA1 (11). Here, we have obtained or designed 54 analogs of CINPA1 and used a ti...

  13. The Structure of an Infectious Human Polyomavirus and Its Interactions with Cellular Receptors.

    Hurdiss, Daniel L; Frank, Martin; Snowden, Joseph S; Macdonald, Andrew; Ranson, Neil A

    2018-04-21

    BK polyomavirus (BKV) causes polyomavirus-associated nephropathy and hemorrhagic cystitis in immunosuppressed patients. These are diseases for which we currently have limited treatment options, but potential therapies could include pre-transplant vaccination with a multivalent BKV vaccine or therapeutics which inhibit capsid assembly or block attachment and entry into target cells. A useful tool in such efforts would be a high-resolution structure of the infectious BKV virion and how this interacts with its full repertoire of cellular receptors. We present the 3.4-Å cryoelectron microscopy structure of native, infectious BKV in complex with the receptor fragment of GT1b ganglioside. We also present structural evidence that BKV can utilize glycosaminoglycans as attachment receptors. This work highlights features that underpin capsid stability and provides a platform for rational design and development of urgently needed pharmacological interventions for BKV-associated diseases. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Effects of fexofenadine and hydroxyzine on brake reaction time during car-driving with cellular phone use.

    Tashiro, Manabu; Horikawa, Etsuo; Mochizuki, Hideki; Sakurada, Yumiko; Kato, Motohisa; Inokuchi, Takatoshi; Ridout, Fran; Hindmarch, Ian; Yanai, Kazuhiko

    2005-10-01

    Antihistamines are a mainstay treatment for allergic rhinitis; however, many older agents cause adverse events, including sedation and central nervous system (CNS) impairment. Research has shown sedating effects of antihistamines on driving; currently, no known study has examined whether cellular phone usage while driving further compounds impairment in individuals administered antihistamines. The aim of this study was to examine this endpoint. In a randomized, double-blind, placebo-controlled, three-way crossover study, healthy volunteers received fexofenadine HCl 120 mg, hydroxyzine HCl 30 mg and placebo. Brake reaction time (BRT) was used to examine driving performance across four conditions: driving only; driving while completing simple calculations; complex calculations; and conversing on a cellular phone. Subjective sedation assessments were also conducted. Brake reaction time with and without cellular phone usage in fexofenadine-treated subjects did not differ significantly from placebo in any condition. In contrast, hydroxyzine-treated subjects were significantly more sedated and had slower BRTs, suggesting slower hazard recognition and brake application, compared with the fexofenadine and placebo groups in all conditions. Importantly, cellular phone operation was an additive factor, increasing BRTs in hydroxyzine-treated volunteers. Fexofenadine did not impair CNS function in subjects involved in a divided attention task of driving and cellular phone operation. Copyright (c) 2005 John Wiley & Sons, Ltd.

  15. The Molecular Interaction of CAR and JAML Recruits the Central Cell Signal Transducer PI3K

    Verdino, Petra; Witherden, Deborah A.; Havran, Wendy L.; Wilson, Ian A. (Scripps)

    2010-11-15

    Coxsackie and adenovirus receptor (CAR) is the primary cellular receptor for group B coxsackieviruses and most adenovirus serotypes and plays a crucial role in adenoviral gene therapy. Recent discovery of the interaction between junctional adhesion molecule-like protein (JAML) and CAR uncovered important functional roles in immunity, inflammation, and tissue homeostasis. Crystal structures of JAML ectodomain (2.2 angstroms) and its complex with CAR (2.8 angstroms) reveal an unusual immunoglobulin-domain assembly for JAML and a charged interface that confers high specificity. Biochemical and mutagenesis studies illustrate how CAR-mediated clustering of JAML recruits phosphoinositide 3-kinase (P13K) to a JAML intracellular sequence motif as delineated for the {alpha}{beta} T cell costimulatory receptor CD28. Thus, CAR and JAML are cell signaling receptors of the immune system with implications for asthma, cancer, and chronic nonhealing wounds.

  16. Purine receptor P2Y_6 mediates cellular response to γ-ray-induced DNA damage

    Ide, Shunta; Nishimaki, Naoko; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2014-01-01

    We previously showed that nucleotide P2 receptor agonists such as ATP and UTP amplify γ-ray-induced focus formation of phosphorylated histone H2A variant H2AX (γH2AX), which is considered to be an indicator of DNA damage so far, by activating purine P2Y_6 and P2Y_1_2 receptors. Therefore, we hypothesized that these P2 receptors play a role in inducing the repair response to γ-ray-induced DNA damage. In the present study, we tested this idea by using human lung cancer A549 cells. First, reverse-transcription polymerase chain reaction (RT-PCR) showed that P2Y_6 receptor is highly expressed in A549 cells, but P2Y_1_2 receptor is only weakly expressed. Next, colony formation assay revealed that P2Y_6 receptor antagonist MRS2578 markedly reduced the survival rate of γ-ray-exposed A549 cells. The survival rate was also significantly reduced in P2Y_6-knock-down cells, compared with scramble siRNA-transfected cells. Since it has reported that phosphorylation of ERK1/2 after activation of EGFR via P2Y_6 and P2Y_1_2 receptors is involved in the repair response to γ-ray-induced DNA damage, we next examined whether γ-ray-induced phosphorylation of ERK1/2 was also inhibited by MRS2578 in A549 cells. We found that it was. Taken together, these findings indicate that purinergic signaling through P2Y_6 receptor, followed by ERK1/2 activation, promotes the cellular repair response to γ-ray-induced DNA damage. (author)

  17. Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity

    Congcong Zhang

    2017-05-01

    Full Text Available Significant progress has been made in recent years toward realizing the potential of natural killer (NK cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future

  18. Cholecystokinin receptor-1 mediates the inhibitory effects of exogenous cholecystokinin octapeptide on cellular morphine dependence

    Wen Di

    2012-06-01

    Full Text Available Abstract Background Cholecystokinin octapeptide (CCK-8, the most potent endogenous anti-opioid peptide, has been shown to regulate the processes of morphine dependence. In our previous study, we found that exogenous CCK-8 attenuated naloxone induced withdrawal symptoms. To investigate the precise effect of exogenous CCK-8 and the role of cholecystokinin (CCK 1 and/or 2 receptors in morphine dependence, a SH-SY5Y cell model was employed, in which the μ-opioid receptor, CCK1/2 receptors, and endogenous CCK are co-expressed. Results Forty-eight hours after treating SH-SY5Y cells with morphine (10 μM, naloxone (10 μM induced a cAMP overshoot, indicating that cellular morphine dependence had been induced. The CCK receptor and endogenous CCK were up-regulated after chronic morphine exposure. The CCK2 receptor antagonist (LY-288,513 at 1–10 μM inhibited the naloxone-precipitated cAMP overshoot, but the CCK1 receptor antagonist (L-364,718 did not. Interestingly, CCK-8 (0.1-1 μM, a strong CCK receptor agonist, dose-dependently inhibited the naloxone-precipitated cAMP overshoot in SH-SY5Y cells when co-pretreated with morphine. The L-364,718 significantly blocked the inhibitory effect of exogenous CCK-8 on the cAMP overshoot at 1–10 μM, while the LY-288,513 did not. Therefore, the CCK2 receptor appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence in SH-SY5Y cells. An additional inhibitory effect of CCK-8 at higher concentrations appears to involve the CCK1 receptor. Conclusions This study reveals the difference between exogenous CCK-8 and endogenous CCK effects on the development of morphine dependence, and provides the first evidence for the participation of the CCK1 receptor in the inhibitory effects of exogenous CCK-8 on morphine dependence.

  19. Neurotransmitter Specific, Cellular-Resolution Functional Brain Mapping Using Receptor Coated Nanoparticles: Assessment of the Possibility

    Forati, Ebrahim; Sabouni, Abas; Ray, Supriyo; Head, Brian; Schoen, Christian; Sievenpiper, Dan

    2015-01-01

    Receptor coated resonant nanoparticles and quantum dots are proposed to provide a cellular-level resolution image of neural activities inside the brain. The functionalized nanoparticles and quantum dots in this approach will selectively bind to different neurotransmitters in the extra-synaptic regions of neurons. This allows us to detect neural activities in real time by monitoring the nanoparticles and quantum dots optically. Gold nanoparticles (GNPs) with two different geometries (sphere and rod) and quantum dots (QDs) with different sizes were studied along with three different neurotransmitters: dopamine, gamma-Aminobutyric acid (GABA), and glycine. The absorption/emission spectra of GNPs and QDs before and after binding of neurotransmitters and their corresponding receptors are reported. The results using QDs and nanorods with diameter 25nm and aspect rations larger than three were promising for the development of the proposed functional brain mapping approach. PMID:26717196

  20. Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity.

    Wang, Dongrui; Aguilar, Brenda; Starr, Renate; Alizadeh, Darya; Brito, Alfonso; Sarkissian, Aniee; Ostberg, Julie R; Forman, Stephen J; Brown, Christine E

    2018-05-17

    Chimeric antigen receptor-modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy.

  1. Activation of Constitutive Androstane Receptor (CAR) in Mice Results in Maintained Biliary Excretion of Bile Acids Despite a Marked Decrease of Bile Acids in Liver.

    Lickteig, Andrew J; Csanaky, Iván L; Pratt-Hyatt, Matthew; Klaassen, Curtis D

    2016-06-01

    Activation of Constitutive Androstane Receptor (CAR) protects against bile acid (BA)-induced liver injury. This study was performed to determine the effect of CAR activation on bile flow, BA profile, as well as expression of BA synthesis and transport genes. Synthetic CAR ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) was administered to mice for 4 days. BAs were quantified by UPLC-MS/MS (ultraperformance liquid chromatography-tandem mass spectrometry). CAR activation decreases total BAs in livers of male (49%) and female mice (26%), largely attributable to decreases of the 12α-hydroxylated BA taurocholic acid (T-CA) (males (M) 65%, females (F) 45%). Bile flow in both sexes was increased by CAR activation, and the increases were BA-independent. CAR activation did not alter biliary excretion of total BAs, but overall BA composition changed. Excretion of muricholic (6-hydroxylated) BAs was increased in males (101%), and the 12α-OH proportion of biliary BAs was decreased in both males (37%) and females (28%). The decrease of T-CA in livers of males and females correlates with the decreased mRNA of the sterol 12α-hydroxylase Cyp8b1 in males (71%) and females (54%). As a response to restore BAs to physiologic concentrations in liver, mRNA of Cyp7a1 is upregulated following TCPOBOP (males 185%, females 132%). In ilea, mRNA of the negative feedback regulator Fgf15 was unaltered by CAR activation, indicating biliary BA excretion was sufficient to maintain concentrations of total BAs in the small intestine. In summary, the effects of CAR activation on BAs in male and female mice are quite similar, with a marked decrease in the major BA T-CA in the liver. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. GLYCAN-DIRECTED CAR-T CELLS.

    Steentoft, Catharina; Migliorini, Denis; King, Tiffany R; Mandel, Ulla; June, Carl H; Posey, Avery D

    2018-01-23

    Cancer immunotherapy is rapidly advancing in the treatment of a variety of hematopoietic cancers, including pediatric acute lymphoblastic leukemia and diffuse large B cell lymphoma, with chimeric antigen receptor (CAR)-T cells. CARs are genetically encoded artificial T cell receptors that combine the antigen specificity of an antibody with the machinery of T cell activation. However, implementation of CAR technology in the treatment of solid tumors has been progressing much slower. Solid tumors are characterized by a number of challenges that need to be overcome, including cellular heterogeneity, immunosuppressive tumor microenvironment (TME), and, in particular, few known cancer-specific targets. Post-translational modifications that differentially occur in malignant cells generate valid cell surface, cancer-specific targets for CAR-T cells. We previously demonstrated that CAR-T cells targeting an aberrant O-glycosylation of MUC1, a common cancer marker associated with changes in cell adhesion, tumor growth, and poor prognosis, could control malignant growth in mouse models. Here, we discuss the field of glycan-directed CAR-T cells and review the different classes of antibodies specific for glycan-targeting, including the generation of high affinity O-glycopeptide antibodies. Finally, we discuss historic and recently investigated glycan targets for CAR-T cells and provide our perspective on how targeting the tumor glycoproteome and/or glycome will improve CAR-T immunotherapy. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Behind the curtain: cellular mechanisms for allosteric modulation of calcium-sensing receptors

    Cavanaugh, Alice; Huang, Ying; Breitwieser, Gerda E

    2012-01-01

    Calcium-sensing receptors (CaSR) are integral to regulation of systemic Ca2+ homeostasis. Altered expression levels or mutations in CaSR cause Ca2+ handling diseases. CaSR is regulated by both endogenous allosteric modulators and allosteric drugs, including the first Food and Drug Administration-approved allosteric agonist, Cinacalcet HCl (Sensipar®). Recent studies suggest that allosteric modulators not only alter function of plasma membrane-localized CaSR, but regulate CaSR stability at the endoplasmic reticulum. This brief review summarizes our current understanding of the role of membrane-permeant allosteric agonists in cotranslational stabilization of CaSR, and highlights additional, indirect, signalling-dependent role(s) for membrane-impermeant allosteric drugs. Overall, these studies suggest that allosteric drugs act at multiple cellular organelles to control receptor abundance and hence function, and that drug hydrophobicity can bias the relative contributions of plasma membrane and intracellular organelles to CaSR abundance and signalling. LINKED ARTICLES This article is part of a themed section on the Molecular Pharmacology of G Protein-Coupled Receptors (GPCRs). To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-6. To view the 2010 themed section on the same topic visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2010.159.issue-5/issuetoc PMID:21470201

  4. The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4 Receptors

    Ramón A. Lorca

    2011-01-01

    Full Text Available Although the physiological function of the cellular prion protein (PrPC remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+.

  5. Restriction of Receptor Movement Alters Cellular Response: Physical Force Sensing by EphA2

    Salaita, Khalid; Nair, Pradeep M; Petit, Rebecca S; Neve, Richard M; Das, Debopriya; Gray, Joe W; Groves, Jay T

    2009-09-09

    Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed. EphA2 transport can be blocked by physical barriers nanofabricated onto the underlying substrate. This physical reorganization of EphA2 alters the cellular response to ephrin-A1, as observed by changes in cytoskeleton morphology and recruitment of a disintegrin and metalloprotease 10. Quantitative analysis of receptor-ligand spatial organization across a library of 26 mammary epithelial cell lines reveals characteristic differences that strongly correlate with invasion potential. These observations reveal a mechanism for spatio-mechanical regulation of EphA2 signaling pathways.

  6. Receptor-mediated endocytosis generates nanomechanical force reflective of ligand identity and cellular property.

    Zhang, Xiao; Ren, Juan; Wang, Jingren; Li, Shixie; Zou, Qingze; Gao, Nan

    2018-08-01

    Whether environmental (thermal, chemical, and nutrient) signals generate quantifiable, nanoscale, mechanophysical changes in the cellular plasma membrane has not been well elucidated. Assessment of such mechanophysical properties of plasma membrane may shed lights on fundamental cellular process. Atomic force microscopic (AFM) measurement of the mechanical properties of live cells was hampered by the difficulty in accounting for the effects of the cantilever motion and the associated hydrodynamic force on the mechanical measurement. These challenges have been addressed in our recently developed control-based AFM nanomechanical measurement protocol, which enables a fast, noninvasive, broadband measurement of the real-time changes in plasma membrane elasticity in live cells. Here we show using this newly developed AFM platform that the plasma membrane of live mammalian cells exhibits a constant and quantifiable nanomechanical property, the membrane elasticity. This mechanical property sensitively changes in response to environmental factors, such as the thermal, chemical, and growth factor stimuli. We demonstrate that different chemical inhibitors of endocytosis elicit distinct changes in plasma membrane elastic modulus reflecting their specific molecular actions on the lipid configuration or the endocytic machinery. Interestingly, two different growth factors, EGF and Wnt3a, elicited distinct elastic force profiles revealed by AFM at the plasma membrane during receptor-mediated endocytosis. By applying this platform to genetically modified cells, we uncovered a previously unknown contribution of Cdc42, a key component of the cellular trafficking network, to EGF-stimulated endocytosis at plasma membrane. Together, this nanomechanical AFM study establishes an important foundation that is expandable and adaptable for investigation of cellular membrane evolution in response to various key extracellular signals. © 2017 Wiley Periodicals, Inc.

  7. Young T cells age during a redirected anti-tumour attack: chimeric antigen receptor (CAR-provided dual costimulation is half the battle.

    Andreas A Hombach

    2013-06-01

    Full Text Available Adoptive therapy with chimeric antigen receptor (CAR-redirected T cells showed spectacular efficacy in the treatment of leukaemia in recent early phase trials. Patient's T cells were ex vivo genetically engineered with a CAR, amplified and re-administered to the patient. While T cells mediating the primary response were predominantly of young effector and central memory phenotype, repetitive antigen engagement irreversible triggers T cell maturation leaving late memory cells with the KLRG-1+ CD57+ CD7- CCR7- phenotype in the long-term. These cells preferentially accumulate in the periphery, are hypo-responsive upon TCR engagement and prone to activation-induced cell death. A recent report indicates that those T cells can be rescued by CAR provided CD28 and OX40 (CD134 stimulation. We discuss the strategy with respect to prolong the anti-tumour response and to improve the over-all efficacy of adoptive cell therapy.

  8. Secreted glyceraldehye-3-phosphate dehydrogenase is a multifunctional autocrine transferrin receptor for cellular iron acquisition.

    Sheokand, Navdeep; Kumar, Santosh; Malhotra, Himanshu; Tillu, Vikas; Raje, Chaaya Iyengar; Raje, Manoj

    2013-06-01

    The long held view is that mammalian cells obtain transferrin (Tf) bound iron utilizing specialized membrane anchored receptors. Here we report that, during increased iron demand, cells secrete the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which enhances cellular uptake of Tf and iron. These observations could be mimicked by utilizing purified GAPDH injected into mice as well as when supplemented in culture medium of model cell lines and primary cell types that play a key role in iron metabolism. Transferrin and iron delivery was evaluated by biochemical, biophysical and imaging based assays. This mode of iron uptake is a saturable, energy dependent pathway, utilizing raft as well as non-raft domains of the cell membrane and also involves the membrane protein CD87 (uPAR). Tf internalized by this mode is also catabolized. Our research demonstrates that, even in cell types that express the known surface receptor based mechanism for transferrin uptake, more transferrin is delivered by this route which represents a hidden dimension of iron homeostasis. Iron is an essential trace metal for practically all living organisms however its acquisition presents major challenges. The current paradigm is that living organisms have developed well orchestrated and evolved mechanisms involving iron carrier molecules and their specific receptors to regulate its absorption, transport, storage and mobilization. Our research uncovers a hidden and primitive pathway of bulk iron trafficking involving a secreted receptor that is a multifunctional glycolytic enzyme that has implications in pathological conditions such as infectious diseases and cancer. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Retinoic acid receptor gamma impacts cellular adhesion, Alpha5Beta1 integrin expression and proliferation in K562 cells.

    Kelley, Melissa D; Phomakay, Raynin; Lee, Madison; Niedzwiedz, Victoria; Mayo, Rachel

    2017-01-01

    The interplay between cellular adhesion and proliferation is complex; however, integrins, particularly the α5β1 subset, play a pivotal role in orchestrating critical cellular signals that culminate in cellular adhesion and growth. Retinoids modify the expression of a variety of adhesive/proliferative signaling proteins including α5β1 integrins; however, the role of specific retinoic acid receptors involved in these processes has not been elucidated. In this study, the effect of all-trans-retinoic acid receptor (RAR) agonists on K562 cellular adhesion, proliferation, and α5β1 integrin cell surface expression was investigated. RARγ agonist exposure increased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin and FN-120 in a time- and concentration dependent manner, while RARα or RARβ agonist treatment had no effect on cellular adhesion. Due to the novel RARγ- dependent cellular adhesion response exhibited by K562 cells, we examined α5 and β1 integrin subunit expression when K562 cells were exposed to retinoid agonists or vehicle for 24, 48, 72 or 96 hours. Our data demonstrates no differences in K562 cell surface expression of the α5 integrin subunit when cells were exposed to RARα, RARβ, or RARγ agonists for all time points tested. In contrast, RARγ agonist exposure resulted in an increase in cell surface β1 integrin subunit expression within 48 hours that was sustained at 72 and 96 hours. Finally, we demonstrate that while exposure to RARα or RARβ agonists have no effect on K562 cellular proliferation, the RARγ agonist significantly dampens K562 cellular proliferation levels in a time- and concentration- dependent manner. Our study is the first to report that treatment with a RARγ specific agonist augments cellular adhesion to α5β1 integrin substrates, increases cell surface levels of the β1 integrin subunit, and dampens cellular proliferation in a time and concentration dependent manner in a human

  10. TNF and TNF Receptor Superfamily Members in HIV infection: New Cellular Targets for Therapy?

    Amit Kumar

    2013-01-01

    Full Text Available Tumor necrosis factor (TNF and TNF receptors (TNFR superfamily members are engaged in diverse cellular phenomena such as cellular proliferation, morphogenesis, apoptosis, inflammation, and immune regulation. Their role in regulating viral infections has been well documented. Viruses have evolved with numerous strategies to interfere with TNF-mediated signaling indicating the importance of TNF and TNFR superfamily in viral pathogenesis. Recent research reports suggest that TNF and TNFRs play an important role in the pathogenesis of HIV. TNFR signaling modulates HIV replication and HIV proteins interfere with TNF/TNFR pathways. Since immune activation and inflammation are the hallmark of HIV infection, the use of TNF inhibitors can have significant impact on HIV disease progression. In this review, we will describe how HIV infection is modulated by signaling mediated through members of TNF and TNFR superfamily and in turn how these latter could be targeted by HIV proteins. Finally, we will discuss the emerging therapeutics options based on modulation of TNF activity that could ultimately lead to the cure of HIV-infected patients.

  11. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

    Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P; Zhu, R; Mayer, B; Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F; Salio, M; Shepherd, D; Polzella, P; Cerundolo, V; Dieudonne, M

    2010-01-01

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ∼ 25 to ∼ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  12. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

    Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P [Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Zhu, R; Mayer, B [Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F [Agilent Technologies Austria GmbH, Aubrunnerweg 11, A-4040 Linz (Austria); Salio, M; Shepherd, D; Polzella, P; Cerundolo, V [Cancer Research UK Tumor Immunology Group, Weatherall Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DS (United Kingdom); Dieudonne, M, E-mail: ferry_kienberger@agilent.com [Agilent Technologies Belgium, Wingepark 51, Rotselaar, AN B-3110 (Belgium)

    2010-03-19

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on {alpha}-galactosylceramide ({alpha}GalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from {approx} 25 to {approx} 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  13. Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

    Aldossari, Abdullah A.; Shannahan, Jonathan H. [The University of Colorado Anschutz Medical Campus, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences (United States); Podila, Ramakrishna [Clemson University, Department of Physics and Astronomy (United States); Brown, Jared M., E-mail: jared.brown@ucdenver.edu [The University of Colorado Anschutz Medical Campus, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences (United States)

    2015-07-15

    Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf-α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

  14. Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

    Aldossari, Abdullah A.; Shannahan, Jonathan H.; Podila, Ramakrishna; Brown, Jared M.

    2015-07-01

    Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf- α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

  15. Sleep deprivation and divergent toll-like receptor-4 activation of cellular inflammation in aging.

    Carroll, Judith E; Carrillo, Carmen; Olmstead, Richard; Witarama, Tuff; Breen, Elizabeth C; Yokomizo, Megumi; Seeman, Teresa; Irwin, Michael R

    2015-02-01

    Sleep disturbance and aging are associated with increases in inflammation, as well as increased risk of infectious disease. However, there is limited understanding of the role of sleep loss on age-related differences in immune responses. This study examines the effects of sleep deprivation on toll-like receptor activation of monocytic inflammation in younger compared to older adults. Community-dwelling adults (n = 70) who were categorized as younger (25-39 y old, n = 21) and older (60-84 y old, n = 49) participants, underwent a sleep laboratory-based experimental partial sleep deprivation (PSD) protocol including adaptation, an uninterrupted night of sleep, sleep deprivation (sleep restricted to 03:00-07:00), and recovery. Blood samples were obtained each morning to measure toll-like receptor-4 activation of monocyte intracellular production of the inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Partial sleep deprivation induced a significant increase in the production of IL-6 and/or TNF-α that persisted after a night of recovery sleep (F(2,121.2) = 3.8, P sleep loss, such that younger adults had an increase in inflammatory cytokine production that was not present in older adults (F(2,121.2) = 4.0, P sleep loss. Whereas sleep loss increases cellular inflammation in younger adults and may contribute to inflammatory disorders, blunted toll-like receptor activation in older adults may increase the risk of infectious disease seen with aging. © 2015 Associated Professional Sleep Societies, LLC.

  16. Enhanced thyroid hormone breakdown in hepatocytes by mutual induction of the constitutive androstane receptor (CAR, NR1I3) and arylhydrocarbon receptor by benzo[a]pyrene and phenobarbital.

    Schraplau, Anne; Schewe, Bettina; Neuschäfer-Rube, Frank; Ringel, Sebastian; Neuber, Corinna; Kleuser, Burkhard; Püschel, Gerhard P

    2015-02-03

    Xenobiotics may interfere with the hypothalamic-pituitary-thyroid endocrine axis by inducing enzymes that inactivate thyroid hormones and thereby reduce the metabolic rate. This induction results from an activation of xeno-sensing nuclear receptors. The current study shows that benzo[a]pyrene, a frequent contaminant of processed food and activator of the arylhydrocarbon receptor (AhR) activated the promoter and induced the transcription of the nuclear receptor constitutive androstane receptor (CAR, NR1I3) in rat hepatocytes. Likewise, phenobarbital induced the AhR transcription. This mutual induction of the nuclear receptors enhanced the phenobarbital-dependent induction of the prototypic CAR target gene Cyp2b1 as well as the AhR-dependent induction of UDP-glucuronosyltransferases. In both cases, the induction by the combination of both xenobiotics was more than the sum of the induction by either substance alone. By inducing the AhR, phenobarbital enhanced the benzo[a]pyrene-dependent reduction of thyroid hormone half-life and the benzo[a]pyrene-dependent increase in the rate of thyroid hormone glucuronide formation in hepatocyte cultures. CAR ligands might thus augment the endocrine disrupting potential of AhR activators by an induction of the AhR. Copyright © 2014. Published by Elsevier Ireland Ltd.

  17. Enhanced thyroid hormone breakdown in hepatocytes by mutual induction of the constitutive androstane receptor (CAR, NR1I3) and arylhydrocarbon receptor by benzo[a]pyrene and phenobarbital

    Schraplau, Anne; Schewe, Bettina; Neuschäfer-Rube, Frank; Ringel, Sebastian; Neuber, Corinna; Kleuser, Burkhard; Püschel, Gerhard P.

    2015-01-01

    Xenobiotics may interfere with the hypothalamic-pituitary-thyroid endocrine axis by inducing enzymes that inactivate thyroid hormones and thereby reduce the metabolic rate. This induction results from an activation of xeno-sensing nuclear receptors. The current study shows that benzo[a]pyrene, a frequent contaminant of processed food and activator of the arylhydrocarbon receptor (AhR) activated the promoter and induced the transcription of the nuclear receptor constitutive androstane receptor (CAR, NR1I3) in rat hepatocytes. Likewise, phenobarbital induced the AhR transcription. This mutual induction of the nuclear receptors enhanced the phenobarbital-dependent induction of the prototypic CAR target gene Cyp2b1 as well as the AhR-dependent induction of UDP-glucuronosyltransferases. In both cases, the induction by the combination of both xenobiotics was more than the sum of the induction by either substance alone. By inducing the AhR, phenobarbital enhanced the benzo[a]pyrene-dependent reduction of thyroid hormone half-life and the benzo[a]pyrene-dependent increase in the rate of thyroid hormone glucuronide formation in hepatocyte cultures. CAR ligands might thus augment the endocrine disrupting potential of AhR activators by an induction of the AhR

  18. Advantages and Applications of CAR-Expressing Natural Killer Cells

    Wolfgang eGlienke

    2015-02-01

    Full Text Available In contrast to donor T cells, natural killer (NK cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD. In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/ on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.

  19. Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S

    1996-01-01

    A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid...

  20. Role of the nuclear xenobiotic receptors CAR and PXR in induction of cytochromes P450 by non-dioxinlike polychlorinated biphenyls in cultured rat hepatocytes

    Gährs, Maike; Roos, Robert; Andersson, Patrik L.; Schrenk, Dieter

    2013-01-01

    Polychlorinated biphenyls (PCBs) are among the most ubiquitously detectable ‘persistent organic pollutants’. In contrast to ‘dioxinlike’ (DL) PCBs, less is known about the molecular mode of action of the larger group of the ‘non-dioxinlike’ (NDL) PCBs. Owing to the life-long exposure of the human population, a carcinogenic, i.e., tumor-promoting potency of NDL-PCBs has to be considered in human risk assessment. A major problem in risk assessment of NDL-PCBs is dioxin-like impurities that can occur in commercially available NDL-PCB standards. In the present study, we analyzed the induction of CYP2B1 and CYP3A1 in primary rat hepatocytes using a number of highly purified NDL-PCBs with various degrees of chlorination and substitution patterns. Induction of these enzymes is mediated by the nuclear xenobiotic receptors CAR (Constitutive androstane receptor) and PXR (Pregnane X receptor). For CYP2B1 induction, concentration–response analysis revealed a very narrow window of EC 50 estimates, being in the range of 1–4 μM for PCBs 28 and 52, and between 0.4 and 1 μM for PCBs 101, 138, 153 and 180. CYP3A1 induction was less sensitive to NDL-PCBs, the most pronounced induction being achieved at 100 μM with the higher chlorinated congeners. Using okadaic acid and small interfering RNAs targeting CAR and PXR, we could demonstrate that CAR plays a major role and PXR a minor role in NDL-PCB-driven induction of CYPs, both effects showing no stringent structure–activity relationship. As the only obvious relevant determinant, the degree of chlorination was found to be positively correlated with the inducing potency of the congeners. - Highlights: • We analyzed six highly purified NDL-PCBs for CYP2B1 and CYP3A1 expression. • CAR plays a major, PXR a minor role in NDL-PCB-driven induction of CYPs. • The degree of chlorination seems to be the major parameter for the inducing potency. • There exists a competition between CAR and PXR. • Activated PXR may

  1. Role of the nuclear xenobiotic receptors CAR and PXR in induction of cytochromes P450 by non-dioxinlike polychlorinated biphenyls in cultured rat hepatocytes

    Gährs, Maike; Roos, Robert [University of Kaiserslautern, Food Chemistry and Toxicology, Erwin-Schroedinger-Str. 52, D-67663 Kaiserslautern (Germany); Andersson, Patrik L. [Umeå University, Department of Chemistry, Linnaeus väg 6, SE-901 87 Umeå (Sweden); Schrenk, Dieter, E-mail: schrenk@rhrk.uni-kl.de [University of Kaiserslautern, Food Chemistry and Toxicology, Erwin-Schroedinger-Str. 52, D-67663 Kaiserslautern (Germany)

    2013-10-01

    Polychlorinated biphenyls (PCBs) are among the most ubiquitously detectable ‘persistent organic pollutants’. In contrast to ‘dioxinlike’ (DL) PCBs, less is known about the molecular mode of action of the larger group of the ‘non-dioxinlike’ (NDL) PCBs. Owing to the life-long exposure of the human population, a carcinogenic, i.e., tumor-promoting potency of NDL-PCBs has to be considered in human risk assessment. A major problem in risk assessment of NDL-PCBs is dioxin-like impurities that can occur in commercially available NDL-PCB standards. In the present study, we analyzed the induction of CYP2B1 and CYP3A1 in primary rat hepatocytes using a number of highly purified NDL-PCBs with various degrees of chlorination and substitution patterns. Induction of these enzymes is mediated by the nuclear xenobiotic receptors CAR (Constitutive androstane receptor) and PXR (Pregnane X receptor). For CYP2B1 induction, concentration–response analysis revealed a very narrow window of EC{sub 50} estimates, being in the range of 1–4 μM for PCBs 28 and 52, and between 0.4 and 1 μM for PCBs 101, 138, 153 and 180. CYP3A1 induction was less sensitive to NDL-PCBs, the most pronounced induction being achieved at 100 μM with the higher chlorinated congeners. Using okadaic acid and small interfering RNAs targeting CAR and PXR, we could demonstrate that CAR plays a major role and PXR a minor role in NDL-PCB-driven induction of CYPs, both effects showing no stringent structure–activity relationship. As the only obvious relevant determinant, the degree of chlorination was found to be positively correlated with the inducing potency of the congeners. - Highlights: • We analyzed six highly purified NDL-PCBs for CYP2B1 and CYP3A1 expression. • CAR plays a major, PXR a minor role in NDL-PCB-driven induction of CYPs. • The degree of chlorination seems to be the major parameter for the inducing potency. • There exists a competition between CAR and PXR. • Activated PXR

  2. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  3. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-01-01

    Highlights: • LPA 5 inhibits the cell growth and motile activities of 3T3 cells. • LPA 5 suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA 5 on the cell motile activities inhibited by LPA 1 in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA 5 in 3T3 cells. • LPA signaling via LPA 5 acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA 1 –LPA 6 ) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA 1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA 5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA 1 and LPA 5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA 5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA 1

  4. Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

    Wilson, Jamie L.; Taylor, Linda; Polgar, Peter

    2012-01-01

    Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [ 3 H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: ► ET- hinders cell proliferation in CHO cells transfected with ETB. ► ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. ► JNK and PI3K appear to be involved in this reduction of DNA synthesis. ► ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. ► In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

  5. Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

    Wilson, Jamie L.; Taylor, Linda; Polgar, Peter, E-mail: peterp@bu.edu

    2012-06-10

    Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [{sup 3}H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: Black-Right-Pointing-Pointer ET- hinders cell proliferation in CHO cells transfected with ETB. Black-Right-Pointing-Pointer ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. Black-Right-Pointing-Pointer JNK and PI3K appear to be involved in this reduction of DNA synthesis. Black-Right-Pointing-Pointer ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. Black-Right-Pointing-Pointer In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

  6. Cellular androgen content influences enzalutamide agonism of F877L mutant androgen receptor

    Coleman, Daniel J.; Van Hook, Kathryn; King, Carly J.; Schwartzman, Jacob; Lisac, Robert; Urrutia, Joshua; Sehrawat, Archana; Woodward, Josha; Wang, Nicholas J.; Gulati, Roman; Thomas, George V.; Beer, Tomasz M.; Gleave, Martin; Korkola, James E.; Gao, Lina; Heiser, Laura M.; Alumkal, Joshi J.

    2016-01-01

    Prostate cancer is the most commonly diagnosed and second-most lethal cancer among men in the United States. The vast majority of prostate cancer deaths are due to castration-resistant prostate cancer (CRPC) – the lethal form of the disease that has progressed despite therapies that interfere with activation of androgen receptor (AR) signaling. One emergent resistance mechanism to medical castration is synthesis of intratumoral androgens that activate the AR. This insight led to the development of the AR antagonist enzalutamide. However, resistance to enzalutamide invariably develops, and disease progression is nearly universal. One mechanism of resistance to enzalutamide is an F877L mutation in the AR ligand-binding domain that can convert enzalutamide to an agonist of AR activity. However, mechanisms that contribute to the agonist switch had not been fully clarified, and there were no therapies to block AR F877L. Using cell line models of castration-resistant prostate cancer (CRPC), we determined that cellular androgen content influences enzalutamide agonism of mutant F877L AR. Further, enzalutamide treatment of AR F877L-expressing cell lines recapitulated the effects of androgen activation of F877L AR or wild-type AR. Because the BET bromodomain inhibitor JQ-1 was previously shown to block androgen activation of wild-type AR, we tested JQ-1 in AR F877L-expressing CRPC models. We determined that JQ-1 suppressed androgen or enzalutamide activation of mutant F877L AR and suppressed growth of mutant F877L AR CRPC tumors in vivo, demonstrating a new strategy to treat tumors harboring this mutation. PMID:27276681

  7. Downregulation of the non-integrin laminin receptor reduces cellular viability by inducing apoptosis in lung and cervical cancer cells.

    Kiashanee Moodley

    Full Text Available The non-integrin laminin receptor, here designated the 37-kDa/67-kDa laminin receptor (LRP/LR, is involved in many physiologically relevant processes, as well as numerous pathological conditions. The overexpression of LRP/LR on various cancerous cell lines plays critical roles in tumour metastasis and angiogenesis. This study investigated whether LRP/LR is implicated in the maintenance of cellular viability in lung and cervical cancer cell lines. Here we show a significant reduction in cellular viability in the aforementioned cell lines as a result of the siRNA-mediated downregulation of LRP. This reduction in cellular viability is due to increased apoptotic processes, reflected by the loss of nuclear integrity and the significant increase in the activity of caspase-3. These results indicate that LRP/LR is involved in the maintenance of cellular viability in tumorigenic lung and cervix uteri cells through the blockage of apoptosis. Knockdown of LRP/LR by siRNA might represent an alternative therapeutic strategy for the treatment of lung and cervical cancer.

  8. Car Sickness

    ... Preventable Diseases Healthy Children > Health Issues > Conditions > Head Neck & Nervous System > Car Sickness Health Issues Listen Español Text Size Email Print Share Car Sickness Page Content ...

  9. Role of toll-like receptors 3, 4 and 7 in cellular uptake and response to titanium dioxide nanoparticles

    Peng Chen, Koki Kanehira and Akiyoshi Taniguchi

    2013-01-01

    Full Text Available Innate immune response is believed to be among the earliest provisional cellular responses, and mediates the interactions between microbes and cells. Toll-like receptors (TLRs are critical to these interactions. We hypothesize that TLRs also play an important role in interactions between nanoparticles (NPs and cells, although little information has been reported concerning such an interaction. In this study, we investigated the role of TLR3, TLR4 and TLR7 in cellular uptake of titanium dioxide NP (TiO2 NP agglomerates and the resulting inflammatory responses to these NPs. Our data indicate that TLR4 is involved in the uptake of TiO2 NPs and promotes the associated inflammatory responses. The data also suggest that TLR3, which has a subcellular location distinct from that of TLR4, inhibits the denaturation of cellular protein caused by TiO2 NPs. In contrast, the unique cellular localization of TLR7 has middle-ground functional roles in cellular response after TiO2 NP exposure. These findings are important for understanding the molecular interaction mechanisms between NPs and cells.

  10. Fine-tuning the CAR spacer improves T-cell potency

    Watanabe, Norihiro; Bajgain, Pradip; Sukumaran, Sujita; Ansari, Salma; Heslop, Helen E.; Rooney, Cliona M.; Brenner, Malcolm K.; Leen, Ann M.; Vera, Juan F.

    2016-01-01

    ABSTRACT The adoptive transfer of genetically engineered T cells expressing chimeric antigen receptors (CARs) has emerged as a transformative cancer therapy with curative potential, precipitating a wave of preclinical and clinical studies in academic centers and the private sector. Indeed, significant effort has been devoted to improving clinical benefit by incorporating accessory genes/CAR endodomains designed to enhance cellular migration, promote in vivo expansion/persistence or enhance safety by genetic programming to enable the recognition of a tumor signature. However, our efforts centered on exploring whether CAR T-cell potency could be enhanced by modifying pre-existing CAR components. We now demonstrate how molecular refinements to the CAR spacer can impact multiple biological processes including tonic signaling, cell aging, tumor localization, and antigen recognition, culminating in superior in vivo antitumor activity. PMID:28180032

  11. Expressions of toll-like receptors 2 and 4, and relative cellular ...

    for regulation of the immune system. Their cellular factors are TNF-α, IFN-γ, IL-2, IL-6 and. IL-10. Th1 cells induce cellular response reaction and inflammatory reaction, but Th2 cell promote immunity of body fluids and resist parasitic infections; these two types of cells maintain balance in the immune system [20]. HIV infection.

  12. CAR Suppresses Hepatic Gluconeogenesis by Facilitating the Ubiquitination and Degradation of PGC1α

    Gao, Jie; Yan, Jiong; Xu, Meishu; Ren, Songrong

    2015-01-01

    The constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) are master regulators of drug metabolism and gluconeogenesis, respectively. In supporting the cross talk between drug metabolism and energy metabolism, activation of CAR has been shown to suppress hepatic gluconeogenesis and ameliorate hyperglycemia in vivo, but the underlying molecular mechanism remains elusive. In this study, we demonstrated that CAR suppressed hepatic gluconeogenic gene expression through posttranslational regulation of the subcellular localization and degradation of PGC1α. Activated CAR translocated into the nucleus and served as an adaptor protein to recruit PGC1α to the Cullin1 E3 ligase complex for ubiquitination. The interaction between CAR and PGC1α also led to their sequestration within the promyelocytic leukemia protein-nuclear bodies, where PGC1α and CAR subsequently underwent proteasomal degradation. Taken together, our findings revealed an unexpected function of CAR in recruiting an E3 ligase and targeting the gluconeogenic activity of PGC1α. Both drug metabolism and gluconeogenesis are energy-demanding processes. The negative regulation of PGC1α by CAR may represent a cellular adaptive mechanism to accommodate energy-restricted conditions. PMID:26407237

  13. Two-color cytofluorometry and cellular properties of the urokinase receptor associated with a human metastatic carcinomatous cell line

    Takahashi, K.; Gojobori, T.; Tanifuji, M.

    1991-01-01

    Purified human urokinase was labeled with either fluorescein isothiocyanate or iodine-125 and used as a probe for binding to the human metastatic carcinomatous cell line, Detroit 562. Cytofluorometry showed that the ligand bound preferentially to cells that had been exposed to acidic pH. The binding was competitive and decreased after mild tryptic digestion. The bound ligand could be removed by restoration of the cells to a low pH. Therefore, the cells had specific binding sites. The bound urokinase was involved in the breakdown of fibrin. Two-color cytofluorometric maps were constructed by counterstaining with propidium iodide. Results suggested that there were different cell populations that had different numbers of receptors and amounts of DNA. We cloned cells and found that single clones had homogeneous levels of receptors with different dissociation constants (from 10(-13) to 10(-11) mol/mg protein) for different clones. Cells of one clone, C5, which had high levels of receptor production, moved characteristically on a glass substratum coated with gold particles and reacted with wheat germ agglutinin, but not with concanavalin A. The receptors were found together with adhesion proteins at the sites where the cells adhered to the substrate. These results and the data obtained by zymography of the cellular proteins suggested that the urokinase-type plasminogen activators were bound to the receptors. The membrane-associated activator may stimulate local proteolysis, facilitating the migration of the tumor cell across the substrate

  14. Two-color cytofluorometry and cellular properties of the urokinase receptor associated with a human metastatic carcinomatous cell line

    Takahashi, K.; Gojobori, T.; Tanifuji, M. (Shimane Medical Univ., Izumo (Japan))

    1991-02-01

    Purified human urokinase was labeled with either fluorescein isothiocyanate or iodine-125 and used as a probe for binding to the human metastatic carcinomatous cell line, Detroit 562. Cytofluorometry showed that the ligand bound preferentially to cells that had been exposed to acidic pH. The binding was competitive and decreased after mild tryptic digestion. The bound ligand could be removed by restoration of the cells to a low pH. Therefore, the cells had specific binding sites. The bound urokinase was involved in the breakdown of fibrin. Two-color cytofluorometric maps were constructed by counterstaining with propidium iodide. Results suggested that there were different cell populations that had different numbers of receptors and amounts of DNA. We cloned cells and found that single clones had homogeneous levels of receptors with different dissociation constants (from 10(-13) to 10(-11) mol/mg protein) for different clones. Cells of one clone, C5, which had high levels of receptor production, moved characteristically on a glass substratum coated with gold particles and reacted with wheat germ agglutinin, but not with concanavalin A. The receptors were found together with adhesion proteins at the sites where the cells adhered to the substrate. These results and the data obtained by zymography of the cellular proteins suggested that the urokinase-type plasminogen activators were bound to the receptors. The membrane-associated activator may stimulate local proteolysis, facilitating the migration of the tumor cell across the substrate.

