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Rutten, Michael J., Kathy D. Bacon, Katie L. Marlink, Mark Stoney, Camie L. Meichsner, Fred P. Lee, Susan A. Hobson et al. « Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells ». American Journal of Physiology-Gastrointestinal and Liver Physiology 277, no 3 (1 septembre 1999) : G662—G670. http://dx.doi.org/10.1152/ajpgi.1999.277.3.g662.
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The purpose of the present study was to determine whether human gastric mucous epithelial cells express a functional Ca2+-sensing receptor (CaR). Human gastric mucous epithelial cells were isolated from surgical tissues and cultured on glass coverslips, plastic dishes, or porous membrane filters. Cell growth was assessed by the MTT assay, CaR localization was detected by immunohistochemistry and confocal microscopy, CaR protein expression was assessed by Western immunoblotting, and intracellular Ca2+ concentration ([Ca2+]i) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the basolateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial cell cultures showed abundant CaR immunofluorescence at the basolateral membrane and little to no CaR immunoreactivity at the apical membrane. Western immunoblot detection of CaR protein in cell culture lysates showed two significant immunoreactive bands of 140 and 120 kDa. Addition of extracellular Ca2+ to preconfluent cultures of human gastric mucous epithelial cells produced a significant proliferative response. Changes in [Ca2+]iwere also observed in response to graded doses of extracellular Ca2+ and Gd3+. The phospholipase C inhibitor U-73122 specifically inhibited Gd3+-induced changes in [Ca2+]iin the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mucous epithelial cells.
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Wilk, Richard R. « Car Cultures:Car Cultures. » American Anthropologist 105, no 1 (mars 2003) : 202–3. http://dx.doi.org/10.1525/aa.2003.105.1.202.
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Borg, Kevin. « Car Cultures (review) ». Technology and Culture 43, no 3 (2002) : 636–37. http://dx.doi.org/10.1353/tech.2002.0105.
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Sheller, Mimi. « Book Review : Car Cultures ». Journal of Consumer Culture 2, no 1 (mars 2002) : 142–44. http://dx.doi.org/10.1177/146954050200200110.
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Chattopadhyay, Naibedya, Shozo Yano, Jacob Tfelt-Hansen, Paul Rooney, Deepthi Kanuparthi, Sanghamitra Bandyopadhyay, Xianghui Ren, Ernest Terwilliger et Edward M. Brown. « Mitogenic Action of Calcium-Sensing Receptor on Rat Calvarial Osteoblasts ». Endocrinology 145, no 7 (1 juillet 2004) : 3451–62. http://dx.doi.org/10.1210/en.2003-1127.
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Abstract The parathyroid calcium-sensing receptor (CaR) plays a nonredundant role in systemic calcium homeostasis. In bone, Ca2+o, a major extracellular factor in the bone microenvironment during bone remodeling, could potentially serve as an extracellular first messenger, acting via the CaR, that stimulates the proliferation of preosteoblasts and their differentiation to osteoblasts (OBs). Primary digests of rat calvarial OBs express the CaR as assessed by RT-PCR, Northern, and Western blot analysis, and immunocolocalization of the CaR with the OB marker cbfa-1. Real-time PCR revealed a significant increase in CaR mRNA in 5- and 7-d cultures compared with 3-d cultures post harvesting. High Ca2+o did not affect the expression of CaR mRNA during this time but up-regulated cyclin D (D1, D2, and D3) genes, which are involved in transition from the G1 to the S phase of the cell cycle, as well as the early oncogenes, c-fos and early growth response-1; high Ca2+o did not, however, alter IGF-I expression, a mitogenic factor for OBs. The high Ca2+o-dependent increase in the proliferation of OBs was attenuated after transduction with a dominant-negative CaR (R185Q), confirming that the effect of high Ca2+o is CaR mediated. Stimulation of proliferation by the CaR involves the Jun-terminal kinase (JNK) pathway, as high Ca2+o stimulated the phosphorylation of JNK in a CaR-mediated manner, and the JNK inhibitor SP600125 abolished CaR-induced proliferation. Our data, therefore, show that the parathyroid/kidney CaR expressed in rat calvarial OBs exerts a mitogenic effect that involves activation of the JNK pathway and up-regulation of several mitogenic genes.
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Matthaeus, Claudia, René Jüttner, Michael Gotthardt et Fritz G. Rathjen. « The IgCAM CAR Regulates Gap Junction-Mediated Coupling on Embryonic Cardiomyocytes and Affects Their Beating Frequency ». Life 13, no 1 (21 décembre 2022) : 14. http://dx.doi.org/10.3390/life13010014.
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The IgCAM coxsackie–adenovirus receptor (CAR) is essential for embryonic heart development and electrical conduction in the mature heart. However, it is not well-understood how CAR exerts these effects at the cellular level. To address this question, we analyzed the spontaneous beating of cultured embryonic hearts and cardiomyocytes from wild type and CAR knockout (KO) embryos. Surprisingly, in the absence of the CAR, cultured cardiomyocytes showed increased frequencies of beating and calcium cycling. Increased beatings of heart organ cultures were also induced by the application of reagents that bind to the extracellular region of the CAR, such as the adenovirus fiber knob. However, the calcium cycling machinery, including calcium extrusion via SERCA2 and NCX, was not disrupted in CAR KO cells. In contrast, CAR KO cardiomyocytes displayed size increases but decreased in the total numbers of membrane-localized Cx43 clusters. This was accompanied by improved cell–cell coupling between CAR KO cells, as demonstrated by increased intercellular dye diffusion. Our data indicate that the CAR may modulate the localization and oligomerization of Cx43 at the plasma membrane, which could in turn influence electrical propagation between cardiomyocytes via gap junctions.
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Aftabizadeh, Maryam, Lovish Bawa, Simiao Wang, Blake Brewster, Dongrui Wang, Xin Wang, Christine Brown et Michael Barish. « EXTH-10. EXPLORATION OF A NOVEL TOXIN-INCORPORATING CAR T CELL : HOW DOES CHLOROTOXIN RECOGNIZE GLIOBLASTOMA CELLS ? » Neuro-Oncology 23, Supplement_6 (2 novembre 2021) : vi165. http://dx.doi.org/10.1093/neuonc/noab196.649.
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Abstract Chlorotoxin, a peptide toxin component of scorpion venom, binds selectively to glioblastoma and other neuroepithelial tumors, with minimal binding to non-malignant cells. We have recently developed a CAR T cell incorporating chlorotoxin (CLTX) as its target recognition domain, and CLTX-CAR T cells are now in clinical trial for recurrent glioblastoma (NCT04214392). We determined in preclinical studies that surface MMP-2 was required for CLTX-CAR T cell killing. However, the precise composition and structure of the cell surface complex recognized by CLTX-CAR T cells remains an unresolved question of increasing importance. Previous investigations have variously proposed matrix metalloprotease-2 (MMP-2), ClC-3 chloride channels, regulators of MMP-2, annexin A2, and neuropilin-1, as receptors, or components of receptors, for CLTX. To approach this question, we have visualized bound Cy5.5-conjugated CLTX peptide (CLTX.Cy5.5) or biotin-conjugated CLTX peptide (CLTX.biotin) on tumor cells in fixed sections of patient resections, on tumor cells in organotypic cultures of patient resections, and on cells of cultured patient-derived glioblastoma tumor lines. At tissue- and cell-level spatial resolution, we saw good correlation of CLTX binding with MMP-2 expression in patient tumor samples and on cultured GBM cells, and between CLTX binding and tumor cell death (cleaved caspase-3) in organotypic GBM cultures. However, at subcellular resolution, surface binding of CLTX was related to, but not precisely overlapping, with MMP-2 or other putative receptors. Rather, on fixed PBT003, PBT030 and PBT106 cells in monolayer cultures, MMP-2 staining was clustered, with diffuse loosely associated CLTX.biotin staining. Diffuse distribution of CLTX.Cy5.5 was also seen in fixed xenograft sections of PBT003-4, PBT1206, PBT030-2, PBT051 and PBT138 tumor cells, and not obviously associated with more discrete staining for IL13Rα2 and EGFR. Ongoing experiments are further examining association of CLTX with other putative components of CLTX receptor complexes, and their redistribution during binding by CLTX and CLTX-CAR T cells.
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Lee, Dong Hoon, Francisco Cervantes-Contreras, Sang Yun Lee, Damian J. Green et Brian G. Till. « Improved Expansion and Function of CAR T Cell Products from Cultures Initiated at Defined CD4:CD8 Ratios ». Blood 132, Supplement 1 (29 novembre 2018) : 3334. http://dx.doi.org/10.1182/blood-2018-99-111576.