  15. Revealing the sequence and resulting cellular morphology of receptor-ligand interactions during Plasmodium falciparum invasion of erythrocytes.

    Greta E Weiss

    2015-02-01

    Full Text Available During blood stage Plasmodium falciparum infection, merozoites invade uninfected erythrocytes via a complex, multistep process involving a series of distinct receptor-ligand binding events. Understanding each element in this process increases the potential to block the parasite's life cycle via drugs or vaccines. To investigate specific receptor-ligand interactions, they were systematically blocked using a combination of genetic deletion, enzymatic receptor cleavage and inhibition of binding via antibodies, peptides and small molecules, and the resulting temporal changes in invasion and morphological effects on erythrocytes were filmed using live cell imaging. Analysis of the videos have shown receptor-ligand interactions occur in the following sequence with the following cellular morphologies; 1 an early heparin-blockable interaction which weakly deforms the erythrocyte, 2 EBA and PfRh ligands which strongly deform the erythrocyte, a process dependant on the merozoite's actin-myosin motor, 3 a PfRh5-basigin binding step which results in a pore or opening between parasite and host through which it appears small molecules and possibly invasion components can flow and 4 an AMA1-RON2 interaction that mediates tight junction formation, which acts as an anchor point for internalization. In addition to enhancing general knowledge of apicomplexan biology, this work provides a rational basis to combine sequentially acting merozoite vaccine candidates in a single multi-receptor-blocking vaccine.

  16. Comparison of Cellular Uptake and Inflammatory Response via Toll-Like Receptor 4 to Lipopolysaccharide and Titanium Dioxide Nanoparticles

    Akiyoshi Taniguchi

    2013-06-01

    Full Text Available The innate immune response is the earliest cellular response to infectious agents and mediates the interactions between microbes and cells. Toll-like receptors (TLRs play an important role in these interactions. We have already shown that TLRs are involved with the uptake of titanium dioxide nanoparticles (TiO2 NPs and promote inflammatory responses. In this paper, we compared role of cellular uptake and inflammatory response via TLR 4 to lipopolysaccharide (LPS and TiO2 NPs. In the case of LPS, LPS binds to LPS binding protein (LBP and CD 14, and then this complex binds to TLR 4. In the case of TiO2 NPs, the necessity of LBP and CD 14 to induce the inflammatory response and for uptake by cells was investigated using over-expression, antibody blocking, and siRNA knockdown experiments. Our results suggested that for cellular uptake of TiO2 NPs, TLR 4 did not form a complex with LBP and CD 14. In the TiO2 NP-mediated inflammatory response, TLR 4 acted as the signaling receptor without protein complex of LPS, LBP and CD 14. The results suggested that character of TiO2 NPs might be similar to the complex of LPS, LBP and CD 14. These results are important for development of safer nanomaterials.

  17. HAVCR1 (CD365) and Its Mouse Ortholog Are Functional Hepatitis A Virus (HAV) Cellular Receptors That Mediate HAV Infection.

    Costafreda, Maria Isabel; Kaplan, Gerardo

    2018-05-01

    The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV. IMPORTANCE HAVCR1, an HAV receptor, is expressed in different cell types, including regulatory immune cells and antigen-presenting cells. How HAV evades the immune response during a long incubation period of up to 4 weeks and the

  18. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    Evgeny Bychkov

    Full Text Available G protein-coupled receptor kinases (GRKs and arrestins mediate desensitization of G protein-coupled receptors (GPCR. Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  19. Expressions of toll-like receptors 2 and 4, and relative cellular ...

    Purpose: To investigate the expressions of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), tumor necrosis factor alpha (TNF-α), IFN-γ (IFN- gamma), interleukin 2 (IL-2), interleukin 6 (IL-6) and interleukin 10 (IL-10) in human immunodeficiency virus (HIV) patients with tuberculosis (TB) infection. Methods: Two groups of ...

  20. The Dopamine D2 Receptor Gene in Lamprey, Its Expression in the Striatum and Cellular Effects of D2 Receptor Activation

    Robertson, Brita; Huerta-Ocampo, Icnelia; Ericsson, Jesper; Stephenson-Jones, Marcus; Pérez-Fernández, Juan; Bolam, J. Paul; Diaz-Heijtz, Rochellys; Grillner, Sten

    2012-01-01

    All basal ganglia subnuclei have recently been identified in lampreys, the phylogenetically oldest group of vertebrates. Furthermore, the interconnectivity of these nuclei is similar to mammals and tyrosine hydroxylase-positive (dopaminergic) fibers have been detected within the input layer, the striatum. Striatal processing is critically dependent on the interplay with the dopamine system, and we explore here whether D2 receptors are expressed in the lamprey striatum and their potential role. We have identified a cDNA encoding the dopamine D2 receptor from the lamprey brain and the deduced protein sequence showed close phylogenetic relationship with other vertebrate D2 receptors, and an almost 100% identity within the transmembrane domains containing the amino acids essential for dopamine binding. There was a strong and distinct expression of D2 receptor mRNA in a subpopulation of striatal neurons, and in the same region tyrosine hydroxylase-immunoreactive synaptic terminals were identified at the ultrastructural level. The synaptic incidence of tyrosine hydroxylase-immunoreactive boutons was highest in a region ventrolateral to the compact layer of striatal neurons, a region where most striatal dendrites arborise. Application of a D2 receptor agonist modulates striatal neurons by causing a reduced spike discharge and a diminished post-inhibitory rebound. We conclude that the D2 receptor gene had already evolved in the earliest group of vertebrates, cyclostomes, when they diverged from the main vertebrate line of evolution (560 mya), and that it is expressed in striatum where it exerts similar cellular effects to that in other vertebrates. These results together with our previous published data (Stephenson-Jones et al. 2011, 2012) further emphasize the high degree of conservation of the basal ganglia, also with regard to the indirect loop, and its role as a basic mechanism for action selection in all vertebrates. PMID:22563388

  1. Spatial biomarker of disease and detection of spatial organization of cellular receptors

    Salaita, Khalid S.; Nair, Pradeep M.; Das, Debopriya; Gray, Joe W.; Groves, John T.

    2017-07-18

    A signature of a condition of a live cell is established in an assay that allows distribution of the receptors on the cell surface in response to binding a ligand. The receptors can be optically detected and quantified to provide a value for the condition, Test drugs can be screened for therapeutic potential in the assay: a potentially efficacious drug is identified by an ability to modulate an established signature. The receptor distribution signature can be corroborated with an mRNA expression profile of several genes, indicating, for example, metastasis.

  2. Phenobarbital and propiconazole toxicogenomic profiles in mice show major similarities consistent with the key role that constitutive androstane receptor (CAR) activation plays in their mode of action

    Currie, Richard A.; Peffer, Richard C.; Goetz, Amber K.; Omiecinski, Curtis J.; Goodman, Jay I.

    2014-01-01

    Toxicogenomics (TGx) is employed frequently to investigate underlying molecular mechanisms of the compound of interest and, thus, has become an aid to mode of action determination. However, the results and interpretation of a TGx dataset are influenced by the experimental design and methods of analysis employed. This article describes an evaluation and reanalysis, by two independent laboratories, of previously published TGx mouse liver microarray data for a triazole fungicide, propiconazole (PPZ), and the anticonvulsant drug phenobarbital (PB). Propiconazole produced an increase incidence of liver tumors in male CD-1 mice only at a dose that exceeded the maximum tolerated dose (2500 ppm). Firstly, we illustrate how experimental design differences between two in vivo studies with PPZ and PB may impact the comparisons of TGx results. Secondly, we demonstrate that different researchers using different pathway analysis tools can come to different conclusions on specific mechanistic pathways, even when using the same datasets. Finally, despite these differences the results across three different analyses also show a striking degree of similarity observed for PPZ and PB treated livers when the expression data are viewed as major signaling pathways and cell processes affected. Additional studies described here show that the postulated key event of hepatocellular proliferation was observed in CD-1 mice for both PPZ and PB, and that PPZ is also a potent activator of the mouse CAR nuclear receptor. Thus, with regard to the events which are hallmarks of CAR-induced effects that are key events in the mode of action (MOA) of mouse liver carcinogenesis with PB, PPZ-induced tumors can be viewed as being promoted by a similar PB-like CAR-dependent MOA. PMID:24675475

  3. Phenobarbital and propiconazole toxicogenomic profiles in mice show major similarities consistent with the key role that constitutive androstane receptor (CAR) activation plays in their mode of action

    Currie, Richard A.; Peffer, Richard C.; Goetz, Amber K.; Omiecinski, Curtis J.; Goodman, Jay I.

    2014-01-01

    Toxicogenomics (TGx) is employed frequently to investigate underlying molecular mechanisms of the compound of interest and, thus, has become an aid to mode of action determination. However, the results and interpretation of a TGx dataset are influenced by the experimental design and methods of analysis employed. This article describes an evaluation and reanalysis, by two independent laboratories, of previously published TGx mouse liver microarray data for a triazole fungicide, propiconazole (PPZ), and the anticonvulsant drug phenobarbital (PB). Propiconazole produced an increase incidence of liver tumors in male CD-1 mice only at a dose that exceeded the maximum tolerated dose (2500 ppm). Firstly, we illustrate how experimental design differences between two in vivo studies with PPZ and PB may impact the comparisons of TGx results. Secondly, we demonstrate that different researchers using different pathway analysis tools can come to different conclusions on specific mechanistic pathways, even when using the same datasets. Finally, despite these differences the results across three different analyses also show a striking degree of similarity observed for PPZ and PB treated livers when the expression data are viewed as major signaling pathways and cell processes affected. Additional studies described here show that the postulated key event of hepatocellular proliferation was observed in CD-1 mice for both PPZ and PB, and that PPZ is also a potent activator of the mouse CAR nuclear receptor. Thus, with regard to the events which are hallmarks of CAR-induced effects that are key events in the mode of action (MOA) of mouse liver carcinogenesis with PB, PPZ-induced tumors can be viewed as being promoted by a similar PB-like CAR-dependent MOA

  4. CD19 CAR T Cells Expressing IL-12 Eradicate Lymphoma in Fully Lymphoreplete Mice through Induction of Host Immunity

    Gray Kueberuwa

    2018-03-01

    Full Text Available Chimeric antigen receptor (CAR T cell therapy represents a significant advancement in cancer therapy. Larger studies have shown ∼90% complete remission rates against chemoresistant and/or refractory CD19+ leukemia or lymphoma. Effective CAR T cell therapy is highly dependent on lymphodepleting preconditioning, which is achieved through chemotherapy or radiotherapy that carries with it significant toxicities. These can exclude patients of low performance status. In order to overcome the need for preconditioning, we constructed fully mouse first and second generation anti-murine CD19 CARs with or without interleukin-12 (IL-12 secretion. To test these CARs, we established a mouse model to reflect the human situation without preconditioning. Murine second generation CAR T cells expressing IL-12 were capable of eradicating established B cell lymphoma with a long-term survival rate of ∼25%. We believe this to be the first study in a truly lymphoreplete model. We provide evidence that IL-12-expressing CAR T cells not only directly kill target CD19+ cells, but also recruit host immune cells to an anti-cancer immune response. This finding is critical because lymphodepletion regimens required for the success of current CAR T cell technology eliminate host immune cells whose anti-cancer activity could otherwise be harnessed by strategies such as IL-12-secreting CAR T cells. Keywords: CD19 CAR T cells, IL-12, immunotherapy, chimeric antigen receptor, adoptive cellular therapy, lymphoma, B cell malignancies, TRUCKs, pre-conditioning

  5. Nuclear receptor CAR specifically activates the two-pore K+ channel Kcnk1 gene in male mouse livers, which attenuates phenobarbital-induced hepatic hyperplasia.

    Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2013-03-01

    KCNK1, a member of the family of two-pore K(+) ion channels, is specifically induced in the livers of male mice after phenobarbital treatment. Here, we have determined the molecular mechanism of this male-specific activation of the Kcnk1 gene and characterized KCNK1 as a phenobarbital-inducible antihyperplasia factor. Upon activation by phenobarbital, nuclear receptor CAR binds the 97-bp response element (-2441/-2345) within the Kcnk1 promoter. This binding is observed in the livers of male mice, but not in the livers of female mice and requires the pituitary gland, because hypophysectomy abrogates it. Hyperplasia further progressed in the livers of Kcnk1 ( -/- ) male mice compared with those of Kcnk1 ( +/+ ) males after phenobarbital treatment. Thus, KCNK1 suppresses phenobarbital-induced hyperplasia. These results indicate that phenobarbital treatment induces KCNK1 to elicit a male-specific and growth-suppressing signal. Thus, KCNK1 and Kcnk1 ( -/- ) mice provide an experimental tool for further investigation into the molecular mechanism of CAR-mediated promotion of the development of hepatocellular carcinoma in mice.

  6. Laminar and Cellular Distribution of Monoamine Receptors in Rat Medial Prefrontal Cortex

    Noemí Santana

    2017-09-01

    Full Text Available The prefrontal cortex (PFC is deeply involved in higher brain functions, many of which are altered in psychiatric conditions. The PFC exerts a top-down control of most cortical and subcortical areas through descending pathways and is densely innervated by axons emerging from the brainstem monoamine cell groups, namely, the dorsal and median raphe nuclei (DR and MnR, respectively, the ventral tegmental area and the locus coeruleus (LC. In turn, the activity of these cell groups is tightly controlled by afferent pathways arising from layer V PFC pyramidal neurons. The reciprocal connectivity between PFC and monoamine cell groups is of interest to study the pathophysiology and treatment of severe psychiatric disorders, such as major depression and schizophrenia, inasmuch as antidepressant and antipsychotic drugs target monoamine receptors/transporters expressed in these areas. Here we review previous reports examining the presence of monoamine receptors in pyramidal and GABAergic neurons of the PFC using double in situ hybridization. Additionally, we present new data on the quantitative layer distribution (layers I, II–III, V, and VI of monoamine receptor-expressing cells in the cingulate (Cg, prelimbic (PrL and infralimbic (IL subfields of the medial PFC (mPFC. The receptors examined include serotonin 5-HT1A, 5-HT2A, 5-HT2C, and 5-HT3, dopamine D1 and D2 receptors, and α1A-, α1B-, and α1D-adrenoceptors. With the exception of 5-HT3 receptors, selectively expressed by layers I–III GABA interneurons, the rest of monoamine receptors are widely expressed by pyramidal and GABAergic neurons in intermediate and deep layers of mPFC (5-HT2C receptors are also expressed in layer I. This complex distribution suggests that monoamines may modulate the communications between PFC and cortical/subcortical areas through the activation of receptors expressed by neurons in intermediate (e.g., 5-HT1A, 5-HT2A, α1D-adrenoceptors, dopamine D1 receptors and deep

  7. Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy.

    Golubovskaya, Vita; Wu, Lijun

    2016-03-15

    This review is focused on different subsets of T cells: CD4 and CD8, memory and effector functions, and their role in CAR-T therapy--a cellular adoptive immunotherapy with T cells expressing chimeric antigen receptor. The CAR-T cells recognize tumor antigens and induce cytotoxic activities against tumor cells. Recently, differences in T cell functions and the role of memory and effector T cells were shown to be important in CAR-T cell immunotherapy. The CD4⁺ subsets (Th1, Th2, Th9, Th17, Th22, Treg, and Tfh) and CD8⁺ memory and effector subsets differ in extra-cellular (CD25, CD45RO, CD45RA, CCR-7, L-Selectin [CD62L], etc.); intracellular markers (FOXP3); epigenetic and genetic programs; and metabolic pathways (catabolic or anabolic); and these differences can be modulated to improve CAR-T therapy. In addition, CD4⁺ Treg cells suppress the efficacy of CAR-T cell therapy, and different approaches to overcome this suppression are discussed in this review. Thus, next-generation CAR-T immunotherapy can be improved, based on our knowledge of T cell subsets functions, differentiation, proliferation, and signaling pathways to generate more active CAR-T cells against tumors.

  8. Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy

    Vita Golubovskaya

    2016-03-01

    Full Text Available This review is focused on different subsets of T cells: CD4 and CD8, memory and effector functions, and their role in CAR-T therapy––a cellular adoptive immunotherapy with T cells expressing chimeric antigen receptor. The CAR-T cells recognize tumor antigens and induce cytotoxic activities against tumor cells. Recently, differences in T cell functions and the role of memory and effector T cells were shown to be important in CAR-T cell immunotherapy. The CD4+ subsets (Th1, Th2, Th9, Th17, Th22, Treg, and Tfh and CD8+ memory and effector subsets differ in extra-cellular (CD25, CD45RO, CD45RA, CCR-7, L-Selectin [CD62L], etc.; intracellular markers (FOXP3; epigenetic and genetic programs; and metabolic pathways (catabolic or anabolic; and these differences can be modulated to improve CAR-T therapy. In addition, CD4+ Treg cells suppress the efficacy of CAR-T cell therapy, and different approaches to overcome this suppression are discussed in this review. Thus, next-generation CAR-T immunotherapy can be improved, based on our knowledge of T cell subsets functions, differentiation, proliferation, and signaling pathways to generate more active CAR-T cells against tumors.

  9. Receptor-independent, vacuolar ATPase-mediated cellular uptake of histamine receptor-1 ligands: Possible origin of pharmacological distortions and side effects

    Morissette, Guillaume; Lodge, Robert; Bouthillier, Johanne; Marceau, Francois

    2008-01-01

    The aims of this study were to investigate whether several histamine receptor agonists and antagonists are subjected to receptor-independent ion trapping into acidic organelles, and whether this sequestration influences their pharmacological or toxicological properties. Vacuolar (V)-ATPase-dependent intracellular sequestration of agonists was recognized as morphological alterations (large fluid-filled vacuoles for betahistine and 1-methylhistamine, granular uptake for fluorescent BODIPY FL histamine) prevented by the specific V-ATPase inhibitor bafilomycin A1 in rabbit vascular smooth muscle cells. Lipophilicity was the major determinant of these cellular effects (order of potency: BODIPY FL histamine > betahistine > 1-methylhistamine > histamine) that occurred at high concentrations. This ranking was dissociable from the potency order for H 1 receptor-mediated contraction of the rabbit aorta, a response uninfluenced by bafilomycin. Antihistamines are inherently more lipophilic and caused vacuolization of a proportion of cells at 5-500 μM. Agonist or antagonist-induced vacuoles were of macroautophagic nature (labeled with GFP-conjugated LC3, Rab7 and CD63; detection of LC3 II). Further, the 2 most lipophilic antihistamines tested, astemizole and terfenadine, were potentiated by V-ATPase blockade in the aortic contractility assay (13- and 3.6-fold more potent, respectively, pA 2 scale), suggesting that V-ATPase-mediated cation trapping sequesters these antagonists from the vicinity of H 1 receptors in the therapeutic concentration range. This potentiation did not apply to less lipophilic antagonists (pyrilamine, diphenhydramine). While some agonists and all tested antagonists of the histamine H 1 receptors induce the V-ATPase-dependent vacuolar and autophagic cytopathology, sequestration affects the pharmacology of only the most lipophilic antagonists, the ones prone to off-target arrhythmogenic side effects

  10. CAR-T cells are serial killers.

    Davenport, Alexander J; Jenkins, Misty R; Ritchie, David S; Prince, H Miles; Trapani, Joseph A; Kershaw, Michael H; Darcy, Phillip K; Neeson, Paul J

    2015-12-01

    Chimeric antigen receptor (CAR) T cells have enjoyed unprecedented clinical success against haematological malignancies in recent years. However, several aspects of CAR T cell biology remain unknown. We recently compared CAR and T cell receptor (TCR)-based killing in the same effector cell and showed that CAR T cells can not only efficiently kill single tumor targets, they can also kill multiple tumor targets in a sequential manner. Single and serial killing events were not sustained long term due to CAR down-regulation after 20 hours.

  11. Interaction of cadmium with atrial natriuretic factor receptors: Ligand binding and cellular processing

    Giridhar, J.; Rathinavelu, A.; Isom, G.E.

    1990-01-01

    ANF is a peptide hormone secreted by the heart and produces potent diuresis and vascular smooth muscle relaxation. It is well known that Cd produces cardiovascular toxicity and is implicated in the pathogenesis of hypertension. Hence the effects of Cd on ANF receptor dynamics and ligand binding were studied in PC12 cells. Receptor internalization using 125 I-ANF as the ligand at 37 degree C displayed a decrease in endocytic rate constants (ERC) when either preincubated with Cd (500 μM for 30 min, ERC = 0.183/min) or coincubated with Cd (500 μM, ERC = 0.196) when compared to control value (ERC = 0.259/min). Ligand binding ( 125 I-ANF) was changed by Cd as reflected by a decrease in the number of binding sites/cell in both Cd preincubated (Kd = 3.81 x 10 -10 M, B max = 1 x 10 -10 M, binding sites/cell = 9333) and coincubated cells (Kd = 1.76 x 10 -10 M, B max = 3.92 x 10 -11 M, binding sites/cell = 5960) from control (Kd = 3.87 x 10 -10 M, B max = 9.58 x 10 -11 M, binding sites/cell = 12141). Photoaffinity labelling with 125 I-ANF as the ligand was used to measure receptor subtype binding. Coincubation of cells with Cd (500 μM) and ligand decreased both high and low mol. wt. receptor binding, whereas preincubation with Cd (500μM) for 60 min produced a slight decrease in binding of both receptor subtypes. These results indicate that the cardiovascular toxicity of Cd may be partially mediated by altered ANF receptor function

  12. An analytical biomarker for treatment of patients with recurrent B-ALL after remission induced by infusion of anti-CD19 chimeric antigen receptor T (CAR-T) cells.

    Zhang, Yajing; Zhang, Wenying; Dai, Hanren; Wang, Yao; Shi, Fengxia; Wang, Chunmeng; Guo, Yelei; Liu, Yang; Chen, Meixia; Feng, Kaichao; Zhang, Yan; Liu, Chuanjie; Yang, Qingming; Li, Suxia; Han, Weidong

    2016-04-01

    Anti-CD19 chimeric antigen receptor-modified T (CAR-T-19) cells have emerged as a powerful targeted immunotherapy for B-cell lineage acute lymphoblastic leukemia with a remarkable clinical response in recent trials. Nonetheless, few data are available on the subsequent clinical monitoring and treatment of the patients, especially those with disease recurrence after CAR-T-19 cell infusion. Here, we analyzed three patients who survived after our phase I clinical trial and who were studied by means of biomarkers reflecting persistence of CAR-T-19 cells in vivo and predictive factors directing further treatment. One patient achieved 9-week sustained complete remission and subsequently received an allogeneic hematopoietic stem cell transplant. Another patient who showed relapse after 20 weeks without detectable leukemia in the cerebrospinal fluid after CAR-T-19 cell treatment was able to achieve a morphological remission under the influence of stand-alone low-dose chemotherapeutic agents. The third patient gradually developed extensive extramedullary involvement in tissues with scarce immune- cell infiltration during a long period of hematopoietic remission after CAR-T-19 cell therapy. Long-term and discontinuous increases in serum cytokines (mainly interleukin 6 and C-reactive protein) were identified in two patients (Nos. 1 and 6) even though only a low copy number of CAR molecules could be detected in their peripheral blood. This finding was suggestive of persistent functional activity of CAR-T-19 cells. Combined analyses of laboratory biomarkers with their clinical manifestations before and after salvage treatment showed that the persistent immunosurveillance mediated by CAR-T-19 cells would inevitably potentiate the leukemia-killing effectiveness of subsequent chemotherapy in patients who showed relapse after CAR-T-19-induced remission.

  13. Clinical trials of CAR-T cells in China

    Bingshan Liu

    2017-10-01

    Full Text Available Abstract Novel immunotherapeutic agents targeting tumor-site microenvironment are revolutionizing cancer therapy. Chimeric antigen receptor (CAR-engineered T cells are widely studied for cancer immunotherapy. CD19-specific CAR-T cells, tisagenlecleucel, have been recently approved for clinical application. Ongoing clinical trials are testing CAR designs directed at novel targets involved in hematological and solid malignancies. In addition to trials of single-target CAR-T cells, simultaneous and sequential CAR-T cells are being studied for clinical applications. Multi-target CAR-engineered T cells are also entering clinical trials. T cell receptor-engineered CAR-T and universal CAR-T cells represent new frontiers in CAR-T cell development. In this study, we analyzed the characteristics of CAR constructs and registered clinical trials of CAR-T cells in China and provided a quick glimpse of the landscape of CAR-T studies in China.

  14. Clinical trials of CAR-T cells in China.

    Liu, Bingshan; Song, Yongping; Liu, Delong

    2017-10-23

    Novel immunotherapeutic agents targeting tumor-site microenvironment are revolutionizing cancer therapy. Chimeric antigen receptor (CAR)-engineered T cells are widely studied for cancer immunotherapy. CD19-specific CAR-T cells, tisagenlecleucel, have been recently approved for clinical application. Ongoing clinical trials are testing CAR designs directed at novel targets involved in hematological and solid malignancies. In addition to trials of single-target CAR-T cells, simultaneous and sequential CAR-T cells are being studied for clinical applications. Multi-target CAR-engineered T cells are also entering clinical trials. T cell receptor-engineered CAR-T and universal CAR-T cells represent new frontiers in CAR-T cell development. In this study, we analyzed the characteristics of CAR constructs and registered clinical trials of CAR-T cells in China and provided a quick glimpse of the landscape of CAR-T studies in China.

  15. The binding of NCAM to FGFR1 induces a specific cellular response mediated by receptor trafficking

    Francavilla, Chiara; Cattaneo, Paola; Berezin, Vladimir

    2009-01-01

    different from that elicited by FGF-2. In contrast to FGF-induced degradation of endocytic FGFR1, NCAM promotes the stabilization of the receptor, which is recycled to the cell surface in a Rab11- and Src-dependent manner. In turn, FGFR1 recycling is required for NCAM-induced sustained activation of various...

  16. Expression of PML tumor suppressor in A 431 cells reduces cellular growth by inhibiting the epidermal growth factor receptor expression

    Vallian, S.; Chang, K.S.

    2004-01-01

    Our previous studies showed that the promyelocytic leukemia, PML, protein functions as a cellular and growth suppressor. Transient expression of PML was also found to repress the activity of the epidermal growth factor receptor gene promoter. In this study we have examined the effects of PML on A431 cells, which express a high level of + protein. The PML gene was introduced into the cells using the adenovirus-mediated gene transfer system. Western blot analysis on the extracts from the cells expressing PML showed a significant repression in the expression of the epidermal growth factor receptor protein. The cells were examined for growth and DNA synthesis. The data showed a marked reduction in both growth and DNA synthesis rate in the cells expressing PML compared with the control cells. Furthermore, in comparison with the controls, the cells expressing PML were found to be more in G1 phase, fewer in S and about the same number in the G2/M phase. This data clearly demonstrated that the repression of epidermal growth factor receptor expression in A 431 cells by PML was associated with inhibition of cell growth and alteration of the cell cycle distribution, suggesting a novel mechanism for the known growth inhibitory effects of PML

  17. Chimeric antigen receptor engineering: a right step in the evolution of adoptive cellular immunotherapy.

    Figueroa, Jose A; Reidy, Adair; Mirandola, Leonardo; Trotter, Kayley; Suvorava, Natallia; Figueroa, Alejandro; Konala, Venu; Aulakh, Amardeep; Littlefield, Lauren; Grizzi, Fabio; Rahman, Rakhshanda Layeequr; Jenkins, Marjorie R; Musgrove, Breeanna; Radhi, Saba; D'Cunha, Nicholas; D'Cunha, Luke N; Hermonat, Paul L; Cobos, Everardo; Chiriva-Internati, Maurizio

    2015-03-01

    Cancer immunotherapy comprises different therapeutic strategies that exploit the use of distinct components of the immune system, with the common goal of specifically targeting and eradicating neoplastic cells. These varied approaches include the use of specific monoclonal antibodies, checkpoint inhibitors, cytokines, therapeutic cancer vaccines and cellular anticancer strategies such as activated dendritic cell (DC) vaccines, tumor-infiltrating lymphocytes (TILs) and, more recently, genetically engineered T cells. Each one of these approaches has demonstrated promise, but their generalized success has been hindered by the paucity of specific tumor targets resulting in suboptimal tumor responses and unpredictable toxicities. This review will concentrate on recent advances on the use of engineered T cells for adoptive cellular immunotherapy (ACI) in cancer.

  18. Cellular and molecular biology of orphan G protein-coupled receptors.

    Oh, Da Young; Kim, Kyungjin; Kwon, Hyuk Bang; Seong, Jae Young

    2006-01-01

    The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

  19. Identification of Aminopeptidase N as a Cellular Receptor for Human Coronavirus-229E

    1992-05-12

    hemagglutinating encephalomyelitis virus (HEV), canine coronavirus (CCV), cat FIPV and feline enteric corona virus (FECV), human CVLPs, mouse...While the cat , dog and pig serve as natural hosts for the other coronavirus group 1 viruses , feline infectious peritonitis virus (FIPV), canine...3 2 . Virus Receptors ••••••••.••••••.....•................ 20 3. Viruses Which Cause Common Colds

  20. Chitinase 3-like 1 Regulates Cellular and Tissue Responses via IL-13 Receptor α2

    Chuan Hua He

    2013-08-01

    Full Text Available Members of the 18 glycosyl hydrolase (GH 18 gene family have been conserved over species and time and are dysregulated in inflammatory, infectious, remodeling, and neoplastic disorders. This is particularly striking for the prototypic chitinase-like protein chitinase 3-like 1 (Chi3l1, which plays a critical role in antipathogen responses where it augments bacterial killing while stimulating disease tolerance by controlling cell death, inflammation, and remodeling. However, receptors that mediate the effects of GH 18 moieties have not been defined. Here, we demonstrate that Chi3l1 binds to interleukin-13 receptor α2 (IL-13Rα2 and that Chi3l1, IL-13Rα2, and IL-13 are in a multimeric complex. We also demonstrate that Chi3l1 activates macrophage mitogen-activated protein kinase, protein kinase B/AKT, and Wnt/β-catenin signaling and regulates oxidant injury, apoptosis, pyroptosis, inflammasome activation, antibacterial responses, melanoma metastasis, and TGF-β1 production via IL-13Rα2-dependent mechanisms. Thus, IL-13Rα2 is a GH 18 receptor that plays a critical role in Chi3l1 effector responses.

  1. Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR target gene Cyp2b10 in the liver of B6C3F1 mice.

    Harri Lempiäinen

    2011-03-01

    Full Text Available Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.

  2. Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice.

    Lempiäinen, Harri; Müller, Arne; Brasa, Sarah; Teo, Soon-Siong; Roloff, Tim-Christoph; Morawiec, Laurent; Zamurovic, Natasa; Vicart, Axel; Funhoff, Enrico; Couttet, Philippe; Schübeler, Dirk; Grenet, Olivier; Marlowe, Jennifer; Moggs, Jonathan; Terranova, Rémi

    2011-03-24

    Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.

  3. Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice

    Brasa, Sarah; Teo, Soon-Siong; Roloff, Tim-Christoph; Morawiec, Laurent; Zamurovic, Natasa; Vicart, Axel; Funhoff, Enrico; Couttet, Philippe; Schübeler, Dirk; Grenet, Olivier; Marlowe, Jennifer; Moggs, Jonathan; Terranova, Rémi

    2011-01-01

    Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis. PMID:21455306

  4. DIFFERENTIAL BINDING OF HUMAN INTERLEUKIN-1 (IL-1) RECEPTOR ANTAGONIST TO NATURAL AND RECOMBINANT SOLUBLE AND CELLULAR IL-1 TYPE-I RECEPTORS

    Svenson, Morten; Nedergaard, Susanne; Heegaard, Peter M. H.

    1995-01-01

    antagonist (IL-1ra). Recombinant soluble human IL-1RI expressed in COS cells (sIL-1RI) consists of the extracellular part of the receptor and binds all three known IL-1 species but preferentially to IL-1ra. We further characterized the sizes and binding of IL-1raBF and sIL-1RI to IL-1ra by polyacrylamide gel...... electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N-glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL-1ra. We also evaluated the binding of IL-1ra to cellular IL-1RI on MRC5...... binding of both molecules to IL-1ra. Both factors blocked binding of IL-1ra to cellular IL-1RI, as did mAb to IL-1ra, but the sites on IL-1ra which bound to the mAb, and to IL-1raBF and sIL-1RI, differed. We conclude that there are important differences between the natural and recombinant forms of soluble...

  5. PTH1 receptor is involved in mediating cellular response to long-chain polyunsaturated fatty acids.

    Jose Candelario

    Full Text Available The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK. From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA and docosahexaenoic fatty acids (DHA caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1-34 in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA and C (PKC, reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC, we detected conformational responses to EPA similar to those caused by PTH(1-34. PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1-34 leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone.

  6. Mouse Hepatitis Virus Strain A59 and Blocking Antireceptor Monoclonal Antibody Bind to the N-Terminal Domain of Cellular Receptor

    Dveksler, Gabriela S.; Pensiero, Michael N.; Dieffenbach, Carl W.; Cardellichio, Christine B.; Basile, Alexis A.; Elia, Patrick E.; Holmes, Kathryn V.

    1993-03-01

    Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

  7. Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification

    Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang; Krause, Mechthild; Yaromina, Ala; Meyer-Staeckling, Soenke; Scherkl, Benjamin; Kriegs, Malte; Brandt, Burkhard; Grenman, Reidar; Petersen, Cordula; Baumann, Michael; Dikomey, Ekkehard

    2011-01-01

    Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

  8. Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival.

    Tomohisa Mori

    Full Text Available The membrane of the endoplasmic reticulum (ER of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.

  9. Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival.

    Mori, Tomohisa; Hayashi, Teruo; Hayashi, Eri; Su, Tsung-Ping

    2013-01-01

    The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.

  10. Modified T-cells (using TCR and CTAs, chimeric antigen receptor (CAR and other molecular tools in recent gene therapy

    A.S. Odiba

    2018-07-01

    Full Text Available T-cell-based cancer immunotherapy by the transfer of cloned TCRs that are isolated from tumor penetrating T-cells becomes a possibility through NY-ESOc259; a human-derived affinity-enhanced TCR that provides a level of sufficiency in long-term safety and efficacy. NY-ESOc259 recognizes a peptide common to CTAs (LAGE-1 and NY-ESO-1 in melanoma. Risks associated with insertion related transformation in gene therapy have been alleviated through strategies that include the engineering of transcription activator like effector nucleases (TALEN, RNA-guided nucleases (CRISPR/Cas9, Zinc-finger nucleases (ZFN. Cancer immunotherapy based on the genetic modification of autologous T-cells (dependent on the engineered autologous CD8+ T-cells, designed to distinguish and destroy cells bearing tumor-specific antigens via a CAR is able to exterminate B-cell leukemias and lymphomas that are resilient to conventional therapies. A tool with a very large reservoir of potentials in molecular therapy strategy is the Pluripotent Stem Cells (PSC, with pluripotency factors that include Klf4, Sox2, c-Myc, Oct4, differentiating into disease-associated cell phenotypes of three germ layers, comprising of mesoderm (e.g. cardiac cells, blood and muscle, endoderm (liver, pancreas and ectoderm (epidermis, neurons. It finds good application in disease modelling as well as therapeutic options in the restoration of CGD by using AAVS1 as the vector where the therapeutic cassette is integrated into the locus to restore superoxide production in the granulocytes. Fascinatingly, Clinical trial involving iPSC are already underway where scientists have plans to use iPSC-derived cells to treat macular degeneration (a devastating age-related eye disease. Application of these findings has redefined incurable diseases disorders as curable. Keywords: Clinical trials, Disorders, Gene therapy, Molecular biology, Pharmacotherapy, Vector

  11. Charomers-Interleukin-6 Receptor Specific Aptamers for Cellular Internalization and Targeted Drug Delivery.

    Hahn, Ulrich

    2017-12-06

    Interleukin-6 (IL-6) is a key player in inflammation and the main factor for the induction of acute phase protein biosynthesis. Further to its central role in many aspects of the immune system, IL-6 regulates a variety of homeostatic processes. To interfere with IL-6 dependent diseases, such as various autoimmune diseases or certain cancers like multiple myeloma or hepatocellular carcinoma associated with chronic inflammation, it might be a sensible strategy to target human IL-6 receptor (hIL-6R) presenting cells with aptamers. We therefore have selected and characterized different DNA and RNA aptamers specifically binding IL-6R. These IL-6R aptamers, however, do not interfere with the IL-6 signaling pathway but are internalized with the receptor and thus can serve as vehicles for the delivery of different cargo molecules like therapeutics. We succeeded in the construction of a chlorin e6 derivatized aptamer to be delivered for targeted photodynamic therapy (PDT). Furthermore, we were able to synthesize an aptamer intrinsically comprising the cytostatic 5-Fluoro-2'-deoxy-uridine for targeted chemotherapy. The α6β4 integrin specific DNA aptamer IDA, also selected in our laboratory is internalized, too. All these aptamers can serve as vehicles for targeted drug delivery into cells. We call them charomers-in memory of Charon, the ferryman in Greek mythology, who ferried the deceased into the underworld.

  12. Charomers—Interleukin-6 Receptor Specific Aptamers for Cellular Internalization and Targeted Drug Delivery

    2017-01-01

    Interleukin-6 (IL-6) is a key player in inflammation and the main factor for the induction of acute phase protein biosynthesis. Further to its central role in many aspects of the immune system, IL-6 regulates a variety of homeostatic processes. To interfere with IL-6 dependent diseases, such as various autoimmune diseases or certain cancers like multiple myeloma or hepatocellular carcinoma associated with chronic inflammation, it might be a sensible strategy to target human IL-6 receptor (hIL-6R) presenting cells with aptamers. We therefore have selected and characterized different DNA and RNA aptamers specifically binding IL-6R. These IL-6R aptamers, however, do not interfere with the IL-6 signaling pathway but are internalized with the receptor and thus can serve as vehicles for the delivery of different cargo molecules like therapeutics. We succeeded in the construction of a chlorin e6 derivatized aptamer to be delivered for targeted photodynamic therapy (PDT). Furthermore, we were able to synthesize an aptamer intrinsically comprising the cytostatic 5-Fluoro-2′-deoxy-uridine for targeted chemotherapy. The α6β4 integrin specific DNA aptamer IDA, also selected in our laboratory is internalized, too. All these aptamers can serve as vehicles for targeted drug delivery into cells. We call them charomers—in memory of Charon, the ferryman in Greek mythology, who ferried the deceased into the underworld. PMID:29211023

  13. Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.).

    Park, S H; Strobel, G A

    1994-01-05

    Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).

  14. Beyond the antigen receptor: editing the genome of T-cells for cancer adoptive cellular therapies

    Angharad eLloyd

    2013-08-01

    Full Text Available Recent early-stage clinical trials evaluating the adoptive transfer of patient CD8+ T-cells re-directed with antigen receptors recognising tumours have shown very encouraging results. These reports provide strong support for further development of the therapeutic concept as a curative cancer treatment. In this respect combining the adoptive transfer of tumour-specific T-cells with therapies that increase their anti-tumour capacity is viewed as a promising strategy to improve treatment outcome. The ex-vivo genetic engineering step that underlies T-cell re-direction offers a unique angle to combine antigen receptor delivery with the targeting of cell intrinsic pathways that restrict T-cell effector functions. Recent progress in genome editing technologies such as protein- and RNA-guided endonucleases raise the possibility of disrupting gene expression in T-cells in order to enhance effector functions or to bypass tumour immune suppression. This approach would avoid the systemic administration of compounds that disrupt immune homeostasis, potentially avoiding autoimmune adverse effects, and could improve the efficacy of T-cell based adoptive therapies.

  15. Charomers—Interleukin-6 Receptor Specific Aptamers for Cellular Internalization and Targeted Drug Delivery

    Ulrich Hahn

    2017-12-01

    Full Text Available Interleukin-6 (IL-6 is a key player in inflammation and the main factor for the induction of acute phase protein biosynthesis. Further to its central role in many aspects of the immune system, IL-6 regulates a variety of homeostatic processes. To interfere with IL-6 dependent diseases, such as various autoimmune diseases or certain cancers like multiple myeloma or hepatocellular carcinoma associated with chronic inflammation, it might be a sensible strategy to target human IL-6 receptor (hIL-6R presenting cells with aptamers. We therefore have selected and characterized different DNA and RNA aptamers specifically binding IL-6R. These IL-6R aptamers, however, do not interfere with the IL-6 signaling pathway but are internalized with the receptor and thus can serve as vehicles for the delivery of different cargo molecules like therapeutics. We succeeded in the construction of a chlorin e6 derivatized aptamer to be delivered for targeted photodynamic therapy (PDT. Furthermore, we were able to synthesize an aptamer intrinsically comprising the cytostatic 5-Fluoro-2′-deoxy-uridine for targeted chemotherapy. The α6β4 integrin specific DNA aptamer IDA, also selected in our laboratory is internalized, too. All these aptamers can serve as vehicles for targeted drug delivery into cells. We call them charomers—in memory of Charon, the ferryman in Greek mythology, who ferried the deceased into the underworld.

  16. Engineering CAR-T cells.

    Zhang, Cheng; Liu, Jun; Zhong, Jiang F; Zhang, Xi

    2017-01-01

    Chimeric antigen receptor redirected T cells (CAR-T cells) have achieved inspiring outcomes in patients with B cell malignancies, and are now being investigated in other hematologic malignancies and solid tumors. CAR-T cells are generated by the T cells from patients' or donors' blood. After the T cells are expanded and genetically modified, they are reinfused into the patients. However, many challenges still need to be resolved in order for this technology to gain widespread adoption. In this review, we first discuss the structure and evolution of chimeric antigen receptors. We then report on the tools used for production of CAR-T cells. Finally, we address the challenges posed by CAR-T cells.

  17. Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research.

    Inoo, Kanako; Inagaki, Ryo; Fujiwara, Kento; Sasawatari, Shigemi; Kamigaki, Takashi; Nakagawa, Shinsaku; Okada, Naoki

    2016-01-01

    We previously reported that tumor vessel-redirected T cells, which were genetically engineered with chimeric antigen receptor (CAR) specific for vascular endothelial growth factor receptor 2 (VEGFR2), demonstrated significant antitumor effects in various murine solid tumor models. In the present study, we prepared anti-VEGFR2 CAR-T cells by CAR-coding mRNA electroporation (mRNA-EP) and analyzed their immunological characteristics and functions for use in clinical research. The expression of anti-VEGFR2 CAR on murine and human T cells was detected with approximately 100% efficiency for a few days, after peaking 6-12 hours after mRNA-EP. Triple transfer of murine anti-VEGFR2 CAR-T cells into B16BL6 tumor-bearing mice demonstrated an antitumor effect comparable to that for the single transfer of CAR-T cells engineered with retroviral vector. The mRNA-EP did not cause any damage or defects to human T-cell characteristics, as determined by viability, growth, and phenotypic parameters. Additionally, two kinds of human anti-VEGFR2 CAR-T cells, which expressed different CAR construction, differentiated to effector phase with cytokine secretion and cytotoxic activity in antigen-specific manner. These results indicate that our anti-VEGFR2 CAR-T cells prepared by mRNA-EP have the potential in terms of quality and performance to offer the prospect of safety and efficacy in clinical research as cellular medicine.

  18. Chimeric Antigen Receptors T Cell Therapy in Solid Tumor: Challenges and Clinical Applications

    Hamid R. Mirzaei

    2017-12-01

    Full Text Available Adoptive cellular immunotherapy (ACT employing engineered T lymphocytes expressing chimeric antigen receptors (CARs has demonstrated promising antitumor effects in advanced hematologic cancers, such as relapsed or refractory acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma, supporting the translation of ACT to non-hematological malignancies. Although CAR T cell therapy has made remarkable strides in the treatment of patients with certain hematological cancers, in solid tumors success has been limited likely due to heterogeneous antigen expression, immunosuppressive networks in the tumor microenvironment limiting CAR T cell function and persistence, and suboptimal trafficking to solid tumors. Here, we outline specific approaches to overcome barriers to CAR T cell effectiveness in the context of the tumor microenvironment and offer our perspective on how expanding the use of CAR T cells in solid tumors may require modifications in CAR T cell design. We anticipate these modifications will further expand CAR T cell therapy in clinical practice.