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Abstract BACKGROUND: Current manufacturing paradigms for chimeric antigen receptor (CAR) modified T cells require ex vivo T cell activation, genetic modification, and expansion in cytokine-containing cell cultures. Most CAR T cell studies infuse cell products generated from unselected cells, in which the CD4:CD8 ratio is determined by what is collected during leukapheresis. The proportion of each subset can vary greatly in these products, reflecting the high heterogeneity of the CD4:CD8 ratio present in patients' (pts) blood at the time of treatment. Preclinical data demonstrate superior in vivo anti-tumor efficacy of cell products consisting of equal numbers of CD4+ and CD8+ T cells, and a recent clinical trial, in which CD19-targeted CAR T cell products were infused at a 1:1 ratio of CD4:CD8 T cells, showed clear dose-response and dose-toxicity relationships. However, CAR T cell manufacturing using parallel CD4+ and CD8+ CAR T cell cultures adds significant cost and complexity compared with a single-stream culture. Additionally, we have found that CD8+ T cell cultures from heavily treated pts often exhibit suboptimal expansion. We therefore evaluated whether CAR T cell products with approximately equal ratios of CD4+ to CD8+ cells could be generated by mixing CD4+ and CD8+ cells at a defined ratio at culture initiation, and whether the presence of ex vivo CD4+ help can improve CD8+ cell expansion. METHODS: CD4+ and CD8+ T cells were isolated from apheresis peripheral blood mononuclear cell products of lymphoma pts (n=15) treated in one of three CD20-targeted CAR T cell trials or healthy donors (n=5) using positive (CD4) and negative (CD8) immunomagnetic bead selection. Cell cultures (1x106 cells each) were established by activating CD4+ and CD8+ cells with anti-CD3/CD28-coated paramagnetic beads, mixing them at various ratios in 24-well plates, and transducing 24 hours later with a lentivirus encoding the 1.5.3-NQ-28-BB-z CD20 CAR. Beads were removed at day 4, and cells were expanded in IL-2 containing medium. At day 7, cells were counted and analyzed by flow cytometry for CD4, CD8, and CAR expression, and then restimulated 1:1 with irradiated CD20+ lymphoblastoid cells to boost growth. On day 14 cells were again counted and analyzed by flow cytometry for CD4, CD8, and CAR expression, as well as immunophenotype. In some cases CAR+CD4+ and CAR+CD8+ cells were flow-sorted from either CD4-only, CD8-only, and mixed cultures, stained with CFSE, and restimulated with irradiated CD20+ Raji tumor cells. Proliferation and cytokine secretion were measured by flow cytometry and Luminex, respectively. In vivo T cell activity was assessed using an NSG mouse model in which T cells were infused 7 days after i.v. injection of Raji-ffLuc cells. Suboptimal doses of T cells were used to distinguish differences between conditions, since higher doses cure most mice. Mice received either: (1) 70:30 mixed CD4:CD8 CAR+ cultures (n=8), (2) 1:1 ratio of CD4:CD8 CAR+ cells grown separately (n=8), (3) Mixed CD4:CD8 empty vector cells (n=5), or (4) no treatment (n=5). RESULTS: An initial CD4:CD8 ratio of 70:30 yielded a median CD4/CD8 ratio of 1.1 for pts (Figure 1A) and 1.3 for donors at day 7, and a ratio of 0.6 and 1.0 for pts and donors, respectively, at day 14. Mixed cultures resulted in CD8+ cell expansion that was significantly higher than in separate cultures. At day 7, mean CD8+ cell expansion was 12.1-fold vs. 4.6-fold for 70:30 mixed vs. separate cultures for donors, and 2.9-fold vs. 0.7-fold for pts (Figure 1B). At day 14 CD8+ mean cell expansion was 105-fold (70:30 mixed) vs. 25-fold (separate) for donors and 40-fold vs. 1.9-fold for pts. CD8+ cells grown in mixed cultures also exhibited higher expression of memory markers (CD62L and CCR7), lower levels of exhaustion markers (Lag-3 and PD-1), and better proliferation compared with CD8 cells grown in separate cultures. In the mice we found that tumor growth inhibition was superior in the 70:30 mixed culture group than in mice receiving a 1:1 ratio of CD4:CD8 cells grown separately (Figure 1C). CONCLUSIONS: A single-stream CAR T cell culture initiated at a defined CD4:CD8 ratio of 70:30 yielded on average approximately equal numbers of CD4+ and CD8+ cells in the final cell product. Mixed CD4+ and CD8+ cultures generated CD8+ T cells with a less differentiated phenotype, and superior expansion, proliferative capacity, and in vivo activity compared with CD4+ and CD8+ cells manufactured in parallel. Disclosures Green: Juno Therapeutics: Patents & Royalties, Research Funding. Till:Mustang Bio: Patents & Royalties, Research Funding.
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Dietterle, Johannes, Henry Oppermann, Annegret Glasow, Karsten Neumann, Jürgen Meixensberger et Frank Gaunitz. « Carnosine increases efficiency of temozolomide and irradiation treatment of isocitrate dehydrogenase-wildtype glioblastoma cells in culture ». Future Oncology 15, no 32 (novembre 2019) : 3683–91. http://dx.doi.org/10.2217/fon-2019-0447.
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Aim: The naturally occurring dipeptide carnosine (CAR) has been considered for glioblastoma therapy. As CAR also protects against ionizing irradiation (IR), we investigated whether it may counteract standard therapy consisting of postsurgery IR and treatment with temozolomide (TMZ). Materials & methods: Four isocitrate dehydrogenase-wildtype primary cell cultures were exposed to different doses of IR and different concentrations of TMZ and CAR. After exposure, viability under the different conditions and combinations of them was determined. Results: All cultures responded to treatment with TMZ and IR with reduced viability. CAR further decreased viability when TMZ and IR were combined. Conclusion: Treatment with CAR does not counteract glioblastoma standard therapy. As the dipeptide also protects nontumor cells from IR, it may reduce deleterious side effects of treatment.
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Song, Hannah, Lipei Shao, Michaela Prochazkova, Adam Cheuk, Ping Jin, David Stroncek, Javed Khan et Steven Highfill. « 145 Comparison of CAR-T cell manufacturing platforms reveals distinct phenotypic and transcriptional profiles ». Journal for ImmunoTherapy of Cancer 9, Suppl 2 (novembre 2021) : A153. http://dx.doi.org/10.1136/jitc-2021-sitc2021.145.
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BackgroundWith the clinical success of chimeric antigen receptor (CAR)-T cells against hematological malignancies, investigators are looking to expand CAR-T therapies to new tumor targets and patient populations. To support translation to the clinic, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity while using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses that can number in the billions. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, it is currently unknown whether the manufacturing platform itself significantly influences the output T cell phenotype and function.MethodsStatic bag culture was compared with 3 widely-used commercial CAR-T manufacturing platforms (Miltenyi CliniMACS Prodigy, Cytiva Xuri W25 rocking platform, and Wilson-Wolf G-Rex gas-permeable bioreactor) to generate CAR-T cells against FGFR4, a promising target for pediatric sarcoma. Selected CD4+CD8+ cells were stimulated with Miltenyi TransAct, transduced with lentiviral vector, and cultured out to 14 days in TexMACS media with serum and IL2.ResultsAs expected, there were significant differences in overall expansion, with bag cultures yielding the greatest fold-expansion while the Prodigy had the lowest (481-fold vs. 84-fold, respectively; G-Rex=175-fold; Xuri=127-fold; average of N=4 donors). Interestingly, we also observed considerable differences in CAR-T phenotype. The Prodigy had the highest percentage of CD45RA+CCR7+ stem/central memory (Tscm)-like cells at 46%, while the bag and G-Rex cultures had the lowest at 16% and 13%, respectively (average N=4 donors). In contrast, the bag, G-Rex, and Xuri cultures were enriched for CD45RO+CCR7- effector memory cells and also had higher expression of exhaustion markers PD1 and LAG3. Gene clustering analysis using a CAR-T panel of 780 genes revealed clusters of genes enriched in Prodigy/de-enriched in bag, and vice versa. We are currently in the process of evaluating T cell function.ConclusionsThis is the first study to our knowledge to benchmark these widely-used bioreactor systems in terms of cellular output, demonstrating that variables inherent to each platform (such as such as nutrient availability, gas exchange, and shear force) significantly influence the final CAR-T cell product. Whether enrichment of Tscm-like cells in the final infusion product correlates with response rate, as has been demonstrated in the setting of CD19 CAR-Ts, remains to be seen and may differ for FGFR4 CAR-Ts and other solid tumors. Overall, our study outlines methods to identify the optimal manufacturing process for future CAR-T cell therapies.
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Holzer, Peter. « Kulturwissenschaftliche Theorien – Basis einer translationsrelevanten Kultur(transfer)kompetenz ». La traduction : formation, compétences, recherches 57, no 1 (10 octobre 2012) : 35–47. http://dx.doi.org/10.7202/1012739ar.
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Les théories et le transfert culturels gagnent en importance dans le cadre de la communication interculturelle ou transculturelle. Cet état de fait ne se limite pas à un secteur culturel spécifique, il comprend le transfert des idées, des produits culturels, des pratiques et des institutions d’un système de société à un autre. Les processus de transfert culturel sont dynamiques, car ils touchent autant le monde matériel et tangible que les sphères intellectuelles et spirituelles. Si l’on se penche sur la traduction d’ouvrages ou sur la transmission d’informations en provenance d’autres cultures ou d’autres espaces culturels, comme dans le cas des nouvelles ou d’autres catégories spécifiques de textes, ces processus sont directement perceptibles et ils peuvent être caractérisés tant sur le plan quantitatif que qualitatif. Les emprunts sont présentés comme des originaux, et une interprétation permet aux objets et aux idées de remplir leur fonction dans un contexte nouveau. Les théories culturelles, les modèles culturels anthropologiques et les théories traductologiques aident à définir plus précisément ce qu’est la « culture », à saisir les différentes facettes du concept et à comprendre les processus de transfert pertinents.
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Carson, Steven D., Kyung-Soo Kim, Samuel J. Pirruccello, Steven Tracy et Nora M. Chapman. « Endogenous low-level expression of the coxsackievirus and adenovirus receptor enables coxsackievirus B3 infection of RD cells ». Journal of General Virology 88, no 11 (1 novembre 2007) : 3031–38. http://dx.doi.org/10.1099/vir.0.82710-0.
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Cells in which the appropriate viral receptor cannot be detected may paradoxically act as a host to the virus. For example, RD cells are often considered to be non-permissive for infection with coxsackievirus and adenovirus receptor (CAR)-dependent group B coxsackieviruses (CVB), insofar as inoculated cell monolayers show little or no cytopathic effect (CPE) and immunohistological assays for CAR have been consistently negative. Supernatants recovered from RD cells exposed to CVB, however, contained more virus than was added in the initial inoculum, indicating that productive virus replication occurred in the monolayer. When infected with a recombinant CVB type 3 (CVB3) chimeric strain expressing S-Tag within the viral polyprotein, 4–11 % of RD cells expressed S-Tag over 48 h. CAR mRNA was detected in RD cells by RT-PCR, and CAR protein was detected on Western blots of RD lysates; both were detected at much lower levels than in HeLa cells. Receptor blockade by an anti-CAR antibody confirmed that CVB3 infection of RD cells was mediated by CAR. These results show that some RD cells in the culture population express CAR and can thereby be infected by CVB, which explains the replication of CAR-dependent CVB in cell types that show little or no CPE and in which CAR has not previously been detected. Cells within cultures of cell types that have been considered non-permissive may express receptor transiently, leading to persistent replication of virus within the cultured population.
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Kerr, Sophie-May, Natascha Klocker et Gordon Waitt. « Diverse Driving Emotions ». Transfers 8, no 2 (1 juin 2018) : 23–43. http://dx.doi.org/10.3167/trans.2018.080203.
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In the industrialized West, cars are considered an essential part of everyday life. Their dominance is underpinned by the challenges of managing complex, geographically stretched daily routines. Drivers’ emotional and embodied relationships with automobiles also help to explain why car cultures are difficult to disrupt. This article foregrounds ethnic diversity to complicate notions of a “love affair” with the car. We report on the mobilities of fourteen Chinese migrants living in Sydney, Australia—many of whom described embodied dispositions against the car, influenced by their life histories. Their emotional responses to cars and driving, shaped by transport norms and infrastructures in their places of origin, ranged from pragmatism and ambivalence to fear and hostility. The lived experiences of these migrants show that multiple cultures of mobility coexist, even in ostensibly car-dependent societies. Migrants’ life histories and contemporary practices provide an opportunity to reflect on fissures in the logic of automobility.
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Hollmann, Julia, Aletéia De Moura Carpes et Thiago Antonio Beuron. « The DaimlerChrysler merger – a cultural mismatch ? » Revista de Administração da UFSM 3, no 3 (27 janvier 2011) : 431–40. http://dx.doi.org/10.5902/198346592506.
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American company Chrysler Corporation due to differences in the organizational cultures involved or due to a so-called ‘clash of culture'. What happens when two successful car producers with different know-how and a different knowledge background, different work processes, different product portfolios and last but not least, completely different corporate cultures decide to merge? Daimler-Benz and Chrysler wanted to strengthen their position during economically difficult times for the car industry by juggling the crisis together and they hoped to be able to combine their strengths. Therefore the two companies decided to fuse in 1998. But not even ten years later Daimler-Benz once again sold all its shares of the Chrysler division. The dream to become the third biggest car producer of the world, behind General Motors and Ford, burst. The expected and wished for synergy effects stayed out. Instead of gaining competitive advantage over their competitors, the merger rushed the two car producers ever deeper into the crisis and did not provide the companies with the necessary tools to overcome the recession. The presented paper deals with the failed merger of the German company Daimler-Benz with the U.S. American company Chrysler Corporation due to differences in the organizational cultures involved or due to a so-called ‘clash of culture'.
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Wasserman, Ilene. « Bumper-Car Moments and Relationship Recovery : Leveraging Strengths in Building Inclusive Cultures ». AI Practitioner 23, no 2 (3 mai 2021) : 62–66. http://dx.doi.org/10.12781/978-1-907549-47-2-10.
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A ‘bumper-car moment’ is the jolt when we discover that the rules and norms we take for granted are not necessarily the same for others. In real life, the effects of the jolt can linger, affecting relationships. A triggering event, or bumper-car moment, provides an opening for learning and change.
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Bohren, Lenora. « Cars, Cultures, and Cures@ : Environmental Education For K-12 ». Practicing Anthropology 23, no 3 (1 juillet 2001) : 38–41. http://dx.doi.org/10.17730/praa.23.3.2414g0622p388602.
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My role as an environmental anthropologist has allowed me to work in many national and international settings. With an interest in culture, technology and the environment, I have focused on environmental issues concerning air pollution and climate change. I have worked with the USEPA managing national surveys and on projects on the US/Mexico border. Most recently, I have worked with the City of Fort Collins, Colorado in an effort to develop an environmental education course that can be delivered to junior high/middle school (preferably 9th grade) students. The purpose of this course is to heighten awareness of personal responsibility as it relates to the automobile and the environment within the context of the American Culture. As an anthropologist, my role was to help the students gain an understanding of the "American Car Culture" and to see how their attitudes and actions reflect their culture and effect their environment. I did this by developing a slide presentation that introduced the concept of the "culture" of the car in America and by including the use of anthropological methods in student assignments assessing attitudes and actions toward the car.
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Mandeville, Taylor K., Cory Mavis, Juan Gu, Scott Olejniczak, Gyorgy Paragh, Prasenjit Dey et Francisco J. Hernandez-Ilizaliturri. « Contribution of Bcl-2 Inhibitor Venetoclax Toward Anti-CD19 CAR T Cell Efficacy in Relapsed/Refractory Diffuse Large B Cell Lymphoma ». Blood 138, Supplement 1 (5 novembre 2021) : 1719. http://dx.doi.org/10.1182/blood-2021-154128.
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Abstract Introduction Clinical outcomes of anti-CD19 chimeric antigen receptor T-cell (CAR-T CD19) therapy for the treatment of relapsed/refractory Diffuse Large B Cell lymphoma (r/r DLBCL) must be improved, as patients undergoing CAR-T CD19 therapy can expect a cure rate of only 41% and a median survival of just 6.3 months if unresponsive. Of patients presenting with naïve DLBCL, 35% will advance to third-line treatment with CAR-T CD19 therapy. Poor clinical responses to CAR-T CD19 therapy and a small window of survival for unresponsive patients highlights the critical need for improvement of CAR-T CD19 therapy in the setting of r/r DLBCL. Overexpression of anti-apoptotic Bcl-2 protein has been characterized in r/r DLBCL, and contributes to the disease's aggressive phenotype. Aberrant expression of the Bcl-2 protein proves targetable by an FDA-approved small molecule inhibitor which mimics the role of pro-apoptotic BH3 proteins, known as Venetoclax. Here, we investigate the effect of Venetoclax on CAR-T CD19 cell immunophenotype, effector function, and cytotoxicity upon co-culture with a lab-generated model of immortalized rituximab/chemo-resistant B cell lymphomas (Raji4RH) which recapitulate the resistant phenotype of r/r DLBCL seen in patients. Methods PBMCs were isolated and activated from apheresis samples from consenting adults (healthy donors) using the Miltenyi Biotec TransACT human CD3/CD28 Kit. Cells were transduced with lentiviral vectors encoding for 2nd generation CAR constructs 24 hours post-activation. Transduced cells were cultured in RPMI media supplemented with hIL-2 (1ng/mL) and hIL-7 (10ng/mL). At day 14, cells were cultured with Raji4RH and Venetoclax in either combined or solitary cultures. Co-culture cytotoxicity assays were performed by pre-labeling target cells with permeant dye (Thermo Fisher Scientific CellTrace Blue Proliferation Kit), then incubating with CAR-T CD19 cells (GFP labeled) at various effector:target ratios and Venetoclax concentrations for 24 hours. Sample wells were stained with viability dye and acquired on BD LSR II Cytometer. CAR-T CD19 cell immunophenotype was identified following 24-hour exposure of Venetoclax to CAR T cell populations at various concentrations. Following exposure, samples were stained with viability dye, CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD25, and CD95. Frequencies of T REG, T Central Memory, T naive, and T Stem Cell Memory populations were calculated. CAR-T CD19 cell effector function was elucidated by activating (24 hours), then exposing CAR-T CD19 cells to concentrations of Venetoclax for 24 hours. Brefeldin A was added to culture for the remaining 4 hours of incubation. Cells were collected and stained intracellularly for IFNγ, Perforin, and Granzyme B; and extracellularly for CD3, CD4, and CD8. Sample acquisition for effector function assay was performed on BD LSRII Cytometer. Results Concurrent in vitro CAR-T CD19 administration and Venetoclax exposure significantly increases Raji4RH cell death as opposed to single agent cohorts. CAR-T CD19 cell viability is unaffected by Venetoclax at concentrations which confer synergy, and a predominantly CD8+ population is favored. Finally, Venetoclax appears to significantly increase the frequency of T N/T SCM and T CM within exposed cultures, while affording a 21% decrease in T REG frequency. Additionally, the notable increase in T N/T SCM and T CM phenotypes following exposure to Venetoclax did not arise as a consequence of decreased cellular viability. Conclusion The combination of Venetoclax exposure and αCD19 CAR T cell administration demonstrates synergy when employed in the context of rituximab-resistant lymphoma cells, which may be explained by the effect of the BH3 mimetic's exposure on CAR-T CD19 cell immunophenotype. The ability to manipulate Bcl-2 protein expression within CAR-T CD19 cells affords insight into the role that the Bcl-2 family pathway plays within CAR-T CD19 cell biology. In addition to answering fundamental questions of CAR T cell biology, the combination of Venetoclax and CAR-T CD19 therapy may provide a solution to the observed clinical gap, in which CAR-T CD19 therapy alone cures less than half of all patients who advance to this third line treatment regimen. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Botterill, Jacqueline. « Mobile Eating : A Cultural Perspective ». International Review of Social Research 7, no 2 (27 novembre 2017) : 71–79. http://dx.doi.org/10.1515/irsr-2017-0009.
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AbstractOver 80 percent of North Americans regularly eat in the car, yet neither mobility literature nor expanding discussions of food cultures focus on the practice. Two studies shed light on eating in the car. First, North American’s distinct, dynamic, and embedded mobile food infrastructure is outlined via discussion of noteworthy innovations - from the 19thcentury dining car to the 21stcentury drive thru - that food entrepreneurs constructed to facilitate eating on the go. Second, four exploratory focus groups investigate the meanings and practices drivers associate with eating in the car. Together findings suggest that eating in the car is compromised by the demands of accelerating modernity. Framing eating in the car as simply another facet of an obesity crisis, as culinary preference, or personal choice and responsibility limits full understanding of the cultural anxieties, environmental and health risks surrounding this widespread food practice.