  19. iNR-Drug: predicting the interaction of drugs with nuclear receptors in cellular networking.

    Fan, Yue-Nong; Xiao, Xuan; Min, Jian-Liang; Chou, Kuo-Chen

    2014-03-19

    Nuclear receptors (NRs) are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called "iNR-Drug" was developed. In the predictor, the drug compound concerned was formulated by a 256-D (dimensional) vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM (support vector machine) algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at http://www.jci-bioinfo.cn/iNR-Drug/, which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well.

  20. iNR-Drug: Predicting the Interaction of Drugs with Nuclear Receptors in Cellular Networking

    Yue-Nong Fan

    2014-03-01

    Full Text Available Nuclear receptors (NRs are closely associated with various major diseases such as cancer, diabetes, inflammatory disease, and osteoporosis. Therefore, NRs have become a frequent target for drug development. During the process of developing drugs against these diseases by targeting NRs, we are often facing a problem: Given a NR and chemical compound, can we identify whether they are really in interaction with each other in a cell? To address this problem, a predictor called “iNR-Drug” was developed. In the predictor, the drug compound concerned was formulated by a 256-D (dimensional vector derived from its molecular fingerprint, and the NR by a 500-D vector formed by incorporating its sequential evolution information and physicochemical features into the general form of pseudo amino acid composition, and the prediction engine was operated by the SVM (support vector machine algorithm. Compared with the existing prediction methods in this area, iNR-Drug not only can yield a higher success rate, but is also featured by a user-friendly web-server established at http://www.jci-bioinfo.cn/iNR-Drug/, which is particularly useful for most experimental scientists to obtain their desired data in a timely manner. It is anticipated that the iNR-Drug server may become a useful high throughput tool for both basic research and drug development, and that the current approach may be easily extended to study the interactions of drug with other targets as well.

  1. Car sick.

    Renner, M G

    1988-01-01

    The automobile is currently seen as the most desirable mode of transportation. However, this view needs to be changed since the proliferation of the automobile worldwide is leading to the poisoning of the environment and people. In the US the number of passenger cars grew 51% between 1971-86 and in the noncommunist industrialized community that figure is 71%. The gasoline and diesel fuel used to power the overwhelming majority of cars creates a variety of problems. The pollution is estimated to have a hidden cost of US $.80/gallon. Others estimate that the pollution causes 30,000 premature deaths annually just in the US. 75% of the carbon monoxide (CO), 48% of nitrogen oxides (NO2), 13% of particulates (P), and 3% of sulfur (S) emissions come from cars in the countries of the Organization for Economic Cooperation and Development (OECD), which includes the US, Canada, Western Europe, Japan, Australia, and New Zealand. 17% of all worldwide carbon dioxide (CO2) emission comes from the production and use of fossil fuels for cars. The single biggest problem associated with cars is the photochemical smog they create in urban areas. In 1986 75 million Americans lived in areas that failed to meet national air quality standards for CO, P, and ozone (03). The only area of major improvement has been the removal of lead from gasoline. It was known to cause problems from the beginning of its use in the 1920s, but remained for 50 years because of auto and oil company pressure. Ground 03 is estimated by the US government to cost US $4 billion in annual losses, just for corn, wheat, soybeans, and peanuts. Acid rain is the other major problem associated with cars, and its damage is estimated at US $5 billion annually. Both these problems are shortterm, their effects occur immediately; the longterm disadvantage is the build up of CO2 and its contribution to the greenhouse effect. While the US is at the forefront of regulation and many other countries are modeling their emission

  2. Clean cars

    Piffaretti, M.

    2008-07-01

    This well-illustrated presentation made at the Swiss 2008 research conference on traffic by the Protoscar company takes a look at research, design, engineering and communication topics in the area of 'clean cars'. The present situation with electrically driven and hybrid-drive cars is reviewed and the chances and problems of the present-day vehicles are examined. New developments and a number of vehicles that should be on the market in the period from 2012 to 2015 are presented. Also, 'clean' specialist vehicles such as trucks and buses are reviewed. Battery systems and associated problems and new developments are looked at. The promotion scheme in Mendrisio, Switzerland is reviewed. Bottom-up and top-down approaches are discussed and future market developments are looked at, as are promotional activities in various countries.

  3. Efficacy and safety of chimeric antigen receptor T-cell (CAR-T) therapy in patients with haematological and solid malignancies: protocol for a systematic review and meta-analysis.

    Grigor, Emma J M; Fergusson, Dean A; Haggar, Fatima; Kekre, Natasha; Atkins, Harold; Shorr, Risa; Holt, Robert A; Hutton, Brian; Ramsay, Tim; Seftel, Matthew; Jonker, Derek; Daugaard, Mads; Thavorn, Kednapa; Presseau, Justin; Lalu, Manoj M

    2017-12-29

    Patients with relapsed or refractory malignancies have a poor prognosis. Immunotherapy with chimeric antigen receptor T (CAR-T) cells redirects a patient's immune cells against the tumour antigen. CAR-T cell therapy has demonstrated promise in treating patients with several haematological malignancies, including acute B-cell lymphoblastic leukaemia and B-cell lymphomas. CAR-T cell therapy for patients with other solid tumours is also being tested. Safety is an important consideration in CAR-T cell therapy given the potential for serious adverse events, including death. Previous reviews on CAR-T cell therapy have been limited in scope and methodology. Herein, we present a protocol for a systematic review to identify CAR-T cell interventional studies and examine the safety and efficacy of this therapy in patients with haematology malignancies and solid tumours. We will search MEDLINE, including In-Process and Epub Ahead of Print, EMBASE and the Cochrane Central Register of Controlled Trials from 1946 to 22 February 2017. Studies will be screened by title, abstract and full text independently and in duplicate. Studies that report administering CAR-T cells of any chimeric antigen receptor construct targeting antigens in patients with haematological malignancies and solid tumours will be eligible for inclusion. Outcomes to be extracted will include complete response rate (primary outcome), overall response rate, overall survival, relapse and adverse events. A meta-analysis will be performed to synthesise the prevalence of outcomes reported as proportions with 95% CIs. The potential for bias within included studies will be assessed using a modified Institute of Health Economics tool. Heterogeneity of effect sizes will be determined using the Cochrane I 2 statistic. The review findings will be submitted for peer-reviewed journal publication and presented at relevant conferences and scientific meetings to promote knowledge transfer. CRD42017075331. © Article author(s) (or

  4. Nanobody Based Dual Specific CARs

    Stijn De Munter

    2018-01-01

    Full Text Available Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR. High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

  5. Nanobody Based Dual Specific CARs.

    De Munter, Stijn; Ingels, Joline; Goetgeluk, Glenn; Bonte, Sarah; Pille, Melissa; Weening, Karin; Kerre, Tessa; Abken, Hinrich; Vandekerckhove, Bart

    2018-01-30

    Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

  6. Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies

    Isabell Lang

    2018-04-01

    Full Text Available Progranulin (PGRN is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1 and TNFR2 and inhibition of tumor necrosis factor (TNF-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold. Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co

  7. Clinical manufacturing of CAR T cells: foundation of a promising therapy

    Wang, Xiuyan; Rivière, Isabelle

    2016-01-01

    The treatment of cancer patients with autologous T cells expressing a chimeric antigen receptor (CAR) is one of the most promising adoptive cellular therapy approaches. Reproducible manufacturing of high-quality, clinical-grade CAR-T cell products is a prerequisite for the wide application of this technology. Product quality needs to be built-in within every step of the manufacturing process. We summarize herein the requirements and logistics to be considered, as well as the state of the art manufacturing platforms available. CAR-T cell therapy may be on the verge of becoming standard of care for a few clinical indications. Yet, many challenges pertaining to manufacturing standardization and product characterization remain to be overcome in order to achieve broad usage and eventual commercialization of this therapeutic modality. PMID:27347557

  8. Clinical manufacturing of CAR T cells: foundation of a promising therapy

    Xiuyan Wang

    2016-01-01

    Full Text Available The treatment of cancer patients with autologous T cells expressing a chimeric antigen receptor (CAR is one of the most promising adoptive cellular therapy approaches. Reproducible manufacturing of high-quality, clinical-grade CAR-T cell products is a prerequisite for the wide application of this technology. Product quality needs to be built-in within every step of the manufacturing process. We summarize herein the requirements and logistics to be considered, as well as the state of the art manufacturing platforms available. CAR-T cell therapy may be on the verge of becoming standard of care for a few clinical indications. Yet, many challenges pertaining to manufacturing standardization and product characterization remain to be overcome in order to achieve broad usage and eventual commercialization of this therapeutic modality.

  9. [Establishment and application of a Vero cell line stably expressing raccoon dog SLAM, the cellular receptor of canine distemper virus].

    Zhao, Jianjun; Yan, Ruxun; Zhang, Hailing; Zhang, Lei; Hu, Bo; Bai, Xue; Shao, Xiqun; Chai, Xiuli; Yan, Xijun; Wu, Wei

    2012-12-04

    The signaling lymphocyte activation molecule (SLAM, also known as CD150), is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM (rSLAM) to efficiently isolate CDV from pathological samples. A eukaryotic expression plasmid, pIRES2-EGFP-rSLAMhis, containing rSLAM gene fused with six histidine-coding sequence, EGFP gene, and neomycin resistance gene was constructed. After transfection with the plasmid, a stable cell line, Vero-rSLAM, was screened from Vero cells with the identification of EGFP reporter and G418 resistance. Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation. Foxes and raccoon dogs were inoculated subcutaneously LN (10)fl strain with 4 x 10(2.39)TCID50 dose to evaluate pathogenicity of CDV isolations. The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay. Three CDV strains were isolated successfully in Vero-rSLAM cells 36 -48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper. By contrast, no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation. Infected foxes and raccoon dogs with LN(10)f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge. Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.

  10. Hepatitis A virus cellular receptor 2 (HAVCR2) is decreased with viral infection and regulates pro-labour mediators OA.

    Liong, Stella; Lim, Ratana; Barker, Gillian; Lappas, Martha

    2017-07-01

    Intrauterine infection caused by viral infection has been implicated to contribute to preterm birth. Hepatitis A virus cellular receptor 2 (HAVCR2) regulates inflammation in non-gestational tissues in response to viral infection. The aims of this study were to determine the effect of: (i) viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) on HAVCR2 expression; and (ii) HAVCR2 silencing by siRNA (siHAVCR2) in primary amnion and myometrial cells on poly(I:C)-induced inflammation. In human foetal membranes and myometrium, HAVCR2 mRNA and protein expression was decreased when exposed to poly(I:C). Treatment of primary amnion and myometrial cells with poly(I:C) significantly increased the expression and release of pro-inflammatory cytokines TNF, IL1A, IL1B and IL6; the expression of chemokines CXCL8 and CCL2; the expression and secretion of adhesion molecules ICAM1 and VCAM1; and PTGS2 and PTGFR mRNA expression and the release of prostaglandin PGF 2α . This increase was significantly augmented in cells transfected with siHAVCR2. Furthermore, mRNA expression of anti-inflammatory cytokines IL4 and IL10 was significantly decreased. Collectively, our data suggest that HAVCR2 regulates cytokines, chemokines, prostaglandins and cell adhesion molecules in the presence of viral infection. This suggests a potential for HAVCR2 activators as therapeutics for the management of preterm birth associated with viral infections. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans.

    Josephson, Anna; Trifunovski, Alexandra; Widmer, Hans Ruedi; Widenfalk, Johan; Olson, Lars; Spenger, Christian

    2002-11-18

    Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. Copyright 2002 Wiley-Liss, Inc.

  12. CAR-T cell therapy in ovarian cancer: from the bench to the bedside.

    Zhu, Xinxin; Cai, Han; Zhao, Ling; Ning, Li; Lang, Jinghe

    2017-09-08

    Ovarian cancer (OC) is the most lethal gynecological malignancy and is responsible for most gynecological cancer deaths. Apart from conventional surgery, chemotherapy, and radiotherapy, chimeric antigen receptor-modified T (CAR-T) cells as a representative of adoptive cellular immunotherapy have received considerable attention in the research field of cancer treatment. CARs combine antigen specificity and T-cell-activating properties in a single fusion molecule. Several preclinical experiments and clinical trials have confirmed that adoptive cell immunotherapy using typical CAR-engineered T cells for OC is a promising treatment approach with striking clinical efficacy; moreover, the emerging CAR-Ts targeting various antigens also exert great potential. However, such therapies have side effects and toxicities, such as cytokine-associated and "on-target, off-tumor" toxicities. In this review, we systematically detail and highlight the present knowledge of CAR-Ts including the constructions, vectors, clinical applications, development challenges, and solutions of CAR-T-cell therapy for OC. We hope to provide new insight into OC treatment for the future.

  13. CD47-CAR-T Cells Effectively Kill Target Cancer Cells and Block Pancreatic Tumor Growth.

    Golubovskaya, Vita; Berahovich, Robert; Zhou, Hua; Xu, Shirley; Harto, Hizkia; Li, Le; Chao, Cheng-Chi; Mao, Mike Ming; Wu, Lijun

    2017-10-21

    CD47 is a glycoprotein of the immunoglobulin superfamily that is often overexpressed in different types of hematological and solid cancer tumors and plays important role in blocking phagocytosis, increased tumor survival, metastasis and angiogenesis. In the present report, we designed CAR (chimeric antigen receptor)-T cells that bind CD47 antigen. We used ScFv (single chain variable fragment) from mouse CD47 antibody to generate CD47-CAR-T cells for targeting different cancer cell lines. CD47-CAR-T cells effectively killed ovarian, pancreatic and other cancer cells and produced high level of cytokines that correlated with expression of CD47 antigen. In addition, CD47-CAR-T cells significantly blocked BxPC3 pancreatic xenograft tumor growth after intratumoral injection into NSG mice. Moreover, we humanized mouse CD47 ScFv and showed that it effectively bound CD47 antigen. The humanized CD47-CAR-T cells also specifically killed ovarian, pancreatic, and cervical cancer cell lines and produced IL-2 that correlated with expression of CD47. Thus, CD47-CAR-T cells can be used as a novel cellular therapeutic agent for treating different types of cancer.

  14. Relationship between peroxisome proliferator-activated receptor alpha activity and cellular concentration of 14 perfluoroalkyl substances in HepG2 cells.

    Rosenmai, Anna Kjerstine; Ahrens, Lutz; le Godec, Théo; Lundqvist, Johan; Oskarsson, Agneta

    2018-02-01

    Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target for perfluoroalkyl substances (PFASs). Little is known about the cellular uptake of PFASs and how it affects the PPARα activity. We investigated the relationship between PPARα activity and cellular concentration in HepG2 cells of 14 PFASs, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates and perfluorooctane sulfonamide (FOSA). Cellular concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry and PPARα activity was determined in transiently transfected cells by reporter gene assay. Cellular uptake of the PFASs was low (0.04-4.1%) with absolute cellular concentrations in the range 4-2500 ng mg -1 protein. Cellular concentration of PFCAs increased with perfluorocarbon chain length up to perfluorododecanoate. PPARα activity of PFCAs increased with chain length up to perfluorooctanoate. The maximum induction of PPARα activity was similar for short-chain (perfluorobutanoate and perfluoropentanoate) and long-chain PFCAs (perfluorododecanoate and perfluorotetradecanoate) (approximately twofold). However, PPARα activities were induced at lower cellular concentrations for the short-chain homologs compared to the long-chain homologs. Perfluorohexanoate, perfluoroheptanoate, perfluorooctanoate, perfluorononanoate (PFNA) and perfluorodecanoate induced PPARα activities >2.5-fold compared to controls. The concentration-response relationships were positive for all the tested compounds, except perfluorooctane sulfonate PFOS and FOSA, and were compound-specific, as demonstrated by differences in the estimated slopes. The relationships were steeper for PFCAs with chain lengths up to and including PFNA than for the other studied PFASs. To our knowledge, this is the first report establishing relationships between PPARα activity and cellular concentration of a broad range of PFASs. Copyright © 2017 John Wiley & Sons, Ltd.

  15. Activated α2 -Macroglobulin Induces Mesenchymal Cellular Migration Of Raw264.7 Cells Through Low-Density Lipoprotein Receptor-Related Protein 1.

    Ferrer, Darío G; Dato, Virginia Actis; Fincati, Javier R Jaldín; Lorenc, Valeria E; Sánchez, María C; Chiabrando, Gustavo A

    2017-07-01

    Distinct modes of cell migration contribute to diverse types of cell movements. The mesenchymal mode is characterized by a multistep cycle of membrane protrusion, the formation of focal adhesion, and the stabilization at the leading edge associated with the degradation of extracellular matrix (ECM) components and with regulated extracellular proteolysis. Both α 2 -Macroglobulin (α 2 M) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1), play important roles in inflammatory processes, by controlling the extracellular activity of several proteases. The binding of the active form of α 2 M (α 2 M*) to LRP1 can also activate different signaling pathways in macrophages, thus inducing extracellular matrix metalloproteinase-9 (MMP-9) activation and cellular proliferation. In the present study, we investigated whether the α 2 M*/LRP1 interaction induces cellular migration of the macrophage-derived cell line, Raw264.7. By using the wound-scratch migration assay and confocal microscopy, we demonstrate that α 2 M* induces LRP1-mediated mesenchymal cellular migration. This migration exhibits the production of enlarged cellular protrusions, MT1-MMP distribution to these leading edge protrusions, actin polymerization, focal adhesion formation, and increased intracellular LRP1/β1-integrin colocalization. Moreover, the presence of calphostin-C blocked the α 2 M*-stimulated cellular protrusions, suggesting that the PKC activation is involved in the cellular motility of Raw264.7 cells. These findings could constitute a therapeutic target for inflammatory processes with deleterious consequences for human health, such as rheumatoid arthritis, atherosclerosis and cancer. J. Cell. Biochem. 118: 1810-1818, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Steroidogenesis and early response gene expression in MA-10 Leydig tumor cells following heterologous receptor down-regulation and cellular desensitization

    Tsuey-Ming Chen

    2016-03-01

    Full Text Available The Leydig tumor cell line, MA-10, expresses the luteinizing hormone receptor, a G protein-coupled receptor that, when activated with luteinizing hormone or chorionic gonadotropin (CG, stimulates cAMP production and subsequent steroidogenesis, notably progesterone. These cells also respond to epidermal growth factor (EGF and phorbol esters with increased steroid biosynthesis. In order to probe the intracellular pathways along with heterologous receptor down-regulation and cellular desensitization, cells were preincubated with EGF or phorbol esters and then challenged with CG, EGF, dibutryl-cyclic AMP, and a phorbol ester. Relative receptor numbers, steroid biosynthesis, and expression of the early response genes, JUNB and c-FOS, were measured. It was found that in all cases but one receptor down-regulation and decreased progesterone production were closely coupled under the conditions used; the exception involved preincubation of the cells with EGF followed by addition of CG where the CG-mediated stimulation of steroidogenesis was considerably lower than the level of receptor down-regulation. In a number of instances JUNB and c-FOS expression paralleled the decreases in receptor number and progesterone production, while in some cases these early response genes were affected little if at all by the changes in receptor number. This finding may indicate that even low levels of activated signaling kinases, e.g. protein kinase A, protein kinase C, or receptor tyrosine kinase, may suffice to yield good expression of JUNB and c-FOS, or it may suggest alternative pathways for regulating expression of these two early response genes.

  17. Expression of Human CAR Splicing Variants in BAC-Transgenic Mice

    Zhang, Yu-Kun Jennifer; Lu, Hong; Klaassen, Curtis D.

    2012-01-01

    The nuclear receptor constitutive androstane receptor (CAR) is a key regulator for drug metabolism in liver. Human CAR (hCAR) transcripts are subjected to alternative splicing. Some hCAR splicing variants (SVs) have been shown to encode functional proteins by reporter assays. However, in vivo research on the activity of these hCAR SVs has been impeded by the absence of a valid model. This study engineered an hCAR-BAC-transgenic (hCAR-TG) mouse model by integrating the 8.5-kbp hCAR gene as wel...

  18. CAR STICKERS

    Access and Control Service

    2004-01-01

    Following to the operational circular No2 title III. Conditions of access, paragraph 21 . Except in the case of exemptions authorized by the Director-General, all drivers must facilitate the identification of their vehicle. For CERN car stickers to be valid in 2004, they must have the numbers 04 printed on them. As of Monday, March 15th, the security agents on duty at the various access points will have no alternative but to refuse entry to vehicles which do not have a valid sticker. Anyone in this situation is requested to follow the regularization procedure either by logging on to the web site, or by going in person to the registration service in bldg. 55, first floor, between 07h30 et 16h30, Monday through Friday. Access and Control Service - FM Group, TS Department

  19. 5-HT2C Receptor Desensitization Moderates Anxiety in 5-HTT Deficient Mice: From Behavioral to Cellular Evidence

    Martin, Cédric BP; Martin, Vincent S.; Trigo, José M.; Chevarin, Caroline; Maldonado, Rafael; Fink, Latham H.; Cunningham, Kathryn A.; Hamon, Michel; Lanfumey, Laurence

    2015-01-01

    Background: Desensitization and blockade of 5-HT2C receptors (5-HT2CR) have long been thought to be central in the therapeutic action of antidepressant drugs. However, besides behavioral pharmacology studies, there is little in vivo data documenting antidepressant-induced 5-HT2CR desensitization in specific brain areas. Methods: Mice lacking the 5-HT reuptake carrier (5-HTT-/-) were used to model the consequences of chronic 5-HT reuptake inhibition with antidepressant drugs. The effect of this mutation on 5-HT2CR was evaluated at the behavioral (social interaction, novelty-suppressed feeding, and 5-HT2CR–induced hypolocomotion tests), the neurochemical, and the cellular (RT-qPCR, mRNA editing, and c-fos–induced expression) levels. Results: Although 5-HTT-/- mice had an anxiogenic profile in the novelty-suppressed feeding test, they displayed less 5-HT2CR–mediated anxiety in response to the agonist m-chlorophenylpiperazine in the social interaction test. In addition, 5-HT2CR–mediated inhibition of a stress-induced increase in 5-HT turnover, measured in various brain areas, was markedly reduced in 5-HTT-/- mutants. These indices of tolerance to 5-HT2CR stimulation were associated neither with altered levels of 5-HT2CR protein and mRNA nor with changes in pre-mRNA editing in the frontal cortex. However, basal c-fos mRNA production in cells expressing 5-HT2CR was higher in 5-HTT-/- mutants, suggesting an altered basal activity of these cells following sustained 5-HT reuptake carrier inactivation. Furthermore, the increased c-fos mRNA expression in 5-HT2CR–like immune-positive cortical cells observed in wild-type mice treated acutely with the 5-HT2CR agonist RO-60,0175 was absent in 5-HTT-/- mutants. Conclusions: Such blunted responsiveness of the 5-HT2CR system, observed at the cell signaling level, probably contributes to the moderation of the anxiety phenotype in 5-HTT-/- mice. PMID:25522398

  20. Driving CAR T-cells forward

    Jackson, Hollie J.; Rafiq, Sarwish; Brentjens, Renier J.

    2017-01-01

    The engineered expression of chimeric antigen receptors (CARs) on the surface of T cells enables the redirection of T-cell specificity. Early clinical trials using CAR T cells for the treatment of patients with cancer showed modest results, but the impressive outcomes of several trials of CD19-targeted CAR T cells in the treatment of patients with B-cell malignancies have generated an increased enthusiasm for this approach. Important lessons have been derived from clinical trials of CD19-specific CAR T cells, and ongoing clinical trials are testing CAR designs directed at novel targets involved in haematological and solid malignancies. In this Review, we discuss these trials and present strategies that can increase the antitumour efficacy and safety of CAR T-cell therapy. Given the fast-moving nature of this field, we only discuss studies with direct translational application currently or soon-to-be tested in the clinical setting. PMID:27000958

  1. Car Seat Safety

    ... Staying Safe Videos for Educators Search English Español Car Seat Safety KidsHealth / For Parents / Car Seat Safety ... certified child passenger safety technician.) Guidelines for Choosing Car Seats Choose a seat with a label that ...

  2. Jet Car Track Site

    Federal Laboratory Consortium — Located in Lakehurst, New Jersey, the Jet Car Track Site supports jet cars with J57 engines and has a maximum jet car thrust of 42,000 pounds with a maximum speed of...

  3. Clinical trials of CAR-T cells in China

    Bingshan Liu; Yongping Song; Delong Liu

    2017-01-01

    Abstract Novel immunotherapeutic agents targeting tumor-site microenvironment are revolutionizing cancer therapy. Chimeric antigen receptor (CAR)-engineered T cells are widely studied for cancer immunotherapy. CD19-specific CAR-T cells, tisagenlecleucel, have been recently approved for clinical application. Ongoing clinical trials are testing CAR designs directed at novel targets involved in hematological and solid malignancies. In addition to trials of single-target CAR-T cells, simultaneous...

  4. Car Club

    Automobile Club

    2012-01-01

    The Car Club wishes all its members Good road and Happy New Year 2012. It is time to think about renewing your subscription for this year, at a cost of 50 CHF, unchanged since several years. For those of you who are regular users of our equipment and who know all the advantages that the club is in a position to offer, it seems pointless to going to more details, as we are sure that many of you have made use of them and are satisfied. Therefore don’t forget to fill in the payment slip to continue to be a part of our large family. We remind you that everyone who works on the CERN site can be members of our club, this includes industrial support personnel and the personnel of companies which have a contract with CERN. If you are not yet a member, come and visit us! We will be happy to welcome you and show you the installations, alternatively you can visit our web site: http://club-acc.web.cern.ch/club-acc/ The use of the club’s installations is strictly reserved for members. Pour t...

  5. Design and cellular kinetics of dansyl-labeled CADA derivatives with anti-HIV and CD4 receptor down-modulating activity.

    Vermeire, Kurt; Lisco, Andrea; Grivel, Jean-Charles; Scarbrough, Emily; Dey, Kaka; Duffy, Noah; Margolis, Leonid; Bell, Thomas W; Schols, Dominique

    2007-08-15

    A new class of anti-retrovirals, cyclotriazadisulfonamide (CADA) and its derivatives, specifically down-regulate CD4, the main receptor of HIV, and prevent HIV infection in vitro. In this work, several CADA derivatives, chemically labeled with a fluorescent dansyl group, were evaluated for their biological features and cellular uptake kinetics. We identified a derivative KKD-016 with antiviral and CD4 down-modulating capabilities similar to those of the parental compound CADA. By using flow cytometry, we demonstrated that the dose-dependent cellular uptake of this derivative correlated with CD4 down-modulation. The uptake and activity of the dansyl-labeled compounds were not dependent on the level of expression of CD4 at the cell surface. Removal of the CADA compounds from the cell culture medium resulted in their release from the cells followed by a complete restoration of CD4 expression. The inability of several fluorescent CADA derivatives to down-modulate CD4 was not associated with their lower cellular uptake and was not reversed by facilitating their cell penetration by a surfactant. These results prove the successful integration of the dansyl fluorophore into the chemical structure of a CD4 down-modulating anti-HIV compound, and show the feasibility of tracking a receptor and its down-modulator simultaneously. These fluorescent CADA analogs with reversible CD4 down-regulating potency can now be applied in further studies on receptor modulation, and in the exploration of their potentials as preventive and therapeutic anti-HIV drugs.

  6. T-cells fighting B-cell lymphoproliferative malignancies: the emerging field of CD19 CAR T-cell therapy

    Heijink, D. M.; Kater, A. P.; Hazenberg, M. D.; Hagenbeek, A.; Kersten, M. J.

    2016-01-01

    CAR T-cells are autologous T-cells transduced with a chimeric antigen receptor (CAR). The CAR contains an antigen recognition part (originating from an antibody), a T-cell receptor transmembrane and cytoplasmic signalling part, and one or more co-stimulatory domains. While CAR T-cells can be

  7. Co-ordinate action of bacterial adhesins and human carcinoembryonic antigen receptors in enhanced cellular invasion by capsulate serum resistant Neisseria meningitidis.

    Rowe, Helen A; Griffiths, Natalie J; Hill, Darryl J; Virji, Mumtaz

    2007-01-01

    Neisseria meningitidis (Nm) is a human specific opportunistic pathogen that occasionally penetrates mucosal barriers via the action of adhesins and invasins and evades host immune mechanisms during further dissemination via capsule expression. From in vitro studies, the primary adhesion of capsulate bacteria is believed to be mediated by polymeric pili, followed by invasion via outer membrane adhesins such as Opa proteins. As the latter requires the surface capsule to be down-modulated, invading bacteria would be serum sensitive and thus avirulent. However, there is recent evidence that capsulate bacteria may interact via Opa proteins when host cells express high levels of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), their target receptors. Such a situation may arise following increased circulation of inflammatory cytokines that upregulate certain adhesion molecules on host cells. In this study, using a tetracycline controlled expression system, we have developed cell lines with inducible CEACAM expression to mimic post-inflammation state of target tissues and analysed the interplay between the three surface components capsule, pili and Opa proteins in cellular interactions. With two distinct cell lines, not only the level but also the rate of adhesion of capsulate Opa-expressing Nm increased concurrently with CEACAM density. Moreover, when threshold levels of receptor were reached, cellular invasion ensued in an Opa-dependent manner. In studies with cell lines intrinsically expressing pilus receptors, notable synergism in cellular interactions between pili and Opa of several meningococcal strains was observed and was independent of capsule type. A number of internalized bacteria were shown to express capsule and when directly isolated from host cells, these bacteria were as serum resistant as the inoculated phenotype. Furthermore, we observed that agents that block Opa-CEACAM binding substantially reduced cellular invasion, while maintaining

  8. The Influence of Receptor-Mediated Interactions on Reaction-Diffusion Mechanisms of Cellular Self-organisation

    Klika, Vá clav; Baker, Ruth E.; Headon, Denis; Gaffney, Eamonn A.

    2011-01-01

    formation, motivating numerous theoretical and experimental studies, though its verification at the molecular level in biological systems has remained elusive. In this work, we consider the influence of receptor-mediated dynamics within the framework

  9. ERK controls epithelial cell death receptor signalling and cellular FLICE-like inhibitory protein (c-FLIP) in ulcerative colitis

    Seidelin, Jakob Benedict; Coskun, Mehmet; Vainer, Ben

    2013-01-01

    Intestinal epithelial cell (IEC) death signalling through the Fas receptor is impaired in active ulcerative colitis (UC). This is possibly due to the activation of cytoprotective pathways resulting in limitation of the tissue injury secondary to inflammation. We hypothesized that inflammatory...... the resistance to receptor mediated epithelial apoptosis in active UC. Oncogenic c-FLIP could promote propagation of DNA-damaged IECs and contribute to cancer development in UC....

  10. Loss of Progesterone Receptor-Mediated Actions Induce Preterm Cellular and Structural Remodeling of the Cervix and Premature Birth

    Yellon, Steven M.; Dobyns, Abigail E.; Beck, Hailey L.; Kurtzman, James T.; Garfield, Robert E.; Kirby, Michael A.

    2013-01-01

    A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone), or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term. PMID:24339918

  11. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-01-01

    Highlights: → A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. → The second zinc finger of LIM domain 1 of testin is critical for interaction. → Testin bound to a region of the receptor tail important for cell signalling. → Testin and receptor interaction was confirmed in mammalian (HEK293) cells. → Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  12. Analysis of Epidermal Growth Factor Receptor Related Gene Expression Changes in a Cellular and Animal Model of Parkinson’s Disease

    In-Su Kim

    2017-02-01

    Full Text Available We employed transcriptome analysis of epidermal growth factor receptor related gene expression changes in cellular and animal models of Parkinson’s disease (PD. We used a well-known Parkinsonian toxin 1-methyl-4-phenylpyridine (MPP+ to induce neuronal apoptosis in the human neuroblastoma SH-SY5Y cell line. The MPP+-treatment of SH-SY5Y cells was capable of inducing neuro-apoptosis, but it remains unclear what kinds of transcriptional genes are affected by MPP+ toxicity. Therefore the pathways that were significantly perturbed in MPP+ treated human neuroblastoma SH-SY5Y cells were identified based on genome-wide gene expression data at two time points (24 and 48 h. We found that the Epidermal Growth Factor Receptor (EGFR pathway-related genes showed significantly differential expression at all time points. The EGFR pathway has been linked to diverse cellular events such as proliferation, differentiation, and apoptosis. Further, to evaluate the functional significance of the altered EGFR related gene expression observed in MPP+-treated SH-SY5Y cells, the EGFR related GJB2 (Cx26 gene expression was analyzed in an MPP+-intoxicated animal PD model. Our findings identify that the EGFR signaling pathway and its related genes, such as Cx26, might play a significant role in dopaminergic (DAergic neuronal cell death during the process of neuro-apoptosis and therefore can be focused on as potential targets for therapeutic intervention.

  13. Time-course comparison of xenobiotic activators of CAR and PPARα in mouse liver

    Ross, Pamela K.; Woods, Courtney G.; Bradford, Blair U.; Kosyk, Oksana; Gatti, Daniel M.; Cunningham, Michael L.; Rusyn, Ivan

    2009-01-01

    Constitutive androstane receptor (CAR) and peroxisome proliferator activated receptor (PPAR)α are transcription factors known to be primary mediators of liver effects, including carcinogenesis, by phenobarbital-like compounds and peroxisome proliferators, respectively, in rodents. Many similarities exist in the phenotypes elicited by these two classes of agents in rodent liver, and we hypothesized that the initial transcriptional responses to the xenobiotic activators of CAR and PPARα will exhibit distinct patterns, but at later time-points these biological pathways will converge. In order to capture the global transcriptional changes that result from activation of these nuclear receptors over a time-course in the mouse liver, microarray technology was used. First, differences in basal expression of liver genes between C57Bl/6J wild-type and Car-null mice were examined and 14 significantly differentially expressed genes were identified. Next, mice were treated with phenobarbital (100 mg/kg by gavage for 24 h, or 0.085% w/w diet for 7 or 28 days), and liver gene expression changes with regards to both time and treatment were identified. While several pathways related to cellular proliferation and metabolism were affected by phenobarbital in wild-type mice, no significant changes in gene expression were found over time in the Car-nulls. Next, we determined commonalities and differences in the temporal response to phenobarbital and WY-14,643, a prototypical activator of PPAR α. Gene expression signatures from livers of wild-type mice C57Bl6/J mice treated with PB or WY-14,643 were compared. Similar pathways were affected by both compounds; however, considerable time-related differences were present. This study establishes common gene expression fingerprints of exposure to activators of CAR and PPARα in rodent liver and demonstrates that despite similar phenotypic changes, molecular pathways differ between classes of chemical carcinogens

  14. Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors.

    Luisier, Raphaëlle; Lempiäinen, Harri; Scherbichler, Nina; Braeuning, Albert; Geissler, Miriam; Dubost, Valerie; Müller, Arne; Scheer, Nico; Chibout, Salah-Dine; Hara, Hisanori; Picard, Frank; Theil, Diethilde; Couttet, Philippe; Vitobello, Antonio; Grenet, Olivier; Grasl-Kraupp, Bettina; Ellinger-Ziegelbauer, Heidrun; Thomson, John P; Meehan, Richard R; Elcombe, Clifford R; Henderson, Colin J; Wolf, C Roland; Schwarz, Michael; Moulin, Pierre; Terranova, Rémi; Moggs, Jonathan G

    2014-06-01

    The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

  15. CAR models: next-generation CAR modifications for enhanced T-cell function

    Daniel Abate-Daga

    2016-01-01

    Full Text Available T cells genetically targeted with a chimeric antigen receptor (CAR to B-cell malignancies have demonstrated tremendous clinical outcomes. With the proof in principle for CAR T cells as a therapy for B-cell malignancies being established, current and future research is being focused on adapting CAR technology to other cancers, as well as enhancing its efficacy and/or safety. The modular nature of the CAR, extracellular antigen-binding domain fused to a transmembrane domain and intracellular T-cell signaling domains, allows for optimization by replacement of the various components. These modifications are creating a whole new class of therapeutic CARs. In this review, we discuss the recent major advances in CAR design and how these modifications will impact its clinical application.

  16. Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.

    Marlène Dreux

    2009-02-01

    Full Text Available HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI, a major receptor of high-density lipoprotein (HDL, the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

  17. Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity

    Campana Dario

    2010-10-01

    Full Text Available Abstract Background The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i the small number of NK cells in peripheral blood, (ii the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii the need to activate the NK cells in order to induce NK cell mediated killing and (iv the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii their ability to kill allogeneic and autologous tumor targets by direct cytotoxitiy and by antibody-mediated cellular cytotoxicity and (iii defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. Methods Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. Results Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. Conclusion These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical

  18. Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.

    Devost, Dominic; Sleno, Rory; Pétrin, Darlaine; Zhang, Alice; Shinjo, Yuji; Okde, Rakan; Aoki, Junken; Inoue, Asuka; Hébert, Terence E

    2017-03-31

    Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gα q , Gα 11 , Gα 12 , and Gα 13 or β-arrestins, we showed that Gα q and Gα 11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

    Kanako Inoo

    2016-01-01

    Full Text Available We previously reported that tumor vessel-redirected T cells, which were genetically engineered with chimeric antigen receptor (CAR specific for vascular endothelial growth factor receptor 2 (VEGFR2, demonstrated significant antitumor effects in various murine solid tumor models. In the present study, we prepared anti-VEGFR2 CAR-T cells by CAR-coding mRNA electroporation (mRNA-EP and analyzed their immunological characteristics and functions for use in clinical research. The expression of anti-VEGFR2 CAR on murine and human T cells was detected with approximately 100% efficiency for a few days, after peaking 6–12 hours after mRNA-EP. Triple transfer of murine anti-VEGFR2 CAR-T cells into B16BL6 tumor-bearing mice demonstrated an antitumor effect comparable to that for the single transfer of CAR-T cells engineered with retroviral vector. The mRNA-EP did not cause any damage or defects to human T-cell characteristics, as determined by viability, growth, and phenotypic parameters. Additionally, two kinds of human anti-VEGFR2 CAR-T cells, which expressed different CAR construction, differentiated to effector phase with cytokine secretion and cytotoxic activity in antigen-specific manner. These results indicate that our anti-VEGFR2 CAR-T cells prepared by mRNA-EP have the potential in terms of quality and performance to offer the prospect of safety and efficacy in clinical research as cellular medicine.

  20. The Influence of Receptor-Mediated Interactions on Reaction-Diffusion Mechanisms of Cellular Self-organisation

    Klika, Václav; Baker, R. E.; Headon, D.; Gaffney, E. A.

    2012-01-01

    Roč. 74, č. 4 (2012), s. 935-957 ISSN 0092-8240 Institutional research plan: CEZ:AV0Z20760514 Keywords : reaction-diffusion * receptor-mediated patterning * turing models Subject RIV: BO - Biophysics Impact factor: 2.023, year: 2012 http://www.springerlink.com/content/9713544x6871w4n6/?MUD=MP

  1. Cellular and behavioural profile of the novel, selective neurokinin1 receptor antagonist, vestipitant: a comparison to other agents.

    Brocco, Mauricette; Dekeyne, Anne; Mannoury la Cour, Clotilde; Touzard, Manuelle; Girardon, Sylvie; Veiga, Sylvie; de Nanteuil, Guillaume; deJong, Trynke R; Olivier, Berend; Millan, Mark J

    2008-10-01

    This study characterized the novel neurokinin (NK)(1) antagonist, vestipitant, under clinical evaluation for treatment of anxiety and depression. Vestipitant possessed high affinity for human NK(1) receptors (pK(i), 9.4), and potently blocked Substance P-mediated phosphorylation of Extracellular-Regulated-Kinase. In vivo, it occupied central NK(1) receptors in gerbils (Inhibitory Dose(50), 0.11 mg/kg). At similar doses, it abrogated nociception elicited by formalin in gerbils, and blocked foot-tapping and locomotion elicited by the NK(1) agonist, GR73632, in gerbils and guinea pigs, respectively. Further, vestipitant attenuated fear-induced foot-tapping in gerbils, separation-induced distress-vocalizations in guinea pigs, marble-burying behaviour in mice, and displayed anxiolytic actions in Vogel conflict and fear-induced ultrasonic vocalization procedures in rats. These actions were mimicked by CP99,994, L733,060 and GR205,171 which acted stereoselectively vs its less active isomer, GR226,206. In conclusion, vestipitant is a potent NK(1) receptor antagonist: its actions support the utility of NK(1) receptor blockade in the alleviation of anxiety and, possibly, depression.

  2. The development of CAR design for tumor CAR-T cell therapy.

    Xu, Dandan; Jin, Guoliang; Chai, Dafei; Zhou, Xiaowan; Gu, Weiyu; Chong, Yanyun; Song, Jingyuan; Zheng, Junnian

    2018-03-02

    In recent years, the chimeric antigen receptor modified T cells (Chimeric antigen receptor T cells, CAR-T) immunotherapy has developed rapidly, which has been considered the most promising therapy. Efforts to enhance the efficacy of CAR-based anti-tumor therapy have been made, such as the improvement of structures of CAR-T cells, including the development of extracellular antigen recognition receptors, intracellular co-stimulatory molecules and the combination application of CARs and synthetic small molecules. In addition, effects on the function of the CAR-T cells that the space distance between the antigen binding domains and tumor targets and the length of the spacer domains have are also being investigated. Given the fast-moving nature of this field, it is necessary to make a summary of the development of CAR-T cells. In this review, we mainly focus on the present design strategies of CAR-T cells with the hope that they can provide insights to increase the anti-tumor efficacy and safety.

  3. Hepatocellular hypertrophy and cell proliferation in Sprague–Dawley rats from dietary exposure to potassium perfluorooctanesulfonate results from increased expression of xenosensor nuclear receptors PPARα and CAR/PXR

    Elcombe, Clifford R.; Elcombe, Barbara M.; Foster, John R.; Chang, Shu-Ching; Ehresman, David J.; Butenhoff, John L.

    2012-01-01

    14,643 increased CYP2B enzyme activity to a slight extent. All test compounds increased the liver cell proliferative index and decreased the liver apoptotic index. No histological changes of the thyroid were noted; however, PB and WY increased thyroid follicular cell proliferation index (seven-day treatment only), while K + PFOS did not. The thyroid follicular cell apoptotic index did not differ between groups. The hepatomegaly and hepatocellular adenoma observed after dietary exposure of Sprague–Dawley rats to K + PFOS likely are due to the increased expression of xenosensor nuclear receptors PPARα and CAR/PXR. Given the markedly lower or absent response of human hepatocytes to the proliferative stimulus from activation of PPARα and CAR/PXR, the hepatocellular proliferative response from activation of these receptors by PFOS observed in rats is not expected to be of human relevance.

  4. The Socialist Car

    Christensen, Lars K.

    2013-01-01

    Review of L.H. Siegelbaum (ed.) The Socialist Car. Automobility in the Eastern Block. Cornell University Press, 2011.......Review of L.H. Siegelbaum (ed.) The Socialist Car. Automobility in the Eastern Block. Cornell University Press, 2011....

  5. Specific receptors for phorbol diesters on freshly isolated human myeloid and lymphoid leukemia cells: comparable binding characteristics despite different cellular responses.

    Goodwin, B J; Moore, J O; Weinberg, J B

    1984-02-01

    Freshly isolated human leukemia cells have been shown in the past to display varying in vitro responses to phorbol diesters, depending on their cell type. Specific receptors for the phorbol diesters have been demonstrated on numerous different cells. This study was designed to characterize the receptors for phorbol diesters on leukemia cells freshly isolated from patients with different kinds of leukemia and to determine if differences in binding characteristics for tritium-labeled phorbol 12,13-dibutyrate (3H-PDBu) accounted for the different cellular responses elicited in vitro by phorbol diesters. Cells from 26 patients with different kinds of leukemia were studied. PDBu or phorbol 12-myristate 13-acetate (PMA) caused cells from patients with acute myeloblastic leukemia (AML), acute promyelocytic (APML), acute myelomonocytic (AMML), acute monocytic (AMoL), acute erythroleukemia (AEL), chronic myelocytic leukemia (CML) in blast crisis (myeloid), acute undifferentiated leukemia (AUL), and hairy cell leukemia (HCL) (n = 15) to adhere to plastic and spread. However, they caused no adherence or spreading and only slight aggregation of cells from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or CML-blast crisis (lymphoid) (n = 11). All leukemia cells studied, irrespective of cellular type, displayed specific receptors for 3H-PDBu. The time courses for binding by all leukemia types were similar, with peak binding at 5-10 min at 37 degrees C and 120 min at 4 degrees C. The binding affinities were similar for patients with ALL (96 +/- 32 nM, n = 4), CLL (126 +/- 32 nM, n = 6), and acute nonlymphoid leukemia (73 +/- 14 nM, n = 11). Likewise, the numbers of specific binding sites/cell were comparable for the patients with ALL (6.2 +/- 1.3 X 10(5) sites/cell, n = 4), CLL (5.0 +/- 2.0 X 10(5) sites/cell, n = 6), and acute nonlymphoid leukemia (4.4 +/- 1.9 X 10(5) sites/cell, n = 11). Thus, the differing responses to phorbol diesters of

  6. Cars, Cycles, and Consumers.

    Idleman, Hillis K. Ed.

    The purpose of this consumer education module is to provide information and skills, and the ability to raise questions and find answers, while seeking the best automobile or motorcycle buy available for the money. The module may be used for a full or part semester course. The five sections (cars and the consumer, renting and leasing cars, cars and…

  7. β-Arrestin-2 knockout prevents development of cellular μ-opioid receptor tolerance but does not affect opioid-withdrawal-related adaptations in single PAG neurons.

    Connor, M; Bagley, E E; Chieng, B C; Christie, M J

    2015-01-01

    Tolerance to the behavioural effects of morphine is blunted in β-arrestin-2 knockout mice, but opioid withdrawal is largely unaffected. The cellular mechanisms of tolerance have been studied in some neurons from β-arrestin-2 knockouts, but tolerance and withdrawal mechanisms have not been examined at the cellular level in periaqueductal grey (PAG) neurons, which are crucial for central tolerance and withdrawal phenomena. μ-Opioid receptor (MOPr) inhibition of voltage-gated calcium channel currents (ICa ) was examined by patch-clamp recordings from acutely dissociated PAG neurons from wild-type and β-arrestin-2 knockout mice treated chronically with morphine (CMT) or vehicle. Opioid withdrawal-induced activation of GABA transporter type 1 (GAT-1) currents was determined using perforated patch recordings from PAG neurons in brain slices. MOPr inhibition of ICa in PAG neurons was unaffected by β-arrestin-2 deletion. CMT impaired coupling of MOPrs to ICa in PAG neurons from wild-type mice, but this cellular tolerance was not observed in neurons from CMT β-arrestin-2 knockouts. However, β-arrestin-2 knockouts displayed similar opioid-withdrawal-induced activation of GAT-1 currents as wild-type PAG neurons. In β-arrestin-2 knockout mice, the central neurons involved in the anti-nociceptive actions of opioids also fail to develop cellular tolerance to opioids following chronic morphine. The results also provide the first cellular physiological evidence that opioid withdrawal is not disrupted by β-arrestin-2 deletion. However, the unaffected basal sensitivity to opioids in PAG neurons provides further evidence that changes in basal MOPr sensitivity cannot account for the enhanced acute nociceptive response to morphine reported in β-arrestin-2 knockouts. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British

  8. A crosstalk between muscarinic and CRF2 receptors regulates cellular adhesion properties of human colon cancer cells.

    Pelissier-Rota, M; Chartier, N T; Bonaz, B; Jacquier-Sarlin, M R

    2017-07-01

    Patients with inflammatory bowel disease often suffer from chronic and relapsing intestinal inflammation that favor the development of colitis associated cancer. An alteration of the epithelial intestinal barrier function observed in IBD is supposed to be a consequence of stress. It has been proposed that corticotrophin-releasing factor receptor (CRF2), one of the two receptors of CRF, the principal neuromediator of stress, acts on cholinergic nerves to induce stress-mediated epithelial barrier dysfunction. Non-neuronal acetylcholine (Ach) and muscarinic receptors (mAchR) also contribute to alterations of epithelial cell functions. In this study, we investigated the mechanisms through which stress and Ach modulate epithelial cell adhesive properties. We show that Ach-induced activation of mAchR in HT-29 cells results in cell dissociation together with changes in cell-matrix contacts, which correlates with the acquisition of invasive potential consistent with a matrix metalloproteinase (MMP) mode of invasion. These processes result from mAchR subsequent stimulation of the cascade of src/Erk and FAK activation. Ach-induced secretion of laminin 332 leads to α3β1 integrin activation and RhoA-dependent reorganization of the actin cytoskeleton. We show that Ach-mediated effects on cell adhesion are blocked by astressin 2b, a CRF2 antagonist, suggesting that Ach action depends partly on CRF2 signaling. This is reinforced by the fact that Ach-mediated activation of mAchR stimulates both the synthesis and the release of CRF2 ligands in HT-29 cells (effects blocked by atropine). In summary, our data provides evidence for a novel intracellular circuit involving mAchR acting on CRF2-signaling that could mediate colonic mucosal barrier dysfunction and exacerbate mucosal inflammation. Copyright © 2017. Published by Elsevier B.V.

  9. Endocrine Disrupters in Human Blood and Breast Milk: Extraction Methodologies, Cellular Uptake and Effect on Key Nuclear Receptor Functions

    Hjelmborg, Philip Sebastian

    2010-01-01

    -products from incineration plants, plastic additives, technical industry products, pesticides from the farming industry and detergent degradation products. Many of these substances can interfere with the hormonal system in organisms. The common name for these compounds is endocrine disrupters (EDCs). Some EDCs...... are persistent to degradation and are also called persistent organic pollutants (POPs). Endocrine disrupters are compounds that can interfere with an organism’s hormone system by interacting with the hormone receptors. Many of an organism’s body functions are controlled by interactions between hormones...

  10. Development of CINPA1 analogs as novel and potent inverse agonists of constitutive androstane receptor.

    Lin, Wenwei; Yang, Lei; Chai, Sergio C; Lu, Yan; Chen, Taosheng

    2016-01-27

    Constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) are master regulators of endobiotic and xenobiotic metabolism and disposition. Because CAR is constitutively active in certain cellular contexts, inhibiting CAR might reduce drug-induced hepatotoxicity and resensitize drug-resistant cancer cells to chemotherapeutic drugs. We recently reported a novel CAR inhibitor/inverse agonist CINPA1 (11). Here, we have obtained or designed 54 analogs of CINPA1 and used a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate their CAR inhibition potency. Many of the 54 analogs showed CAR inverse agonistic activities higher than those of CINPA1, which has an IC50 value of 687 nM. Among them, 72 has an IC50 value of 11.7 nM, which is about 59-fold more potent than CINPA1 and over 10-fold more potent than clotrimazole (an IC50 value of 126.9 nM), the most potent CAR inverse agonist in a biochemical assay previously reported by others. Docking studies provide a molecular explanation of the structure-activity relationship (SAR) observed experimentally. To our knowledge, this effort is the first chemistry endeavor in designing and identifying potent CAR inverse agonists based on a novel chemical scaffold, leading to 72 as the most potent CAR inverse agonist so far. The 54 chemicals presented are novel and unique tools for characterizing CAR's function, and the SAR information gained from these 54 analogs could guide future efforts to develop improved CAR inverse agonists. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  11. Fc Gamma Receptor 3B (FCGR3Bc.233C>A-rs5030738) Polymorphism Modifies the Protective Effect of Malaria Specific Antibodies in Ghanaian Children

    Adu, Bright; Jepsen, Micha Phill Grønholm; Gerds, Thomas A

    2014-01-01

    Immunoglobulin G (IgG) cross-linking with Fc gamma receptor IIIB (FcγRIIIB) triggers neutrophil degranulation, releasing reactive oxygen species with high levels associated with protection against malaria. The FCGR3B-c.233C>A polymorphism thought to influence the interaction between IgG and Fcγ...

  12. The Influence of Receptor-Mediated Interactions on Reaction-Diffusion Mechanisms of Cellular Self-organisation

    Klika, Václav

    2011-11-10

    Understanding the mechanisms governing and regulating self-organisation in the developing embryo is a key challenge that has puzzled and fascinated scientists for decades. Since its conception in 1952 the Turing model has been a paradigm for pattern formation, motivating numerous theoretical and experimental studies, though its verification at the molecular level in biological systems has remained elusive. In this work, we consider the influence of receptor-mediated dynamics within the framework of Turing models, showing how non-diffusing species impact the conditions for the emergence of self-organisation. We illustrate our results within the framework of hair follicle pre-patterning, showing how receptor interaction structures can be constrained by the requirement for patterning, without the need for detailed knowledge of the network dynamics. Finally, in the light of our results, we discuss the ability of such systems to pattern outside the classical limits of the Turing model, and the inherent dangers involved in model reduction. © 2011 Society for Mathematical Biology.

  13. 10 Years of Car-2-X Communication - a Success Story?

    Wischhof, Lars; Ebner, André

    2012-05-01

    For more than ten years, car-2-x communication has been a major topic of research in the scientific community and an important development focus for the automotive industry. First, this article takes a retrospective look at the evolution of car-2-x and the two different communication paradigms: decentralized car-2-car communication and centralized cellular solutions. Afterwards, a comparison of their technical advantages and limitations is presented, respectively. The result shows that in order to implement safety-relevant applications, car-2-car communication has strong advantages compared to cellular technologies but requires high market penetration. However, its introduction solely for safety applications is difficult since the required penetration will not be achieved until several years after initial deployment. Therefore, car-2-car communication must provide a benefit to the customer, even in the phase of market introduction. For this purpose, the article outlines an approach called SODAD (Segment-Oriented Data Abstraction and Dissemination). It offers a possibility to introduce decentralized vehicular applications with early customer benefit, in order to enable safety applications based on car-2-car communication on a long term.

  14. The interaction of hepatitis A virus (HAV with soluble forms of its cellular receptor 1 (HAVCR1 share the physiological requirements of infectivity in cell culture

    Kaplan Gerardo G

    2009-10-01

    Full Text Available Abstract Background Hepatitis A virus (HAV, an atypical Picornaviridae that causes acute hepatitis in humans, usurps the HAV cellular receptor 1 (HAVCR1 to infect cells. HAVCR1 is a class 1 integral membrane glycoprotein that contains two extracellular domains: a virus-binding immunoglobulin-like (IgV domain and a mucin-like domain that extends the IgV from the cell membrane. Soluble forms of HAVCR1 bind, alter, and neutralize cell culture-adapted HAV, which is attenuated for humans. However, the requirements of the HAV-HAVCR1 interaction have not been fully characterized, and it has not been determined whether HAVCR1 also serves as a receptor for wild-type (wt HAV. Here, we used HAV soluble receptor neutralization and alteration assays to study the requirements of the HAV-HAVCR1 interaction and to determine whether HAVCR1 is also a receptor for wt HAV. Results Treatment of HAV with a soluble form of HAVCR1 that contained the IgV and two-thirds of the mucin domain fused to the Fc fragment of human IgG1 (D1 muc-Fc, altered particles at 37°C but left a residual level of unaltered particles at 4°C. The kinetics of neutralization of HAV by D1 muc-Fc was faster at 37°C than at 4°C. Alteration of HAV particles by D1 muc-Fc required Ca, which could not be replaced by Li, Na, Mg, Mn, or Zn. Neutralization of HAV by D1 muc-Fc occurred at pH 5 to 8 but was more efficient at pH 6 to 7. D1 muc-Fc neutralized wt HAV as determined by a cell culture system that allows the growth of wt HAV. Conclusion The interaction of HAV with soluble forms of HAVCR1 shares the temperature, Ca, and pH requirements for infectivity in cell culture and therefore mimics the cell entry process of HAV. Since soluble forms of HAVCR1 also neutralized wt HAV, this receptor may play a significant role in pathogenesis of HAV.

  15. Aryl Hydrocarbon Receptor Antagonists Mitigate the Effects of Dioxin on Critical Cellular Functions in Differentiating Human Osteoblast-Like Cells

    Chawon Yun

    2018-01-01

    Full Text Available The inhibition of bone healing in humans is a well-established effect associated with cigarette smoking, but the underlying mechanisms are still unclear. Recent work using animal cell lines have implicated the aryl hydrocarbon receptor (AhR as a mediator of the anti-osteogenic effects of cigarette smoke, but the complexity of cigarette smoke mixtures makes understanding the mechanisms of action a major challenge. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin is a high-affinity AhR ligand that is frequently used to investigate biological processes impacted by AhR activation. Since there are dozens of AhR ligands present in cigarette smoke, we utilized dioxin as a prototype ligand to activate the receptor and explore its effects on pro-osteogenic biomarkers and other factors critical to osteogenesis using a human osteoblast-like cell line. We also explored the capacity for AhR antagonists to protect against dioxin action in this context. We found dioxin to inhibit osteogenic differentiation, whereas co-treatment with various AhR antagonists protected against dioxin action. Dioxin also negatively impacted cell adhesion with a corresponding reduction in the expression of integrin and cadherin proteins, which are known to be involved in this process. Similarly, the dioxin-mediated inhibition of cell migration correlated with reduced expression of the chemokine receptor CXCR4 and its ligand, CXCL12, and co-treatment with antagonists restored migratory capacity. Our results suggest that AhR activation may play a role in the bone regenerative response in humans exposed to AhR activators, such as those present in cigarette smoke. Given the similarity of our results using a human cell line to previous work done in murine cells, animal models may yield data relevant to the human setting. In addition, the AhR may represent a potential therapeutic target for orthopedic patients who smoke cigarettes, or those who are exposed to secondhand smoke or other

  16. Tank car leaks gasoline

    Anon.

    1997-01-01

    On January 27, 1994, a Canadian National (CN) tank car loaded with gasoline began to leak from a crack in the tank shell on the end of the car near the stub sill. The tank car had been damaged from impact switching. A part of the tank car was sent for laboratory analysis which concluded that: (1) the fracture originated in two locations in welds, (2) the cracks propagated in a symmetrical manner and progressed into the tank plate, (3) the fracture surface revealed inadequate weld fusion. A stress analysis of the tank car was conducted to determine the coupling force necessary to cause the crack. It was noted that over the last decade several problems have occurred pertaining to stub sill areas of tank cars that have resulted in hazardous material spills. An advisory was sent to Transport Canada outlining many examples where tank cars containing serious defects had passed CN inspections that were specifically designed to identify such defects. 4 figs

  17. Variation in the binding of 125I-labeled interferon-beta ser to cellular receptors during growth of human renal and bladder carcinoma cells in vitro

    Ruzicka, F.J.; Schmid, S.M.; Groveman, D.S.; Cummings, K.B.; Borden, E.C.

    1987-01-01

    Studies of various established human bladder and renal carcinoma cell lines cultured in vitro demonstrated the presence of specific, saturable, high affinity binding sites for 125 I-labeled human interferon Beta ser IFN-beta ser). This recombinant produced interferon labeled with approximately one atom of 125 I/molecule of IFN expressed minimal or no loss of antiviral activity. A single class of binding sites (1000-2000/cell) with an affinity constant of 10(10)-10(11) L/M was measured at 4 degrees C for cells exhibiting widely different sensitivity to the antiproliferative effect of IFN-beta ser. Major fluctuations in the binding of 125 I-labeled IFN-beta ser to cellular receptors were observed during in vitro proliferation of four of five cell lines examined. A significant decrease (P less than 0.001) in specific binding was observed 48 h after cultures were established. Cell cycle analysis suggested that within the first 24 h and in the very late log and stationary phase of growth of ACHN (human renal carcinoma) cells, variations in the binding of 125 I-labeled IFN-beta ser were partially attributable to binding fluctuations during the mitotic cycle. The 2- to 3-fold decline 24 h following plating of ACHN cells corresponded to a 70% decrease in the number of cells in G0-G1. T24 (human transitional cell carcinoma) and ACHN cells, synchronized by serum starvation, demonstrated increased binding of 125 I-labeled IFN-beta ser 4-16 h following serum replenishment. This increase in receptor binding occurred prior to the onset of DNA and protein synthesis and was followed by a decline immediately prior to cell division. Binding site analysis indicated that the increased binding prior to DNA synthesis was due to a 5- to 6-fold increase in receptor affinity for the radiolabeled ligand

  18. Comparative genomic analysis of buffalo (Bubalus bubalis NOD1 and NOD2 receptors and their functional role in in-vitro cellular immune response.

    Biswajit Brahma

    Full Text Available Nucleotide binding and oligomerization domain (NOD-like receptors (NLRs are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo--a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1 and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.

  19. Establishing guidelines for CAR-T cells: challenges and considerations.

    Wang, Wei; Qin, Di-Yuan; Zhang, Bing-Lan; Wei, Wei; Wang, Yong-Sheng; Wei, Yu-Quan

    2016-04-01

    T cells, genetically modified by chimeric antigen receptors (CAR-T), are endowed with specificity to a desired antigen and are cytotoxic to cells expressing the targeted antigen. CAR-T-based cancer immunotherapy is a promising therapy for curing hematological malignancy, such as acute lymphoid leukemia, and is promising for extending their efficacy to defeat solid tumors. To date, dozens of different CAR-T cells have been evaluated in clinical trials to treat tumors; this necessitates the establishment of guidelines for the production and application of CAR-T cells. However, it is challenging to standardize CAR-T cancer therapy because it involves a combination of gene therapy and cell therapy. In this review, we compare the existing guidelines for CAR-T cells and discuss the challenges and considerations for establishing guidance for CAR-T-based cancer immunotherapy.

  20. CARs in the Lead Against Multiple Myeloma

    Ormhøj, Maria; Bedoya, Felipe; Frigault, Matthew J.

    2017-01-01

    The recent clinical success of CD19-directed chimeric antigen receptor (CAR) T cell therapy in chronic and acute leukemia has led to increased interest in broadening this technology to other hematological malignancies and solid tumors. Now, advances are being made using CAR T cell technology...... to target myeloma antigens such as B cell maturation antigen (BCMA), CD138, and kappa-light chain as well as CD19 on putative myeloma stem cells. To date, only a limited number of multiple myeloma patients have received CAR T cell therapy but preliminary results have been encouraging. In this review, we...... summarize the recently reported results of clinical trials conducted utilizing CAR T cell therapy in multiple myeloma (MM)....

  1. High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

    Martinez, Emily M; Klebanoff, Samuel D; Secrest, Stephanie; Romain, Gabrielle; Haile, Samuel T; Emtage, Peter C R; Gilbert, Amy E

    2018-04-01

    High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

  2. CAR2 - Czech Database of Car Speech

    P. Sovka

    1999-12-01

    Full Text Available This paper presents new Czech language two-channel (stereo speech database recorded in car environment. The created database was designed for experiments with speech enhancement for communication purposes and for the study and the design of a robust speech recognition systems. Tools for automated phoneme labelling based on Baum-Welch re-estimation were realised. The noise analysis of the car background environment was done.

  3. CAR2 - Czech Database of Car Speech

    Pollak, P.; Vopicka, J.; Hanzl, V.; Sovka, Pavel

    1999-01-01

    This paper presents new Czech language two-channel (stereo) speech database recorded in car environment. The created database was designed for experiments with speech enhancement for communication purposes and for the study and the design of a robust speech recognition systems. Tools for automated phoneme labelling based on Baum-Welch re-estimation were realised. The noise analysis of the car background environment was done.

  4. Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

    Tang, Xiang-Jun; Sun, Xu-Yong; Huang, Kuan-Ming; Zhang, Li; Yang, Zhuo-Shun; Zou, Dan-Dan; Wang, Bin; Warnock, Garth L; Dai, Long-Jun; Luo, Jie

    2015-12-29

    Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

  5. Growth Hormone Receptor Antagonist Transgenic Mice Have Increased Subcutaneous Adipose Tissue Mass, Altered Glucose Homeostasis and No Change in White Adipose Tissue Cellular Senescence.

    Comisford, Ross; Lubbers, Ellen R; Householder, Lara A; Suer, Ozan; Tchkonia, Tamara; Kirkland, James L; List, Edward O; Kopchick, John J; Berryman, Darlene E

    2016-01-01

    Growth hormone (GH)-resistant/deficient mice experience improved glucose homeostasis and substantially increased lifespan. Recent evidence suggests that long-lived GH-resistant/deficient mice are protected from white adipose tissue (WAT) dysfunction, including WAT cellular senescence, impaired adipogenesis and loss of subcutaneous WAT in old age. This preservation of WAT function has been suggested to be a potential mechanism for the extended lifespan of these mice. The objective of this study was to examine WAT senescence, WAT distribution and glucose homeostasis in dwarf GH receptor antagonist (GHA) transgenic mice, a unique mouse strain having decreased GH action but normal longevity. 18-month-old female GHA mice and wild-type (WT) littermate controls were used. Prior to dissection, body composition, fasting blood glucose as well as glucose and insulin tolerance tests were performed. WAT distribution was determined by weighing four distinct WAT depots at the time of dissection. Cellular senescence in four WAT depots was assessed using senescence-associated β-galactosidase staining to quantify the senescent cell burden, and real-time qPCR to quantify gene expression of senescence markers p16 and IL-6. GHA mice had a 22% reduction in total body weight, a 33% reduction in lean mass and a 10% increase in body fat percentage compared to WT controls. GHA mice had normal fasting blood glucose and improved insulin sensitivity; however, they exhibited impaired glucose tolerance. Moreover, GHA mice displayed enhanced lipid storage in the inguinal subcutaneous WAT depot (p < 0.05) and a 1.7-fold increase in extra-/intraperitoneal WAT ratio compared to controls (p < 0.05). Measurements of WAT cellular senescence showed no difference between GHA mice and WT controls. Similar to other mice with decreased GH action, female GHA mice display reduced age-related lipid redistribution and improved insulin sensitivity, but no change in cellular senescence. The similar abundance of

  6. Growth hormone receptor antagonist (GHA) transgenic mice have increased subcutaneous adipose tissue mass, altered glucose homeostasis, and no change in white adipose tissue cellular senescence

    Comisford, Ross; Lubbers, Ellen R.; Householder, Lara; Suer, Ozan; Tchkonia, Tamara; Kirkland, James L.; List, Edward O.; Kopchick, John J.; Berryman, Darlene E.

    2015-01-01

    Background Growth hormone (GH) resistant/deficient mice experience improved glucose homeostasis and substantially increased lifespan. Recent evidence suggests long-lived GH resistant/deficient mice are protected from white adipose tissue (WAT) dysfunction, including WAT cellular senescence, impaired adipogenesis and loss of subcutaneous WAT in old age. This preservation of WAT function has been suggested to be a potential mechanism for the extended lifespan of these mice. OBJECTIVE The objective of this study was to examine white adipose tissue (WAT) senescence, WAT distribution, and glucose homeostasis in dwarf growth hormone receptor antagonist (GHA) transgenic mice, a unique mouse strain having decreased GH action but normal longevity. METHODS 18mo old female GHA mice and wild type (WT) littermate controls were used. Prior to dissection, body composition, fasting blood glucose, and glucose and insulin tolerance tests were performed. WAT distribution was determined by weighing four distinct WAT depots at the time of dissection. Cellular senescence in four WAT depots was assessed using senescence-associated β-galactosidase (SA-β-gal) staining to quantify the senescent cell burden and real time qPCR to quantify gene expression of senescence markers p16 and IL-6. RESULTS GHA mice had a 22% reduction in total body weight, 33% reduction in lean mass, and a 10% increase in body fat percentage compared to WT controls. GHA mice had normal fasting blood glucose and improved insulin sensitivity; however, they exhibited impaired glucose tolerance. Moreover, GHA mice displayed enhanced lipid storage in the inguinal subcutaneous WAT depot (p<.05) and a 1.7 fold increase in extra-/intraperitoneal WAT ratio compared to controls (p<.05). Measurements of WAT cellular senescence showed no difference between GHA mice and WT controls. CONCLUSIONS Similar to other mice with decreased GH action, female GHA mice display reduced age-related lipid redistribution and improved insulin

  7. Helix 11 Dynamics is Critical for Constitutive Androstane Receptor Activity

    Wright, Edward; Busby, Scott A.; Wisecarver, Sarah; Vincent, Jeremy; Griffin, Patrick R.; Fernandez, Elias J.

    2011-01-01

    The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizi...

  8. Pentoxifylline regulates the cellular adhesion and its allied receptors to extracellular matrix components in breast cancer cells.

    Goel, Peeyush N; Gude, Rajiv P

    2014-02-01

    Pentoxifylline (PTX) is a methylxanthine derivative that improves blood flow by decreasing its viscosity. Being an inhibitor of platelet aggregation, it can thus reduce the adhesiveness of cancer cells prolonging their circulation time. This delay in forming secondary tumours makes them more prone to immunological surveillance. Recently, we have evaluated its anti-metastatic efficacy against breast cancer, using MDA-MB-231 model system. In view of this, we had ascertained the effect of PTX on adhesion of MDA-MB-231 cells to extracellular matrix components (ECM) and its allied receptors such as the integrins. PTX affected adhesion of breast cancer cells to matrigel, collagen type IV, fibronectin and laminin in a dose dependent manner. Further, PTX showed a differential effect on integrin expression profile. The experimental metastasis model using NOD-SCID mice showed lesser tumour island formation when treated with PTX compared to the control. These findings further substantiate the anti-adhesive potential of PTX in breast cancer and warrant further insights into the functional regulation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  9. CD19 CAR-targeted T cells induce long-term remission and B Cell Aplasia in an immunocompetent mouse model of B cell acute lymphoblastic leukemia.

    Marco L Davila

    Full Text Available Although many adults with B cell acute lymphoblastic leukemia (B-ALL are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.

  10. Electric Car Special

    Zoethout, T.; Belin, H.; Verwijs, H.; Nicola, S.; De Saint Jacob, Y.; Gatermann, R.

    2009-09-15

    In six articles, two columns and two interviews a part of this issue is dedicated to electric car developments: about winners and losers in the electric car race; a unique business model to rolling out the electric car by the electric battery company Better Place and the automobile industry Renault Nissan; interview with entrepreneur Shai Agassi of the Indian company Better Place; the development of electric cars in Germany; interview with Jean-Jacques Chanaron, an economist specialising in innovation management and a firm believer in electric cars; start of mass production of electric vehicles at the Japanese Nissan automobile industry; the constraints in Sweden in developing fuel-efficient automobiles; plans for 1 million electric or hybrid cars by 2025 in the Netherlands.

  11. Breaking car use habits

    Thøgersen, John; Møller, Berit Thorup

    2008-01-01

    Based on calls for innovative ways of reducing car traffic and research indicating that car driving is often the result of habitual decision-making and choice processes, this paper reports on a field experiment designed to test a tool aimed to entice drivers to skip the habitual choice of the car...... and consider using-or at least trying-public transport instead. About 1,000 car drivers participated in the experiment either as experimental subjects, receiving a free one-month travelcard, or as control subjects. As predicted, the intervention had a significant impact on drivers' use of public transport...... and it also neutralized the impact of car driving habits on mode choice. However, in the longer run (i.e., four months after the experiment) experimental subjects did not use public transport more than control subjects. Hence, it seems that although many car drivers choose travel mode habitually, their final...

  12. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J N; Platt, Jesse M; Johnson, F Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C; June, Carl H

    2015-04-01

    This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. ©2015 American Association for Cancer Research.

  13. Car stickers for 2011

    GS Department

    2010-01-01

    The 2011 car stickers are now available. Holders of blue car stickers will receive their 2011 car stickers by internal mail as of 15 December.   Holders of red car stickers are kindly requested to come to the Registration Service (Building 55,1st floor) to renew their 2011 stickers. This service is open from Monday to Friday from 7.30 am to 5.30 pm non-stop. Documents for the vehicles concerned must be presented. Reception and Access Control Service – GS/ISG/SIS General Infrastructure Services Department

  14. Car stickers for 2012

    GS Department

    2011-01-01

    The 2012 car stickers are now available. Holders of blue car stickers will receive by internal mail their 2012 car stickers as of 5 December. Holders of red car stickers are kindly requested to come to the Registration Service (Building 55,1st floor) to renew their 2011 stickers. This service is open from Monday to Friday from 7.30 am to 5.30 pm non-stop. Documents related to the vehicles concerned are mandatory. Reception and Access Control Service – GS/IS/SIS General Infrastructure Services Department

  15. Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor.

    Jeske, Judith; Windshügel, Björn; Thasler, Wolfgang E; Schwab, Matthias; Burk, Oliver

    2017-06-01

    Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named "CAR inhibitor not PXR activator 1" (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.

  16. P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach.

    France Massicot

    Full Text Available Limited information is available regarding the cellular mechanisms of oxaliplatin-induced painful neuropathy during exposure of patients to this drug. We therefore determined oxidative stress in cultured cells and evaluated its occurrence in C57BL/6 mice. Using both cultured neuroblastoma (SH-SY5Y and macrophage (RAW 264.7 cell lines and also brain tissues of oxaliplatin-treated mice, we investigated whether oxaliplatin (OXA induces oxidative stress and apoptosis. Cultured cells were treated with 2-200 µM OXA for 24 h. The effects of pharmacological inhibitors of oxidative stress or inflammation (N-acetyl cysteine, ibuprofen, acetaminophen were also tested. Inhibitors were added 30 min before OXA treatment and then in combination with OXA for 24 h. In SH-SY5Y cells, OXA caused a significant dose-dependent decrease in viability, a large increase in ROS and NO production, lipid peroxidation and mitochondrial impairment as assessed by a drop in mitochondrial membrane potential, which are deleterious for the cell. An increase in levels of negatively charged phospholipids such as cardiolipin but also phosphatidylserine and phosphatidylinositol, was also observed. Additionally, OXA caused concentration-dependent P2X7 receptor activation, increased chromatin condensation and caspase-3 activation associated with TNF-α and IL-6 release. The majority of these toxic effects were equally observed in Raw 264.7 which also presented high levels of PGE2. Pretreatment of SH-SY5Y cells with pharmacological inhibitors significantly reduced or blocked all the neurotoxic OXA effects. In OXA-treated mice (28 mg/kg cumulated dose significant cold hyperalgesia and oxidative stress in the tested brain areas were shown. Our study suggests that targeting P2X7 receptor activation and mitochondrial impairment might be a potential therapeutic strategy against OXA-induced neuropathic pain.

  17. Stimulation of toll-like receptor 2 with bleomycin results in cellular activation and secretion of pro-inflammatory cytokines and chemokines

    Razonable, Raymund R.; Henault, Martin; Paya, Carlos V.

    2006-01-01

    The clinical use of bleomycin results in systemic and pulmonary inflammatory syndromes that are mediated by the production of cytokines and chemokines. In this study, we demonstrate that cell activation is initiated upon the recognition of bleomycin as a pathogen-associated molecular pattern by toll-like receptor (TLR) 2. The THP1 human monocytic cell line, which constitutively expresses high levels of TLR2, secretes interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α during bleomycin exposure. The TLR2-dependent nature of cell activation and cytokine secretion is supported by (1) the inability of TLR2-deficient human embryonic kidney (HEK) 293 cells to exhibit nuclear factor-kappa B (NF-κB) activation and secrete IL-8 in response to bleomycin; (2) the acquired ability of HEK293 to exhibit NF-κB activation and secrete IL-8 upon experimental expression of TLR2; and (3) the inhibition of cell activation in TLR2-expressing HEK293 and THP1 by anti-TLR2 monoclonal antibody. Collectively, these observations identify TLR2 activation as a critical event that triggers NF-κB activation and secretion of cytokines and chemokines during bleomycin exposure. Our in vitro findings could serve as a molecular mechanism underlying the pro-inflammatory toxicity associated with bleomycin. Whether bleomycin engages with other cellular receptors that results in activation of alternate signaling pathways and whether the TLR2-agonist activity of bleomycin contribute to its anti-neoplastic property deserve further study

  18. UV exposure in cars.

    Moehrle, Matthias; Soballa, Martin; Korn, Manfred

    2003-08-01

    There is increasing knowledge about the hazards of solar and ultraviolet (UV) radiation to humans. Although people spend a significant time in cars, data on UV exposure during traveling are lacking. The aim of this study was to obtain basic information on personal UV exposure in cars. UV transmission of car glass samples, windscreen, side and back windows and sunroof, was determined. UV exposure of passengers was evaluated in seven German middle-class cars, fitted with three different types of car windows. UV doses were measured with open or closed windows/sunroof of Mercedes-Benz E 220 T, E 320, and S 500, and in an open convertible car (Mercedes-Benz CLK). Bacillus subtilis spore film dosimeters (Viospor) were attached to the front, vertex, cheeks, upper arms, forearms and thighs of 'adult' and 'child' dummies. UV wavelengths longer than >335 nm were transmitted through car windows, and UV irradiation >380 nm was transmitted through compound glass windscreens. There was some variation in the spectral transmission of side windows according to the type of glass. On the arms, UV exposure was 3-4% of ambient radiation when the car windows were shut, and 25-31% of ambient radiation when the windows were open. In the open convertible car, the relative personal doses reached 62% of ambient radiation. The car glass types examined offer substantial protection against short-wave UV radiation. Professional drivers should keep car windows closed on sunny days to reduce occupational UV exposure. In individuals with polymorphic light eruption, produced by long-wave UVA, additional protection by plastic films, clothes or sunscreens appears necessary.

  19. Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene.

    Casucci, Monica; Falcone, Laura; Camisa, Barbara; Norelli, Margherita; Porcellini, Simona; Stornaiuolo, Anna; Ciceri, Fabio; Traversari, Catia; Bordignon, Claudio; Bonini, Chiara; Bondanza, Attilio

    2018-01-01

    Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

  20. Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

    Monica Casucci

    2018-03-01

    Full Text Available Chimeric antigen receptor (CAR-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR. We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6. We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

  1. Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

    Casucci, Monica; Falcone, Laura; Camisa, Barbara; Norelli, Margherita; Porcellini, Simona; Stornaiuolo, Anna; Ciceri, Fabio; Traversari, Catia; Bordignon, Claudio; Bonini, Chiara; Bondanza, Attilio

    2018-01-01

    Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting. PMID:29619024

  2. Increased anaerobic metabolism is a distinctive signature in a colorectal cancer cellular model of resistance to antiepidermal growth factor receptor antibody.

    Monteleone, Francesca; Rosa, Roberta; Vitale, Monica; D'Ambrosio, Chiara; Succoio, Mariangela; Formisano, Luigi; Nappi, Lucia; Romano, Maria Fiammetta; Scaloni, Andrea; Tortora, Giampaolo; Bianco, Roberto; Zambrano, Nicola

    2013-03-01

    Cetuximab is a chimeric antibody approved for the treatment of metastatic colorectal cancer that selectively targets epidermal growth factor receptor (EGFR) signaling. Treatment efficacy with this drug is often impaired by acquired resistance and poor information has been accumulated on the mechanisms underlying such a phenomenon. By taking advantage of a syngenic cellular system of sensitivity and acquired resistance to anti-EGFR therapy in the colorectal carcinoma GEO cell line, we profiled protein expression differences between Cetuximab-sensitive and -resistant cells. Combined 2D DIGE and MS analyses revealed a main proteomic signature resulting from selective deregulation of various metabolic enzymes, including glucose-6-phosphate dehydrogenase, transketolase, lactate dehydrogenase B, and pyruvate dehydrogenase E1, which was also confirmed by Western blotting experiments. Lactate dehydrogenase B downregulation has been already related to an increased anaerobic utilization of glucose by tumor cells; accordingly, we verified that Cetuximab-resistant cells have a significantly higher production of lactate. Resistant cells also showed decreased nicotinamide adenine dinucleotide phosphate (NADPH) levels. Observed protein deregulations were not related to functional alterations of the hypoxia-inducible factor 1-associated pathways. Our data demonstrate that increased anaerobic metabolism is a prominent feature observed in the GEO syngenic model of acquired resistance to anti-EGFR therapy in colorectal cancer. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The Electric Cars Challenge

    Roman, Harry T.

    2011-01-01

    Over 100 years ago, the great inventor Thomas Edison warned that gasoline cars would pollute the environment and lead to gasoline shortages. He preferred the use of clean electric vehicles. He also put his money where his mouth was and developed an entirely new alkaline storage battery system for his beloved cars, the nickel-iron storage battery.…

  4. Collaborative Car Pooling System

    João Ferreira; Paulo Trigo; Porfírio Filipe

    2009-01-01

    This paper describes the architecture for a collaborative Car Pooling System based on a credits mechanism to motivate the cooperation among users. Users can spend the accumulated credits on parking facilities. For this, we propose a business model to support the collaboration between a car pooling system and parking facilities. The Portuguese Lisbon-s Metropolitan area is used as application scenario.

  5. Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells.

    van der Mark, Vincent A; Rudi de Waart, D; Shevchenko, Valery; Elferink, Ronald P J Oude; Chamuleau, Robert A F M; Hoekstra, Ruurdtje

    2017-01-01

    Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  6. City Car = The City Car / Andres Sevtshuk

    Sevtshuk, Andres, 1981-

    2008-01-01

    Massachusettsi Tehnoloogiainstituudi (MIT) meedialaboratooriumi juures tegutseva Targa Linna Grupi (Smart City Group) ja General Motorsi koostööna sündinud kaheistmelisest linnasõbralikust elektriautost City Car. Nimetatud töögrupi liikmed (juht William J. Mitchell, töögruppi kuulus A. Sevtshuk Eestist)

  7. Car Covers | Outdoor Covers Canada

    Covers, Outdoor

    2018-01-01

    Protect your car from the elements with Ultimate Touch Car Cover. The multi-layer non-woven fabric is soft on the finish and offers 4 seasons all weather protection.https://outdoorcovers.ca/car-covers/

  8. Cellular therapies: Day by day, all the way.

    Atilla, Erden; Kilic, Pelin; Gurman, Gunhan

    2018-04-18

    Tremendous effort and knowledge have elucidated a new era of 'cellular therapy,' also called "live" or "living" drugs. There are currently thousands of active clinical trials that are ongoing, seeking hope for incurable conditions thanks to the increased accessibility and reliability of gene editing and cellular reprogramming. Accomplishments in various adoptive T cell immunotherapies and chimeric antigen receptor (CART) T cell therapies oriented researchers to the field. Cellular therapies are believed to be the next generation of curative therapeutics in many ways, the classification and nomenclature for these applications have not yet reached a consensus. Trends in recent years are moving towards making tissues and cell processes only in centers with production permits. It is quite promising that competent authorities have increased licensing activities of tissue and cell establishments in their countries, under good practice (GxP) rules, and preclinical and clinical trials involving cell-based therapies have led to significant investments. Despite the initiatives undertaken and the large budgets that have been allocated, only limited success has been achieved in cellular therapy compared to conventional drug development. Cost, and cost effectiveness, are important issues for these novel therapies to meet unmet clinical needs, and there are still many scientific, translational, commercializational, and ethical questions that do not have answers. The main objectives of this review is to underline the current position of cellular therapies in research, highlight the timely topic of immunotherapy and chimeric antigen receptor (CAR) T-cell treatment, and compile information related to regulations and marketing of cellular therapeutic approaches worldwide. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Mitochondrial-targeted aryl hydrocarbon receptor and the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cellular respiration and the mitochondrial proteome

    Hwang, Hye Jin [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824 (United States); Dornbos, Peter [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Steidemann, Michelle [Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Dunivin, Taylor K. [Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States); Rizzo, Mike [Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Cell and Molecular Biology Graduate Program, Michigan State University, East Lansing, MI 48824 (United States); LaPres, John J., E-mail: lapres@msu.edu [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824 (United States)

    2016-08-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor within the Per-Arnt-Sim (PAS) domain superfamily. Exposure to the most potent AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is associated with various pathological effects including metabolic syndrome. While research over the last several years has demonstrated a role for oxidative stress and metabolic dysfunction in AHR-dependent TCDD-induced toxicity, the role of the mitochondria in this process has not been fully explored. Our previous research suggested that a portion of the cellular pool of AHR could be found in the mitochondria (mitoAHR). Using a protease protection assay with digitonin extraction, we have now shown that this mitoAHR is localized to the inter-membrane space (IMS) of the organelle. TCDD exposure induced a degradation of mitoAHR similar to that of cytosolic AHR. Furthermore, siRNA-mediated knockdown revealed that translocase of outer-mitochondrial membrane 20 (TOMM20) was involved in the import of AHR into the mitochondria. In addition, TCDD altered cellular respiration in an AHR-dependent manner to maintain respiratory efficiency as measured by oxygen consumption rate (OCR). Stable isotope labeling by amino acids in cell culture (SILAC) identified a battery of proteins within the mitochondrial proteome influenced by TCDD in an AHR-dependent manner. Among these, 17 proteins with fold changes ≥ 2 are associated with various metabolic pathways, suggesting a role of mitochondrial retrograde signaling in TCDD-mediated pathologies. Collectively, these studies suggest that mitoAHR is localized to the IMS and AHR-dependent TCDD-induced toxicity, including metabolic dysfunction, wasting syndrome, and hepatic steatosis, involves mitochondrial dysfunction. - Highlights: • The mitoAHR is localized in the mitochondrial intermembrane space. • TOMM20 participates in mitoAHR translocation. • AHR contributes to the maintenance of respiratory control ratio following

  10. Restoration of the cellular secretory milieu overrides androgen dependence of in vivo generated castration resistant prostate cancer cells overexpressing the androgen receptor.

    Patki, Mugdha; Huang, Yanfang; Ratnam, Manohar

    2016-07-22

    It is believed that growth of castration resistant prostate cancer (CRPC) cells is enabled by sensitization to minimal residual post-castrate androgen due to overexpression of the androgen receptor (AR). Evidence is derived from androgen-induced colony formation in the absence of cell-secreted factors or from studies involving forced AR overexpression in hormone-dependent cells. On the other hand, standard cell line models established from CRPC patient tumors (e.g., LNCaP and VCaP) are hormone-dependent and require selection pressure in castrated mice to re-emerge as CRPC cells and the resulting tumors then tend to be insensitive to the androgen antagonist enzalutamide. Therefore, we examined established CRPC model cells produced by castration of mice bearing hormone-dependent cell line xenografts including CRPC cells overexpressing full-length AR (C4-2) or co-expressing wtAR and splice-variant AR-V7 that is incapable of ligand binding (22Rv1). In standard colony formation assays, C4-2 cells were shown to be androgen-dependent and sensitive to enzalutamide whereas 22Rv1 cells were incapable of colony formation under identical conditions. However, both C4-2 and 22Rv1 cells formed colonies in conditioned media derived from the same cells or from HEK293 fibroblasts that were proven to lack androgenic activity. This effect was (i) not enhanced by androgen, (ii) insensitive to enzalutamide, (iii) dependent on AR (in C4-2) and on AR-V7 and wtAR (in 22Rv1) and (iv) sensitive to inhibitors of several signaling pathways, similar to androgen-stimulation. Therefore, during progression to CRPC in vivo, coordinate cellular changes accompanying overexpression of AR may enable cooperation between hormone-independent activity of AR and actions of cellular secretory factors to completely override androgen-dependence and sensitivity to drugs targeting hormonal factors. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT expression in an ELISA-based system.

    Philip Wing-Lok Ho

    Full Text Available Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA, and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT is transcriptionally regulated by estrogen via estrogen receptor (ER. Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP, and di-n-butyl phthalate (DBP. Cells were exposed to either these plasticizers or 17β-estradiol (E2 in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10(-9-10(-7M dose-dependently reduced COMT expression (p<0.05, which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different

  12. Novel chimeric virus-like particles vaccine displaying MERS-CoV receptor-binding domain induce specific humoral and cellular immune response in mice.