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Hofstede, Gert Jan, Catholijn M. Jonker, Tim Verwaart et Neil Yorke-Smith. « The Lemon Car Game Across Cultures : Evidence of Relational Rationality ». Group Decision and Negotiation 28, no 5 (18 juillet 2019) : 849–77. http://dx.doi.org/10.1007/s10726-019-09630-9.
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Hämeenaho, Pilvi. « Daily Life on Wheels : Global (Car) Cultures and Local Living ». Journal of Finnish Studies 22, no 1-2 (1 janvier 2019) : 137–54. http://dx.doi.org/10.5406/28315081.22.1.2.08.
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Abstract Ordinary tasks, like working, taking part in social events, leisure time activities, and consumerism create a need to move. The location of residence determines the means and ways of performing these activities. When studying life in remote rural areas, researchers inevitably confront the question of daily traveling. In Finland, remote rural areas are truly distant from municipal centers and far from cities, workplaces, and public activities. Distances are long, and it takes time and money to move from home villages with no public transport available. How can we analyze the meaning that these distances have for the people living their everyday lives in remote rural areas? In this article, I analyze the most common feature of mobility in rural Finland: private motoring and its multifaceted meaning to residents of rural places. My research, conducted via fieldwork, shows how the body of a car becomes a confluence for daily activities and emotions, a private space where duties, leisure, caring of family members and neighbors, and pleasure gained from the living environment join. By focusing on private motoring, I am also able to open up the wider picture about rural (im) mobility, the nature of rural life, and significance of rural environment as a source of well-being.
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Nohilly, Margaret. « Cultures of Care in Primary Schools in Ireland that Support Child Protection Work ». Child Abuse Review 28, no 4 (juillet 2019) : 261–72. http://dx.doi.org/10.1002/car.2575.
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Joy, Joash Dominic, Beatrice Malacrida, Florian Laforêts, Panoraia Kotantaki, Eleni Maniati, Sarah Hopkins, Ianire Calleja et al. « Abstract 693 : TGFβ-mediated targeting of the extracellular matrix enhances the migration and cytotoxicity of CAR-T cells in 3D models of ovarian cancer ». Cancer Research 82, no 12_Supplement (15 juin 2022) : 693. http://dx.doi.org/10.1158/1538-7445.am2022-693.
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Abstract We have assessed the utility of 3-dimensional (3D) in vitro human cell models to understand barriers to chimeric antigen receptor (CAR)-T cell activity in high-grade serous ovarian cancer, (HGSOC) a disease with a poor response to immunotherapy. We defined mucin-1 (MUC1) as a potential target in HGSOC biopsies and the HGSOC cell lines, OvCAR3 and G164. We then generated CAR-T cells against MUC1 and tested them in spheroid and collagen gel cultures. In spheroids, although CAR-T cells killed OvCAR3 cells, G164 cells failed to induce CAR-T cell activation or cytotoxicity. However, when we added primary omental fibroblasts from ovarian cancer patients to G164 spheroids, CAR-T cells were activated and cytotoxic. Fibroblast conditioned medium also activated CAR-T cells to kill G164 cells in spheroids and this was due to their production of C-C motif chemokine ligand 2 (CCL2). Further experiments revealed that CCL2 produced by fibroblast stimulated CCR2/4 positive CAR-T cells to a higher state of activation, which enhanced the cytotoxicity of CAR-T cells against G164 cells. We then investigated CAR-T cell activity in co-cultures of OvCAR3 or G164 cells and primary fibroblasts embedded in collagen. CAR-T cells migrated into OvCAR3 gels and killed the malignant cells during a three-day period. However, CAR-T cells failed to migrate into gels with G164 cells and there was no malignant cell killing. Gels containing G164 cells had denser extracellular matrix (ECM) than OvCAR3 gels, as measured by staining for collagens and fibronectin. Previously, we showed that transforming growth factor-beta (TGFβ) secreted by HGSOC cells acted on fibroblasts to induce the production of ECM in collagen gels.1 Treating G164 gels with the TGFβ receptor inhibitor SB431542 reduced ECM density, stimulated CAR-T cell migration and restored CAR-T cell cytotoxicity against G164 cells. Using these different human 3D models we have demonstrated that malignant cell intrinsic factors can cause resistance to CAR-T cells. Sensitivity to CAR-T cell killing can be modulated both positively and negatively by fibroblasts. Targeting ECM along with CAR-T cell therapy might improve the efficiency of CAR-T cells in solid tumors. 1Delaine-Smith et al, iScience, 2021 Citation Format: Joash Dominic Joy, Beatrice Malacrida, Florian Laforêts, Panoraia Kotantaki, Eleni Maniati, Sarah Hopkins, Ianire Calleja, Sara Brett, Takis Athanasopoulos, Sadfer Ali, Peter Emery-Billcliff, Ida Ricciardelli, Charlotte Kay, Jayne Colebrook, Magda Ali, Katherine Strong, Frances Balkwill. TGFβ-mediated targeting of the extracellular matrix enhances the migration and cytotoxicity of CAR-T cells in 3D models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 693.
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Mögele, Michael, et Henrike Rau. « Cultivating the “car state” : a culturally sensitive analysis of car-centric discourses and mobility cultures in Southern Germany ». Sustainability : Science, Practice and Policy 16, no 1 (3 juin 2020) : 15–28. http://dx.doi.org/10.1080/15487733.2020.1756188.
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Mirza, Momina, Cecilia Petersen, Katarina Nordqvist et Kerstin Sollerbrant. « Coxsackievirus and Adenovirus Receptor Is Up-Regulated in Migratory Germ Cells during Passage of the Blood-Testis Barrier ». Endocrinology 148, no 11 (1 novembre 2007) : 5459–69. http://dx.doi.org/10.1210/en.2007-0359.
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The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule expressed in epithelial tight junctions and other cell-cell contacts. Using indirect immunofluorescence, quantitative RT-PCR, and Western blots, the expression and distribution of CAR in developing and adult testis are examined. CAR is highly expressed in both Sertoli and germ cells during perinatal and postnatal development, followed by a rapid down-regulation of both mRNA and protein levels. Interestingly, we find that CAR is a previously unknown downstream target for FSH because CAR mRNA levels were induced in primary cultures of FSH-stimulated Sertoli cells. In contrast to other epithelia, CAR is not a general component of tight junctions in the seminiferous epithelium, and Sertoli cells in the adult testis do not express CAR. Instead, CAR expression is stage dependent and specifically found in migratory germ cells. RT-PCR also demonstrated the presence of junctional adhesion molecule-like (JAML) in the testis. JAML was previously reported by others to form a functional complex with CAR regulating transepithelial migration of leukocytes. The expression of JAML in the testis suggests that a similar functional complex might be present during germ cell migration across the blood-testis barrier. Finally, an intermediate compartment occupied by CAR-positive, migrating germ cells and flanked by two occludin-containing junctions is identified. Together, these results implicate a function for CAR in testis morphogenesis and in migration of germ cells across the blood-testis barrier during spermatogenesis.
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van Caeneghem, Yasmine, Glenn Goetgeluk, Karin Weening, Greet Verstichel, Sarah Bonte, Tom Taghon, Georges Leclercq et al. « Chimeric Antigen Receptor Transgenic, T Cell Receptor/CD3 Negative Monospecific T Cells Generated from Cord Blood CD34 Positive Cells ». Blood 126, no 23 (3 décembre 2015) : 3087. http://dx.doi.org/10.1182/blood.v126.23.3087.3087.
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Abstract Autologous T cells transduced to express chimeric antigen receptors (CAR) directed against CD19, a B cell antigen, are reported to induce complete remission in patients with leukemia or lymphoma of the B cell lineage. Although potentially very effective, this treatment strategy has major drawbacks. First, CAR therapy is based on autologous T cells and therefore dependent on the nature and quality of T cells present in the peripheral blood of these patients at the time of treatment. Poor quality of the T cells may cause treatment failure in some patients. In addition, therapy based on autologous cells is tailor-made i.e. CAR+ T cells have to be generated de novo for every patient. Finally, autologous cell therapy requires different, more complicated logistics than conventional therapy. We therefore investigate whether a general purpose, allogeneic CAR therapy based on HLA-matched cord blood obtained from cord blood banks can be devised. Here, we investigated whether functional CAR+ T cells can be generated in vitro that do not express an endogenous T cell receptor to avoid alloreactivity causing graft versus host reactions. We compared carcino-embryonic antigen (CEA)- specific CARs of the first generation (intracellular CD3ζ signaling chain), of the 2nd generation (intracellular CD3ζ and CD28 signaling chain) and of the 3rd generation (intracellular CD3ζ, CD28 and OX40 signaling chain). CD34+ progenitor cells were isolated from human cord blood or postnatal thymus and subsequently transduced with one of the three green fluorescent protein (GFP)-encoding CAR constructs. Transduced cells were subsequently co-cultured on OP9DL1 in the presence of stem cell factor, Flt3-ligand and interleukin-7. Unlike T cell receptor transduced precursors (1), expansion was not enhanced by transduction of the chimeric receptor. Expansion was highest with first generation CARs whereas second and third generation CARs displayed only restricted expansion. Similar to T cell receptor transduced progenitors, CAR transduced cells show an accelerated differentiation during co-culture compared to the non-transduced cells: first committed CD5+ CD7+ T precursors appear, then CD4+ CD8+ double positive cells (DP) and finally CD1- CD27- single positive or double negative (DN) mature T cells. In cultures transduced with 2nd and 3rd generation CARs, few transduced cells passed through the proliferative DP pathway but rather differentiated to mature CD1- CD27- non-proliferative DN cells without passing through the DP stage. This phenomenon is responsible for the limited expansion seen with precursors transgenic for 2nd or 3rd generation CARs. However, in all cultures CAR+ DP cells were generated and, as shown for TCR transgenic cells (1), we were able to induce CEA specific maturation after co-culturing these DP cells with a cell line expressing CEA or by antibody-induced cross-linking of the CAR, giving rise to CD1- CD27+ matured cells. The observations described above are compatible with data obtained in mice showing that strong T cell receptor (TCR) activation during thymocyte differentiation inhibits the generation of DP cells and induces maturation to DN cells. Both the spontaneously and induced mature CAR+ cells were TCR and CD3 negative, suggesting that the expression of a CAR in early T cell precursors shuts down rearrangements of the endogenous TCR chains. Moreover, these cells lack NK marker expression (CD56, NKG2D) and show expression of T cell markers (CD5, CD7, CD2), confirming their T cell nature. In conclusion, the CAR+ CD3/TCR negative cells are T cells as these are derived from T cell precursors (CD5+, DP cells) and express various membrane and nuclear T cell markers. Mature CD1- CD27- CAR+ cells can be expanded to large cell numbers using T cell expansion protocols. They displayed cytokine production specific for CEA+ tumor lines as well as specific cytotoxicity. Moreover, the 2nd and 3rd generation CAR expressing cells showed increased specific cytokine production when compared to the first generation CAR expressing cells. These results show that the cord blood-derived CAR+ cells have potent functional activity similar to peripheral blood derived CAR+ T cells. We believe that these in vitro generated CAR+ cells developed from HLA-matched cord blood progenitors may be ideal as an adjunct to cord blood transplantation. (1) Snauwaert et al, Leukemia, 2014 Disclosures No relevant conflicts of interest to declare.