    Wang, Chong; Zheng, Xuexing; Gai, Weiwei; Wong, Gary; Wang, Hualei; Jin, Hongli; Feng, Na; Zhao, Yongkun; Zhang, Weijiao; Li, Nan; Zhao, Guoxing; Li, Junfu; Yan, Jinghua; Gao, Yuwei; Hu, Guixue; Yang, Songtao; Xia, Xianzhu

    2017-04-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) has continued spreading since its emergence in 2012 with a mortality rate of 35.6%, and is a potential pandemic threat. Prophylactics and therapies are urgently needed to address this public health problem. We report here the efficacy of a vaccine consisting of chimeric virus-like particles (VLP) expressing the receptor binding domain (RBD) of MERS-CoV. In this study, a fusion of the canine parvovirus (CPV) VP2 structural protein gene with the RBD of MERS-CoV can self-assemble into chimeric, spherical VLP (sVLP). sVLP retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to CPV virions. Immunization with sVLP induced RBD-specific humoral and cellular immune responses in mice. sVLP-specific antisera from these animals were able to prevent pseudotyped MERS-CoV entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. IFN-γ, IL-4 and IL-2 secreting cells induced by the RBD were detected in the splenocytes of vaccinated mice by ELISpot. Furthermore, mice inoculated with sVLP or an adjuvanted sVLP vaccine elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. Our study demonstrates that sVLP displaying the RBD of MERS-CoV are promising prophylactic candidates against MERS-CoV in a potential outbreak situation. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Distortionary company car taxation: deadweight losses through increased car ownership

    van Ommeren, J.N.; Gutierrez Puigarnau, E.

    2013-01-01

    We analyse the effects of distortionary company car taxation through increased household car consumption for the Netherlands. We use several identification strategies and demonstrate that for about 20 % of households company car possession increases car ownership. The annual welfare loss of

  14. CAR: no longer forgotten?

    Toby Lanzer

    2007-07-01

    Full Text Available The international community is waking up to the strategic importance of the Central African Republic (CAR in the crisis over Darfur. Will current interest endure long enough to help the people of CARCAR beyond the immediate future?

  15. The Electric Car Challenge.

    Diehl, Brian E.

    1997-01-01

    Describes the Electric Car Challenge during which students applied methods of construction to build lightweight, strong vehicles that were powered by electricity. The activity required problem solving, sheet metal work, electricity, design, and construction skills. (JOW)

  16. Car-use habits

    Møller, Berit Thorup; Thøgersen, John

    2008-01-01

    It is often claimed that many drivers use their private car rather habitually. The claim gains credibility from the fact that travelling to many everyday destinations fulfils all the prerequisites for habit formation: it is recurring, performed under stable circumstances and produces rewarding...... consequences. Since the decision is made quite automatically and only one choice alternative is considered (the habitually chosen one), behaviour guided by habit is difficult to change. The implications of car use habits for converting drivers to commuters using public transportation is analysed based...... to do so, car use habit, and the interaction between the two, confirms the theory-derived hypothesis that car use habits act as an obstacle to the transformation of intentions to commute by public transportation into action....

  17. Chimeric Antigen Receptors in Different Cell Types: New Vehicles Join the Race.

    Harrer, Dennis C; Dörrie, Jan; Schaft, Niels

    2018-05-01

    Adoptive cellular therapy has evolved into a powerful force in the battle against cancer, holding promise for curative responses in patients with advanced and refractory tumors. Autologous T cells, reprogrammed to target malignant cells via the expression of a chimeric antigen receptor (CAR) represent the frontrunner in this approach. Tremendous clinical regressions have been achieved using CAR-T cells against a variety of cancers both in numerous preclinical studies and in several clinical trials, most notably against acute lymphoblastic leukemia, and resulted in a very recent United States Food and Drug Administration approval of the first CAR-T-cell therapy. In most studies CARs are transferred to conventional αβT cells. Nevertheless, transferring a CAR into different cell types, such as γδT cells, natural killer cells, natural killer T cells, and myeloid cells has yet received relatively little attention, although these cell types possess unique features that may aid in surmounting some of the hurdles CAR-T-cell therapy currently faces. This review focuses on CAR therapy using effectors beyond conventional αβT cells and discusses those strategies against the backdrop of developing a safe, powerful, and durable cancer therapy.

  18. Xenosensor CAR mediates down-regulation of miR-122 and up-regulation of miR-122 targets in the liver

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Mostovich, Lyudmila A. [The Institute of Molecular Biology and Biophysics, Timakova str., 2/12, Novosibirsk 630117 (Russian Federation); Pustylnyak, Yuliya A. [Novosibirsk State University, Pirogova str., 2, Novosibirsk 630090 (Russian Federation); Pustylnyak, Vladimir O., E-mail: pustylnyak@ngs.ru [The Institute of Molecular Biology and Biophysics, Timakova str., 2/12, Novosibirsk 630117 (Russian Federation); Novosibirsk State University, Pirogova str., 2, Novosibirsk 630090 (Russian Federation); The Institute International Tomography Center of the Russian Academy of Sciences, Institutskaya str. 3-A, Novosibirsk 630090 (Russian Federation)

    2015-10-01

    MiR-122 is a major hepatic microRNA, accounting for more than 70% of the total liver miRNA population. It has been shown that miR-122 is associated with liver diseases, including hepatocellular carcinoma. Mir-122 is an intergenic miRNA with its own promoter. Pri-miR-122 expression is regulated by liver-enriched transcription factors, mainly by HNF4α, which mediates the expression via the interaction with a specific DR1 site. It has been shown that phenobarbital-mediated activation of constitutive androstane receptor (CAR), xenobiotic nuclear receptor, is associated with a decrease in miR-122 in the liver. In the present study, we investigated HNF4α–CAR cross-talk in the regulation of miR-122 levels and promitogenic signalling in mouse livers. The level of miR-122 was significantly repressed by treatment with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), which is an agonist of mouse CAR. ChIP assays demonstrated that TCPOBOP-activated CAR inhibited HNF4α transactivation by competing with HNF4α for binding to the DR1 site in the pri-miR-122 promoter. Such transcription factor replacement was strongly correlated with miR-122 down-regulation. Additionally, the decrease in miR-122 levels produced by CAR activation is accompanied by an increase in mRNA and cellular protein levels of E2f1 and its accumulation on the target cMyc gene promoter. The increase in accumulation of E2f1 on the target cMyc gene promoter is accompanied by an increase in cMyc levels and transcriptional activity. Thus, our results provide evidence to support the conclusion that CAR activation decreases miR-122 levels through suppression of HNF4α transcriptional activity and indirectly regulates the promitogenic protein cMyc. HNF4α–CAR cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments. - Highlights: • CAR activation decreased the level of miR-122 in mouse livers. • CAR decreases

  19. Xenosensor CAR mediates down-regulation of miR-122 and up-regulation of miR-122 targets in the liver

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Mostovich, Lyudmila A.; Pustylnyak, Yuliya A.; Pustylnyak, Vladimir O.

    2015-01-01

    MiR-122 is a major hepatic microRNA, accounting for more than 70% of the total liver miRNA population. It has been shown that miR-122 is associated with liver diseases, including hepatocellular carcinoma. Mir-122 is an intergenic miRNA with its own promoter. Pri-miR-122 expression is regulated by liver-enriched transcription factors, mainly by HNF4α, which mediates the expression via the interaction with a specific DR1 site. It has been shown that phenobarbital-mediated activation of constitutive androstane receptor (CAR), xenobiotic nuclear receptor, is associated with a decrease in miR-122 in the liver. In the present study, we investigated HNF4α–CAR cross-talk in the regulation of miR-122 levels and promitogenic signalling in mouse livers. The level of miR-122 was significantly repressed by treatment with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), which is an agonist of mouse CAR. ChIP assays demonstrated that TCPOBOP-activated CAR inhibited HNF4α transactivation by competing with HNF4α for binding to the DR1 site in the pri-miR-122 promoter. Such transcription factor replacement was strongly correlated with miR-122 down-regulation. Additionally, the decrease in miR-122 levels produced by CAR activation is accompanied by an increase in mRNA and cellular protein levels of E2f1 and its accumulation on the target cMyc gene promoter. The increase in accumulation of E2f1 on the target cMyc gene promoter is accompanied by an increase in cMyc levels and transcriptional activity. Thus, our results provide evidence to support the conclusion that CAR activation decreases miR-122 levels through suppression of HNF4α transcriptional activity and indirectly regulates the promitogenic protein cMyc. HNF4α–CAR cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments. - Highlights: • CAR activation decreased the level of miR-122 in mouse livers. • CAR decreases

  20. Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75(NTR)

    Skeldal, Sune; Kjaergaard, Maj M; Alwasel, Saleh

    2015-01-01

    the vps10p domain receptor sortilin and the neurotrophin receptor p75(NTR). However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay...

  1. NREL Model Car Competitions | NREL

    work together building cars with guidance from a parent, teacher, or coach to compete in race and Solar and Lithium Ion model car races in Colorado. Building solar- and battery-powered cars requires listPDF for Junior Solar Sprint and Lithium-Ion Battery model cars. Junior Solar Sprint Solar Made Pitsco

  2. Cellular dosimetry

    Humm, J.L.; Chin, L.M.

    1989-01-01

    Radiation dose is a useful predictive parameter for describing radiation toxicity in conventional radiotherapy. Traditionally, in vitro radiation biology dose-effect relations are expressed in the form of cell survival curves, a semilog plot of cell survival versus dose. However, the characteristic linear or linear quadratic survival curve shape, for high- and low-LET radiations respectively, is only strictly valid when the radiation dose is uniform across the entire target population. With an external beam of 60 Co gamma rays or x-rays, a uniform field may be readily achievable. When radionuclides are incorporated into a cell milieu, several new problems emerge which can result in a departure from uniformity in energy deposition throughout a cell population. This nonuniformity can have very important consequences for the shape of the survival curve. Cases in which perturbations of source uniformity may arise include: 1. Elemental sources may equilibrate in the cell medium with partition coefficients between the extracellular, cytosol, and nuclear compartments. The effect of preferential cell internalization or binding to cell membrane of some radionuclides can increase or decrease the slope of the survival curve. 2. Radionuclides bound to antibodies, hormones, metabolite precursors, etc., may result in a source localization pattern characteristic of the carrier agent, i.e., the sources may bind to cell surface receptors or antigens, be internalized, bind to secreted antigen concentrated around a fraction of the cell population, or become directly incorporated into the cell DNA. We propose to relate the distribution of energy deposition in cell nuclei to biological correlates of cellular inactivation. The probability of each cell's survival is weighted by its individual radiation burden, and the summation of these probabilities for the cell population can be used to predict the number or fraction of cell survivors

  3. Dynamic imaging for CAR-T-cell therapy.

    Emami-Shahri, Nia; Papa, Sophie

    2016-04-15

    Chimaeric antigen receptor (CAR) therapy is entering the mainstream for the treatment of CD19(+)cancers. As is does we learn more about resistance to therapy and the role, risks and management of toxicity. In solid tumour CAR therapy research the route to the clinic is less smooth with a wealth of challenges facing translating this, potentially hugely valuable, therapeutic option for patients. As we strive to understand our successes, and navigate the challenges, having a clear understanding of how adoptively transferred CAR-T-cells behavein vivoand in human trials is invaluable. Harnessing reporter gene imaging to enable detection and tracking of small numbers of CAR-T-cells after adoptive transfer is one way by which we can accomplish this. The compatibility of certain reporter gene systems with tracers available routinely in the clinic makes this approach highly useful for future appraisal of CAR-T-cell success in humans. © 2016 Authors; published by Portland Press Limited.

  4. New development in CAR-T cell therapy.

    Wang, Zhenguang; Wu, Zhiqiang; Liu, Yang; Han, Weidong

    2017-02-21

    Chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) have yielded unprecedented efficacy in B cell malignancies, most remarkably in anti-CD19 CAR-T cells for B cell acute lymphoblastic leukemia (B-ALL) with up to a 90% complete remission rate. However, tumor antigen escape has emerged as a main challenge for the long-term disease control of this promising immunotherapy in B cell malignancies. In addition, this success has encountered significant hurdles in translation to solid tumors, and the safety of the on-target/off-tumor recognition of normal tissues is one of the main reasons. In this mini-review, we characterize some of the mechanisms for antigen loss relapse and new strategies to address this issue. In addition, we discuss some novel CAR designs that are being considered to enhance the safety of CAR-T cell therapy in solid tumors.

  5. New development in CAR-T cell therapy

    Zhenguang Wang

    2017-02-01

    Full Text Available Abstract Chimeric antigen receptor (CAR-engineered T cells (CAR-T cells have yielded unprecedented efficacy in B cell malignancies, most remarkably in anti-CD19 CAR-T cells for B cell acute lymphoblastic leukemia (B-ALL with up to a 90% complete remission rate. However, tumor antigen escape has emerged as a main challenge for the long-term disease control of this promising immunotherapy in B cell malignancies. In addition, this success has encountered significant hurdles in translation to solid tumors, and the safety of the on-target/off-tumor recognition of normal tissues is one of the main reasons. In this mini-review, we characterize some of the mechanisms for antigen loss relapse and new strategies to address this issue. In addition, we discuss some novel CAR designs that are being considered to enhance the safety of CAR-T cell therapy in solid tumors.

  6. Aerodynamics of Race Cars

    Katz, Joseph

    2006-01-01

    Race car performance depends on elements such as the engine, tires, suspension, road, aerodynamics, and of course the driver. In recent years, however, vehicle aerodynamics gained increased attention, mainly due to the utilization of the negative lift (downforce) principle, yielding several important performance improvements. This review briefly explains the significance of the aerodynamic downforce and how it improves race car performance. After this short introduction various methods to generate downforce such as inverted wings, diffusers, and vortex generators are discussed. Due to the complex geometry of these vehicles, the aerodynamic interaction between the various body components is significant, resulting in vortex flows and lifting surface shapes unlike traditional airplane wings. Typical design tools such as wind tunnel testing, computational fluid dynamics, and track testing, and their relevance to race car development, are discussed as well. In spite of the tremendous progress of these design tools (due to better instrumentation, communication, and computational power), the fluid dynamic phenomenon is still highly nonlinear, and predicting the effect of a particular modification is not always trouble free. Several examples covering a wide range of vehicle shapes (e.g., from stock cars to open-wheel race cars) are presented to demonstrate this nonlinear nature of the flow field.

  7. Cellular localization of kinin B1 receptor in the spinal cord of streptozotocin-diabetic rats with a fluorescent [Nα-Bodipy]-des-Arg9-bradykinin

    Gaudreau Pierrette

    2009-03-01

    Full Text Available Abstract Background The kinin B1 receptor (B1R is upregulated by pro-inflammatory cytokines, bacterial endotoxins and hyperglycaemia-induced oxidative stress. In animal models of diabetes, it contributes to pain polyneuropathy. This study aims at defining the cellular localization of B1R in thoracic spinal cord of type 1 diabetic rats by confocal microscopy with the use of a fluorescent agonist, [Nα-Bodipy]-des-Arg9-BK (BdABK and selective antibodies. Methods Diabetes was induced by streptozotocin (STZ; 65 mg/kg, i.p.. Four days post-STZ treatment, B1R expression was confirmed by quantitative real-time PCR and autoradiography. The B1R selectivity of BdABK was determined by assessing its ability to displace B1R [125I]-HPP-desArg10-Hoe140 and B2R [125I]-HPP-Hoe 140 radioligands. The in vivo activity of BdABK was also evaluated on thermal hyperalgesia. Results B1R was increased by 18-fold (mRNA and 2.7-fold (binding sites in the thoracic spinal cord of STZ-treated rats when compared to control. BdABK failed to displace the B2R radioligand but displaced the B1R radioligand (IC50 = 5.3 nM. In comparison, IC50 values of B1R selective antagonist R-715 and B1R agonist des-Arg9-BK were 4.3 nM and 19 nM, respectively. Intraperitoneal BdABK and des-Arg9-BK elicited dose-dependent thermal hyperalgesia in STZ-treated rats but not in control rats. The B1R fluorescent agonist was co-localized with immunomarkers of microglia, astrocytes and sensory C fibers in the spinal cord of STZ-treated rats. Conclusion The induction and up-regulation of B1R in glial and sensory cells of the spinal cord in STZ-diabetic rats reinforce the idea that kinin B1R is an important target for drug development in pain processes.

  8. receptores

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  9. Connected Car: Quantified Self becomes Quantified Car

    Melanie Swan

    2015-02-01

    Full Text Available The automotive industry could be facing a situation of profound change and opportunity in the coming decades. There are a number of influencing factors such as increasing urban and aging populations, self-driving cars, 3D parts printing, energy innovation, and new models of transportation service delivery (Zipcar, Uber. The connected car means that vehicles are now part of the connected world, continuously Internet-connected, generating and transmitting data, which on the one hand can be helpfully integrated into applications, like real-time traffic alerts broadcast to smartwatches, but also raises security and privacy concerns. This paper explores the automotive connected world, and describes five killer QS (Quantified Self-auto sensor applications that link quantified-self sensors (sensors that measure the personal biometrics of individuals like heart rate and automotive sensors (sensors that measure driver and passenger biometrics or quantitative automotive performance metrics like speed and braking activity. The applications are fatigue detection, real-time assistance for parking and accidents, anger management and stress reduction, keyless authentication and digital identity verification, and DIY diagnostics. These kinds of applications help to demonstrate the benefit of connected world data streams in the automotive industry and beyond where, more fundamentally for human progress, the automation of both physical and now cognitive tasks is underway.

  10. Car stickers for 2009

    TS Department

    2008-01-01

    All members of the personnel holding a valid contract (except owners of cars with green or CD plates) can come to the Registration Service (Building 55, 1st floor) to obtain their 2009 car sticker, Mondays to Fridays from 7.30 a.m. to 4.00 p.m. non-stop. Please ensure you bring with you the documents relating to the vehicles(s) concerned. If you only wish to register one vehicle, you can obtain the 2009 sticker using the request form on the Web (via internet Explorer only). NB: This notice only applies to members of the personnel who obtained one or several blue car stickers for 2008. Reception and Access Control Service – TS/FM

  11. Car use within the household

    de Borger, Bruno; Mulalic, Ismir; Rouwendal, Jan

    2013-01-01

    In this paper we study the demand for car kilometres in two-car households, focusing on the substitution between cars in response to fuel price changes. We use a large sample of detailed Danish data on two-car households to estimate—for each car owned by the household—own and cross-price effects...... of increases in fuel costs per kilometre. The empirical results show that failure to capture substitution between cars within the household can result in substantial misspecification biases. Ignoring substitution, we estimate fuel price elasticities of –0.81 and -0.65 for the primary and secondary cars...... efficient car, finding partial support for the underlying hypothesis. More importantly, the results of this extended model emphasize the importance of behavioural differences related to the position of the most fuel efficient car in the household, suggesting that households’ fuel efficiency choices...

  12. What is CAR doing in the middle of the adult neurogenic road?

    Junyent, Felix; Coré, Nathalie; Cremer, Harold

    2017-01-01

    ABSTRACT The molecular and cellular basis of adult neurogenesis has attracted considerable attention for fundamental and clinical applications because neural stem cells and newborn neurons may, one day, be harnessed to replace neurons and allow cognitive improvement in the diseased brain. In rodents, neural progenitors are located in the dentate gyrus and the sub/periventricular zone. In the dentate gyrus the generation of newborn neurons is associated with plasticity, including regulation of memory. The role of subventricular zone neural precursors that migrate to the olfactory bulb is less characterized. Identifying factors that impact neural stem cell proliferation, migration and differentiation is therefore sine qua non before we can harness their potential. Here, we expand upon our recent results showing that CAR, the coxsackievirus and adenovirus receptor, is among the developing list of key players when it comes to the complex process of integrating newborn neurons into existing circuits in the mature brain. PMID:28516108

  13. The market for gasoline cars and diesel cars

    Verboven, F.

    1999-01-01

    In Europe the tax tariff is much lower for diesel fuel than for gasoline. This benefit is used by manufacturers to increase the price of diesel-fueled cars, which limits the possibility to control the use of diesel cars by means of a fiscal policy (tax incidence). Attention is paid to the impact of fiscal advantages for diesel cars on the purchasing behavior of the consumer and the pricing policy (price discrimination) of the car manufacturers. 1 ref

  14. Gas Tank for Cars

    Peter Lorenz

    2011-09-01

    Full Text Available In this work the development of a highly efficient pressure vessel for liquid petroleum gas (LPG in integral design is described. The pressure vessel can be customized in an optimal available installation space and thus means that the suitable for everyday use of existing modified cars or trucks can be increased.

  15. Modeling the Mousetrap Car

    Jumper, William D.

    2012-01-01

    Many high school and introductory college physics courses make use of mousetrap car projects and competitions as a way of providing an engaging hands-on learning experience incorporating Newton's laws, conversion of potential to kinetic energy, dissipative forces, and rotational mechanics. Presented here is a simple analytical and finite element…

  16. Race Car Rally.

    Anthony, Joan L.

    1994-01-01

    Describes an activity where teams of parents and children work together to solve problems involving matchbox-sized race cars. The teams collect, record, and analyze data; measure distances in metric; and explore concepts related to mass, friction, and force. (PR)

  17. Lego Car Race

    Preston, Christine

    2017-01-01

    Familiar toys can be used to scaffold young children's learning about basic physics as well as guide scientific inquiry. Teachers looking for resources to engage young children and develop science inquiry skills need look no further than the toy box. In this two-part activity, children first construct a Lego® car and use it to explore the effects…

  18. Motor car driving; Kraftfahrzeugfuehrung

    Juergensohn, T. [Technische Univ. Berlin (Germany). ISS-Fahrzeugtechnik; Timpe, K.P. (eds.) [Technische Univ. Berlin (DE). Zentrum Mensch-Maschine-Systeme (ZMMS)

    2001-07-01

    This is the first comprehensive book on motor car driving, i.e. all aspects of motor car technology that cannot be looked at separately from the needs, characteristics and limitations of the human driver. This includes ergonomics as well as the design of the driver interface in consideration of the findings of cognitive science, problems of driving simulation in the context of simulation of technical systems, problems relating to optimal car automation up to traffic psychology. The book is in honour of Prof. Dr. Willumeit who died in summer 2000. Prof. Willumeit was one of the few scientists in Germany who had been an expert on all aspects of motor car driving for many years. [German] Erstmalig wird das Thema der Fahrzeugfuehrung geschlossen dargestellt. Die Thematik der 'Kraftfahrzeugfuehrung' umfasst in diesem Zusammenhang alle Aspekte der Kraftfahrzeugtechnik, die nicht isoliert von den Erfordernissen, Eigenschaften und Grenzen des menschlichen Fahrers betrachtet werden koennen. Dies beinhaltet u.a. Probleme der Ergonomie, aber auch Fragen nach einer kognitionswissenschaftlich unterstuetzten Schnittstellengestaltung, Fragen der Simulation des Fahrverhalten im Kontext der Simulation technischer Systeme oder Fragen einer optimalen Fahrzeugautomatisierung bis hin zu verkehrspsychologischen Aspekten. Das Buch ist als Gedenkband fuer Prof. Dr. Willumeit konzipiert, der im Sommer 2000 verstarb. Prof. Willumeit war einer der wenigen Wissenschaftler in Deutschland, der ueber viele Jahre diese Thematik der Kraftfahrzeugfuehrung in ihrer vollen Breite verfolgte. (orig.)

  19. Cellular gravity

    F.C. Gruau; J.T. Tromp (John)

    1999-01-01

    textabstractWe consider the problem of establishing gravity in cellular automata. In particular, when cellular automata states can be partitioned into empty, particle, and wall types, with the latter enclosing rectangular areas, we desire rules that will make the particles fall down and pile up on

  20. Misuse of car safety seats.

    Bull, M J; Stroup, K B; Gerhart, S

    1988-01-01

    Correct use of car seats for small children is essential to prevent serious injuries and death from automotive accidents. Failure to use a car seat properly can contribute to serious injury or death of a child. A case study in which misuse of a car seat occurred is reported. The infant died of hemorrhage and shock secondary to liver laceration which resulted from excessive pressure over the abdomen sustained on impact. Surveys of car seat use for small children prior to and following a child restraint law are also reported. Observers noted types of car seats and specific forms of misuse. Survey results suggest that parents are more likely to misuse car seats for infants than toddlers. Medical professionals can reinforce the importance of proper car seat use by incorporating specific car seat use questions into the patient interview and by providing educational materials.

  1. Design of an intelligent car

    Na, Yongyi

    2017-03-01

    The design of simple intelligent car, using AT89S52 single chip microcomputer as the car detection and control core; The metal sensor TL - Q5MC induction to iron, to detect the way to send feedback to the signal of single chip microcomputer, make SCM according to the scheduled work mode to control the car in the area according to the predetermined speed, and the operation mode of the microcontroller choose different also can control the car driving along s-shaped iron; Use A44E hall element to detect the car speeds; Adopts 1602 LCD display time of car driving, driving the car to stop, take turns to show the car driving time, distance, average speed and the speed of time. This design has simple structure and is easy to implement, but are highly intelligent, humane, to a certain extent reflects the intelligence.

  2. Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia

    Wei, Li; Tokizane, Kyohei; Konishi, Hiroyuki; Yu, Hua-Rong; Kiyama, Hiroshi

    2017-01-01

    Background Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood...

  3. Our Car as Power Plant

    Van Wijk, A.J.M.; Verhoef, L.

    2014-01-01

    Fuel cell cars can provide more efficient and cleaner transportation. However, we use our cars for transportation only 5% of the time. When parked, the fuel cell in the car can produce electricity from hydrogen, which is cleaner and more efficient than the current electricity system, generating

  4. DOES ELECTRIC CAR PRODUCE EMISSIONS?

    Vladimír RIEVAJ

    2017-03-01

    Full Text Available This article focuses on the comparison of the amount of emissions produced by vehicles with a combustion engine and electric cars. The comparison, which is based on the LCA factor results, indicates that an electric car produces more emissions than a vehicle with combustion engine. The implementation of electric cars will lead to an increase in the production of greenhouse gases.

  5. IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor.

    Adachi, Keishi; Kano, Yosuke; Nagai, Tomohiko; Okuyama, Namiko; Sakoda, Yukimi; Tamada, Koji

    2018-04-01

    Infiltration, accumulation, and survival of chimeric antigen receptor T (CAR-T) cells in solid tumors is crucial for tumor clearance. We engineered CAR-T cells to express interleukin (IL)-7 and CCL19 (7 × 19 CAR-T cells), as these factors are essential for the maintenance of T-cell zones in lymphoid organs. In mice, 7 × 19 CAR-T cells achieved complete regression of pre-established solid tumors and prolonged mouse survival, with superior anti-tumor activity compared to conventional CAR-T cells. Histopathological analyses showed increased infiltration of dendritic cells (DC) and T cells into tumor tissues following 7 × 19 CAR-T cell therapy. Depletion of recipient T cells before 7 × 19 CAR-T cell administration dampened the therapeutic effects of 7 × 19 CAR-T cell treatment, suggesting that CAR-T cells and recipient immune cells collaborated to exert anti-tumor activity. Following treatment of mice with 7 × 19 CAR-T cells, both recipient conventional T cells and administered CAR-T cells generated memory responses against tumors.

  6. Transcriptional activation of PPARalpha by phenobarbital in the absence of CAR and PXR.

    Tamasi, Viola; Juvan, Peter; Beer, Markus; Rozman, Damjana; Meyer, Urs A

    2009-01-01

    The nuclear receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor) mediate the effects of phenobarbital on gene transcription. To investigate the relative contribution of these nuclear receptors to the expression of specific genes we studied the effect of phenobarbital in livers of wild type, CAR(-/-), PXR(-/-) and CAR/PXR(-/-) knockout mice. Spotted Steroltalk v1 cDNA arrays were applied containing probes for genes involved in drug metabolism, sterol biosynthesis, steroid synthesis/transport and heme synthesis. In the absence of CAR and PXR, phenobarbital unexpectedly induced mRNAs of several nuclear receptors, including PPARalpha and its target genes Cyp4a10 and Cyp4a14. Interestingly, in primary cultures of hepatocytes isolated from CAR/PXR(-/-) knockout mice, phenobarbital increased HNF-4alpha levels. In further experiments in these hepatocyte cultures we provide evidence that phenobarbital directly induces transcription of the PPARalpha gene via its HNF-4alpha response element, and indirectly by lack of inhibitory crosstalk of AMPK, CAR and PXR with HNF-4alpha. Our results provide further insight into CAR and PXR-independent effects of phenobarbital and the crosstalk between different nuclear receptor signaling pathways.

  7. Calcium-Induced Activation of a Mutant G-Protein-Coupled Receptor Causes In Vitro Transformation of NIH/3T3 Cells

    Ana O. Hoff

    1999-12-01

    Full Text Available The calcium-sensing receptor (CaR is a G-proteincoupled receptor that is widely expressed, has tissuespecific functions, regulates cell growth. Activating mutations of this receptor cause autosomal dominant hypocalcemia, a syndrome characterized by hypocalcemia and hypercalciuria. The identification of a family with an activating mutation of the CaR (Thr151 Met in which hypocalcemia cosegregates with several unusual neoplasms led us to examine the transforming effects of this mutant receptor. Transfection of NIH/3T3 cells with the mutant but not the normal receptor supported colony formation in soft agar at subphysiologic calcium concentrations. The mutant CaR causes a calcium-dependent activation of the extracellular signal-regulated protein kinase (ERK 1/2 and Jun-N-terminal kinase/stress-activated (JNK/ SAPK pathways, but not P38 MAP kinase. These findings contribute to a growing body of information suggesting that this receptor plays a role in the regulation of cellular proliferation, that aberrant activation of the mutant receptor in this family may play a role in the unusual neoplastic manifestations.

  8. CERN car stickers 2005

    Reception and Access Control Service - TS Department

    2004-01-01

    Please note that the car stickers for 2005 are now available. If you have not received the new one by internal mail, you should go along in person to the Registration Service (bldg. 55 - 1st floor), open non-stop from 7:30 to 16:30, taking with you your 2004 sticker and the log-book of your vehicle. Thank you for your collaboration. Reception and Access Control Service - TS Department.

  9. THE EFFECT OF CELLULAR PHONE USE ON DRIVING PERFORMANCE

    Toshiro ISHIDA

    2001-01-01

    Many experiments using driving simulators or real roads have shown that using a cellular phone while driving may cause an accident because it delays visual information processing by the driver. In this research, we examined the influence on driving performance of cellular phone use on a course that simulated streets. Driving conditions were driving only, listening to the car radio, hands-free cellular phone use and using a cellular phone with the left hand. Driving performance measurements in...

  10. CuseCar--community car-sharing program : car sharing lessons learned.

    2011-08-01

    CuseCar of Syracuse launched services in December 2008 with 3 Toyota Prius Hybrids. CuseCar initially, due to : concerns about availability, limited membership to Origination Sponsor Locations, which in turn developed few : members. In 2009 CuseCar o...

  11. Individual differences in ethanol locomotor sensitization are associated with dopamine D1 receptor intra-cellular signaling of DARPP-32 in the nucleus accumbens.

    Karina Possa Abrahao

    Full Text Available In mice there are clear individual differences in the development of behavioral sensitization to ethanol, a progressive potentiation of its psychomotor stimulant effect. Variability in the behavioral responses to ethanol has been associated with alcohol preference. Here we investigated if the functional hyperresponsiveness of D1 receptors observed in ethanol sensitized mice leads to an increased activation of DARPP-32, a central regulatory protein in medium spiny neurons, in the nucleus accumbens - a brain region known to play a role in drug reinforcement. Swiss Webster mice received ethanol (2.2 g/kg/day or saline i.p. administrations for 21 days and were weekly evaluated regarding their locomotor activity. From those treated with ethanol, the 33% with the highest levels of locomotor activity were classified as "sensitized" and the 33% with the lowest levels as "non-sensitized". The latter presented similar locomotor levels to those of saline-treated mice. Different subgroups of mice received intra-accumbens administrations of saline and, 48 h later, SKF-38393, D1 receptor agonist 0.1 or 1 µg/side. Indeed, sensitized mice presented functional hyperresponsiveness of D1 receptors in the accumbens. Two weeks following the ethanol treatment, other subgroups received systemic saline or SKF 10 mg/kg, 20 min before the euthanasia. The nucleus accumbens were dissected for the Western Blot analyses of total DARPP-32 and phospho-Thr34-DARPP-32 expression. D1 receptor activation induced higher phospho-Thr34-DARPP-32 expression in sensitized mice than in non-sensitized or saline. The functionally hyperresponsiveness of D1 receptors in the nucleus accumbens is associated with an increased phospho-Thr34-DARPP-32 expression after D1 receptor activation. These data suggest that an enduring increase in the sensitivity of the dopamine D1 receptor intracellular pathway sensitivity represents a neurobiological correlate associated with the development of

  12. Development of CAR T cells designed to improve antitumor efficacy and safety

    Jaspers, Janneke E.; Brentjens, Renier J.

    2017-01-01

    Chimeric antigen receptor (CAR) T cell therapy has shown promising efficacy against hematologic malignancies. Antitumor activity of CAR T cells, however, needs to be improved to increase therapeutic efficacy in both hematologic and solid cancers. Limitations to overcome are ‘on-target, off-tumor’ toxicity, antigen escape, short CAR T cell persistence, little expansion, trafficking to the tumor and inhibition of T cell activity by an inhibitory tumor microenvironment. Here we will discuss how ...

  13. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor.

    Wu, Chia-Yung; Roybal, Kole T; Puchner, Elias M; Onuffer, James; Lim, Wendell A

    2015-10-16

    There is growing interest in using engineered cells as therapeutic agents. For example, synthetic chimeric antigen receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, these engineered T cells can exhibit excessive activity that is difficult to control and can cause severe toxicity. We designed "ON-switch" CARs that enable small-molecule control over T cell therapeutic functions while still retaining antigen specificity. In these split receptors, antigen-binding and intracellular signaling components assemble only in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate cell-autonomous recognition and user control. Copyright © 2015, American Association for the Advancement of Science.

  14. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor

    Wu, Chia-Yung; Roybal, Kole T.; Puchner, Elias M.; Onuffer, James; Lim, Wendell A.

    2016-01-01

    There is growing promise in using engineered cells as therapeutic agents. For example, synthetic Chimeric Antigen Receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, excessive activity and poor control over such engineered T cells can cause severe toxicities. We present the design of “ON-switch” CARs that enable small molecule-control over T cell therapeutic functions, while still retaining antigen specificity. In these split receptors, antigen binding and intracellular signaling components only assemble in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate both cell autonomous recognition and user control. PMID:26405231

  15. Small-molecule modulators of PXR and CAR

    Chai, Sergio C.; Cherian, Milu T.; Wang, Yue-Ming; Chen, Taosheng

    2016-01-01

    Two nuclear receptors, the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), participate in the xenobiotic detoxification system by regulating the expression of drug-metabolizing enzymes and transporters in order to degrade and excrete foreign chemicals or endogenous metabolites. This review aims to expand the perceived relevance of PXR and CAR beyond their established role as master xenosensors to disease-oriented areas, emphasizing their modulation by small molecules. Structural studies of these receptors have provided much-needed insight into the nature of their binding promiscuity and the important elements that lead to ligand binding. Reports of species- and isoform-selective activation highlight the need for further scrutiny when extrapolating from animal data to humans, as animal models are at the forefront of early drug discovery. PMID:26921498

  16. [Current Status and Challenges of CAR-T Immunotherapy in Hematologic Malignancies -Review].

    Cheng, Xin; Wang, Ya-Jie; Feng, Shuai; Wu, Ya-Yun; Yang, Tong-Hua; Lai, Xun

    2018-04-01

    The chimeric antigen receptor (CAR) T cell therapy has gradually became a new trend in the treatment of refractory and relapsed hematologic malignancies by developing for 30 years. With the exciting development of genetic engineering, CAR-T technology has subjected to 4 generations of innovation. Structure of CAR-T started from a single signal molecule to 2 or more than 2 co-stimulatory molecules, and then coding the CAR gene or promoter. CAR-T can specifically recognize tumor antigens, and does not be restricted by major histocompatibility complex (MHC), thus making a breakthrough in clinical treatment. In this review, the history, structure and mechanism of action of CAR-T, as well as the current status and challenges of CAR-T immunotherapy in acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia and multiple myeloma are summarized.

  17. CAR-T Cell Therapies From the Transfusion Medicine Perspective.

    Fesnak, Andrew; Lin, ChieYu; Siegel, Don L; Maus, Marcela V

    2016-07-01

    The use of chimeric antigen receptor (CAR)-T cell therapy for the treatment of hematologic malignancies has generated significant excitement over the last several years. From a transfusion medicine perspective, the implementation of CAR-T therapy as a potential mainstay treatment for not only hematologic but also solid-organ malignancies represents a significant opportunity for growth and expansion. In this review, we will describe the rationale for the development of genetically redirected T cells as a cancer therapeutic, the different elements that are required to engineer these cells, as well as an overview of the process by which patient cells are harvested and processed to create and subsequently validate CAR-T cells. Finally, we will briefly describe some of the toxicities and clinical efficacy of CAR-T cells in the setting of patients with advanced malignancy. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Toxicity and management in CAR T-cell therapy

    Challice L Bonifant

    2016-01-01

    Full Text Available T cells can be genetically modified to target tumors through the expression of a chimeric antigen receptor (CAR. Most notably, CAR T cells have demonstrated clinical efficacy in hematologic malignancies with more modest responses when targeting solid tumors. However, CAR T cells also have the capacity to elicit expected and unexpected toxicities including: cytokine release syndrome, neurologic toxicity, “on target/off tumor” recognition, and anaphylaxis. Theoretical toxicities including clonal expansion secondary to insertional oncogenesis, graft versus host disease, and off-target antigen recognition have not been clinically evident. Abrogating toxicity has become a critical step in the successful application of this emerging technology. To this end, we review the reported and theoretical toxicities of CAR T cells and their management.

  19. Towards safe and effective CD38-CAR T cell therapy for myeloma

    Drent, Esther

    2018-01-01

    Immunotherapy is a promising field within cancer therapy. The recent progresses resulted in 'Immunotherapy for the treatment of cancer' as break-through of the year in 2013. This was partly due to the great successes with Chimeric Antigen Receptor (CAR) T cell therapy. With CAR T cells, recognition

  20. Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

    Kebriaei, Partow; Singh, Harjeet; Huls, M. Helen; Figliola, Matthew J.; Bassett, Roland; Olivares, Simon; Jena, Bipulendu; Dawson, Margaret J.; Kumaresan, Pappanaicken R.; Su, Shihuang; Maiti, Sourindra; Dai, Jianliang; Moriarity, Branden; Forget, Marie-Andrée; Senyukov, Vladimir

    2016-01-01

    BACKGROUND. T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR.

  1. Building a CAR Garage: Preparing for the Delivery of Commercial CAR T Cell Products at Memorial Sloan Kettering Cancer Center.

    Perica, Karlo; Curran, Kevin J; Brentjens, Renier J; Giralt, Sergio A

    2018-03-01

    Two commercial chimeric antigen receptor (CAR) T cell therapies for CD19-expressing B cell malignancies, Kymriah and Yescarta, have recently been approved by the Food and Drug Administration. The administration of CAR T cells is a complex endeavor involving cell manufacture, tracking and shipping of apheresis products, and management of novel and severe toxicities. At Memorial Sloan Kettering Cancer Center, we have identified 8 essential tasks that define the CAR T cell workflow. In this review, we discuss practical aspects of CAR T cell program development, including clinical, administrative, and regulatory challenges for successful implementation. Copyright © 2018 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  2. The human angiotensin AT(1) receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation

    Hansen, Jakob Lerche; Aplin, Mark; Hansen, Jonas Tind

    2008-01-01

    The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensi...

  3. Deficient p75 low-affinity neurotrophin receptor expression does alter the composition of cellular infiltrate in experimental autoimmune encephalomyelitis in C57BL/6 mice

    Kust, B; Mantingh-Otter, [No Value; Boddeke, E; Copray, S

    We have shown earlier that induction of experimental autoimmune encephalomyelitis (EAE)-a model for the human disease multiple sclerosis-in C5713L/6 wild-type mice resulted in the expression of the p75 low-affinity neurotrophin receptor (p75(NTR)) in endothelial cells in the CNS. In comparison to

  4. Molecular chaperones enhance the degradation of expanded polyglutamine repeat androgen receptor in a cellular model of spinal and bulbar muscular atrophy

    Bailey, CK; Andriola, IFM; Kampinga, HH; Merry, DE

    2002-01-01

    Spinal and bulbar muscular atrophy (SBMA) is one of a growing number of neurodegenerative diseases caused by a polyglutamine-encoding CAG trinucleotide repeat expansion, and is caused by an expansion within exon 1 of the androgen receptor (AR) gene. The family of polyglutamine diseases is

  5. Proliferation of NS0 cells in protein-free medium: the role of cell-derived proteins, known growth factors and cellular receptors.

    Spens, Erika; Häggström, Lena

    2009-05-20

    NS0 cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may open for the novel approaches to improve animal cell processes. The following proteins were identified in NS0 conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6, TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11, IL-25, MIF, TGF-beta and TNF-alpha as well as the known growth factors EGF, IGF-I/II, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NS0 cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NS0 cell membranes since TGF-beta(1) caused cell death, and IGF-I/II and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NS0 cells as demonstrated by siRNA gene silencing.

  6. Car stickers for 2010

    GS Department

    The 2010 car stickers are now available. Holders of blue stickers will receive their 2010 stickers through the internal mail from 1st December onwards. Holders of red stickers are required to go to the Registration Service (Building 55, first floor), which is open non-stop from 7.30 a.m. to 4.00 p.m. Mondays to Fridays, in order to obtain their new stickers. They will be asked to present documents relating to the vehicles concerned. Owners of vehicles registered on green and CD plates should disregard this message. Reception and Access Control Service – GS/SEM/LS

  7. Car monitoring information systems

    Alica KALAŠOVÁ

    2008-01-01

    Full Text Available The objective of this contribution is to characterize alternatives of information systems used for managing, processing and evaluation of information related to company vehicles. Especially we focus on logging, transferring and processing of on-road vehicle movement information in inland and international transportation. This segment of company information system has to monitor the car movement – actively or passively – according to demand of the company and after the processing it has to evaluate and give the complex monitoring of a situation of all the company vehicles to the controller.

  8. Flying car design and testing

    Klein, S.; Smrcek, L.

    2009-01-01

    This paper is primarily concerned with the inverted design process and manufacture of a flying car prototype which can overcome the problem of traffic management in the world today. A possible solution to the problem of overcrowded roads would be to design a flying or hovering car. Given technological advances in aircraft construction, navigation and operation, flying cars or personal aircraft are now a feasible proposition. The viability of such a concept was investigated in terms of produci...

  9. Electric Cars and Oil Prices

    Azar, Jose

    2009-01-01

    This paper studies the joint dynamics of oil prices and interest in electric cars, measured as the volume of Google searches for related phrases. Not surprisingly, I find that oil price shocks predict increases in Google searches for electric cars. Much more surprisingly, I also find that an increase in Google searches predicts declines in oil prices. The high level of public interest in electric cars between April and August of 2008 can explain approximately half of the decline in oil prices...

  10. Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma

    Jelena Skuljec

    2017-09-01

    Full Text Available Cellular therapy with chimeric antigen receptor (CAR-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR and a chronic, T helper-2 (Th2 cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma.

  11. Product declaration for cars

    Gruetter, J.M.

    2000-01-01

    This reports for the Swiss Federal Office of Energy (SFOE) presents the results of a study made on the possible ways of declaring product information on cars. The basic elements of such a declaration are discussed and a recommendation for an energy label for cars is presented. The report discusses the fundamental questions posed such as how long a label should be valid, if comparisons should be made and if it is to be based on CO 2 -emissions or on fuel consumption. Also, the criteria to be used for comparisons - such as vehicle weight, size or power - are looked at and methods of classification are examined along with data fundamentals. Further, the expectations placed on the product declarations with respect to their energetic and economic impact are discussed. The design of the label and the legislature on which it is based are discussed and initial reactions of the automobile industry are noted. The report is rounded off by a discussion of the effects of the declaration in relation to other instruments that have been proposed

  12. EP3 receptors inhibit antidiuretic-hormone-dependent sodium transport across frog skin epithelium

    Rytved, Klaus A.; Nielsen, Robert

    1999-01-01

    Antidiuretic hormone; tight epithelium; prostaglandin receptors; sulprostone; misoprostol; cAMP; cellular Ca2+......Antidiuretic hormone; tight epithelium; prostaglandin receptors; sulprostone; misoprostol; cAMP; cellular Ca2+...

  13. D-serine deficiency attenuates the behavioral and cellular effects induced by the hallucinogenic 5-HT(2A) receptor agonist DOI

    Santini, Martin A; Balu, Darrick T; Puhl, Matthew D

    2014-01-01

    Both the serotonin and glutamate systems have been implicated in the pathophysiology of schizophrenia, as well as in the mechanism of action of antipsychotic drugs. Psychedelic drugs act through the serotonin 2A receptor (5-HT2AR), and elicit a head-twitch response (HTR) in mice, which directly...... correlates to 5-HT2AR activation and is absent in 5-HT2AR knockout mice. The precise mechanism of this response remains unclear, but both an intrinsic cortico-cortical pathway and a thalamo-cortical pathway involving glutamate release have been proposed. Here, we used a genetic model of NMDAR hypofunction......RNA. These altered functional responses in SRKO mice were not associated with changes in cortical or hippocampal 5-HT levels or in 5-HT2AR and metabotropic glutamate-2 receptor (mGluR2) mRNA and protein expression. Together, these findings suggest that D-serine-dependent NMDAR activity is involved in mediating...

  14. Proton - Malaysia's national car project

    Fleming, Daniel; Søborg, Henrik

    2017-01-01

    The rise and development of the Malaysian national car project. How this project has become an esential part of the industrial development in Malaysia and how it has underpinned a growing middle class consumption culture with house and car as it pivotal goods.......The rise and development of the Malaysian national car project. How this project has become an esential part of the industrial development in Malaysia and how it has underpinned a growing middle class consumption culture with house and car as it pivotal goods....

  15. Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia.

    Wei, Li; Tokizane, Kyohei; Konishi, Hiroyuki; Yu, Hua-Rong; Kiyama, Hiroshi

    2017-10-03

    Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood. We used endogenous, natural, and surrogate agonists for GPR84 (capric acid, embelin, and 6-OAU, respectively) and examined their effect on mouse primary cultured microglia in vitro. 6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 gene. GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury.

  16. Background free CARS imaging by phase sensitive heterodyne CARS

    Jurna, M.; Korterik, Jeroen P.; Otto, Cornelis; Herek, Jennifer Lynn; Offerhaus, Herman L.

    2008-01-01

    In this article we show that heterodyne CARS, based on a controlled and stable phase-preserving chain, can be used to measure amplitude and phase information of molecular vibration modes. The technique is validated by a comparison of the imaginary part of the heterodyne CARS spectrum to the

  17. Quarter Century of Anti-HIV CAR T Cells.

    Wagner, Thor A

    2018-04-01

    A therapy that might cure HIV is a very important goal for the 30-40 million people living with HIV. Chimeric antigen receptor T cells have recently had remarkable success against certain leukemias, and there are reasons to believe they could be successful for HIV. This manuscript summarizes the published research on HIV CAR T cells and reviews the current anti-HIV chimeric antigen receptor strategies. Research on anti-HIV chimeric antigen receptor T cells has been going on for at least the last 25 years. First- and second-generation anti-HIV chimeric antigen receptors have been developed. First-generation anti-HIV chimeric antigen receptors were studied in clinical trials more than 15 years ago, but did not have meaningful clinical efficacy. There are some reasons to be optimistic about second-generation anti-HIV chimeric antigen receptor T cells, but they have not yet been tested in vivo.

  18. Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy

    Yeku, Oladapo O.; Brentjens, Renier J.

    2016-01-01

    Chimaeric antigen receptor (CAR) T-cells are T-cells that have been genetically modified to express an artificial construct consisting of a synthetic T-cell receptor (TCR) targeted to a predetermined antigen expressed on a tumour. Coupling the T-cell receptor to a CD3ζ signalling domain paved the way for first generation CAR T-cells that were efficacious against cluster of differentiation (CD)19-expressing B-cell malignancies. Optimization with additional signalling domains such as CD28 or 4-...

  19. Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals.

    Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T; Ackerman, Margaret E; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

    2011-07-05

    In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Decreased Fc-Receptor expression on innate immune cells is associated with impaired antibody mediated cellular phagocytic activity in chronically HIV-1 infected individuals

    Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T.; Ackerman, Margaret E.; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

    2011-01-01

    In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular-phagocytosis (ADCP), antibody dependent cellular-cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. PMID:21565376

  1. CAR-T therapy for leukemia: progress and challenges.

    Wang, Xin; Xiao, Qing; Wang, Zhe; Feng, Wen-Li

    2017-04-01

    Despite the rapid development of therapeutic strategies, leukemia remains a type of difficult-to-treat hematopoietic malignancy that necessitates introduction of more effective treatment options to improve life expectancy and quality of patients. Genetic engineering in adoptively transferred T cells to express antigen-specific chimeric antigen receptors (CARs) has proved highly powerful and efficacious in inducing sustained responses in patients with refractory malignancies, as exemplified by the success of CD19-targeting CAR-T treatment in patients with relapsed acute lymphoblastic leukemia. Recent strategies, including manipulating intracellular activating domains and transducing viral vectors, have resulted in better designed and optimized CAR-T cells. This is further facilitated by the rapid identification of an accumulating number of potential leukemic antigens that may serve as therapeutic targets for CAR-T cells. This review will provide a comprehensive background and scrutinize recent important breakthrough studies on anti-leukemia CAR-T cells, with focus on recently identified antigens for CAR-T therapy design and approaches to overcome critical challenges. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Regulation of G-protein coupled receptor traffic by an evolutionary conserved hydrophobic signal.

    Angelotti, Tim; Daunt, David; Shcherbakova, Olga G; Kobilka, Brian; Hurt, Carl M

    2010-04-01

    Plasma membrane (PM) expression of G-protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c-adrenergic receptors (alpha(2c)-ARs), heterologous expression in non-native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric alpha(2a)/alpha(2c)-AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for alpha(2c)-AR subtype-specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into alpha(2a)-ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within alpha(2c)-ARs serves as a regulator of GPCR trafficking.

  3. Cellular metabolism

    Hildebrand, C.E.; Walters, R.A.

    1977-01-01

    Progress is reported on the following research projects: chromatin structure; the use of circular synthetic polydeoxynucleotides as substrates for the study of DNA repair enzymes; human cellular kinetic response following exposure to DNA-interactive compounds; histone phosphorylation and chromatin structure in cell proliferation; photoaddition products induced in chromatin by uv light; pollutants and genetic information transfer; altered RNA metabolism as a function of cadmium accumulation and intracellular distribution in cultured cells; and thymidylate chromophore destruction by water free radicals

  4. Frontline Science: Functionally impaired geriatric CAR-T cells rescued by increased α5β1 integrin expression.

    Guha, Prajna; Cunetta, Marissa; Somasundar, Ponnandai; Espat, N Joseph; Junghans, Richard P; Katz, Steven C

    2017-08-01

    Chimeric antigen receptor expressing T cells (CAR-T) are a promising form of immunotherapy, but the influence of age-related immune changes on CAR-T production remains poorly understood. We showed that CAR-T cells from geriatric donors (gCAR-T) are functionally impaired relative to CAR-T from younger donors (yCAR-T). Higher transduction efficiencies and improved cell expansion were observed in yCAR-T cells compared with gCAR-T. yCAR-T demonstrated significantly increased levels of proliferation and signaling activation of phosphorylated (p)Erk, pAkt, pStat3, and pStat5. Furthermore, yCAR-T contained higher proportions of CD4 and CD8 effector memory (EM) cells, which are known to have enhanced cytolytic capabilities. Accordingly, yCAR-T demonstrated higher levels of tumor antigen-specific cytotoxicity compared with gCAR-T. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin and granzyme B. yCAR-T had increased α5β1 integrin expression, a known mediator of retroviral transduction. We found that treatment with M-CSF or TGF-β1 rescued the impaired transduction efficiency of the gCAR-T by increasing the α5β1 integrin expression. Neutralization of α5β1 confirmed that this integrin was indispensable for CAR expression. Our study suggests that the increase of α5β1 integrin expression levels enhances CAR expression and thereby improves tumor killing by gCAR-T. © Society for Leukocyte Biology.

  5. Effects of insulin analogs and glucagon-like peptide-1 receptor agonists on proliferation and cellular energy metabolism in papillary thyroid cancer

    He L

    2017-11-01

    Full Text Available Liang He,1,* Siliang Zhang,2,* Xiaowen Zhang,3 Rui Liu,2 Haixia Guan,2 Hao Zhang1 1Department of Thyroid Surgery, The First Hospital of China Medical University, Shenyang, Liaoning, 2Department of Endocrinology and Metabolism, The Endocrine Institute and The Liaoning Provincial Key Laboratory of Endocrine Diseases, The First Hospital of China Medical University, Shenyang, Liaoning, 3Department of Endocrinology and Metabolism, Drum Tower Hospital Affiliated to Nanjing University Medical School, Nanjing, People’s Republic of China *These authors contributed equally to this work Purpose: This study was aimed to investigate the expressions of the insulin receptor (IR, insulin-like growth factor receptor (IGF-1R, and glucagon-like peptide-1 receptor (GLP-1R in normal thyroid tissue, papillary thyroid cancer (PTC tissues, and PTC cells, and to examine the possible role of insulin analogs and GLP-1R agonists in cell proliferation and energy metabolism in PTC cells.Methods: The expressions of IR, IGF-1R, and GLP-1R in PTC tissues and PTC cell lines were detected by immunohistochemistry and western blotting, respectively. Cell proliferation was evaluated by the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay. Levels of members of the phosphoinositol-3 kinase/AKT serine/threonine kinase (Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk signaling pathways were measured by western blotting. Energy metabolism of PTC cell lines was analyzed using a Seahorse Extracellular Flux analyzer.Results: Three receptors could be detected in both PTC tissues and PTC cell lines. Expressions of IGF-1R and GLP-1R were more obvious in PTC than in normal thyroid cells. Neither insulin, four insulin analogs, and two GLP-1R agonists showed significant effects on the proliferation of PTC cells, nor did they influence the levels of Akt/p-Akt and Erk/p-Erk. None of these antidiabetic agents could change the mitochondrial

  6. Initial heating in cold cars

    Daanen, H.A.M.; Teunissen, L.P.J.; Hoogh, I.M. de

    2012-01-01

    During the initial minutes after entering a cold car, people feel uncomfortably cold. Six different warming systems were investigated in a small car in order to find out how to improve the feeling of comfort using 16 volunteers. The methods were: no additional warming next to a standard heating

  7. Energy Use of Passenger Cars

    Jørgensen, Kaj

    1998-01-01

    Analysis of the Danish sale and stock of passenger cars, focusing particularly on aspects influencing energy use. The project has tracked the development of vehicle weight, power and fuel economy for both the sale of new cars (from 1980 to 1997)and the stock. In addition, the energy use...

  8. Shopping for a safer car

    2010-01-01

    This brochure provides some helpful tips on what to look for when shopping for a safer car. Automakers are increasingly advertising the safety features of their cars. The problem is sorting out their claims and zeroing in on the safety features that ...

  9. FUEL PRICES AND CAR SALES

    Vlad Cârstea

    2008-01-01

    Automotive industry is a very important economic sector that is highly responsive to changes in the world economy. The fuel price is the biggest enemy of car manufacturers. This is a compared analysis between Europe and Romania regarding new car registrations.

  10. Panorama 2014 - Car-sharing

    Vinot, Simon

    2013-10-01

    Car-sharing is a new mode of transportation that consists of multiple users sharing the same vehicle. This type of service is expanding with the arrival of larger players, such as traditional car rental companies, automotive manufacturers, and large firms specializing in transportation. This new mode of transportation offers real potential and is currently finding its users, in France and worldwide. (author)

  11. New Approaches in CAR-T Cell Immunotherapy for Breast Cancer.

    Wang, Jinghua; Zhou, Penghui

    2017-01-01

    Despite significant advances in surgery, chemotherapy, radiotherapy, endocrine therapy, and molecular-targeted therapy, breast cancer remains the leading cause of death from malignant tumors among women. Immunotherapy has recently become a critical component of breast cancer treatment with encouraging activity and mild safety profiles. CAR-T therapy using genetically modifying T cells with chimeric antigen receptors (CAR) is the most commonly used approach to generate tumor-specific T cells. It has shown good curative effect for a variety of malignant diseases, especially for hematological malignancies. In this review, we briefly introduce the history and the present state of CAR research. Then we discuss the barriers of solid tumors for CARs application and possible strategies to improve therapeutic response with a focus on breast cancer. At last, we outlook the future directions of CAR-T therapy including managing toxicities and developing universal CAR-T cells.

  12. CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells.

    Zhang, Yongping; Zhang, Xingying; Cheng, Chen; Mu, Wei; Liu, Xiaojuan; Li, Na; Wei, Xiaofei; Liu, Xiang; Xia, Changqing; Wang, Haoyi

    2017-12-01

    T cells engineered with chimeric antigen receptor (CAR) have been successfully applied to treat advanced refractory B cell malignancy. However, many challenges remain in extending its application toward the treatment of solid tumors. The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CAR-T efficacy. One negative regulator of Tcell activity is lymphocyte activation gene-3 (LAG-3). We successfully generated LAG-3 knockout Tand CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture. LAG-3 knockout CAR-T cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model, which is comparable to standard CAR-T cells. Our study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.

  13. Hepatitis C virus E2 protein promotes human hepatoma cell proliferation through the MAPK/ERK signaling pathway via cellular receptors

    Zhao Lanjuan; Wang Lu; Ren Hao; Cao Jie; Li Li; Ke Jinshan; Qi Zhongtian

    2005-01-01

    Dysregulation of mitogen-activated protein kinase (MAPK) signaling pathways by various viruses has been shown to be responsible for viral pathogenicity. The molecular mechanism by which hepatitis C virus (HCV) infection caused human liver diseases has been investigated on the basis of abnormal intracellular signal events. Current data are very limited involved in transmembrane signal transduction triggered by HCV E2 protein. Here we explored regulation of the MAPK/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway by E2 expressed in Chinese hamster oval cells. In human hepatoma Huh-7 cells, E2 specifically activated the MAPK/ERK pathway including downstream transcription factor ATF-2 and greatly promoted cell proliferation. CD81 and low density lipoprotein receptor (LDLR) on the cell surface mediated binding of E2 to Huh-7 cells. The MAPK/ERK activation and cell proliferation driven by E2 were suppressed by blockage of CD81 as well as LDLR. Furthermore, pretreatment with an upstream kinase MEK1/2 inhibitor U0126 also impaired the MAPK/ERK activation and cell proliferation induced by E2. Our results suggest that the MAPK/ERK signaling pathway triggered by HCV E2 via its receptors maintains survival and growth of target cells

  14. Synergistic Effects of Cabozantinib and EGFR-Specific CAR-NK-92 Cells in Renal Cell Carcinoma

    Qing Zhang

    2017-01-01

    Full Text Available The chimeric antigen receptor-modified immune effector cell (CAR-T and CAR-NK therapies are newly developed adoptive treatments of cancers. However, their therapeutic efficacy against solid tumors is limited. Combining CAR-T or CAR-NK cells with chemotherapeutic drugs to treat solid tumor may be a promising strategy. We developed an epidermal growth factor- (EGFR- specific third-generation CAR. NK-92 cells were modified with the CAR by lentivirus infection. The specific killing ability of the CAR-modified NK-92 cells (CAR-NK-92 against renal cell carcinoma (RCC cell lines was confirmed in vitro. The synergistic effects of cabozantinib and EGFR-specific CAR-NK-92 cells were investigated in vitro and in vivo. Our results showed that the CAR-NK-92 cells lyse RCC cells in an EGFR-specific manner. Treatment with cabozantinib could increase EGFR and decrease PD-L1 membrane surface expression in RCC cells and enhance the killing ability of CAR-NK-92 cells against the RCC cells in vitro. Furthermore, the CAR-NK-92 cells show synergistic therapeutic efficacy with cabozantinib against human RCC xenograft models. Our results provided the basis for combination with chemotherapy as a novel strategy for enhancing the therapeutic efficacy of CAR-modified immune effector cells for solid tumors.

  15. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  16. 49 CFR 172.330 - Tank cars and multi-unit tank car tanks.

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Tank cars and multi-unit tank car tanks. 172.330..., TRAINING REQUIREMENTS, AND SECURITY PLANS Marking § 172.330 Tank cars and multi-unit tank car tanks. (a... material— (1) In a tank car unless the following conditions are met: (i) The tank car must be marked on...

  17. 49 CFR 1247.1 - Annual Report of Cars Loaded and Cars Terminated.

    2010-10-01

    ... 49 Transportation 9 2010-10-01 2010-10-01 false Annual Report of Cars Loaded and Cars Terminated... TRANSPORTATION BOARD, DEPARTMENT OF TRANSPORTATION (CONTINUED) ACCOUNTS, RECORDS AND REPORTS REPORT OF CARS LOADED AND CARS TERMINATED § 1247.1 Annual Report of Cars Loaded and Cars Terminated. Beginning with the...

  18. Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy.

    Shou-Hui Du

    Full Text Available Gamma delta (γδ T cells and cytokine-induced killer (CIK cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs using Zometa, interferon-gamma (IFN-γ, interleukin 2 (IL-2, anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR, anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer.

  19. AAP Updates Recommendations on Car Seats

    ... Size Email Print Share AAP Updates Recommendations on Car Seats Page Content Article Body Children should ride ... of approved car safety seats. Healthy Children Radio: Car Seat Safety Dennis Durbin, MD, FAAP, lead author ...

  20. Regional CAR-T cell infusions for peritoneal carcinomatosis are superior to systemic delivery.

    Katz, S C; Point, G R; Cunetta, M; Thorn, M; Guha, P; Espat, N J; Boutros, C; Hanna, N; Junghans, R P

    2016-05-01

    Metastatic spread of colorectal cancer (CRC) to the peritoneal cavity is common and difficult to treat, with many patients dying from malignant bowel obstruction. Chimeric antigen receptor T cell (CAR-T) immunotherapy has shown great promise, and we previously reported murine and phase I clinical studies on regional intrahepatic CAR-T infusion for CRC liver metastases. We are now studying intraperitoneal (IP) delivery of CAR-Ts for peritoneal carcinomatosis. Regional IP infusion of CAR-T resulted in superior protection against carcinoembryonic antigen (CEA+) peritoneal tumors, when compared with systemically infused CAR-Ts. IP CAR-Ts also provided prolonged protection against IP tumor re-challenges and demonstrated an increase in effector memory phenotype over time. IP CAR-Ts provided protection against tumor growth at distant subcutaneous (SC) sites in association with increases in serum IFNγ levels. Given the challenges posed by immunoinhibitory pathways in solid tumors, we combined IP CAR-T treatment with suppressor cell targeting. High frequencies of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) were found within the IP tumors, with MDSC expressing high levels of immunosuppressive PD-L1. Combinatorial IP CAR-T treatment with depleting antibodies against MDSC and Treg further improved efficacy against peritoneal metastases. Our data support further development of combinatorial IP CAR-T immunotherapy for peritoneal malignancies.

  1. CERN CAR CLUB

    Automobile club

    2009-01-01

    You are cordially invited to the next General Assembly of the CERN Car Club Tuesday 12 January 2010 at 5:45pm Bldg. 593 / room 11 As the end of 2009 is approaching, it is time to think about renewing your subscription. Therefore next time you are on the CERN-Meyrin site or at the Post Office counter don’t forget to fill in the payment slip to continue to be a part of our large family. The fee remains unchanged: 50 CHF. For those of you who are regular users of our equipment and who know of all the advantages that the club is in a position to offer, it seems pointless to give details, we are sure that many of you have made use of them and are satisfied. We remind you everyone working on CERN site is entitled to become a member of our club, this includes industrial support personnel and staff of companies which have a contract with CERN. If you are not yet a member, come and visit us! We will be happy to welcome you and show you the facilities, or you can visit our web site. The use of the club&...

  2. CERN CAR STICKERS

    Service Accueil et Controle d'Accès; ST Division

    1999-01-01

    In accordance with Operational Circular n¡ 2, paragraph 21, CERN car stickers are to be renewed. The new stickers are now available and will be valid for a year.Youare therefore requested:either to obtain them from the distribution points for new stickers (see below); or to send us the application form below, duly completed, via the internal mail; or to complete the application form directly via the Web at the address: http://cern.ch/registration-stickers. Each vehicle has to carry a sticker and needs a separate application form.Vehicles bearing CERN diplomatic plates (CD07, 431K and CD series) do not need a sticker for access to the CERN areas.Thank you.List of distribution points:Registration Service (bldg 55 1st floor), open from 07h30 to 16h30. Building 33 (entrance hall), open from 08h00 to 18h00. Building 120 (ground floor), outside working hours.Name Surname CERN identification number Vehicle registration plates Country issuing the plates Vehicle ma...

  3. Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor

    Cherian, Milu T.; Yang, Lei; Chai, Sergio C.; Lin, Wenwei; Chen, Taosheng

    2016-01-01

    The constitutive androstane receptor (CAR) regulates the expression of genes involved in drug metabolism and other processes. A specific inhibitor of CAR is critical for modulating constitutive CAR activity. We recently described a specific small-molecule inhibitor of CAR, CINPA1 (ethyl (5-(diethylglycyl)-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)carbamate), which is capable of reducing CAR-mediated transcription by changing the coregulator recruitment pattern and reducing CAR occupancy at the...

  4. Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

    Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

    2000-11-15

    Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

  5. [Study of androgen receptor and phosphoglycerate kinase gene polymorphism in major cellular components of the so-called pulmonary sclerosing hemangioma].

    Qi, Feng-jie; Zhang, Xiu-wei; Zhang, Yong-xing; Dai, Shun-dong; Wang, En-hua

    2006-05-01

    To study the clonality of polygonal cells and surface cuboidal cells in the so-called pulmonary sclerosing hemangioma (PSH). 17 female surgically resected PSH were found. The polygonal cells and surface cuboidal cells of the 17 PSH cases were microdissected from routine hematoxylin and eosin-stained sections. Genomic DNA was extracted, pretreated through incubation with methylation-sensitive restrictive endonuclease HhaI or HpaII, and amplified by nested polymerase chain reaction for X chromosome-linked androgen receptor (AR) and phosphoglycerate kinase (PGK) genes. The length polymorphism of AR gene was demonstrated by denaturing polyacrylamide gel electrophoresis and silver staining. The PGK gene products were treated with Bst XI and resolved on agarose gel. Amongst the 17 female cases of PSH, 15 samples were successfully amplified for AR and PGK genes. The rates of polymorphism were 53% (8/15) and 27% (4/15) for AR and PGK genes respectively. Polygonal cells and surface cuboidal cells of 10 cases which were suitable for clonality study, showed the same loss of alleles (clonality ratio = 0) or unbalanced methylation pattern (clonality ratio < 0.25). The polygonal cells and surface cuboidal cells in PSH demonstrate patterns of monoclonal proliferation, indicating that both represent true neoplastic cells.

  6. Chimeric antigen receptor T-cell therapy for solid tumors

    Kheng Newick

    2016-01-01

    Full Text Available Chimeric antigen receptor (CAR T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias. This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment.

  7. Dwelling on Everyday Car Journeys

    Tølbøll, Lene; Jensen, Hanne Louise

    different traffic conditions as well as the emotional states related to the drivers’ thoughts about work and family issues, the materiality of the car and the recreational activities inside the car. Analyses are based on a web-based questionnaire, sent to 373 participating drivers in the Big Data research...... project Intelligent Transportation System Platform North Denmark (Lahrmann 2012). In that project data on e.g. position and speed was collected via an On Board Unit from more than 400 cars in 2012-2014 (Tøfting et. al. 2014). The full dataset includes a driven distance of approximately 14 million km...... experiences related to commuting. The findings will be discussed using theoretical inspiration from Sheller (2004), Bull (2003) and Thrift (2004) and we will suggest that the various emotional experiences of the commuters are of great importance for their ability to use of the car as a dwelling place....

  8. CHINA ACCOUNTING REVIEW(CAR)

    2007-01-01

    China Accounting Review(CAR)is a new accounting journal in Chinese,spon- sored by Peking University,Tsinghua University,Beijing National Accounting Insti- tute and ten more universities,and published by the Peking University Press.

  9. Adaptation Model for Corporate Car Sharing in the Car Rental Industry

    Matthes, Martin

    2016-01-01

    The focus of this thesis was on developing an adaption model for implementing a corporate car sharing service within the existing infrastructure of a car rental company. The investigated case companies were a leading Finnish car rental franchisee and an international car sharing subsidiary, largely owned by the car rental franchisor, which offers corporate car sharing solutions in major European countries. Adapting this new service in Finland will help the car rental franchisee to assert its ...

  10. Passenger car fuel consumption survey

    1984-03-01

    This survey originated from a proposal to monitor the fuel consumption and fuel economy of personal use passenger cars operated in Canada. Its purpose is to establish a data base which would contain information on total distance travelled, total amount of fuel consumed, average distance obtained per unit of fuel, total expenditures on fuel, and seasonal fluctuations in fuel consumption and in distance travelled. Among the needs served by this data base are the monitoring of passenger car fuel economy standards and the estimation of pasenger car fuel requirements in conditions involving fuel shortages. Survey methodology is by telephone interview to trace selected vehicles to the registered owners, at which time a fuel purchase diary is then mailed to the principal driver of the car. The results are tabulated on a quarterly basis and to be released as they become available in bulletins similar to this. Data are presented for each province and the total for Canada is given. During the fourth quarter of 1982, it is estimated that there were 7.3 million personal use passenger cars operated in Canada. These cars were driven 28 billion kilometers and consumed 4.3 billion litres of fuel. Their average litres/100 kilometres and the average fuel consumption was 590 litres. 8 tabs.

  11. A guide to manufacturing CAR T cell therapies.

    Vormittag, Philipp; Gunn, Rebecca; Ghorashian, Sara; Veraitch, Farlan S

    2018-02-17

    In recent years, chimeric antigen receptor (CAR) modified T cells have been used as a treatment for haematological malignancies in several phase I and II trials and with Kymriah of Novartis and Yescarta of KITE Pharma, the first CAR T cell therapy products have been approved. Promising clinical outcomes have yet been tempered by the fact that many therapies may be prohibitively expensive to manufacture. The process is not yet defined, far from being standardised and often requires extensive manual handling steps. For academia, big pharma and contract manufacturers it is difficult to obtain an overview over the process strategies and their respective advantages and disadvantages. This review details current production processes being used for CAR T cells with a particular focus on efficacy, reproducibility, manufacturing costs and release testing. By undertaking a systematic analysis of the manufacture of CAR T cells from reported clinical trial data to date, we have been able to quantify recent trends and track the uptake of new process technology. Delivering new processing options will be key to the success of the CAR-T cells ensuring that excessive manufacturing costs do not disrupt the delivery of exciting new therapies to the wide possible patient cohort. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Engineering an Affordable Self-Driving Car

    Budisteanu, Alexandru Ionut

    2018-01-01

    for affordable self-driving cars and he designed a low-cost self-driving car. The car's roof has cameras and low-resolution 3D LiDAR equipment to detect traffic lanes, other cars, curbs and obstacles, such as people crossing by. To process this dizzying amount

  13. 49 CFR 174.615 - Cleaning cars.

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Cleaning cars. 174.615 Section 174.615... Requirements for Division 6.1 (Poisonous) Materials § 174.615 Cleaning cars. (a) [Reserved] (b) After Division 6.1 (poisonous) materials are unloaded from a rail car, that car must be thoroughly cleaned unless...

  14. 49 CFR 231.6 - Flat cars.

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Flat cars. 231.6 Section 231.6 Transportation... TRANSPORTATION RAILROAD SAFETY APPLIANCE STANDARDS § 231.6 Flat cars. (Cars with sides 12 inches or less above the floor may be equipped the same as flat cars.) (a) Hand brakes—(1) Number. Same as specified for...

  15. Art Cars: Transformations of the Mundane

    Stienecker, Dawn

    2010-01-01

    The automobile itself is often understood as an extension of oneself, where individuals may manipulate the interior and exterior of cars and trucks, decorating them through detailing, stickers, custom colors, and so on. Others go further and change their cars into unique works of art called art cars. Such cars break away from the banality of mass…

  16. 49 CFR 215.203 - Restricted cars.

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than 50...

  17. 49 CFR 1037.2 - Cars.

    2010-10-01

    ... 49 Transportation 8 2010-10-01 2010-10-01 false Cars. 1037.2 Section 1037.2 Transportation Other... GENERAL RULES AND REGULATIONS BULK GRAIN AND GRAIN PRODUCTS-LOSS AND DAMAGE CLAIMS § 1037.2 Cars. A car is... railroad-leased cars. [57 FR 54334, Nov. 18, 1992] ...

  18. Cloning, expression, cellular distribution, and role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in a nonmammalian species

    Boshra, Hani; Li, Jun; Peters, Rodney; Hansen, John; Matlapudi, Anjan; Sunyer, J. Oriol

    2004-01-01

    C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.

  19. Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice

    Park, Young Joo [Department of Internal Medicine, Seoul National University College of Medicine (Korea, Republic of); Seoul National University Bundang Hospital, Seoul (Korea, Republic of); Lee, Eun Kyung [Department of Internal Medicine, Seoul National University College of Medicine (Korea, Republic of); Lee, Yoon Kwang [Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Park, Do Joon; Jang, Hak Chul [Department of Internal Medicine, Seoul National University College of Medicine (Korea, Republic of); Moore, David D., E-mail: moore@bcm.edu [Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States)

    2012-09-01

    Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR{sup −/−} mice, but not in hypothyroid PXR{sup −/−} mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR{sup −/−} hypothyroid mice, and this induction was abolished in CAR{sup −/−} mice and in and CAR{sup −/−} PXR{sup −/−} double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR{sup −/−} mice and lowest in WT and PXR{sup −/−} mice. Hypothyroid WT or PXR{sup −/−} mice survived chronic CBZ treatment, but all hypothyroid CAR{sup −/−} and CAR{sup −/−} PXR{sup −/−} mice died, with CAR{sup −/−}PXR{sup −/−} mice surviving longer than CAR{sup −/−} mice (12.3 ± 3.3 days vs. 6.3 ± 2.1 days, p = 0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge. -- Highlights: ► Hypothyroid status activates CAR in mice and induces Cyp2b10 expression. ► Hypothyroid status suppresses PXR activity in mice and represses Cyp3a11 expression. ► These responses balance each other out in normal mice.

  20. Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice

    Park, Young Joo; Lee, Eun Kyung; Lee, Yoon Kwang; Park, Do Joon; Jang, Hak Chul; Moore, David D.

    2012-01-01

    Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR −/− mice, but not in hypothyroid PXR −/− mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR −/− hypothyroid mice, and this induction was abolished in CAR −/− mice and in and CAR −/− PXR −/− double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR −/− mice and lowest in WT and PXR −/− mice. Hypothyroid WT or PXR −/− mice survived chronic CBZ treatment, but all hypothyroid CAR −/− and CAR −/− PXR −/− mice died, with CAR −/− PXR −/− mice surviving longer than CAR −/− mice (12.3 ± 3.3 days vs. 6.3 ± 2.1 days, p = 0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge. -- Highlights: ► Hypothyroid status activates CAR in mice and induces Cyp2b10 expression. ► Hypothyroid status suppresses PXR activity in mice and represses Cyp3a11 expression. ► These responses balance each other out in normal mice. ► Hypothyroidism sensitizes CAR null mice to toxic effects of carbamazepine.

  1. CD19-Targeted CAR T Cells as Novel Cancer Immunotherapy for Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia

    Davila, Marco L.; Brentjens, Renier J.

    2016-01-01

    Immunotherapy has demonstrated significant potential for the treatment of patients with chemotherapy-resistant hematologic malignancies and solid tumors. One type of immunotherapy involves the adoptive transfer of T cells that have been genetically modified with a chimeric antigen receptor (CAR) to target a tumor. These hybrid proteins are composed of the antigen-binding domains of an antibody fused to T-cell receptor signaling machinery. CAR T cells that target CD19 recently have made the ju...

  2. CCR research lays foundation for FDA approval of CAR T cell therapy Yescarta | Center for Cancer Research

    Decades ago, the use of chimeric antigen receptor (CAR)-expressing T cells as an effective form of immunotherapy was a speculative idea. In 2010, a breakthrough clinical trial conducted by Steven Rosenberg, M.D., Ph.D., and his clinical team showed that CAR T cells recognizing the CD19 receptor were useful in the treatment of some types of B-cell malignancies. Read more...

  3. CAR T Cell Therapy for Glioblastoma: Recent Clinical Advances and Future Challenges.

    Bagley, Stephen J; Desai, Arati S; Linette, Gerald P; June, Carl H; O'Rourke, Donald M

    2018-03-02

    In patients with certain hematologic malignancies, the use of autologous T cells genetically modified to express chimeric antigen receptors (CARs) has led to unprecedented clinical responses. Although progress in solid tumors has been elusive, recent clinical studies have demonstrated the feasibility and safety of CAR T cell therapy for glioblastoma. In addition, despite formidable barriers to T cell localization and effector function in glioblastoma, signs of efficacy have been observed in select patients. In this review, we begin with a discussion of established obstacles to systemic therapy in glioblastoma and how these may be overcome by CAR T cells. We continue with a summary of previously published CAR T cell trials in GBM, and end by outlining the key therapeutic challenges associated with the use of CAR T cells in this disease.

  4. Hurdles of CAR-T cell-based cancer immunotherapy directed against solid tumors.

    Zhang, Bing-Lan; Qin, Di-Yuan; Mo, Ze-Ming; Li, Yi; Wei, Wei; Wang, Yong-Sheng; Wang, Wei; Wei, Yu-Quan

    2016-04-01

    Recent reports on the impressive efficacy of chimeric antigen receptor (CAR)-modified T cells against hematologic malignancies have inspired oncologists to extend these efforts for the treatment of solid tumors. Clinical trials of CAR-T-based cancer immunotherapy for solid tumors showed that the efficacies are not as remarkable as in the case of hematologic malignancies. There are several challenges that researchers must face when treating solid cancers with CAR-T cells, these include choosing an ideal target, promoting efficient trafficking and infiltration, overcoming the immunosuppressive microenvironment, and avoiding associated toxicity. In this review, we discuss the obstacles imposed by solid tumors on CAR-T cell-based immunotherapy and strategies adopted to improve the therapeutic potential of this approach. Continued investigations are necessary to improve therapeutic outcomes and decrease the adverse effects of CAR-T cell therapy in patients with solid malignancies in the future.

  5. Insights into cytokine release syndrome and neurotoxicity after CD19-specific CAR-T cell therapy.

    Gauthier, Jordan; Turtle, Cameron J

    2018-04-03

    T-cells engineered to express CD19-specific chimeric antigen receptors (CD19 CAR-T cells) can achieve high response rates in patients with refractory/relapsed (R/R) CD19+ hematologic malignancies. Nonetheless, the efficacy of CD19-specific CAR-T cell therapy can be offset by significant toxicities, such as cytokine release syndrome (CRS) and neurotoxicity. In this report of our presentation at the 2018 Second French International Symposium on CAR-T cells (CAR-T day), we describe the clinical presentations of CRS and neurotoxicity in a cohort of 133 adults treated with CD19 CAR-T cells at the Fred Hutchinson Cancer Research Center, and provide insights into the mechanisms contributing to these toxicities. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  6. CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

    Cherian, Milu T; Lin, Wenwei; Wu, Jing

    2015-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023

  7. CINPA1 is an inhibitor of constitutive androstane receptor that does not activate pregnane X receptor.

    Cherian, Milu T; Lin, Wenwei; Wu, Jing; Chen, Taosheng

    2015-05-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  8. Car sharing à la carte

    Anaïs Schaeffer

    2012-01-01

    Do you want to make your commute to CERN easier, while saving money at the same time? Would you prefer not to spend a quarter of an hour crawling round the CERN car parks looking for a space? If so, read on: this article might well be of great interest to you.   We would like to draw your attention to a well established, albeit sadly under-used, method of transport: car sharing. To promote car-sharing, the GS Department has stepped in to call on the services of the Swiss firm Green Monkeys which specialises in this user-friendly and intelligent transport scheme. The company’s slogan is:  “Car-sharing as you want, when you want and as much as you want”. The principle is very straightforward. To use this car-sharing facility, you simply complete your free online registration with Green Monkeys, providing the following details: your journey, departure time, arrival time and days of the week, and indicating whether you are a passenger or driver or both. &a...

  9. Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts

    Cartellieri, M; Feldmann, A; Koristka, S; Arndt, C; Loff, S; Ehninger, A; Bonin, M von; Bejestani, E P; Ehninger, G; Bachmann, M P

    2016-01-01

    The adoptive transfer of CD19-specific chimeric antigen receptor engineered T cells (CAR T cells) resulted in encouraging clinical trials in indolent B-cell malignancies. However, they also show the limitations of this fascinating technology: CAR T cells can lead to even life-threatening off-tumor, on-target side effects if CAR T cells crossreact with healthy tissues. Here, we describe a novel modular universal CAR platform technology termed UniCAR that reduces the risk of on-target side effects by a rapid and reversible control of CAR T-cell reactivity. The UniCAR system consists of two components: (1) a CAR for an inert manipulation of T cells and (2) specific targeting modules (TMs) for redirecting UniCAR T cells in an individualized time- and target-dependent manner. UniCAR T cells can be armed against different tumor targets simply by replacement of the respective TM for (1) targeting more than one antigen simultaneously or subsequently to enhance efficacy and (2) reducing the risk for development of antigen-loss tumor variants under treatment. Here we provide ‘proof of concept' for retargeting of UniCAR T cells to CD33- and/or CD123-positive acute myeloid leukemia blasts in vitro and in vivo

  10. Efficacy and toxicity management of CAR-T-cell immunotherapy: a matter of responsiveness control or tumour-specificity?

    Alonso-Camino, Vanesa; Harwood, Seandean Lykke; Álvarez-Méndez, Ana; Alvarez-Vallina, Luis

    2016-04-15

    Chimaeric antigen receptor (CAR)-expressing T-cells have demonstrated potent clinical efficacy in patients with haematological malignancies. However, the use of CAR-T-cells targeting solid tumour-associated antigens (TAAs) has been limited by organ toxicities related to activation of T-cell effector functions through the CAR. Most existing CARs recognize TAAs, which are also found in normal tissues. CAR-T-cell-mediated destruction of normal tissues constitutes a major roadblock to CAR-T-cell therapy, and must be avoided or mitigated. There is a broad range of strategies for modulating antigen responsiveness of CAR-T-cells, with varying degrees of complexity. Some of them might ameliorate the acute and chronic toxicities associated with current CAR constructs. However, further embellishments to CAR therapy may complicate clinical implementation and possibly create new immunogenicity issues. In contrast, the development of CARs targeting truly tumour-specific antigens might circumvent on-target/off-tumour toxicities without adding additional complexity to CAR-T-cell therapies, but these antigens have been elusive and may require novel selection strategies for their discovery. © 2016 Authors; published by Portland Press Limited.

  11. Cellular communication through light.

    Daniel Fels

    Full Text Available Information transfer is a fundamental of life. A few studies have reported that cells use photons (from an endogenous source as information carriers. This study finds that cells can have an influence on other cells even when separated with a glass barrier, thereby disabling molecule diffusion through the cell-containing medium. As there is still very little known about the potential of photons for intercellular communication this study is designed to test for non-molecule-based triggering of two fundamental properties of life: cell division and energy uptake. The study was performed with a cellular organism, the ciliate Paramecium caudatum. Mutual exposure of cell populations occurred under conditions of darkness and separation with cuvettes (vials allowing photon but not molecule transfer. The cell populations were separated either with glass allowing photon transmission from 340 nm to longer waves, or quartz being transmittable from 150 nm, i.e. from UV-light to longer waves. Even through glass, the cells affected cell division and energy uptake in neighboring cell populations. Depending on the cuvette material and the number of cells involved, these effects were positive or negative. Also, while paired populations with lower growth rates grew uncorrelated, growth of the better growing populations was correlated. As there were significant differences when separating the populations with glass or quartz, it is suggested that the cell populations use two (or more frequencies for cellular information transfer, which influences at least energy uptake, cell division rate and growth correlation. Altogether the study strongly supports a cellular communication system, which is different from a molecule-receptor-based system and hints that photon-triggering is a fine tuning principle in cell chemistry.

  12. Effect of CAR activation on selected metabolic pathways in normal and hyperlipidemic mouse livers.

    Rezen, Tadeja; Tamasi, Viola; Lövgren-Sandblom, Anita; Björkhem, Ingemar; Meyer, Urs A; Rozman, Damjana

    2009-08-19

    Detoxification in the liver involves activation of nuclear receptors, such as the constitutive androstane receptor (CAR), which regulate downstream genes of xenobiotic metabolism. Frequently, the metabolism of endobiotics is also modulated, resulting in potentially harmful effects. We therefore used 1,4-Bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) to study the effect of CAR activation on mouse hepatic transcriptome and lipid metabolome under conditions of diet-induced hyperlipidemia. Using gene expression profiling with a dedicated microarray, we show that xenobiotic metabolism, PPARalpha and adipocytokine signaling, and steroid synthesis are the pathways most affected by TCPOBOP in normal and hyperlipidemic mice. TCPOBOP-induced CAR activation prevented the increased hepatic and serum cholesterol caused by feeding mice a diet containing 1% cholesterol. We show that this is due to increased bile acid metabolism and up-regulated removal of LDL, even though TCPOBOP increased cholesterol synthesis under conditions of hyperlipidemia. Up-regulation of cholesterol synthesis was not accompanied by an increase in mature SREBP2 protein. As determined by studies in CAR -/- mice, up-regulation of cholesterol synthesis is however CAR-dependent; and no obvious CAR binding sites were detected in promoters of cholesterogenic genes. TCPOBOP also affected serum glucose and triglyceride levels and other metabolic processes in the liver, irrespective of the diet. Our data show that CAR activation modulates hepatic metabolism by lowering cholesterol and glucose levels, through effects on PPARalpha and adiponectin signaling pathways, and by compromising liver adaptations to hyperlipidemia.

  13. Substitution between Cars within the Household

    de Borger, Bruno; Mulalic, Ismir; Rouwendal, Jan

    In this paper we study the demand for car kilometres in two-car households, focusing on the substitution between cars in response to fuel price changes. We use a large sample of detailed Danish data on two-car households to estimate—for each car owned by the household—own and cross-price effects...... of increases in fuel costs per kilometre. The empirical results show that failure to capture substitution between cars within the household can result in substantial misspecification biases. Ignoring substitution, we estimate fuel price elasticities of –0.81 and -0.65 for the primary and secondary cars...... efficient car, finding partial support for the underlying hypothesis. More importantly, the results of this extended model emphasize the importance of behavioural differences related to the position of the most fuel efficient car in the household, suggesting that households’ fuel efficiency choices...

  14. Positive and negative spillover effects from electric car purchase to car use

    Kløckner, Christian; Nayum, Alim; Mehmetoglu, Mehmet

    2013-01-01

    This study reports the results of two online surveys conducted on buyers of conventional combustion engine cars compared to those of electric vehicles in Norway. The results show that electric cars are generally purchased as additional cars, do not contribute to a decrease in annual mileage if the old car is not substituted, and that electric car buyers use the car more often for their everyday mobility. Psychological determinants derived from the theory of planned behavior and the norm-activ...