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Williams, Alan M., Ken Hayama, Yijia Pan, Brian Groff, Rina Mbofung, Lauren Fong, Nicholas Brookhouser et al. « Abstract 2828 : A novel synthetic stealth receptor that redirects host immune cell alloreactivity and potentiates functional persistence of adoptively transferred off-the-shelf cell-based cancer therapy ». Cancer Research 82, no 12_Supplement (15 juin 2022) : 2828. http://dx.doi.org/10.1158/1538-7445.am2022-2828.
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Abstract Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies and have shown significant potential in solid tumor indications. However, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility. Many of these challenges can be overcome with an allogeneic cellular product, but immune cell-mediated rejection of allogeneic cellular therapies remains a significant concern. Both allogeneic and autologous cell therapies currently rely on lymphodepleting conditioning to modulate the immune system and create greater access to homeostatic cytokines. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Therefore, an ideal allogeneic cell therapy would be able to avoid immune rejection while reducing or eliminating the need for chemotherapeutic conditioning to deplete host lymphocytes. To address many of these challenges, we engineered our novel alloimmune defense receptor (ADR) that targets 41BB+ alloreactive immune cells while providing a CD3z signaling boost into our off-the-shelf iPSC derived NK cells expressing anti-CD19 CAR (CAR iNK). The ability of ADR+ CAR iNK cells to resist alloimmune rejection was tested by co-culturing ADR+ CAR iNK cells with allogeneic PBMCs from ten donors in a mixed lymphocyte reaction assay. Notably, ADR+ CAR iNK cells co-cultured with allogeneic PBMCs persisted to similar levels as the PBMC-free culture while ADR negative CAR iNK cells were eliminated when co-cultured with allogeneic PBMCs. Furthermore, all PBMC donors screened in ADR+ CAR iNK cell co-cultures showed ablation of reactive 41BB+ NK and T cells, with non-activated T cells remaining intact. CAR iNK cells +/- ADR were then compared in a Nalm6 disseminated in vivo model for anti-tumor efficacy. ADR+ CAR iNK cells and their ADR negative counterparts were found to equivalently control tumor. Building on this tumor model, we co-infused allogeneic T cells to mimic an immuno-competent setting. The data demonstrated that ADR negative CAR iNK cells were depleted and were unable to control tumor growth while significant levels of allogeneic T cells persisted. In contrast, ADR+ CAR iNK cells were able to resist allogeneic T cell attack, control tumor, and persist when compared to the ADR negative control. Collectively, our preliminary data suggest that ADR-armed CAR iNK cells withstand immune cell-mediated rejection with uncompromised effector function. We are actively developing models to confirm our initial finding that ADR+ effector cells also benefit from their engagement with alloreactive cells in immuno-competent settings to promote enhanced anti-tumor responses, proliferation, and persistence. Citation Format: Alan M. Williams, Ken Hayama, Yijia Pan, Brian Groff, Rina Mbofung, Lauren Fong, Nicholas Brookhouser, Berhan Mandefro, Ramzey Abujarour, Tom Lee, Quirin Hammer, Karl-Johan Malmberg, Maksim Mamonkin, Ryan Bjordahl, Jode Goodridge, Bahram Valamehr. A novel synthetic stealth receptor that redirects host immune cell alloreactivity and potentiates functional persistence of adoptively transferred off-the-shelf cell-based cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2828.
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Dolnikov, Alla, Shen Sylvie, Ning Xu et Tracey O'Brien. « Stem Cell Approach to Generate Chimeric Antigen Receptor Modified Immune Effector Cells to Treat Cancer ». Blood 124, no 21 (6 décembre 2014) : 2437. http://dx.doi.org/10.1182/blood.v124.21.2437.2437.
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Abstract Allogeneic stem cell transplantation is a curative therapy for refractory, relapsed and high risk leukaemia and lymphoma. Despite improvements in leukaemia treatment the disease relapse occurs in 30-50% of patients and remains unacceptably high. There is thus a clear need for novel agents in the treatment of leukaemia relapse. Leukaemia specific T cells genetically modified with Chimeric Antigen Receptor (CAR) generated in the laboratory to target malignant cells is a novel approach with proven success in early phase human trials. Patient's own T cells can be genetically modified in the laboratory to target tumour associated antigens through the introduction of CAR. CAR-modified T cells are amplified ex vivo to numbers suitable for adoptive cell therapy and administered to the patient upon preconditioning. Unfortunately, CAR-modified mature T cells often rapidly differentiate into short-lived effector cells that exhibit limited anti-tumour activity in vivo. Here we propose to use CAR-modified haematopoietic stem cells (HSC) to generate CART- cells. We hypothesise that CAR-modified HSC will provide long-lasting supply of CAR-T cells mediating sustained anti-tumour activity. Umbilical cord blood-derived CD34+ cells enriched with HSC were genetically modified to express CAR targeting CD19 antigen(CAR19) widely expressed on B-cell malignancies. An optimised gamma-retroviral gene transfer method was used to transduce CD34+ stem cells with CAR19. CAR19-transduced stem cells generate CD33+ myeloid, CD56+ natural killer and CD34-CD7+ T cell precursors expressing CAR19 and undergo normal proliferation and differentiation in vitro in colony forming unit (CFU) assay. B cell development analysed in co-cultures with bone marrow stroma MS5 cells, however, was completely suppressed. CAR19-transduced CD34+ stem cells were transplanted to immunocompromised NOD-SCID IL2Rg-/- (NSG) mice to generate multilineage immune effector cells exhibited delayed early engraftment. It is relevant that CD19+ B cells regenerate first in this mouse model. Thus mice transplanted with CAR19-transduced stem cells exhibited severe CD19+ B cell depletion confirming the functionality of CAR19 expressed in human B cells. To promote T cell regeneration, CD34+ HSCs were co-cultured with Notch ligand DL4 for 1 week before infusion. CD3+T cells were detectable in the blood of mice infused with DL4-pre-cultured CD34+ HSCs as soon as 6 weeks post-transplant while control CD34+ stem cells that were not co-cultured without DL4 exhibited significantly delayed T cell regeneration. Importantly, reduced leukaemia burden was observed in mice reconstituted with CAR19-modified stem cells and infused with human CD19+ leukaemia NALM6 cells. These data proves the feasibility of the stem cell approach to generate potent immune effector cells capable of fighting cancer. Further analysis aimed to identify the role of different multilineage immune effector cells in mediating anti-tumour activity in stem cell reconstituted mice is currently being conducted. Disclosures No relevant conflicts of interest to declare.
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Wang, Dairong, Shao-Jian An, Wen-Hui Wang, John C. McGiff et Nicholas R. Ferreri. « CaR-mediated COX-2 expression in primary cultured mTAL cells ». American Journal of Physiology-Renal Physiology 281, no 4 (1 octobre 2001) : F658—F664. http://dx.doi.org/10.1152/ajprenal.2001.281.4.f658.
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Primary cultures of medullary thick ascending limb (mTAL) cells retain the capacity to express calcium-sensing receptor (CaR) mRNA and protein. Increases in cyclooxygenase-2 (COX-2) mRNA accumulation, protein expression, and PGE2 synthesis were observed in a dose- and time-dependent manner after exposure of these cells to extracellular calcium (Ca[Formula: see text]). Moreover, transfection of mTAL cells with a CaR overexpression vector significantly enhanced COX-2 expression and PGE2 production in response to calcium compared with cells transfected with an empty vector. Challenge with the CaR-selective agonist poly-l-arginine (PLA) also increased COX-2 mRNA accumulation, protein expression, and PGE2 synthesis. Furthermore, Ca[Formula: see text]- and PLA-mediated PGE2production was abolished in the presence of NS-398 or nimesulide, two different COX-2-selective inhibitors. These data suggest that intracellular signaling mechanisms initiated via activation of CaR contribute to COX-2-dependent PGE2 synthesis in the mTAL. Because Ca[Formula: see text] concentration varies along Henle's loop, calcium may contribute to salt and water balance via a COX-2- and CaR-dependent mechanism. Thus novel calcimimetics might be useful in conditions such as hypertension in which manipulation of extracellular fluid volume provides beneficial effects.