  15. Chimeric Antigen Receptor T-Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia

    Ciprian Tomuleasa

    2018-02-01

    Full Text Available Chimeric antigen receptor (CAR T-cell technology has seen a rapid development over the last decade mostly due to the potential that these cells may have in treating malignant diseases. It is a generally accepted principle that very few therapeutic compounds deliver a clinical response without treatment-related toxicity, and studies have shown that CAR T-cells are not an exception to this rule. While large multinational drug companies are currently investigating the potential role of CAR T-cells in hematological oncology, the potential of such cellular therapies are being recognized worldwide as they are expected to expand in the patient to support the establishment of the immune memory, provide a continuous surveillance to prevent and/or treat a relapse, and keep the targeted malignant cell subpopulation in check. In this article, we present the possible advantages of using CAR T-cells in treating acute lymphoblastic leukemia, presenting the technology and the current knowledge in their preclinical and early clinical trial use. Thus, this article first presents the main present-day knowledge on the standard of care for acute lymphoblastic leukemia. Afterward, current knowledge is presented about the use of CAR T-cells in cancer immunotherapy, describing their design, the molecular constructs, and the preclinical data on murine models to properly explain the background for their clinical use. Last, but certainly not least, this article presents the use of CAR T-cells for the immunotherapy of B-cell acute lymphoblastic leukemia, describing both their potential clinical advantages and the possible side effects.

  16. Chimeric Antigen Receptor Expressing Natural Killer Cells for the Immunotherapy of Cancer

    Rohtesh S. Mehta

    2018-02-01

    Full Text Available Adoptive cell therapy has emerged as a powerful treatment for advanced cancers resistant to conventional agents. Most notable are the remarkable responses seen in patients receiving autologous CD19-redirected chimeric antigen receptor (CAR T cells for the treatment of B lymphoid malignancies; however, the generation of autologous products for each patient is logistically cumbersome and has restricted widespread clinical use. A banked allogeneic product has the potential to overcome these limitations, yet allogeneic T-cells (even if human leukocyte antigen-matched carry a major risk of graft-versus-host disease (GVHD. Natural killer (NK cells are bone marrow-derived innate lymphocytes that can eliminate tumors directly, with their activity governed by the integration of signals from activating and inhibitory receptors and from cytokines including IL-15, IL-12, and IL-18. NK cells do not cause GVHD or other alloimmune or autoimmune toxicities and thus, can provide a potential source of allogeneic “off-the-shelf” cellular therapy, mediating major anti-tumor effects without inducing potentially lethal alloreactivity such as GVHD. Given the multiple unique advantages of NK cells, researchers are now exploring the use of CAR-engineered NK cells for the treatment of various hematological and non-hematological malignancies. Herein, we review preclinical data on the development of CAR-NK cells, advantages, disadvantages, and current obstacles to their clinical use.

  17. Substitution between cars within the household

    De Borger, Bruno; Mulalic, Ismir; Rouwendal, Jan

    The purpose of this paper is to study to what extent two-car households substitute the use of their less fuel efficient car by the use of their more fuel efficient car after an increase in fuel prices. Based on a simple theoretical framework we use a large sample of detailed Danish data on two-car...... households to estimate, for each car owned by the household, own and cross-price effects of increases in fuel costs per kilometer. The empirical results point at important substitution effects, so that models that estimate responses to fuel prices on the implicit or explicit assumption of one car per...

  18. Optimizing EphA2-CAR T Cells for the Adoptive Immunotherapy of Glioma

    Zhongzhen Yi

    2018-06-01

    Full Text Available Glioblastoma is the most aggressive primary brain tumor in humans and is virtually incurable with conventional therapies. Chimeric antigen receptor (CAR T cell therapy targeting the glioblastoma antigen EphA2 is an attractive approach to improve outcomes because EphA2 is expressed highly in glioblastoma but only at low levels in normal brain tissue. Building upon our previous findings in this area, we generated and evaluated a panel of EphA2-specific CARs. We demonstrate here that T cells expressing CD28.ζ and 41BB.ζ CARs with short spacers had similar effector function, resulting in potent antitumor activity. In addition, incorporating the 41BB signaling domain into CD28.ζ CARs did not improve CAR T cell function. While we could not determine functional differences between CD28.ζ, 41BB.ζ, and CD28.41BB.ζ CAR T cells, we selected CD28.ζ CAR T cells for further clinical development based on safety consideration. Keywords: GBM, CAR T cells, EphA2, brain tumor

  19. Cancer Immunotherapy Using CAR-T Cells: From the Research Bench to the Assembly Line.

    Gomes-Silva, Diogo; Ramos, Carlos A

    2018-02-01

    The focus of cancer treatment has recently shifted toward targeted therapies, including immunotherapy, which allow better individualization of care and are hoped to increase the probability of success for patients. Specifically, T cells genetically modified to express chimeric antigen receptors (CARs; CAR-T cells) have generated exciting results. Recent clinical successes with this cutting-edge therapy have helped to push CAR-T cells toward approval for wider use. However, several limitations need to be addressed before the widespread use of CAR-T cells as a standard treatment. Here, a succinct background on adoptive T-cell therapy (ATCT)is given. A brief overview of the structure of CARs, how they are introduced into T cells, and how CAR-T cell expansion and selection is achieved in vitro is then presented. Some of the challenges in CAR design are discussed, as well as the difficulties that arise in large-scale CAR-T cell manufacture that will need to be addressed to achieve successful commercialization of this type of cell therapy. Finally, developments already on the horizon are discussed. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. CAR T-cell therapy for glioblastoma: ready for the next round of clinical testing?

    Prinzing, Brooke L; Gottschalk, Stephen M; Krenciute, Giedre

    2018-05-01

    The outcome for patients with glioblastoma (GBM) remains poor, and there is an urgent need to develop novel therapeutic approaches. T cells genetically modified with chimeric antigen receptors (CARs) hold the promise to improve outcomes since they recognize and kill cells through different mechanisms than conventional therapeutics. Areas covered: This article reviews CAR design, tumor associated antigens expressed by GBMs that can be targeted with CAR T cells, preclinical and clinical studies conducted with CAR T cells, and genetic approaches to enhance their effector function. Expert commentary: While preclinical studies have highlighted the potent anti-GBM activity of CAR T cells, the initial foray of CAR T-cell therapies into the clinic resulted only in limited benefits for GBM patients. Additional genetic modification of CAR T cells has resulted in a significant increase in their anti-GBM activity in preclinical models. We are optimistic that clinical testing of these enhanced CAR T cells will be safe and result in improved anti-glioma activity in GBM patients.

  1. Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA+ Metastatic Colorectal Cancers.

    Zhang, Chengcheng; Wang, Zhe; Yang, Zhi; Wang, Meiling; Li, Shiqi; Li, Yunyan; Zhang, Rui; Xiong, Zhouxing; Wei, Zhihao; Shen, Junjie; Luo, Yongli; Zhang, Qianzhen; Liu, Limei; Qin, Hong; Liu, Wei; Wu, Feng; Chen, Wei; Pan, Feng; Zhang, Xianquan; Bie, Ping; Liang, Houjie; Pecher, Gabriele; Qian, Cheng

    2017-05-03

    Chimeric antigen receptor T (CAR-T) cells have shown promising efficacy in treatment of hematological malignancies, but its applications in solid tumors need further exploration. In this study, we investigated CAR-T therapy targeting carcino-embryonic antigen (CEA)-positive colorectal cancer (CRC) patients with metastases to evaluate its safety and efficacy. Five escalating dose levels (DLs) (1 × 10 5 to 1 × 10 8 /CAR + /kg cells) of CAR-T were applied in 10 CRC patients. Our data showed that severe adverse events related to CAR-T therapy were not observed. Of the 10 patients, 7 patients who experienced progressive disease (PD) in previous treatments had stable disease after CAR-T therapy. Two patients remained with stable disease for more than 30 weeks, and two patients showed tumor shrinkage by positron emission tomography (PET)/computed tomography (CT) and MRI analysis, respectively. Decline of serum CEA level was apparent in most patients even in long-term observation. Furthermore, we observed persistence of CAR-T cells in peripheral blood of patients receiving high doses of CAR-T therapy. Importantly, we observed CAR-T cell proliferation especially in patients after a second CAR-T therapy. Taken together, we demonstrated that CEA CAR-T cell therapy was well tolerated in CEA + CRC patients even in high doses, and some efficacy was observed in most of the treated patients. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  2. The Speeding Car Design Challenge

    Roman, Harry T.

    2009-01-01

    All too often, one reads about high-speed police chases in pursuit of stolen cars that result in death and injury to people and innocent bystanders. Isn't there another way to accomplish the apprehension of the thieves that does not put people at such great risk? This article presents a classroom challenge to use technology to remotely shutdown…

  3. Automated Car Park Management System

    Fabros, J. P.; Tabañag, D.; Espra, A.; Gerasta, O. J.

    2015-06-01

    This study aims to develop a prototype for an Automated Car Park Management System that will increase the quality of service of parking lots through the integration of a smart system that assists motorist in finding vacant parking lot. The research was based on implementing an operating system and a monitoring system for parking system without the use of manpower. This will include Parking Guidance and Information System concept which will efficiently assist motorists and ensures the safety of the vehicles and the valuables inside the vehicle. For monitoring, Optical Character Recognition was employed to monitor and put into list all the cars entering the parking area. All parking events in this system are visible via MATLAB GUI which contain time-in, time-out, time consumed information and also the lot number where the car parks. To put into reality, this system has a payment method, and it comes via a coin slot operation to control the exit gate. The Automated Car Park Management System was successfully built by utilizing microcontrollers specifically one PIC18f4550 and two PIC16F84s and one PIC16F628A.

  4. Restoring a Classic Electric Car

    Kraft, Thomas E.

    2012-01-01

    One hundred years ago, automobiles were powered by steam, electricity, or internal combustion. Female drivers favored electric cars because, unlike early internal-combustion vehicles, they did not require a crank for starting. Nonetheless, internal-combustion vehicles came to dominate the industry and it's only in recent years that the electrics…

  5. CAR-T cells and allogeneic hematopoietic stem cell transplantation for relapsed/refractory B-cell acute lymphoblastic leukemia.

    Liu, Jun; Zhang, Xi; Zhong, Jiang F; Zhang, Cheng

    2017-10-01

    Relapsed/refractory acute lymphoblastic leukemia (ALL) has a low remission rate after chemotherapy, a high relapse rate and poor long-term survival even when allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed. Chimeric antigen receptors redirected T cells (CAR-T cells) can enhance disease remission with a favorable outcome for relapsed/refractory ALL, though some cases quickly relapsed after CAR-T cell treatment. Thus, treatment with CAR-T cells followed by allo-HSCT may be the best way to treat relapsed/refractory ALL. In this review, we first discuss the different types of CAR-T cells. We then discuss the treatment of relapsed/refractory ALL using only CAR-T cells. Finally, we discuss the use of CAR-T cells, followed by allo-HSCT, for the treatment of relapsed/refractory ALL.

  6. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition

    Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S.; Jones, David R.; Sadelain, Michel; Adusumilli, Prasad S.

    2016-01-01

    Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1–mediated (PD-1–mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB–based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies. PMID:27454297

  7. Transit Car Performance Comparison, State-of-the-Art Car vs. PATCO Transit Car, NYCTA R-46, MBTA Silverbirds

    1978-02-01

    The first phase of this contract authorized the design, development, and demonstration of two State-Of-The-Art Cars (SOAC). This document reports on the gathering of comparative test data on existing in-service transit cars. The three transit cars se...

  8. Interactions of Rodent Coronaviruses with Cellular Receptors

    2016-05-08

    determinants of cell and tissue tropism. For example, it has been shown using immunofluorescence and electron microscopy, that Epstein Barr Virus (EBV) can...ligation reaction were used to transform DH5a competent cells (GIBCO-BRL, Gaithersburg, MO) and plasm ids containing the desired insert were

  9. Induction of a central memory and stem cell memory phenotype in functionally active CD4+ and CD8+ CAR T cells produced in an automated good manufacturing practice system for the treatment of CD19+ acute lymphoblastic leukemia.

    Blaeschke, Franziska; Stenger, Dana; Kaeuferle, Theresa; Willier, Semjon; Lotfi, Ramin; Kaiser, Andrew Didier; Assenmacher, Mario; Döring, Michaela; Feucht, Judith; Feuchtinger, Tobias

    2018-03-31

    Relapsed/refractory B-precursor acute lymphoblastic leukemia (pre-B ALL) remains a major therapeutic challenge. Chimeric antigen receptor (CAR) T cells are promising treatment options. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to promote sustained proliferation and persistence after T-cell therapy, constituting essential preconditions for treatment efficacy. Therefore, we set up a protocol for anti-CD19 CAR T-cell generation aiming at high Tcm/Tscm numbers. 100 ml peripheral blood from pediatric pre-B ALL patients was processed including CD4 + /CD8 + -separation, T-cell activation with modified anti-CD3/-CD28 reagents and transduction with a 4-1BB-based second generation CAR lentiviral vector. The process was performed on a closed, automated device requiring additional manual/open steps under clean room conditions. The clinical situation of these critically ill and refractory patients with leukemia leads to inconsistent cellular compositions at start of the procedure including high blast counts and low T-cell numbers with exhausted phenotype. Nevertheless, a robust T-cell product was achieved (mean CD4 +  = 50%, CD8 +  = 39%, transduction = 27%, Tcm = 50%, Tscm = 46%). Strong proliferative potential (up to > 100-fold), specific cytotoxicity and low expression of co-inhibitory molecules were documented. CAR T cells significantly released TH1 cytokines IFN-γ, TNF-α and IL-2 upon target-recognition. In conclusion, partly automated GMP-generation of CAR T cells from critically small blood samples was feasible with a new stimulation protocol that leads to high functionality and expansion potential, balanced CD4/CD8 ratios and a conversion to a Tcm/Tscm phenotype.

  10. Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.

    Arai, Yasuyuki; Choi, Uimook; Corsino, Cristina I; Koontz, Sherry M; Tajima, Masaki; Sweeney, Colin L; Black, Mary A; Feldman, Steven A; Dinauer, Mary C; Malech, Harry L

    2018-05-02

    We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (c-kit + population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue. Published by Elsevier Inc.

  11. Consumer Behavior towards Safer Car Purchasing Decisions

    Khairil Anwar Abu Kassim; Mohd Hafzi Md Isa; Yahaya Ahmad; Intan Osman; Lawrence Arokiasamy

    2016-01-01

    In Malaysia, the car safety level has been elevated through regulations and a consumer-based approach, i.e. the New Car Assessment Program in Southeast Asian Countries (ASEAN NCAP). Nevertheless, the availability of information on consumers' car purchasing decisions towards safety is still limited in Malaysia. Thus, this study was aimed at evaluating consumers' purchasing decisions of their present cars and investigating their awareness of ASEAN NCAP. Self-administered questionnaires were dis...

  12. Ground effect aerodynamics of racing cars

    Zhang, Xin; Toet, Willem; Zerihan, Jonathan

    2006-01-01

    We review the progress made during the last thirty years on ground effect aerodynamics associated with race cars, in particular open wheel race cars. Ground effect aerodynamics of race cars is concerned with generating downforce, principally via low pressure on the surfaces nearest to the ground. The “ground effected” parts of an open wheeled car's aerodynamics are the most aerodynamically efficient and contribute less drag than that associated with, for example, an upper rear wing. Whilst dr...

  13. CAR SECURITY ENHANCEMENT IN PARKING AREAS

    NANYONGA BERINDA; AYESIGA LINDSEY PATRA; BYEKWASO FAISAL; NATULINDA LADAN

    2017-01-01

    Over time, car thefts have been reported within Kampala parking areas. This has been majorly due to inefficient security measures of the available parking systems which focus mainly on the car and not the driver, making parking management a challenge. The focus of this survey was to explore the requirements of a new system called Car to Driver Matching Security System to enhance security of cars in Kampala, in particular, from the experience of 15 people. The data collected was then analyzed ...

  14. Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer.

    Priceman, Saul J; Gerdts, Ethan A; Tilakawardane, Dileshni; Kennewick, Kelly T; Murad, John P; Park, Anthony K; Jeang, Brook; Yamaguchi, Yukiko; Yang, Xin; Urak, Ryan; Weng, Lihong; Chang, Wen-Chung; Wright, Sarah; Pal, Sumanta; Reiter, Robert E; Wu, Anna M; Brown, Christine E; Forman, Stephen J

    2018-01-01

    Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with "on-target off-tumor" activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies.

  15. Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer

    Priceman, Saul J.; Gerdts, Ethan A.; Tilakawardane, Dileshni; Kennewick, Kelly T.; Murad, John P.; Park, Anthony K.; Jeang, Brook; Yamaguchi, Yukiko; Urak, Ryan; Weng, Lihong; Chang, Wen-Chung; Wright, Sarah; Pal, Sumanta; Reiter, Robert E.; Brown, Christine E.; Forman, Stephen J.

    2018-01-01

    ABSTRACT Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies. PMID:29308300

  16. Active deceleration support in car following

    Mulder, M.; Pauwelussen, J.J.A.; Paassen, M.M. van; Mulder, M.; Abbink, D.A.

    2010-01-01

    A haptic gas pedal feedback system is developed that provides car-following information via haptic cues from the gas pedal. During normal car-following situations, the haptic feedback (HF) cues were sufficient to reduce control activity and improve car-following performance. However, in more

  17. 49 CFR 174.57 - Cleaning cars.

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Cleaning cars. 174.57 Section 174.57... and Loading Requirements § 174.57 Cleaning cars. All hazardous material which has leaked from a package in any rail car or on other railroad property must be carefully removed. ...

  18. Trial products of solar cars; Solar car no shisaku

    Shimizu, A; Hatakeyama, S; Sugiura, S; Shinoda, S; Daigo, Y; Fujihara, Y; Yano, K; Kasuga, M [Yamanashi University, Yamanashi (Japan). Faculty of Engineering

    1997-11-25

    A solar car was trially manufactured installing solar panels on a motor-wheelchair for the old (senior car). It is a car for one person with maximum speed of 6km/h, motor of 360w, two of storage battery of 12Vtimes29AH, and two of solar cell of 20Vtimes3A. The output of solar cell is about 100W, which may not be enough to drive a 360W motor. However, if action time per day is about 2 hours, the required power 700WH, and the sunshine duration 7 hours per day, solar cells of 100W can generate 700WH. This is stored in battery, and when it is short, it is supplemented by nighttime power. Product prices are 200,000-250,000 yen. A solar go-cart was trially manufactured remodeling the gasoline-run go-cart. It is a solar go-cart for one person with maximum speed of 30km/h, a motor of 600W, four of storage battery of 12Vtimes29AH, and four of solar cell of 20Vtimes3A. The output of solar battery at 200W is a third of the motor power, with battery charged three times the travel time. More than 1000 persons trially rode the go-cart. 2 figs.

  19. Target Antigen Density Governs the Efficacy of Anti-CD20-CD28-CD3 zeta Chimeric Antigen Receptor-Modified Effector CD8(+) T Cells

    Watanabe, Keisuke; Terakura, Seitaro; Martens, Anton C.; van Meerten, Tom; Uchiyama, Susumu; Imai, Misa; Sakemura, Reona; Goto, Tatsunori; Hanajiri, Ryo; Imahashi, Nobuhiko; Shimada, Kazuyuki; Tomita, Akihiro; Kiyoi, Hitoshi; Nishida, Tetsuya; Naoe, Tomoki; Murata, Makoto

    2015-01-01

    The effectiveness of chimeric Ag receptor (CAR)-transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second-and later-generation CARs simultaneously transmit costimulatory signals with CD3 zeta signals upon ligation, but may lead to severe adverse effects

  20. An improved car-following model accounting for the preceding car's taillight

    Zhang, Jian; Tang, Tie-Qiao; Yu, Shao-Wei

    2018-02-01

    During the deceleration process, the preceding car's taillight may have influences on its following car's driving behavior. In this paper, we propose an extended car-following model with consideration of the preceding car's taillight. Two typical situations are used to simulate each car's movement and study the effects of the preceding car's taillight on the driving behavior. Meanwhile, sensitivity analysis of the model parameter is in detail discussed. The numerical results show that the proposed model can improve the stability of traffic flow and the traffic safety can be enhanced without a decrease of efficiency especially when cars pass through a signalized intersection.

  1. CAR-T cell therapy in gastrointestinal tumors and hepatic carcinoma: From bench to bedside.

    Zhang, Qi; Zhang, Zimu; Peng, Meiyu; Fu, Shuyu; Xue, Zhenyi; Zhang, Rongxin

    2016-01-01

    The chimeric antigen receptor (CAR) is a genetically engineered receptor that combines a scFv domain, which specifically recognizes the tumor-specific antigen, with T cell activation domains. CAR-T cell therapies have demonstrated tremendous efficacy against hematologic malignancies in many clinical trials. Recent studies have extended these efforts to the treatment of solid tumors. However, the outcomes of CAR-T cell therapy for solid tumors are not as remarkable as the outcomes have been for hematologic malignancies. A series of hurdles has arisen with respect to CAR-T cell-based immunotherapy, which needs to be overcome to target solid tumors. The major challenge for CAR-T cell therapy in solid tumors is the selection of the appropriate specific antigen to demarcate the tumor from normal tissue. In this review, we discuss the application of CAR-T cells to gastrointestinal and hepatic carcinomas in preclinical and clinical research. Furthermore, we analyze the usefulness of several specific markers in the study of gastrointestinal tumors and hepatic carcinoma.

  2. CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

    Cherian, Milu T; Lin, Wenwei; Wu, Jing; Chen, Taosheng

    2015-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug ...

  3. SMART SECURITY SYSTEM FOR CARS

    Akshay V. Balki*, Ankit A. Ramteke, Akshay Dhankar, Prof. Nilesh S. Panchbudhe

    2017-01-01

    This propose work is an attempt to model design an smart advance vehicle security system that uses biometric scanner and RFID card reader to give ignition pulse using two main module and to prevent theft. The system contains biometric scanner, RFID card reader, alcohol sensor, vibration sensor, GSM module, microcontroller (8051), relay switch, high voltage mesh..The safety of car is exceptionally essential. It provides pulse to ignition system by synchronizing driver’s data from license and t...

  4. Car insurance information management system

    Sun, Yu

    2015-01-01

    A customer information system is a typical information management system. It involves three aspects, the backstage database establishment, the application development and the system maintenance. A car insurance information management system is based on browser/server structure. Microsoft SQL Server establishes the backstage database. Active Server Pages, from Microsoft as well is used as the interface layer. The objective of this thesis was to apply ASP to the dynamic storage of a web page...

  5. Solar Powered Heat Control System for Cars

    Abin John; Jithin Thomas

    2014-01-01

    It takes times for an air-conditioner to effectively start cooling the passenger compartment in the car. So the passenger of the car will feel the heat in the car extremely before the air-conditioner fully cooling the interior of the car. Excessive heat can also damage an automobile's interior as well as personal property kept in the passenger compartment. So, a system to reduce this excessive heat by pumping out hot air and allowing cooler ambient air to enter the car by mean...

  6. Receptor tyrosine kinase signaling: a view from quantitative proteomics

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2009-01-01

    Growth factor receptor signaling via receptor tyrosine kinases (RTKs) is one of the basic cellular communication principals found in all metazoans. Extracellular signals are transferred via membrane spanning receptors into the cytoplasm, reversible tyrosine phosphorylation being the hallmark of all...

  7. Consumer Behavior towards Safer Car Purchasing Decisions

    Khairil Anwar Abu Kassim

    2016-08-01

    Full Text Available In Malaysia, the car safety level has been elevated through regulations and a consumer-based approach, i.e. the New Car Assessment Program in Southeast Asian Countries (ASEAN NCAP. Nevertheless, the availability of information on consumers’ car purchasing decisions towards safety is still limited in Malaysia. Thus, this study was aimed at evaluating consumers’ purchasing decisions of their present cars and investigating their awareness of ASEAN NCAP. Self-administered questionnaires were distributed among consumers visiting different car showrooms and dealer shops. The findings suggest that safety was considered as one of the top three factors by the respondents when purchasing their present cars. Awareness of ASEAN NCAP has increased as compared to a previous study. This information is essential for policy makers, manufacturers and other stakeholders to assist in setting priorities with regard to the promotion of car safety in the country.

  8. Substitution between cars within the household

    De Borger, Bruno; Mulalic, Ismir; Rouwendal, Jan

    2016-01-01

    In this paper we study the demand for car kilometres in two-car households, focusing on the substitution between cars of different fuel efficiency in response to fuel price changes. We use a large sample of detailed Danish data on two-car households to estimate – for each car owned by the household...... – own and cross-price effects of increases in fuel costs per kilometre. The empirical results show that failure to capture substitution between cars within the household can result in substantial misspecification biases. Ignoring substitution, the basic model yielded fuel price elasticities of 0.......98 and 1.41 for the primary and secondary cars, respectively. Accounting for substitution effects, these figures reduce to, respectively, 0.32 and 0.45. Consistent with substitution behaviour, we find that the fuel price elasticity of fuel demand exceeds the elasticity of kilometre demands with respect...

  9. Preferences for Alternative Fuel Vehicles of Company Car Drivers

    Koetse, M.J.; Hoen, A.

    2014-01-01

    Costs of car ownership for company cars drivers and private car owners are very different. Car use, car choice decisions and preferences for car characteristics may therefore differ substantially between these two markets. In this paper, we present results of a study on the preferences of company

  10. 49 CFR 215.121 - Defective car body.

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Defective car body. 215.121 Section 215.121..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Freight Car Components Car Bodies § 215.121 Defective car body. A railroad may not place or continue in service a car, if: (a) Any portion of...

  11. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

    Davenport, A J; Cross, R S; Watson, K A; Liao, Y; Shi, W; Prince, H M; Beavis, P A; Trapani, J A; Kershaw, M H; Ritchie, D S; Darcy, P K; Neeson, P J; Jenkins, M R

    2018-02-27

    Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. Copyright © 2018 the Author(s). Published by PNAS.

  12. Biology and clinical application of CAR T cells for B cell malignancies.

    Davila, Marco L; Sadelain, Michel

    2016-07-01

    Chimeric antigen receptor (CAR)-modified T cells have generated broad interest in oncology following a series of dramatic clinical successes in patients with chemorefractory B cell malignancies. CAR therapy now appears to be on the cusp of regulatory approval as a cell-based immunotherapy. We review here the T cell biology and cell engineering research that led to the development of second generation CARs, the selection of CD19 as a CAR target, and the preclinical studies in animal models that laid the foundation for clinical trials targeting CD19+ malignancies. We further summarize the status of CD19 CAR clinical therapy for non-Hodgkin lymphoma and B cell acute lymphoblastic leukemia, including their efficacy, toxicities (cytokine release syndrome, neurotoxicity and B cell aplasia) and current management in humans. We conclude with an overview of recent pre-clinical advances in CAR design that argues favorably for the advancement of CAR therapy to tackle other hematological malignancies as well as solid tumors.

  13. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells.

    Kawalekar, Omkar U; O'Connor, Roddy S; Fraietta, Joseph A; Guo, Lili; McGettigan, Shannon E; Posey, Avery D; Patel, Prachi R; Guedan, Sonia; Scholler, John; Keith, Brian; Snyder, Nathaniel W; Snyder, Nathaniel; Blair, Ian A; Blair, Ian; Milone, Michael C; June, Carl H

    2016-02-16

    Chimeric antigen receptors (CARs) redirect T cell cytotoxicity against cancer cells, providing a promising approach to cancer immunotherapy. Despite extensive clinical use, the attributes of CAR co-stimulatory domains that impact persistence and resistance to exhaustion of CAR-T cells remain largely undefined. Here, we report the influence of signaling domains of coreceptors CD28 and 4-1BB on the metabolic characteristics of human CAR T cells. Inclusion of 4-1BB in the CAR architecture promoted the outgrowth of CD8(+) central memory T cells that had significantly enhanced respiratory capacity, increased fatty acid oxidation and enhanced mitochondrial biogenesis. In contrast, CAR T cells with CD28 domains yielded effector memory cells with a genetic signature consistent with enhanced glycolysis. These results provide, at least in part, a mechanistic insight into the differential persistence of CAR-T cells expressing 4-1BB or CD28 signaling domains in clinical trials and inform the design of future CAR T cell therapies. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Minimum datasets to establish a CAR-mediated mode of action for rodent liver tumors.

    Peffer, Richard C; LeBaron, Matthew J; Battalora, Michael; Bomann, Werner H; Werner, Christoph; Aggarwal, Manoj; Rowe, Rocky R; Tinwell, Helen

    2018-07-01

    Methods for investigating the Mode of Action (MoA) for rodent liver tumors via constitutive androstane receptor (CAR) activation are outlined here, based on current scientific knowledge about CAR and feedback from regulatory agencies globally. The key events (i.e., CAR activation, altered gene expression, cell proliferation, altered foci and increased adenomas/carcinomas) can be demonstrated by measuring a combination of key events and associative events that are markers for the key events. For crop protection products, a primary dataset typically should include a short-term study in the species/strain that showed the tumor response at dose levels that bracket the tumorigenic and non-tumorigenic dose levels. The dataset may vary depending on the species and the test compound. As examples, Case Studies with nitrapyrin (in mice) and metofluthrin (in rats) are described. Based on qualitative differences between the species, the key events leading to tumors in mice or rats by this MoA are not operative in humans. In the future, newer approaches such as a CAR biomarker signature approach and/or in vitro CAR3 reporter assays for mouse, rat and human CAR may eventually be used to demonstrate a CAR MoA is operative, without the need for extensive additional studies in laboratory animals. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Posttransplant chimeric antigen receptor therapy.

    Smith, Melody; Zakrzewski, Johannes; James, Scott; Sadelain, Michel

    2018-03-08

    Therapeutic T-cell engineering is emerging as a powerful approach to treat refractory hematological malignancies. Its most successful embodiment to date is based on the use of second-generation chimeric antigen receptors (CARs) targeting CD19, a cell surface molecule found in most B-cell leukemias and lymphomas. Remarkable complete remissions have been obtained with autologous T cells expressing CD19 CARs in patients with relapsed, chemo-refractory B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Allogeneic CAR T cells may also be harnessed to treat relapse after allogeneic hematopoietic stem cell transplantation. However, the use of donor T cells poses unique challenges owing to potential alloreactivity. We review different approaches to mitigate the risk of causing or aggravating graft-versus-host disease (GVHD), including CAR therapies based on donor leukocyte infusion, virus-specific T cells, T-cell receptor-deficient T cells, lymphoid progenitor cells, and regulatory T cells. Advances in CAR design, T-cell selection and gene editing are poised to enable the safe use of allogeneic CAR T cells without incurring GVHD. © 2018 by The American Society of Hematology.

  16. ErbB-targeted CAR T-cell immunotherapy of cancer.

    Whilding, Lynsey M; Maher, John

    2015-01-01

    Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 years and is now a promising new treatment modality in the field of cancer immunotherapy. The approach involves genetically engineering T cells to target malignant cells through expression of a bespoke fusion receptor that couples an HLA-independent antigen recognition domain to one or more intracellular T-cell activating modules. Multiple clinical trials are now underway in several centers to investigate CAR T-cell immunotherapy of diverse hematologic and solid tumor types. The most successful results have been achieved in the treatment of patients with B-cell malignancies, in whom several complete and durable responses have been achieved. This review focuses on the preclinical and clinical development of CAR T-cell immunotherapy of solid cancers, targeted against members of the ErbB family.

  17. CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O.

    2014-01-01

    Highlights: • CAR activation decreased the level of Foxo1 in mouse livers. • CAR activation decreased the level of p21 in mouse livers. • CAR activation inhibited Foxo1 transcriptional activity in mouse livers. - Abstract: 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments

  18. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients.

    Turtle, Cameron J; Hanafi, Laïla-Aïcha; Berger, Carolina; Gooley, Theodore A; Cherian, Sindhu; Hudecek, Michael; Sommermeyer, Daniel; Melville, Katherine; Pender, Barbara; Budiarto, Tanya M; Robinson, Emily; Steevens, Natalia N; Chaney, Colette; Soma, Lorinda; Chen, Xueyan; Yeung, Cecilia; Wood, Brent; Li, Daniel; Cao, Jianhong; Heimfeld, Shelly; Jensen, Michael C; Riddell, Stanley R; Maloney, David G

    2016-06-01

    T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. ClinicalTrials.gov NCT01865617. R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

  19. Prediction of future car forms based on historical trends

    Bijendra Kumar

    2016-09-01

    Full Text Available Cars are one of the most important products that affects our daily life. Manufacturers of cars are inclined to know factors that affect the sales of cars and how to influence them. Car is a very competitive product whose technology is already matured. Thus, purchase decisions of a car depend on factors such as, aesthetics, ergonomics, features available and price. Exterior form and colour of a car are the most important factors that influence likeness of the car. We did a case study on car aesthetics (form, colour, shape, and user focus with more than 500 car advertisements over the past 70 years, appearing in various car magazines. Results show that form of cars has changed from sharp to smooth over the years, and white colour cars are becoming more popular. Additionally, car size is becoming smaller and increasingly focused towards family. Thus, manufacturers are recommended to develop compact, efficient and hybrid cars.

  20. Research of braking peculiarities of used cars

    V. Mitunevičius

    2002-06-01

    Full Text Available This paper briefly describes some analysis of a car braking process - the peculiarities of car wheel-to-road adhesion, the influence of distribution of braking forces on car stability between front and rear axles. The requirements of EU Directive 71/320/EEC to braking force coefficients of car front and rear axles are exposed. Structural designs of braking systems are analyzed with respect to their meeting the EU standards. Experimental measurements of braking force coefficients for some models of cars which are used in Lithuania, are presented with the analysis how these coefficients meet the EU standards. The analysis of test results, suggestions for the ratio of braking forces of car front and rear axles are presented.

  1. MODERN ELECTRIC CARS OF TESLA MOTORS COMPANY

    O. F. Vynakov

    2016-08-01

    Full Text Available This overview article shows the advantages of a modern electric car as compared with internal combustion cars by the example of the electric vehicles of Tesla Motors Company. It (в смысле- статья describes the history of this firm, provides technical and tactical characteristics of three modifications of electric vehicles produced by Tesla Motors. Modern electric cars are not less powerful than cars with combustion engines both in speed and acceleration amount. They are reliable, economical and safe in operation. With every year the maximum range of an electric car is increasing and its battery charging time is decreasing.Solving the problem of environmental safety, the governments of most countries are trying to encourage people to switch to electric cars by creating subsidy programs, lending and abolition of taxation. Therefore, the advent of an electric vehicle in all major cities of the world is inevitable.

  2. Benefits of magnesium wheels for consumer cars

    Frishfelds, Vilnis; Timuhins, Andrejs; Bethers, Uldis

    2018-05-01

    Advantages and disadvantages of magnesium wheels are considered based on a mechanical model of a car. Magnesium wheels are usually applied to racing cars as they provide slightly better strength/weight ratio than aluminum alloys. Do they provide notable benefits also for the everyday user when the car speeds do not exceed allowed speed limit? Distinct properties of magnesium rims are discussed. Apart from lighter weight of magnesium alloys, they are also good in dissipating the energy of vibrations. The role of energy dissipation in the rim of a wheel is estimated by a quarter car model. Improvements to safety by using the magnesium wheels are considered. Braking distance and responsiveness of the car is studied both with and without using an Anti Blocking System (ABS). Influence of rim weight on various handling parameters of the car is quantitatively tested.

  3. Should Diesel cars in Europe be discouraged?

    Mayeres, Inge; Proost, Stef

    2001-01-01

    This paper examines the rationale for the different tax treatment of gasoline and diesel cars currently observed in Europe. First, we analyse possible justifications for a different tax treatment: pure tax revenue considerations, externality cons0iderations and constraints on the tax instruments used for cars and trucks. Next, an applied general equilibrium model is used to assess the welfare effects of revenue neutral changes in the vehicle and fuel taxes on diesel and gasoline cars. The mod...

  4. PLC Based Automatic Multistoried Car Parking System

    Swanand S .Vaze; Rohan S. Mithari

    2014-01-01

    This project work presents the study and design of PLC based Automatic Multistoried Car Parking System. Multistoried car parking is an arrangement which is used to park a large number of vehicles in least possible place. For making this arrangement in a real plan very high technological instruments are required. In this project a prototype of such a model is made. This prototype model is made for accommodating twelve cars at a time. Availability of the space for parking is detecte...

  5. Alcohol Fuel in Passenger Car

    Adam Polcar

    2016-01-01

    Full Text Available The present article studies the effects of combustion of high-percentage mixture of bioethanol and gasoline on the output parameters of a passenger car engine. The car engine has not been structurally modified for the combustion of fuels with higher ethanol content. The mixture used consisted of E85 summer blend and Natural 95 gasoline in a ratio of 50:50. The parameters monitored during the experiment included the air-fuel ratio in exhaust gasses, the power output and torque of the engine and also the specific energy consumption and efficiency of the engine. As is apparent from the results, E85+N95 (50:50 mixture combustion results in lean-burn (λ > 1 due to the presence of oxygen in bioethanol. The lean-burn led to a slight decrease in torque and power output of the engine. However, due to the positive physicochemical properties of bioethanol, the decrease has not been as significant as would normally be expected from the measured air-fuel ratio. These findings are further confirmed by the calculated energy required to produce 1 kWh of energy, and by the higher efficiency of the engine during the combustion of a 50% bioethanol mixture.

  6. Air quality inside passenger cars

    Joanna Faber

    2017-02-01

    Full Text Available Vehicle interior is a specific environment of relatively small volume, with variety of materials placed inside, including hard and soft plastics, adhesives, paints, lubricants and many others. As a result, particularly in case of newly produced vehicles, large amounts and numbers of volatile species, especially volatile organic compounds (VOCs, may be emitted and have influence vehicle interior air quality (VIAQ. Despite the fact that many of these compounds may not be harmful for human health, some of them may be toxic, and this is the reason for increasing concern of vehicle manufacturers and users recently. The level of contamination varies from one vehicle to another and may be influenced by atmospheric conditions, external pollution, user habits, quality of materials used and others. The main aim of this paper was to present current knowledge status on VIAQ, with indication of main air pollutants and their concentrations. Vehicle interior air quality is discussed on the basis of studies on new and used cars in different conditions and locations. Main sources of VOCs presence inside car cabin are discussed in this paper with additional information regarding materials emissions. Differences in sampling and analytical methodologies were not debated, however, since the results differs largely in the scope of both number and amount of VOCs, a need of testing methods harmonization is indicated. Presented data may be helpful for legislative requirements introduction.

  7. Distinguishing Truncated and Normal MUC1 Glycoform Targeting from Tn-MUC1-Specific CAR T Cells

    Posey, Avery D; Clausen, Henrik; June, Carl H

    2016-01-01

    Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate potent clinical antitumor effects in a variety of blood cancers. However, clinical activity in solid tumors has been disappointing and toxicity has been a serious concern (Lamers et al., 2013; Morgan et al., 2010......). We recently found that a CAR composed of a scFv antibody fragment specific for the Tn-glycoform of MUC1 had potent activity in preclinical models of blood cancer and adenocarcinoma (Posey et al., 2016)....

  8. Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.

    Masihzadeh, Omid; Ammar, David A; Kahook, Malik Y; Lei, Tim C

    2013-05-01

    To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques. Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm. The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes. CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.

  9. MODERN ELECTRIC CARS OF TESLA MOTORS COMPANY

    O. F. Vynakov; E. V. Savolova; A. I. Skrynnyk

    2016-01-01

    This overview article shows the advantages of a modern electric car as compared with internal combustion cars by the example of the electric vehicles of Tesla Motors Company. It (в смысле- статья) describes the history of this firm, provides technical and tactical characteristics of three modifications of electric vehicles produced by Tesla Motors. Modern electric cars are not less powerful than cars with combustion engines both in speed and acceleration amount. They are reliable, economical ...

  10. Stock-car racing makes intuitive physicists

    Gwynne, Peter

    2008-03-01

    Formula One races involve cars festooned with gadgets and complex electronic devices, in which millions of dollars are spent refining a vehicle's aerodynamics and reducing its weight. But in events run by America's National Association of Stock Car Auto Racing (NASCAR), cars hurtle round an oval track at speeds of about 300 km h-1 without the help of the complex sensors that are employed in Formula One cars. To avoid crashing, drivers must make their own adjustments to track conditions, engine problems and the traffic around them.

  11. Overexpression of the cAMP Receptor 1 in Growing Dictyostelium Cells

    Johnson, Ronald L.; Vaughan, Roxanne A.; Caterina, Michael J.; Haastert, Peter J.M. van; Devreotes, Peter N.

    1991-01-01

    cAR1, the cAMP receptor expressed normally during the early aggregation stage of the Dictyostelium developmental program, has been expressed during the growth stage, when only low amounts of endogenous receptors are present. Transformants expressing cAR1 have 7-40 times over growth stage and

  12. Car allocation between household heads in car deficient households : A decision model

    Anggraini, Renni; Arentze, Theo A.; Timmermans, Harry J P

    2008-01-01

    This paper considers car allocation choice behaviour in car-deficient households explicitly in the context of an activity-scheduling process, focusing on work activities. A decision tree induction method is applied to derive a decision tree for the car allocation decision in automobile deficient

  13. The PXR/CAR nuclear receptor sytem and antimalaria chemotherapy

    Piedade, Rita

    2010-01-01

    Malaria is one of the oldest diseases know to mankind, still having devastating consequences; killing 1 million people/year. The efforts to control this disease have been focused on the control of its transmission and, most importantly, its clinical management by effective chemotherapy. Concerning the latter, several antimalarials have been developed in the last decades. They can be subdivided into four structural classes: aminoquinolines, antifolates, artemisinin derived compounds and naphto...

  14. CAR T Cells Targeting Podoplanin Reduce Orthotopic Glioblastomas in Mouse Brains.

    Shiina, Satoshi; Ohno, Masasuke; Ohka, Fumiharu; Kuramitsu, Shunichiro; Yamamichi, Akane; Kato, Akira; Motomura, Kazuya; Tanahashi, Kuniaki; Yamamoto, Takashi; Watanabe, Reiko; Ito, Ichiro; Senga, Takeshi; Hamaguchi, Michinari; Wakabayashi, Toshihiko; Kaneko, Mika K; Kato, Yukinari; Chandramohan, Vidyalakshmi; Bigner, Darell D; Natsume, Atsushi

    2016-03-01

    Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults with a 5-year overall survival rate of less than 10%. Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, expressed in the lymphatic endothelium. Several solid tumors overexpress PDPN, including the mesenchymal type of GBM, which has been reported to present the worst prognosis among GBM subtypes. Chimeric antigen receptor (CAR)-transduced T cells can recognize predefined tumor surface antigens independent of MHC restriction, which is often downregulated in gliomas. We constructed a lentiviral vector expressing a third-generation CAR comprising a PDPN-specific antibody (NZ-1-based single-chain variable fragment) with CD28, 4-1BB, and CD3ζ intracellular domains. CAR-transduced peripheral blood monocytes were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. CAR T-cell therapy that targets PDPN would be a promising adoptive immunotherapy to treat mesenchymal GBM. ©2016 American Association for Cancer Research.

  15. Paralleled comparison of vectors for the generation of CAR-T cells.

    Qin, Di-Yuan; Huang, Yong; Li, Dan; Wang, Yong-Sheng; Wang, Wei; Wei, Yu-Quan

    2016-09-01

    T-lymphocytes genetically engineered with the chimeric antigen receptor (CAR-T) have shown great therapeutic potential in cancer treatment. A variety of preclinical researches and clinical trials of CAR-T therapy have been carried out to lay the foundation for future clinical application. In these researches, several gene-transfer methods were used to deliver CARs or other genes into T-lymphocytes, equipping CAR-modified T cells with a property of recognizing and attacking antigen-expressing tumor cells in a major histocompatibility complex-independent manner. Here, we summarize the gene-transfer vectors commonly used in the generation of CAR-T cell, including retrovirus vectors, lentivirus vectors, the transposon/transposase system, the plasmid-based system, and the messenger RNA electroporation system. The following aspects were compared in parallel: efficiency of gene transfer, the integration methods in the modified T cells, foreground of scale-up production, and application and development in clinical trials. These aspects should be taken into account to generate the optimal CAR-gene vector that may be suitable for future clinical application.