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Peters, Mario. « Automobilität in Lateinamerika – eine historiographische Analyse ». Anuario de Historia de América Latina 56 (20 décembre 2019) : 369–95. http://dx.doi.org/10.15460/jbla.56.152.
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Although car-ownership matters to many Latin Americans and cars are nearly omnipresent in daily life in Latin American societies, very little is known about important aspects of the social and cultural histories of automobility in Latin America. However, in the last ten years, several historians have begun to approach the meanings of automobility in Latin American countries. This trend is closely connected to recent developments and new approaches in the international research on mobility, the latter of which I discuss in the first part of this essay. To proceed, I analyze the state of the art on the history of automobility in Latin America, focusing on the following aspects: the emergence of early Latin American car cultures, car and traffic-related social conflicts, and road building. In the last part I ponder on the question of how future studies might advance the state of research on automobility and offer new perspectives on central themes in Latin American history.Although car-ownership matters to many Latin Americans and cars are nearly omnipresent in daily life in Latin American societies, very little is known about important aspects of the social and cultural histories of automobility in Latin America. However, in the last ten years, several historians have begun to approach the meanings of automobility in Latin American countries. This trend is closely connected to recent developments and new approaches in the international research on mobility, the latter of which I discuss in the first part of this essay. To proceed, I analyze the state of the art on the history of automobility in Latin America, focusing on the following aspects: the emergence of early Latin American car cultures, car and traffic-related social conflicts, and road building. In the last part I ponder on the question of how future studies might advance the state of research on automobility and offer new perspectives on central themes in Latin American history.
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Qin, Yuan, Anna Qin, Anna Musket, Joseph Lee, Zhi Yao, Giedre Krenciute et Qian Xie. « 136 Targeting MET with chimeric antigen receptor T cells in hepatocellular carcinoma ». Journal for ImmunoTherapy of Cancer 8, Suppl 3 (novembre 2020) : A149. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0136.
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BackgroundHepatocellular carcinoma (HCC) is the leading cause of cancer mortality worldwide. While HBV/HCV infection is the primary cause of HCC, overexpression of MET, the receptor of hepatocyte growth factor (HGF), occurs in 50% HCC patients, and is an indicator of poor prognosis. Although the multi-target MET tyrosine kinase inhibitor cabozantinib is FDA approved for treating advanced HCC, the long-term efficacy versus toxicity remains unknown. Our study is to develop specific MET-targeting chimeric antigen receptor T (CAR-T) cells for treating HCC with MET overexpression.MethodsBased on a well-established anti-MET monoclonal antibody, we synthesized and cloned the single-chain variable fragment (ScFv) sequence into two retroviral based 2nd generation CAR vectors (MET-CAR.CD28.ζ. and MET-CAR.4-1BB.ζ.). A MET-CAR without CD3ζ domain (MET-CARΔ) served as a negative control. To produce MET-CAR-T cells, healthy PBMCs were stimulated with anti-CD3/CD28 antibodies in the presence of IL-7/IL-15 followed by transduction with MET-CAR viral particles. T cell transduction efficacy was determined using flow cytometry. HCC cell lines with variable MET expression from high/positive (MHCC97H, C3A, and JHH5) to MET low/negative (SNU398) were used to determine MET-specific CAR T cells specificity and effector function using MTS assay. We also collected media from the tumor-T cell co-cultures and determined IL-2 and IFNγ secretion using ELISA. Finally, real-time confocal imaging (24 h) was performed to record the progress of MET-CAR T cell mediated killing activity against MHCC97H/mCherry cells.ResultsWe show that both MET-CAR.CD28.ζ and MET-CAR.4-1BB.ζ -T cells significantly killed MHCC97H, C3A, and JHH5 cells in antigen dependent manner. MET-CAR T cell killing is MET dependent as we observed no killing of MET-negative SNU398 cells. In addition, MET-CAR.4-1BB.ζ and MET-CAR.CD28.ζ- T cells secreted IL-2 and IFNγ when co-cultured with MHCC97H, C3A, JHH5 cells, but not SNU398. Confocal imaging studies showed that both MET-specific CAR T cells migrated toward MHCC97H/mCherry cells, formed aggregations, and induced tumor cell death, while MET-CARΔ T cells failed to do so.ConclusionsHere we demonstrate that MET-CAR.4-1BB.ζ and MET-CAR.CD28.ζ- T cells specifically recognize and kill MET-positive HCC cells in vitro. While animal studies are required to validate the efficacy in vivo, our study has produced a novel therapeutic CAR T cell target for treating malignant HCC and other type of cancers with MET overexpression.AcknowledgementsThis independent research was supported by the Gilead Sciences Research Scholars Program in Liver Disease- The Americas, and Department of Defense (DoD) Ideal Award (to QX)Ethics ApprovalThe study was approved by East Tennessee State University’s Ethics Board, approval number #0619.3s.
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Imam, Shahnawaz. « OR22-4 Homing of Antigen-specific Engineered Regulatory T Cells To Human Pancreatic Islets ». Journal of the Endocrine Society 6, Supplement_1 (1 novembre 2022) : A456. http://dx.doi.org/10.1210/jendso/bvac150.949.
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Abstract The therapeutic application of regulatory T cells (Tregs) for the treatment of autoimmune disorders, although efficacious, has been limited by the scarcity of antigen-specific Tregs. If antigen-specific Tregs, capable of reaching the desired autoimmune target, could be produced on demand, antigen-specific immune suppression of autoimmune diseases would be achievable. One approach to endow T cells with a desired antigen-specificity uses chimeric T cell antigen-receptors (CAR) with antibody-type specificity. Adoptive cell transfer therapies with CAR-re-directed cytotoxic T cells, have shown impressive efficacy in the treatment of hematologic malignancies. Accordingly, employing such technology to re-direct Tregs to sites of autoimmune attack may be a useful therapeutic approach to alleviate a broad spectrum of diseases in which uncontrolled auto and alloimmune responses play a major role. We recently developed pancreatic beta-cell, antigen-specific, CAR Tregs (1) and explored their therapeutic potential against T1D in our humanized mouse model (2). Ours is the first successful, antigen-specific CAR-Treg treatment of T1D in a humanized mouse model that closely resembles the human disease. Based on our mice data, we believe treatment with pancreatic beta-cell, antigen-specific CAR-Tregs will allow for recovery and reconstitution of beta cells in human T1D patients as well. The purpose of this study was to determine if antigen-specific human CAR Tregs could also identify target and home to human pancreatic islets in culture as they do in mice. The study involved drawing 10 cc of blood 1-2 weeks prior to pancreas surgery; followed by collection of a small piece of pancreas (5 cc wedge) once the pancreas was removed for a clinical indication (cancer, pancreatitis). We first isolated Tregs from peripheral blood of these human donors and expanded them in vitro. Treg cells were genetically modified to express either a beta-cell antigen-specific (GAD65) CAR or an irrelevant (EPCAM) CAR construct together with GFP marker. CAR Tregs were selectively expanded in the presence of rhGAD65 antigen. Once pancreas tissue became available, it was processed for islet separation using the collagenase method. Pancreatic islets were then co-cultured with syngeneic CAR Tregs for 7 days. IncuCyte S3 Live-cell System (Sartorius) is a real-time system that automatically acquires and analyzes HD, phase and fluorescent images of cell cultures, around the clock, for 7 days, while cells remain undisturbed. Live immunofluorescence microscopy demonstrated the distinct homing of GAD65 CAR Tregs to islets as compared to control EPCAM CAR Tregs as early as 24 hours of co-culture. Importantly, proliferation of GAD65 CAR Tregs was clearly demonstrated within 72 hours. GAD65 CAR Treg cytokine profile from co-culture supernatant was characteristic of activated Tregs. 1 PATENT #WO2020097546 2 PATENT #US20200037586 Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.
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Hartawidjaja, Gabriele Faustine, et Anna Amalyah Agus. « Influencer’s Trustworthiness for Car Purchase ». Quantitative Economics and Management Studies 4, no 1 (12 janvier 2023) : 168–74. http://dx.doi.org/10.35877/454ri.qems1446.
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Determine the effect of trustworthiness, customer participation behaviour, customer citizenship behaviour, and expected brand value towards purchase intention in high involvement product. This research uses purposive sampling, quantitative approach and SEM-PLS method with the Smart-PLS version 4 software. All direct effects are proven as significant and affective, except for trustworthiness effect against customer participation behaviour, and customer participation behaviour against brand expected value. All mediation effects are not significant, except for expected brand value’s mediation variables which mediate trustworthiness with purchase intention. There is no moderation effect by para-social relationship between neither trustworthiness and customer participation behaviour, nor trustworthiness and customer citizenship behaviour. Novelty of this research can be found in the research object itself, which is influencer's influence on products' high involvement with independent trustworthiness as variable, where automotive influencer is used as research object. Moreover, this research is conducted in a different country than previous research. Building trust is one of the most important things in ensuring the success of customer citizenship behavior, by giving key updates via various social media or brand influencer. Brand owners could also increase their own involvement in exploring social media, one of the examples is by doing campaign to communicate and interact with customers, reviews, comments, and votes. Brand owners could also consider whether Fitra Eri is the most suitable automotive influencer to promote their brand, in accordance with their objectives. The conclusions taken from the research in another countries, cultures, or influencers cannot be generalized. Followers’ intention of following the influencer is not necessarily related with their purchase intention.
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Alexeeva, Ekaterina. « Contacts de langues dans la communauté allemande de la Volga dans les années 1910-1930 ». Cahiers du Centre de Linguistique et des Sciences du Langage, no 35 (18 septembre 2013) : 45–57. http://dx.doi.org/10.26034/la.cdclsl.2013.761.