  16. Augmentation of Antitumor Immunity by Human and Mouse CAR T Cells Secreting IL-18

    Biliang Hu

    2017-09-01

    Full Text Available The effects of transgenically encoded human and mouse IL-18 on T cell proliferation and its application in boosting chimeric antigen receptor (CAR T cells are presented. Robust enhancement of proliferation of IL-18-secreting human T cells occurred in a xenograft model, and this was dependent on TCR and IL-18R signaling. IL-18 augmented IFN-γ secretion and proliferation of T cells activated by the endogenous TCR. TCR-deficient, human IL-18-expressing CD19 CAR T cells exhibited enhanced proliferation and antitumor activity in the xenograft model. Antigen-propelled activation of cytokine helper ensemble (APACHE CAR T cells displayed inducible expression of IL-18 and enhanced antitumor immunity. In an intact mouse tumor model, CD19-IL-18 CAR T cells induced deeper B cell aplasia, significantly enhanced CAR T cell proliferation, and effectively augmented antitumor effects in mice with B16F10 melanoma. These findings point to a strategy to develop universal CAR T cells for patients with solid tumors.

  17. 49 CFR 238.311 - Single car test.

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Single car test. 238.311 Section 238.311... Requirements for Tier I Passenger Equipment § 238.311 Single car test. (a) Except for self-propelled passenger cars, single car tests of all passenger cars and all unpowered vehicles used in passenger trains shall...

  18. 49 CFR 180.507 - Qualification of tank cars.

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Qualification of tank cars. 180.507 Section 180... QUALIFICATION AND MAINTENANCE OF PACKAGINGS Qualification and Maintenance of Tank Cars § 180.507 Qualification of tank cars. (a) Each tank car marked as meeting a “DOT” specification or any other tank car used...

  19. CERN car stickers for 2014

    2013-01-01

    The stickers on your vehicles will cease to be valid at the end of 2013. We kindly request that you inform us as soon as possible if you no longer own a vehicle that is in our records. In particular, please inform the CERN Registration Service (Building 55, first floor) if you receive a sticker for a vehicle that you no longer own.   Stickers for 2014 are valid immediately and can be displayed as soon as you receive them. The Guards Service will continue to allow cars displaying a 2013 sticker into the CERN site until no later than 31 January 2014. After that date, the Guards Service will be obliged to deny access to any vehicles not displaying a valid sticker. Please see Operational Circular No. 2 for more details. We wish you a pleasant day and happy holidays, GS/DI security and access control service

  20. Reactions of Met-Cars

    Castleman, A.W. Jr.; Guo, B.C.

    1993-01-01

    A new class of metal-carbon complexes, termed metallo-carbohedrenes (Met-Cars), have been discovered to form in a plasma reactor in which early transition metals are vaporized into a stream carrying small hydrocarbon molecules. The initial discovery involved the species Ti 8 c 12 + , while subsequent studies revealed the stability of the anion and, most importantly, the neutral species. Subsequent investigations show that similar molecules, predicted to have a pentagonal dodecahedral structure, can also be formed with vanadium, hafnium, and zirconium. In the case of the latter, more recent investigations have displaced an interesting growth pattern. In particular, pentagonal dodecahedrons with dangling carbon atoms can undergo further growth, adding at least a second and third cage. The latest results on the properties and reactivities of these new cage-like molecular clusters will be discussed

  1. Vibration in car repair work.

    Hansson, J E; Eklund, L; Kihlberg, S; Ostergren, C E

    1987-03-01

    The main objective of the study was to find efficient hand tools which caused only minor vibration loading. Vibration measurements were carried out under standardised working conditions. The time during which car body repairers in seven companies were exposed to vibration was determined. Chisel hammers, impact wrenches, sanders and saws were the types of tools which generated the highest vibration accelerations. The average daily exposure at the different garages ranged from 22 to 70 min. The risk of vibration injury is currently rated as high. The difference between the highest and lowest levels of vibration was considerable in most tool categories. Therefore the choice of tool has a major impact on the magnitude of vibration exposure. The importance of choosing the right tools and working methods is discussed and a counselling service on vibration is proposed.

  2. TNFα promotes CAR-dependent migration of leukocytes across epithelial monolayers

    Morton, Penny E.; Hicks, Alexander; Ortiz-Zapater, Elena; Raghavan, Swetavalli; Pike, Rosemary; Noble, Alistair; Woodfin, Abigail; Jenkins, Gisli; Rayner, Emma; Santis, George; Parsons, Maddy

    2016-01-01

    Trans-epithelial migration (TEpM) of leukocytes during inflammation requires engagement with receptors expressed on the basolateral surface of the epithelium. One such receptor is Coxsackie and Adenovirus Receptor (CAR) that binds to Junctional Adhesion Molecule-like (JAM-L) expressed on leukocytes. Here we provide the first evidence that efficient TEpM of monocyte-derived THP-1 cells requires and is controlled by phosphorylation of CAR. We show that TNFα acts in a paracrine manner on epithelial cells via a TNFR1-PI3K-PKCδ pathway leading to CAR phosphorylation and subsequent transmigration across cell junctions. Moreover, we show that CAR is hyper-phosphorylated in vivo in acute and chronic lung inflammation models and this response is required to facilitate immune cell recruitment. This represents a novel mechanism of feedback between leukocytes and epithelial cells during TEpM and may be important in controlling responses to pro-inflammatory cytokines in pathological settings. PMID:27193388

  3. Expression of CAR in SW480 and HepG2 cells during G1 is associated with cell proliferation

    Osabe, Makoto; Sugatani, Junko; Takemura, Akiko; Yamazaki, Yasuhiro; Ikari, Akira; Kitamura, Naomi; Negishi, Masahiko; Miwa, Masao

    2008-01-01

    Constitutive androstane receptor (CAR) is a transcription factor to regulate the expression of several genes related to drug-metabolism. Here, we demonstrate that CAR protein accumulates during G1 in human SW480 and HepG2 cells. After the G1/S phase transition, CAR protein levels decreased, and CAR was hardly detected in cells by the late M phase. CAR expression in both cell lines was suppressed by RNA interference-mediated suppression of CDK4. Depletion of CAR by RNA interference in both cells and by hepatocyte growth factor treatment in HepG2 cells resulted in decreased MDM2 expression that led to p21 upregulation and repression of HepG2 cell growth. Thus, our results demonstrate that CAR expression is an early G1 event regulated by CDK4 that contributes to MDM2 expression; these findings suggest that CAR may influence the expression of genes involved in not only the metabolism of endogenous and exogenous substances but also in the cell proliferation

  4. Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice

    Hillerdal, Victoria; Ramachandran, Mohanraj; Leja, Justyna; Essand, Magnus

    2014-01-01

    Adoptive transfer of T cells genetically engineered with a chimeric antigen receptor (CAR) has successfully been used to treat both chronic and acute lymphocytic leukemia as well as other hematological cancers. Experimental therapy with CAR-engineered T cells has also shown promising results on solid tumors. The prostate stem cell antigen (PSCA) is a protein expressed on the surface of prostate epithelial cells as well as in primary and metastatic prostate cancer cells and therefore a promising target for immunotherapy of prostate cancer. We developed a third-generation CAR against PSCA including the CD28, OX-40 and CD3 ζ signaling domains. T cells were transduced with a lentivirus encoding the PSCA-CAR and evaluated for cytokine production (paired Student’s t-test), proliferation (paired Student’s t-test), CD107a expression (paired Student’s t-test) and target cell killing in vitro and tumor growth and survival in vivo (Log-rank test comparing Kaplan-Meier survival curves). PSCA-CAR T cells exhibit specific interferon (IFN)-γ and interleukin (IL)-2 secretion and specific proliferation in response to PSCA-expressing target cells. Furthermore, the PSCA-CAR-engineered T cells efficiently kill PSCA-expressing tumor cells in vitro and systemic treatment with PSCA-CAR-engineered T cells significantly delays subcutaneous tumor growth and prolongs survival of mice. Our data confirms that PSCA-CAR T cells may be developed for treatment of prostate cancer

  5. 49 CFR 174.110 - Car magazine.

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Car magazine. 174.110 Section 174.110...) Materials § 174.110 Car magazine. When specially authorized by the carrier, Division 1.1 or 1.2 (explosive... packages of Class 1 (explosive) materials are placed in a “magazine” box made of sound lumber not less than...

  6. Gates Auto Door Car With Lights Modulated

    Lina Carolina; Luyung Dinani, Skom, MMSi

    2002-01-01

    In scientific writing wi ll be explained about automatic gates with modulated headlights, where to find the car lights were adjusted by the relative frequency darker because of this background that the author alleviate human task in performing daily activities by using an automatic gate with the car lights modulated.

  7. The Hungarian car insurance cartel saga

    Cseres, K.J.; Szilágyi, P.; Rodger, B.

    2013-01-01

    his chapter discusses the landmark Hungarian case relating to the car insurance and repair markets, which involved both vertical and horizontal agreements. The case concerned the horizontal relationship between Hungary’s two largest insurance companies and their vertical relationships with car

  8. Rear-facing car seat (image)

    A rear-facing car seat position is recommended for a child who is very young. Extreme injury can occur in an accident because ... child. In a frontal crash a rear-facing car seat is best, because it cradles the head, ...

  9. Modeling human learning involved in car driving

    Wewerinke, P.H.

    1994-01-01

    In this paper, car driving is considered at the level of human tracking and maneuvering in the context of other traffic. A model analysis revealed the most salient features determining driving performance and safety. Learning car driving is modelled based on a system theoretical approach and based

  10. Car Stopping Distance on a Tabletop

    Haugland, Ole Anton

    2013-01-01

    Stopping distances in car braking can be an intriguing topic in physics teaching. It illustrates some basic principles of physics, and sheds valuable light on students' attitude towards aggressive driving. Due to safety considerations, it can be difficult to make experiments with actual car braking. (Contains 2 figures.)

  11. Modelling of fire spread in car parks

    Noordijk, L.M.; Lemaire, A.D.

    2005-01-01

    Currently, design codes assume that in a car park fire at most 3-4 vehicles are on fire at the same time. Recent incidents in car parks have drawn international attention to such assumptions and have raised questions as to the fire spreading mechanism and the resulting fire load on the structure.

  12. Automated Coal-Mine Shuttle Car

    Collins, E. R., Jr.

    1984-01-01

    Cable-guided car increases efficiency in underground coal mines. Unmanned vehicle contains storage batteries in side panels for driving traction motors located in wheels. Batteries recharged during inactive periods or slid out as unit and replaced by fresh battery bank. Onboard generator charges batteries as car operates.

  13. A Radio-Controlled Car Challenge

    Roman, Harry T.

    2010-01-01

    Watching a radio-controlled car zip along a sidewalk or street has become a common sight. Within this toy are the basic ingredients of a mobile robot, used by industry for a variety of important and potentially dangerous tasks. In this challenge, students consider modifying an of-the-shelf, radio-controlled car, adapting it for a robotic task.

  14. Engineering an Affordable Self-Driving Car

    Budisteanu, Alexandru Ionut

    2018-01-17

    "More than a million people die in car accidents each year, and most of those accidents are the result of human errorヤ Alexandru Budisteanu is 23 years old and owns a group of startups including Autonomix, an Artificial Intelligence software for affordable self-driving cars and he designed a low-cost self-driving car. The car\\'s roof has cameras and low-resolution 3D LiDAR equipment to detect traffic lanes, other cars, curbs and obstacles, such as people crossing by. To process this dizzying amount of data, Alexandru employed Artificial Intelligence algorithms to extract information from the visual data and plot a safe route for the car. Then, he built a manufacturing facility in his garage from Romania to assembly affordable VisionBot Pick and Place robots that are used to produce electronics. During this lecture, Alexandru will talk about this autonomous self-driving car prototype, for which he received the grand prize of the Intel International Science and Engineering Fair, and was nominated by TIME magazine as one of the worldメs most influential teens of 2013.

  15. Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors

    Robert Moot

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs. VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition.

  16. FCγ Chimeric Receptor-Engineered T Cells: Methodology, Advantages, Limitations, and Clinical Relevance

    Giuseppe Sconocchia

    2017-04-01

    Full Text Available For many years, disappointing results have been generated by many investigations, which have utilized a variety of immunologic strategies to enhance the ability of a patient’s immune system to recognize and eliminate malignant cells. However, in recent years, immunotherapy has been used successfully for the treatment of hematologic and solid malignancies. The impressive clinical responses observed in many types of cancer have convinced even the most skeptical clinical oncologists that a patient’s immune system can recognize and reject his tumor if appropriate strategies are implemented. The success immunotherapy is due to the development of at least three therapeutic strategies. They include tumor-associated antigen (TAA-specific monoclonal antibodies (mAbs, T cell checkpoint blockade, and TAA-specific chimeric antigen receptors (CARs T cell-based immunotherapy. However, the full realization of the therapeutic potential of these approaches requires the development of strategies to counteract and overcome some limitations. They include off-target toxicity and mechanisms of cancer immune evasion, which obstacle the successful clinical application of mAbs and CAR T cell-based immunotherapies. Thus, we and others have developed the Fc gamma chimeric receptors (Fcγ-CRs-based strategy. Like CARs, Fcγ-CRs are composed of an intracellular tail resulting from the fusion of a co-stimulatory molecule with the T cell receptor ζ chain. In contrast, the extracellular CAR single-chain variable fragment (scFv, which recognizes the targeted TAA, has been replaced with the extracellular portion of the FcγRIIIA (CD16. Fcγ-CR T cells have a few intriguing features. First, given in combination with mAbs, Fcγ-CR T cells mediate anticancer activity in vitro and in vivo by an antibody-mediated cellular cytotoxicity mechanism. Second, CD16-CR T cells can target multiple cancer types provided that TAA-specific mAbs with the appropriate specificity are available

  17. The Green City Car. A holistic approach for NVH abatement of city cars

    Bein, Thilo; Mayer, Dirk; Elliott, Steve; Ferrali, Leonardo; Casella, Mauro; Saemann, Ernst-Ulrich; Kropp, Wolfgang; Nielsen, Finn Kryger; Meschke, Jens; Pisano, Emanuel

    2014-01-01

    Pursuing the different passive and active concepts in a holistic approach, the FP7 project Green City Car demonstrates the feasibility of applying active systems to NVH-related problems light city cars from a system point-of view. During the project, a city car equipped with a small engine has been considered equipped with the latest technology in terms of safety aspects related to pedestrian’s impact and car-to-car compatibility, which are of major importance in an urban environment. The noi...

  18. The kinematic advantage of electric cars

    Meyn, Jan-Peter

    2015-11-01

    Acceleration of a common car with with a turbocharged diesel engine is compared to the same type with an electric motor in terms of kinematics. Starting from a state of rest, the electric car reaches a distant spot earlier than the diesel car, even though the latter has a better specification for engine power and average acceleration from 0 to 100 km h-1. A three phase model of acceleration as a function of time fits the data of the electric car accurately. The first phase is a quadratic growth of acceleration in time. It is shown that the tenfold higher coefficient for the first phase accounts for most of the kinematic advantage of the electric car.

  19. Flex cars and the alcohol price

    Ferreira, Alex Luiz; Da Silveira, Jaylson Jair; De Almeida Prado, Fernando Pigeard

    2009-01-01

    We build a model that incorporates the effect of the innovative 'flex' car, an automobile that is able to run with either gasoline or alcohol, on the dynamics of fuel prices in Brazil. Our model shows that differences regarding fuel prices will now depend on the proportions of alcohol, gasoline and flex cars in the total stock. Conversely, the demand for each type of car will also depend on the expected future prices of alcohol and gasoline (in addition to the car prices). The model reflects our findings that energy prices are tied in the long run and that causality runs stronger from gasoline to alcohol. The estimated error correction parameter is stable, implying that the speed of adjustment towards equilibrium remains unchanged. The latter result is probably due to a still small fraction of flex cars in the total stock (approx. 5%), despite the fact that its sales nearly reached 100% in 2006. (author)

  20. The first case of synchronous cellular angiofibromas of the scrotum

    Chih-Chen Hsu

    2016-06-01

    Full Text Available Cellular angiofibroma is a rare benign tumor which is found in the vulvovaginal or inguinoscrotal region. To the best of our knowledge, there have been no reports that describe two cellular angiofibromas on the same side of the scrotum. Here, we report the case of an 89-year-old male patient with two scrotal tumors which were proven to be cellular angiofibromas after the biopsy specimen was examined. The tumor markers were CD34 (+, focally positive for S-100, actin (−, desmin (−, estrogen receptor (−, progesterone receptor (−, beta-catenin (−, CD99 (−, and B-cell lymphoma 2 (−.

  1. Modelling and optimization of car-to-car compatibility - Modellierung und optimierung von pkw-pkw-kompatibilität

    Mooi, H.G.; Nastic, T.; Huibers, J.H.A.M.

    1999-01-01

    In this paper simple and more detailed MADYMO multibody models were used to simulate the car structure for improving the car-to-car compatibility of the whole car fleet. As a first step, survey studies were performed to develop a method for the optimization of car design with respect to frontal and

  2. 77 FR 3482 - Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen...

    2012-01-24

    ... Exclusive License: Development of T Cell Receptors and Chimeric Antigen Receptors Into Therapeutics for.... 61/473,409 entitled ``Anti-epidermal growth factor receptor variant III chimeric antigen receptors... EGFRvIII chimeric antigen (CARs) and methods of using these engineered T cells to treat and/or prevent...

  3. Wireless Cellular Mobile Communications

    Zalud, V.

    2002-01-01

    In this article is briefly reviewed the history of wireless cellular mobile communications, examined the progress in current second generation (2G) cellular standards and discussed their migration to the third generation (3G). The European 2G cellular standard GSM and its evolution phases GPRS and EDGE are described somewhat in detail. The third generation standard UMTS taking up on GSM/GPRS core network and equipped with a new advanced access network on the basis of code division multiple ac...

  4. Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

    Liu, Hong; Xu, Yiyang; Xiang, Jingyi; Long, Li; Green, Shon; Yang, Zhiyuan; Zimdahl, Bryan; Lu, Jingwei; Cheng, Neal; Horan, Lucas H; Liu, Bin; Yan, Su; Wang, Pei; Diaz, Juan; Jin, Lu; Nakano, Yoko; Morales, Javier F; Zhang, Pengbo; Liu, Lian-Xing; Staley, Binnaz K; Priceman, Saul J; Brown, Christine E; Forman, Stephen J; Chan, Vivien W; Liu, Cheng

    2017-01-15

    The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP 158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01 + /AFP + while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP 158 -expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. Biomechanics of cellular solids.

    Gibson, Lorna J

    2005-03-01

    Materials with a cellular structure are widespread in nature and include wood, cork, plant parenchyma and trabecular bone. Natural cellular materials are often mechanically efficient: the honeycomb-like microstructure of wood, for instance, gives it an exceptionally high performance index for resisting bending and buckling. Here we review the mechanics of a wide range of natural cellular materials and examine their role in lightweight natural sandwich structures (e.g. iris leaves) and natural tubular structures (e.g. plant stems or animal quills). We also describe two examples of engineered biomaterials with a cellular structure, designed to replace or regenerate tissue in the body.

  6. First results from car-to-car and car-to-infrastructure radio channel measurements at 5.2GHZ

    Paier, Alexander; Kåredal, Johan; Czink, Nicolai; Hofstetter, Helmut; Dumard, Charlotte; Zemen, Thomas; Tufvesson, Fredrik; Mecklenbräuker, Christoph; Molisch, Andreas

    2007-01-01

    Car-to-car and car-to-infrastructure (henceforth called C2X) communications are constantly gaining importance for road-safety and other applications. In order to design efficient C2X systems, an understanding of realistic C2X propagation channels is required, but currently, only few measurements have been published. This paper presents a description of an extensive measurement campaign recently conducted in an urban scenario, a rural scenario, and on a highway. We focused on 4 ÿ 4 multiple-in...

  7. Possible Relevance of Receptor-Receptor Interactions between Viral- and Host-Coded Receptors for Viral-Induced Disease

    Luigi F. Agnati

    2007-01-01

    Full Text Available It has been demonstrated that some viruses, such as the cytomegalovirus, code for G-protein coupled receptors not only to elude the immune system, but also to redirect cellular signaling in the receptor networks of the host cells. In view of the existence of receptor-receptor interactions, the hypothesis is introduced that these viral-coded receptors not only operate as constitutively active monomers, but also can affect other receptor function by interacting with receptors of the host cell. Furthermore, it is suggested that viruses could also insert not single receptors (monomers, but clusters of receptors (receptor mosaics, altering the cell metabolism in a profound way. The prevention of viral receptor-induced changes in host receptor networks may give rise to novel antiviral drugs that counteract viral-induced disease.

  8. Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-mediated Contractility via Activation of Focal Adhesion Kinase and Extra Cellular-regulated Kinase 1/2 in Cerebral Arteries from Rat

    Spray, Stine; Rasmussen, Marianne N P; Skovsted, Gry F

    2016-01-01

    that rapid and sustained reduction in wall tension/stretch is a possible trigger mechanism for this vascular remodelling. Isolated rat middle cerebral artery (MCA) segments were incubated in a wire-myograph with or without mechanical stretch, prior to assessment of their contractile response to the selective...... expression to SMC expression and 2) an increased calcium sensitivity of the SMCs due to an increased expression of the calcium channel transient receptor potential canonical 1. Collectively, our results present a possible mechanism linking lack of vessel wall stretch/tension to changes in ETB receptor...

  9. Emergency information systems for cars

    Thirunavukkarasu, M.; Vani Manasa, N.; Kumar, K. Rajesh; Sundar, S.

    2017-11-01

    The main objective of this work is to create a Health Care monitoring and Guidance system for persons who are travelling in outdoor environments like cars. GSM (Global System for Mobile Communications) and GPS (Global Positioning System) technologies are separately and combined today in many applications in our day to day life. The GSM module will send a message along with the GPS location to the end user through text, and a call is initiated to the user for further instructions. The Global Positioning System (GPS) will give the location of the interested vehicle. This system helps the doctor or anyone to monitor the accident who is outdoor and has less help. This will help the hospital to monitor the accident as well as guide the injured through difficult situations. Using a buzzer, the persons nearby will come to know that the person is in danger or in poor health conditions. This project provides a good two-way communication with the injured and the hospital to assist them to give first aid before an ambulance arrives. So, this paper devices a novel technique to assist the people who just met with accident through GPS and GSM.

  10. Chimeric antigen receptor-modified T cells for the treatment of solid tumors: Defining the challenges and next steps☆

    Beatty, Gregory L.; O’Hara, Mark

    2016-01-01

    Chimeric antigen receptor (CAR) T cell therapy has shown promise in CD19 expressing hematologic malignancies, but how to translate this success to solid malignancies remains elusive. Effective translation of CAR T cells to solid tumors will require an understanding of potential therapeutic barriers, including factors that regulate CAR T cells expansion, persistence, trafficking, and fate within tumors. Herein, we describe the current state of CAR T cells in solid tumors; define key barriers t...

  11. Charging electric cars from solar energy

    Liang, Xusheng; Tanyi, Elvis; Zou, Xin

    2016-01-01

    Before vehicles were heavily relied on coal, fossil fuels and wind for power.  Now, they are rapidly being replaced by electric vehicles and or plug-in hybrid electric cars. But these electric cars are still faced with the problem of energy availability because they rely on energy from biomass, hydro power and wind turbines for power generation. The abundance of solar radiation and its use as solar energy as a power source in driving these rapidly increasing electric cars is not only an impor...

  12. PC-based car license plate reader

    Hwang, Chung-Mu; Shu, Shyh-Yeong; Chen, Wen-Yu; Chen, Yie-Wern; Wen, Kuang-Pu

    1992-11-01

    A car license plate reader (CLPR) using fuzzy inference and neural network algorithm has been developed in Industrial Technology Research Institute (ITRI) and installed in highway toll stations to identify stolen cars. It takes an average of 0.7 seconds to recognize a car license plate by using a PC with 80486-50 CPU. The recognition rate of the system is about 97%. The techniques of CLPR include vehicle sensing, image grab control, optic pre- processing, lighting, and optic character recognition (OCR). The CLPR can be used in vehicle flow statistics, the checking of stolen vehicles, automatic charging systems in parking lots or garage management, and so on.

  13. Will Brazil's cars go on the wagon?

    Homewood, B.

    1993-01-01

    The use of ethanol as an alternative fuel for cars in Brazil, may shortly be reduced. Falling world oil prices have meant that ethanol, derived from sugar cane, following a fourteen year research program, has ceased to be a financially viable replacement for petrol. Although about a third of Brazil's cars are at present powered by ethanol, only substantial government subsidies could reinstate this fuel despite its reduced pollutant status. Government officials now predict that ethanol will become merely a petrol additive and production of ethanol cars will have stopped by the year 2000. (UK)

  14. Electric cars as mobile power storage systems

    Herzog, B.

    2010-01-01

    This article discusses the use of electric cars as a means of optimising the use of renewable energy sources. Charging the cars' batteries during periods when cheap electricity prices prevail and then using excess capacity to supply the mains with electricity during periods of peak demand is discussed. The possible use of wind for power generation is discussed and a system proposed by a leading supplier of electrical apparatus and systems is examined. Two examples of electric cars and associated power chains are looked at and tests in everyday practice are described

  15. THEORETICAL BASIS OF MOTOR CAR QUALITY EVALUTION

    T. Bazhinova

    2016-12-01

    Full Text Available Comprehensive assessment of the quality of cars, which is based on the integral parameters of comfort, reliability, safety, environmental and technical solutions are considered and defined. The amount of parameters that define the quality level of the car use throughout the country is defined. Mathematical formulas and quality indicators of the integral index are developed. The integral quality index of vehicles allows comparing the vehicles of different classes based on external operating conditions. The numerical values of the integral index determines the quality of the car.

  16. Density and temperature measurement using CARS spectroscopy

    Hirth, A.; Vollrath, K.

    1979-01-01

    Coherent Anti Stokes Raman Scattering (CARS) a technique derived from nonlinear optics offers two major advantages compared with the spontaneous Raman method: improved scattering efficiency and spatial coherence of the scattered signal. The theory of the coherent mixing in resonant media serves as a quantitative background of the CARS technique. A review of several applications on plasma physics and gasdynamics is given, which permits to consider the CARS spectroscopy as a potential method for nonintrusive measurement of local concentration and temperature in gas flows and reactive media. (Auth.)

  17. Linearizable cellular automata

    Nobe, Atsushi; Yura, Fumitaka

    2007-01-01

    The initial value problem for a class of reversible elementary cellular automata with periodic boundaries is reduced to an initial-boundary value problem for a class of linear systems on a finite commutative ring Z 2 . Moreover, a family of such linearizable cellular automata is given

  18. CD19-Targeted CAR T cells as novel cancer immunotherapy for relapsed or refractory B-cell acute lymphoblastic leukemia.

    Davila, Marco L; Brentjens, Renier J

    2016-10-01

    Immunotherapy has demonstrated significant potential for the treatment of patients with chemotherapy-resistant hematologic malignancies and solid tumors. One type of immunotherapy involves the adoptive transfer of T cells that have been genetically modified with a chimeric antigen receptor (CAR) to target a tumor. These hybrid proteins are composed of the antigen-binding domains of an antibody fused to T-cell receptor signaling machinery. CAR T cells that target CD19 recently have made the jump from the laboratory to the clinic, and the results have been remarkable. CD19-targeted CAR T cells have induced complete remissions of disease in up to 90% of patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL), who have an expected complete response rate of 30% in response to chemotherapy. The high efficacy of CAR T cells in B-ALL suggests that regulatory approval of this therapy for this routinely fatal leukemia is on the horizon. We review the preclinical development of CAR T cells and their early clinical application for lymphoma. We also provide a comprehensive analysis of the use of CAR T cells in patients with B-ALL. In addition, we discuss the unique toxicities associated with this therapy and the management schemes that have been developed.

  19. A compound chimeric antigen receptor strategy for targeting multiple myeloma.

    Chen, K H; Wada, M; Pinz, K G; Liu, H; Shuai, X; Chen, X; Yan, L E; Petrov, J C; Salman, H; Senzel, L; Leung, E L H; Jiang, X; Ma, Y

    2018-02-01

    Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

  20. T cell receptor-engineered T cells to treat solid tumors: T cell processing toward optimal T cell fitness

    C.H.J. Lamers (Cor); S. van Steenbergen-Langeveld (Sabine); M. van Brakel (Mandy); C.M. Groot-van Ruijven (Corrien); P.M.M.L. van Elzakker (Pascal); B.A. van Krimpen (Brigitte); S. Sleijfer (Stefan); J.E.M.A. Debets (Reno)

    2014-01-01

    textabstractTherapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T

  1. Long Terminal Repeat CRISPR-CAR-Coupled "Universal" T Cells Mediate Potent Anti-leukemic Effects.

    Georgiadis, Christos; Preece, Roland; Nickolay, Lauren; Etuk, Aniekan; Petrova, Anastasia; Ladon, Dariusz; Danyi, Alexandra; Humphryes-Kirilov, Neil; Ajetunmobi, Ayokunmi; Kim, Daesik; Kim, Jin-Soo; Qasim, Waseem

    2018-03-06

    Gene editing can be used to overcome allo-recognition, which otherwise limits allogeneic T cell therapies. Initial proof-of-concept applications have included generation of such "universal" T cells expressing chimeric antigen receptors (CARs) against CD19 target antigens combined with transient expression of DNA-targeting nucleases to disrupt the T cell receptor alpha constant chain (TRAC). Although relatively efficient, transgene expression and editing effects were unlinked, yields variable, and resulting T cell populations heterogeneous, complicating dosing strategies. We describe a self-inactivating lentiviral "terminal" vector platform coupling CAR expression with CRISPR/Cas9 effects through incorporation of an sgRNA element into the ΔU3 3' long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid ΔU3-sgRNA, delivery of Cas9 mRNA resulted in targeted TRAC locus cleavage and allowed the enrichment of highly homogeneous (>96%) CAR + (>99%) TCR - populations by automated magnetic separation. Molecular analyses, including NGS, WGS, and Digenome-seq, verified on-target specificity with no evidence of predicted off-target events. Robust anti-leukemic effects were demonstrated in humanized immunodeficient mice and were sustained longer than by conventional CAR + TCR + T cells. Terminal-TRAC (TT) CAR T cells offer the possibility of a pre-manufactured, non-HLA-matched CAR cell therapy and will be evaluated in phase 1 trials against B cell malignancies shortly. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  2. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region

    Jiang, Y P; Wang, H; D'Eustachio, P

    1993-01-01

    We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology, ...

  3. CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation.

    Bajgain, Pradip; Tawinwung, Supannikar; D'Elia, Lindsey; Sukumaran, Sujita; Watanabe, Norihiro; Hoyos, Valentina; Lulla, Premal; Brenner, Malcolm K; Leen, Ann M; Vera, Juan F

    2018-05-10

    The adoptive transfer of T cells redirected to tumor via chimeric antigen receptors (CARs) has produced clinical benefits for the treatment of hematologic diseases. To extend this approach to breast cancer, we generated CAR T cells directed against mucin1 (MUC1), an aberrantly glycosylated neoantigen that is overexpressed by malignant cells and whose expression has been correlated with poor prognosis. Furthermore, to protect our tumor-targeted cells from the elevated levels of immune-inhibitory cytokines present in the tumor milieu, we co-expressed an inverted cytokine receptor linking the IL4 receptor exodomain with the IL7 receptor endodomain (4/7ICR) in order to transform the suppressive IL4 signal into one that would enhance the anti-tumor effects of our CAR T cells at the tumor site. First (1G - CD3ζ) and second generation (2G - 41BB.CD3ζ) MUC1-specific CARs were constructed using the HMFG2 scFv. Following retroviral transduction transgenic expression of the CAR±ICR was assessed by flow cytometry. In vitro CAR/ICR T cell function was measured by assessing cell proliferation and short- and long-term cytotoxic activity using MUC1+ MDA MB 468 cells as targets. In vivo anti-tumor activity was assessed using IL4-producing MDA MB 468 tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. In the IL4-rich tumor milieu, 1G CAR.MUC1 T cells failed to expand or kill MUC1+ tumors and while co-expression of the 4/7ICR promoted T cell expansion, in the absence of co-stimulatory signals the outgrowing cells exhibited an exhausted phenotype characterized by PD-1 and TIM3 upregulation and failed to control tumor growth. However, by co-expressing 2G CAR.MUC1 (signal 1 - activation + signal 2 - co-stimulation) and 4/7ICR (signal 3 - cytokine), transgenic T cells selectively expanded at the tumor site and produced potent and durable tumor control in vitro and in vivo. Our findings demonstrate the feasibility of targeting breast

  4. Computer Security: your car, my control

    Stefan Lueders, Computer Security Team

    2015-01-01

    We have discussed the Internet of Things (IoT) and its security implications already in past issues of the CERN Bulletin, for example in “Today’s paranoia, tomorrow’s reality” (see here). Unfortunately, tomorrow has come. At this years's Black Hat conference researchers presented their findings on how easily your car can be hacked and controlled remotely. Sigh.   While these researchers have just shown that they can wirelessly hijack a Jeep Cherokee, others have performed similar studies with SmartCars, Fords, a Tesla, a Corvette, BMWs, Chryslers and Mercedes! With the increasing computerisation of cars, the engine management system, air conditioning, anti-lock braking system, electronic stability programme, etc. are linked to the infotainment, navigation and communication systems, opening the door for these vehicles to be hacked remotely. The now prevalent Bluetooth connection with smartphones is one entry vector to attack your car remotely...

  5. Design of two wheel self balancing car

    He, Chun-hong; Ren, Bin

    2018-02-01

    This paper proposes a design scheme of the two-wheel self-balancing dolly, the integration of the gyroscope and accelerometer MPU6050 constitutes the car position detection device.System selects 32-bit MCU stmicroelectronics company as the control core, completed the processing of sensor signals, the realization of the filtering algorithm, motion control and human-computer interaction. Produced and debugging in the whole system is completed, the car can realize the independent balance under the condition of no intervention. The introduction of a suitable amount of interference, the car can adjust quickly to recover and steady state. Through remote control car bluetooth module complete forward, backward, turn left and other basic action..

  6. Papers on vehicle size : cars and trucks

    1988-06-01

    The four papers in this volume describe analyses of car size : trends and truck occupant injuries and fatalities. All four were : written between June 1985 and December 1987. The topics : addressed include where heavy truck accidents occur, injury an...

  7. CARS diagnostics of high pressure discharges

    Uhlenbusch, J.

    2001-01-01

    After a short description of the principles of the CARS, RECARS and POLCARS techniques and a discussion of setups for CARS experiments some experimental results are summarized. The results concern mainly plasma under atmospheric pressure, in particular the determination of temperature in a CO 2 laser-induced pyrolysis flame burning in a silane-acetylene gas mixture, the measurements of N 2 vibrational and rotational temperatures as well as the electron density by CARS and of an NO minority by POLCARS in an atmospheric microwave discharge, and finally RECARS experiments on indium iodide, Which is present in metal halide discharge lamps. Guided by these examples some problems and difficulties arising when performing CARS measurements are discussed

  8. Design consideration of solar powered cars

    Koten, Hasan; Yilmaz, Mustafa; Zafer Gul, M. [Marmara University Mechanical Engineering Department (Turkey)], E-mail: hasan.koten@marmara.edu.tr

    2011-07-01

    With the coming shortage of fossil fuels and the rising concerns over the environment, it is important to develop new technologies that reduce both energy consumption and pollution at the same time. Using solar energy is a good solution which could meet the world's energy needs. The aim of this study is to present the design process in the production of a solar powered car. Designing a solar powered car is a difficult task as there are strict requirements in term of efficiency: the car must have low drag resistance, be light-weight, and have low rolling resistance. In addition this paper presents the use of the solar powered Stirling engine technology rather than a photovoltaic conversion system for vehicle propulsion. This study presented a design process in the construction of a solar powered car and is expected to provide a new topic of research in the transportation field.

  9. SODA-IIoT4ConnectedCars: Spread updates between cars with limited Internet access

    Boudguiga , Aymen; Quesnel , Flavien; Bouzerna , Nabil

    2017-01-01

    International audience; A blockchain infrastructure, combined with cryptographic signatures, can improve availability and accountability for the deployment of IoT updates.However, cars with limited or intermittent Internet access may have difficulties in downloading full updates fromthe blockchain. Therefore, we allow cars that successfully downloaded updates to share them with other cars by means of a Peer-to-Peer (P2P) mechanism.

  10. Uncertainty quantification and race car aerodynamics

    Bradford, J; Montomoli, F; D'Ammaro, A

    2014-01-01

    28.04.15 KB. Ok to add accepted version to spiral, embargo expired Car aerodynamics are subjected to a number of random variables which introduce uncertainty into the downforce performance. These can include, but are not limited to, pitch variations and ride height variations. Studying the effect of the random variations in these parameters is important to predict accurately the car performance during the race. Despite their importance the assessment of these variations is difficult and it...

  11. Driving Cars by Means of Genetic Algorithms

    Sáez Achaerandio, Yago; Pérez, Diego; Sanjuan, Óscar; Isasi Viñuela, Pedro

    2008-01-01

    Proceedings of: 10th International Conference on Parallel Problem Solving From Nature, PPSN 2008. Dortmund, Germany, September 13-17, 2008 The techniques and the technologies supporting Automatic Vehicle Guidance are an important issue. Automobile manufacturers view automatic driving as a very interesting product with motivating key features which allow improvement of the safety of the car, reducing emission or fuel consumption or optimizing driver comfort during long journeys. Car raci...

  12. Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis.

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Feng, Kuan; Zhang, Shuangyan; Huang, Jiefang; Miao, Xiang; Baggi, Fulvio; Ostrom, Rennolds S; Zhang, Yanyun; Chen, Xiangjun; Xu, Congfeng

    2017-01-01

    Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.

  13. Situations of car-to-pedestrian contact.

    Matsui, Yasuhiro; Hitosugi, Masahito; Takahashi, Kunio; Doi, Tsutomu

    2013-01-01

    To reduce the severity of injuries and the number of pedestrian deaths in traffic accidents, active safety devices providing pedestrian detection are considered effective countermeasures. The features of car-to-pedestrian collisions need to be known in detail to develop such safety devices. Because information on real-world accidents is limited, this study investigated near-miss situations captured by drive recorders installed in passenger cars. We showed similarities of the contact situation between near-miss incidents and real-world fatal pedestrian accidents in Japan. We analyzed the near-miss incident data via video capturing pedestrians crossing the road in front of forward-moving cars. Using a video frame captured by a drive recorder, the time to collision (TTC) was calculated from the car velocity and the distance between the car and pedestrian at the moment that the pedestrian initially appeared. The average TTC in the cases where pedestrians were not using a pedestrian crossing was shorter than that in the cases where pedestrians were using a pedestrian crossing. The average TTC in the cases where pedestrians emerged from behind obstructions was shorter than that in the cases where drivers had unobstructed views of the pedestrians. We propose that the specifications of the safety device for pedestrian detection and automatic braking should reflect the severe approach situation for a pedestrian and car including the TTC observed for near-miss incidents.

  14. Car radiator burns: a prevention issue.

    Rabbitts, Angela; Alden, Nicole E; Conlin, Tara; Yurt, Roger W

    2004-01-01

    Scald burns continue to be the major cause of injury to patients admitted to the burn center. Scald burns occurring from car radiator fluid comprise a significant subgroup. Although manufacturer warning labels have been placed on car radiators, these burns continue to occur. This retrospective review looks at all patients admitted to our burn center who suffered scald burns from car radiator fluid to assess the extent of this problem. During the study period, 86 patients were identified as having suffered scald burns as a result of contact with car radiator fluid. Seventy-one percent of the burn injuries occurred in the summer months. The areas most commonly burned were the head and upper extremities. Burn prevention efforts have improved greatly over the years; however, this study demonstrates that scald burns from car radiator fluid continue to cause physical, emotional, and financial devastation. The current radiator warning labels alone are not effective. The National Highway Traffic Safety Administration has proposed a new federal motor vehicle safety standard to aid in decreasing the number of scald burns from car radiators. The results of this study were submitted to the United States Department of Transportation for inclusion in a docket for federal legislation supporting these safety measures.

  15. New Strategies for the Treatment of Solid Tumors with CAR-T Cells.

    Zhang, Hao; Ye, Zhen-Long; Yuan, Zhen-Gang; Luo, Zheng-Qiang; Jin, Hua-Jun; Qian, Qi-Jun

    2016-01-01

    Recent years, we have witnessed significant progresses in both basic and clinical studies regarding novel therapeutic strategies with genetically engineered T cells. Modification with chimeric antigen receptors (CARs) endows T cells with tumor specific cytotoxicity and thus induce anti-tumor immunity against malignancies. However, targeting solid tumors is more challenging than targeting B-cell malignancies with CAR-T cells because of the histopathological structure features, specific antigens shortage and strong immunosuppressive environment of solid tumors. Meanwhile, the on-target/off-tumor toxicity caused by relative expression of target on normal tissues is another issue that should be reckoned. Optimization of the design of CAR vectors, exploration of new targets, addition of safe switches and combination with other treatments bring new vitality to the CAR-T cell based immunotherapy against solid tumors. In this review, we focus on the major obstacles limiting the application of CAR-T cell therapy toward solid tumors and summarize the measures to refine this new cancer therapeutic modality.

  16. Heterogeneous cellular networks

    Hu, Rose Qingyang

    2013-01-01

    A timely publication providing coverage of radio resource management, mobility management and standardization in heterogeneous cellular networks The topic of heterogeneous cellular networks has gained momentum in industry and the research community, attracting the attention of standardization bodies such as 3GPP LTE and IEEE 802.16j, whose objectives are looking into increasing the capacity and coverage of the cellular networks. This book focuses on recent progresses,  covering the related topics including scenarios of heterogeneous network deployment, interference management i

  17. Cellular decomposition in vikalloys

    Belyatskaya, I.S.; Vintajkin, E.Z.; Georgieva, I.Ya.; Golikov, V.A.; Udovenko, V.A.

    1981-01-01

    Austenite decomposition in Fe-Co-V and Fe-Co-V-Ni alloys at 475-600 deg C is investigated. The cellular decomposition in ternary alloys results in the formation of bcc (ordered) and fcc structures, and in quaternary alloys - bcc (ordered) and 12R structures. The cellular 12R structure results from the emergence of stacking faults in the fcc lattice with irregular spacing in four layers. The cellular decomposition results in a high-dispersion structure and magnetic properties approaching the level of well-known vikalloys [ru

  18. Cellular Reflectarray Antenna

    Romanofsky, Robert R.

    2010-01-01

    The cellular reflectarray antenna is intended to replace conventional parabolic reflectors that must be physically aligned with a particular satellite in geostationary orbit. These arrays are designed for specified geographical locations, defined by latitude and longitude, each called a "cell." A particular cell occupies nominally 1,500 square miles (3,885 sq. km), but this varies according to latitude and longitude. The cellular reflectarray antenna designed for a particular cell is simply positioned to align with magnetic North, and the antenna surface is level (parallel to the ground). A given cellular reflectarray antenna will not operate in any other cell.

  19. Efficiency of cellular information processing

    Barato, Andre C; Hartich, David; Seifert, Udo

    2014-01-01

    We show that a rate of conditional Shannon entropy reduction, characterizing the learning of an internal process about an external process, is bounded by the thermodynamic entropy production. This approach allows for the definition of an informational efficiency that can be used to study cellular information processing. We analyze three models of increasing complexity inspired by the Escherichia coli sensory network, where the external process is an external ligand concentration jumping between two values. We start with a simple model for which ATP must be consumed so that a protein inside the cell can learn about the external concentration. With a second model for a single receptor we show that the rate at which the receptor learns about the external environment can be nonzero even without any dissipation inside the cell since chemical work done by the external process compensates for this learning rate. The third model is more complete, also containing adaptation. For this model we show inter alia that a bacterium in an environment that changes at a very slow time-scale is quite inefficient, dissipating much more than it learns. Using the concept of a coarse-grained learning rate, we show for the model with adaptation that while the activity learns about the external signal the option of changing the methylation level increases the concentration range for which the learning rate is substantial. (paper)

  20. Visualization of cholinoceptive neurons in the rat neocortex : colocalization of muscarinic and nicotinic acetylcholine receptors

    Zee, E.A. van der; Streefland, C.; Strosberg, A.D.; Schröder, H.; Luiten, P.G.M.

    The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of