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Le but de cet article est d’étudier le contact des langues et des cultures (russe/allemande) dans la communauté allemande de la région de la Volga dans les années 1910-1930.Les recherches sur le bilinguisme des Allemands de la Volga ne sont pas nombreuses, car leur patrimoine linguistique et culturel est resté dans l’oubli jusqu’aux années 1990. Le rôle de leur langue maternelle ainsi que les mutations qu’elle a subies restent peu étudiés.Les contacts linguistiques sur le territoire de la communauté allemande de la Volga dans les années 1910-1930 posent aux linguistes des questions concernant le bilinguisme, le rôle des langues des minorités nationales en URSS, les relations entre les langues et les nations.
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Tuckman, Alan. « Jack Saunders, Assembling Cultures : Workplace Activism, Labour Militancy and Culture Change in Britain’s Car Factories, 1945–82 ». Journal of Labor and Society 25, no 1 (28 janvier 2022) : 154–58. http://dx.doi.org/10.1163/24714607-bja10053.
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Dowling, Robyn. « Cultures of mothering and car use in suburban Sydney : a preliminary investigation ». Geoforum 31, no 3 (août 2000) : 345–53. http://dx.doi.org/10.1016/s0016-7185(99)00048-2.
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Karalewitz, Andrew, Natalie Czeryba, Yewei Xing, Derrik Germain, Philip Lapinski, Sheri Barnes, Mark Cameron et al. « Abstract 2835 : Chimeric antigen receptor (CAR) T-cell generation for therapeutic testing in the disseminated NALM6 human B-cell acute lymphoblastic leukemia mouse model ». Cancer Research 82, no 12_Supplement (15 juin 2022) : 2835. http://dx.doi.org/10.1158/1538-7445.am2022-2835.
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Abstract Cancer immunotherapies reprogram the patient’s immune system to mount a coordinated response against a malignant target. T cells engineered to express Chimeric Antigen Receptors (CARs) through transduction with a lentiviral vector represent an effective strategy to specifically eliminate cancerous cells from a patient. Currently, five CAR T cell therapies are approved by the FDA for the treatment of hematological malignancies. With the recent clinical and regulatory success of CAR T cell therapies, the next generation of CAR T cells are undergoing preclinical development. Labcorp Drug Development produces CAR T cells to support the development of new preclinical strategies. Here, the CAR T Cell Generation Service is demonstrated using an anti-CD19 CAR T cell as an example. Using peripheral blood mononuclear cells from healthy, human donors as a T-cell source, CAR T cells were produced by lentivirus transduction. Flow cytometry featuring a CAR-specific monoclonal antibody was used to determine transduction efficiency. To assess in vitro activity, co-cultures of T cells and CD19-expressing NALM6 cells were used to measure cytotoxicity. Proinflammatory cytokine production was analyzed using a Meso Scale Discovery V-PLEX assay. For evaluation of in vivo efficacy, a disseminated NALM6-Luc-mCh-Puro human acute B cell lymphoblastic leukemia model in female NSG mice was conducted. After T cells were treated with lentivirus, approximately 25% of total cells were CD3+/CAR+. Greater than 95% of all target NALM6 cells were killed by a high dose of anti-CD19 CAR T cells. In contrast, only 33% of target cells were killed by untransduced (UTD) T cells at the highest concentration tested. Cytotoxicity against the negative control MV-4-11 cells was equal for a high dose of UTD and a high dose of anti-CD19 CAR T cells. When co-cultured with CD19-expressing NALM6 cells, anti-CD19 CAR T cells produced proinflammatory cytokines including IFN-γ. Relative to mock-treated control mice, treatment with anti-CD19 CAR T cells at high, medium, and low doses increased the time of in vivo disease progression by 175%, 125%, and 16%, respectively. Anti-CD19 CAR T cells generated in our lab were specifically active against CD19-expressing target cells. As a premier contract research organization, Labcorp Drug Development is experienced in producing, handling and culturing T cells and CAR T cells for in vitro and in vivo early discovery studies. Preclinical screening of multiple CAR T cell candidates, alone or in combination with other agents, would facilitate the identification and selection of CARs with the most favorable activity profile for progression through the drug development pipeline. By providing a reliable source of CAR T cells, we are supporting early discovery studies and the development of therapeutic strategies in oncology. Citation Format: Andrew Karalewitz, Natalie Czeryba, Yewei Xing, Derrik Germain, Philip Lapinski, Sheri Barnes, Mark Cameron, Daniel Saims, Amber Rowse, Heidi Nielsen, Scott Wise. Chimeric antigen receptor (CAR) T-cell generation for therapeutic testing in the disseminated NALM6 human B-cell acute lymphoblastic leukemia mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2835.
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Sun, Hongxing, Shan He, Lijun Meng, Ying Wang, Hanghang Zhang, Yongnian Liu, Jian Wang et al. « Engineering of CD19-Specific Chimeric Antigen Receptor T Cells with the Integrin CD103 Results in Augmented Therapeutic Efficacy Against Human Lymphoma in a Preclinical Model ». Blood 132, Supplement 1 (29 novembre 2018) : 2050. http://dx.doi.org/10.1182/blood-2018-99-117354.
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Abstract Whether tumor-reactive T cells can infiltrate into the tumor to execute effector function is essential for controlling tumor growth. CD103 is an integrin protein (αE) that binds integrin β7 to form the heterodimeric integrin complex αEβ7. CD103 is important for T cell retention in peripheral tissues by interacting with E-cadherin and a promising prognosis biomarker for assessment of tumor-reactive T cells infiltrating in the tumor from various types of cancer, such as lung cancer, ovarian cancer and cervical cancers. However, CD103 is not expressed on the surface of circulating peripheral blood T cells that are genetically modified to express a chimeric antigen receptor (CAR) for adoptive T cell therapy. Whether CD103 expression on the surface of tumor-reactive CAR T cells is functionally important for their anti-tumor activity has not been previously determined. Using a preclinical model of human lymphoma expressing E-cadherin, we demonstrate that engineering of CD19-specific human CAR T cells with CD103 significantly improves their therapeutic effects on eliminating pre-established human lymphoma in immune deficient NSG mice (NOD.scid.Il2Rγcnull). We synthesized a codon optimized CD19-specific CAR containing 4-1BB and CD3zeta intracellular signaling domains (named CD19-BBz-CAR), cloned it into lentiviral vector and infected human T cells. As expected, the resultant human CD19-BBz-CAR T cells possessed potent capacity to cure human B cell leukemia in NSG mice that had been intravenously inoculated with Raji leukemic/lymphoma cells. Notably, while approximately 10% of non-CAR T cells produced high levels of CD103 from these NSG mice, CD19-BBz-CAR T cells failed to upregulate CD103, suggesting that the expression of CD19-BBz-CAR inhibits the induction of CD103 in vivo. Ex vivo assay confirmed that CD19-BBz-CAR caused dose-dependent decrease of CD103 expression in human T cells cultured in the presence of TGF-β1. This effect was mediated by the expression of costimulatory molecule 41BB, which is known essential for sustaining CD19-BBz-CAR T cells in vivo. To circumvent the repression effect of 41BB on induction of CD103, we incorporated the gene encoding integrin αE into the CAR structure to generate CD103-CD19-BBz-CAR T cells. Intriguingly, as compared to conventional CD19-BBz-CAR T cells, CD103-CD19-BBz-CAR T cells expressed high levels of CD62L and CD45RA, which resemble less differentiated T cells, produced higher levels of IL-2, which is crucial for promoting T cell expansion and function, and underwent greater expansion in cultures. Upon adoptive transfer into NSG mice that had subcutaneous human Raji lymphoma, CD103-engineering of CD19-BBz-CAR T cells dramatically decreased the distal metastasis of lymphoma, increased the infiltration of CAR T cells into the solid lymphoma, and improved the in vivo persistence of tumor-reactive CAR T cells. As a result, transfer of CD103-CD19-BBz-CAR T cells significantly increased overall survival rate of lymphoma mice compared to conventional CD19-BBz-CAR T cells (40% versus 10%, p<0.05). Our findings suggest that engineering tumor-reactive T cell with CD103 may represent a novel strategy to improve their anti-tumor efficacy. Moreover, this newly established CD103-CAR structure may have broad implication in the solid tumor treatment. Disclosures Barta: Merck, Takeda, Celgene, Seattle Genetics, Bayer: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.
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Berdejo, Daniel, Beatriz Chueca, Elisa Pagán, Adriana Renzoni, William Kelley, Rafael Pagán et Diego Garcia-Gonzalo. « Sub-Inhibitory Doses of Individual Constituents of Essential Oils Can Select for Staphylococcus aureus Resistant Mutants ». Molecules 24, no 1 (4 janvier 2019) : 170. http://dx.doi.org/10.3390/molecules24010170.
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Increased bacterial resistance to food preservation technologies represents a risk for food safety and shelf-life. The use of natural antimicrobials, such as essential oils (EOs) and their individual constituents (ICs), has been proposed to avoid the generation of antimicrobial resistance. However, prolonged application of ICs might conceivably lead to the emergence of resistant strains. Hence, this study was aimed toward applying sub-inhibitory doses of the ICs carvacrol, citral, and (+)-limonene oxide to Staphylococcus aureus USA300, in order to evaluate the emergence of resistant strains and to identify the genetic modifications responsible for their increased resistance. Three stable-resistant strains, CAR (from cultures with carvacrol), CIT (from cultures with citral), and OXLIM (from cultures with (+)-limonene oxide) were isolated, showing an increased resistance against the ICs and a higher tolerance to lethal treatments by ICs or heat. Whole-genome sequencing revealed in CAR a large deletion in a region that contained genes encoding transcriptional regulators and metabolic enzymes. CIT showed a single missense mutation in aroC (N187K), which encodes for chorismate synthase; and in OXLIM a missense mutation was detected in rpoB (A862V), which encodes for RNA polymerase subunit beta. This study provides a first detailed insight into the mechanisms of action and S. aureus resistance arising from exposure to carvacrol, citral, and (+)-limonene oxide.
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Martins, Tomás A., Marie-Françoise Ritz, Tala Shekarian, Philip Schmassmann, Deniz Kaymak, Valentina Bicvic et Gregor Hutter. « IMMU-45. COMBINATION OF EGFRVIII CAR T-CELL THERAPY AND PARACRINE GAM MODULATION FOR THE TREATMENT OF GBM ». Neuro-Oncology 23, Supplement_6 (2 novembre 2021) : vi102—vi103. http://dx.doi.org/10.1093/neuonc/noab196.404.
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Abstract The GBM immune tumor microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). We have previously shown that blockade of CD47, a ‘don't eat me’-signal overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, monotherapy with CD47 blockade has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local CAR T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. We generated humanized EGFRvIII CAR T-cells by lentiviral transduction of healthy donor human T-cells and engineered them to constitutively release a soluble SIRPγ-related protein (SGRP) with high affinity towards CD47. Tumor viability and CAR T-cell proliferation were assessed by timelapse imaging analysis in co-cultures with endogenous EGFRvIII-expressing BS153 cells. Tumor-induced CAR T-cell activation and degranulation were confirmed by flow cytometry. CAR T-cell secretomes were analyzed by liquid chromatography-mass spectrometry. Immunocompromised mice were orthotopically implanted with EGFRvIII+ BS153 cells and treated intratumorally with a single CAR T-cell injection. EGFRvIII and EGFRvIII-SGRP CAR T-cells killed tumor cells in a dose-dependent manner (72h-timepoint; complete cytotoxicity at effector-target ratio 1:1) compared to CD19 controls. CAR T-cells proliferated and specifically co-expressed CD25 and CD107a in the presence of tumor antigen (24h-timepoint; EGFRvIII: 59.3±3.00%, EGFRvIII-SGRP: 52.6±1.42%, CD19: 0.1±0.07%). Differential expression analysis of CAR T-cell secretomes identified SGRP from EGFRvIII-SGRP CAR T-cell supernatants (-Log10qValue/Log2fold-change= 3.84/6.15). Consistent with studies of systemic EGFRvIII CAR T-cell therapy, our data suggest that intratumoral EGFRvIII CAR T-cells were insufficient to eliminate BS153 tumors with homogeneous EGFRvIII expression in mice (Overall survival; EGFRvIII-treated: 20%, CD19-treated: 0%, n= 5 per group). Our current work focuses on the functional characterization of SGRP binding, SGRP-mediated phagocytosis, and on the development of a translational preclinical model of heterogeneous EGFRvIII expression to investigate an additive effect of CAR T-cell therapy and GAM modulation.
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Chalifoux, Jean-Jacques. « Culture : une notion polémique ? » Service social 42, no 1 (12 avril 2005) : 11–23. http://dx.doi.org/10.7202/706597ar.
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La notion de culture est paradoxale, car elle renvoie à la fois à un concept, la culture, et à des choses, les cultures. L'utilisation de la notion de culture n'est pas neutre, elle s'est développée dans un contexte sociopolitique polémique, tout comme son usage actuel. Appliquée à l'hétérogénéité ethnique, la perspective culturaliste peut être réductionniste si elle ne tient pas compte adéquatement des contextes sociaux plus larges.
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Ayed-Boussema, Imen, Jean-Marc Pascussi, Patrick Maurel, Hassen Bacha et Wafa Hassen. « Effect of Aflatoxin B1 on Nuclear Receptors PXR, CAR, and AhR and Their Target Cytochromes P450 mRNA Expression in Primary Cultures of Human Hepatocytes ». International Journal of Toxicology 31, no 1 (12 octobre 2011) : 86–93. http://dx.doi.org/10.1177/1091581811422453.
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Aflatoxin B1 (AFB1), one of the most common mycotoxins found in human foods and animal feed, is principally hepatotoxic and hepatocarcinogenic. The aim of the present study was to explore the effect of AFB1 on messenger RNA (mRNA) expression of pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) and some of their target cytochromes using primary cultures of human hepatocytes. Our results showed that AFB1, at noncytotoxic increasing concentrations, caused a significant upregulation of cytochrome P 2B6 (CYP2B6), CYP3A5, and to a lesser extent CYP3A4 and CYP2C9. Pregnane X receptor and CAR mRNA expression increased in the 3 treated livers. Aflatoxin B1 was found also to induce an overexpression of CYP1A1 and CYP1A2 genes accompanied by an increase in AhR mRNA expression. These findings suggest that AFB1 could activate PXR, CAR, and AhR; however, further investigations are needed to confirm nuclear receptor activation by AFB1.
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Krug, Adrien, Adriana Martinez-Turtos et Els Verhoeyen. « Importance of T, NK, CAR T and CAR NK Cell Metabolic Fitness for Effective Anti-Cancer Therapy : A Continuous Learning Process Allowing the Optimization of T, NK and CAR-Based Anti-Cancer Therapies ». Cancers 14, no 1 (30 décembre 2021) : 183. http://dx.doi.org/10.3390/cancers14010183.
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Chimeric antigen receptor (CAR) T and CAR NK cell therapies opened new avenues for cancer treatment. Although original successes of CAR T and CAR NK cells for the treatment of hematological malignancies were extraordinary, several obstacles have since been revealed, in particular their use for the treatment of solid cancers. The tumor microenvironment (TME) is competing for nutrients with T and NK cells and their CAR-expressing counterparts, paralyzing their metabolic effective and active states. Consequently, this can lead to alterations in their anti-tumoral capacity and persistence in vivo. High glucose uptake and the depletion of key amino acids by the TME can deprive T and NK cells of energy and building blocks, which turns them into a state of anergy, where they are unable to exert cytotoxic activity against cancer cells. This is especially true in the context of an immune-suppressive TME. In order to re-invigorate the T, NK, CAR T and CAR NK cell-mediated antitumor response, the field is now attempting to understand how metabolic pathways might change T and NK responses and functions, as well as those from their CAR-expressing partners. This revealed ways to metabolically rewire these cells by using metabolic enhancers or optimizing pre-infusion in vitro cultures of these cells. Importantly, next-generation CAR T and CAR NK products might include in the future the necessary metabolic requirements by improving their design, manufacturing process and other parameters. This will allow the overcoming of current limitations due to their interaction with the suppressive TME. In a clinical setting, this might improve their anti-cancer effector activity in synergy with immunotherapies. In this review, we discuss how the tumor cells and TME interfere with T and NK cell metabolic requirements. This may potentially lead to therapeutic approaches that enhance the metabolic fitness of CAR T and CAR NK cells, with the objective to improve their anti-cancer capacity.
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Christodoulou, Ilias, Ruyan Rahnama, Wesley J. Ravich, Jaesung Seo, Sergey Zolov, Andrew Marple, David M. Markovitz et Challice L. Bonifant. « CAR-NK Cells Targeting Sars-Cov-2 Glycosites As COVID-19 Treatment ». Blood 138, Supplement 1 (5 novembre 2021) : 2803. http://dx.doi.org/10.1182/blood-2021-146865.
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Abstract Introduction: Banana Lectin (BanLec) is a glycoprotein-binding lectin derived from banana fruit that has antiviral activity. BanLec binds high mannose glycans expressed on the viral envelopes of HIV, Ebola, influenza, and coronaviruses. BanLec mitogenicity can be divorced from antiviral activity via a single amino acid change (H84T). The SARS-CoV-2 spike (S) protein is decorated with high mannose N-glycosites that are in close proximity to the viral receptor binding domain (RBD). Our goal was to use the H84T-BanLec as the extracellular targeting domain of a chimeric antigen receptor (CAR). We hypothesized that engineering NK cells to express an H84T-BanLec CAR would specifically direct antiviral cytotoxicity against SARS-CoV-2. Methods: H84T-BanLec was synthesized and added to a 4-1BB.ζ CAR by subcloning into an existing retroviral vector. To modify primary human NK cells, CD3-depleted peripheral blood mononuclear cells were first activated with lethally irradiated feeder cells (K562.mbIL15.4-1BBL), then transduced with transiently produced replication incompetent γ-retrovirus carrying the H84T-BanLec.4-1BB.ζ CAR construct. Vector Copy Number (VCN) per cell was measured and CAR protein expression detected with Western blotting. 293T cells were engineered to express human ACE2 (hACE2.293T), the binding receptor for SARS-CoV-2. CAR expression on NK cells and SARS-CoV-2 S-protein binding to hACE2.293T were measured using FACS. S-protein pseudotyped lentivirus carrying a firefly Luciferase (ffLuc) reporter was produced. Viral infectivity was measured using bioluminescence (BL) detection in virally transduced cells. H84T-BanLec CAR NK cells were added to our S-protein pseudotyped lentiviral infectivity assay and degree of inhibited transduction was measured. NK cell activation was assessed with detection of IFNγ and TNFα secretion using ELISA. Results: A median of 4.5 integrated H84T-BanLec CAR copies per cell was measured (range 3.5-7.45, n=4). The CAR was detected by Western blot in NK cell lysates using antibodies to TCRζ and H84T-BanLec. Surface expression of the CAR on primary NK cells was recorded on day 4 after transduction (median [range], 67.5% CAR-positive [64.7-75%], n=6; Fig. 1). CAR expression was maintained on NK cells in culture for 14 days (58.9% CAR-positive [43.6-66.7%], n=6; Fig. 1). ACE2 expression and binding of recombinant S-proteins to hACE2 on hACE2.293T but not parental 293Ts was verified. S-protein pseudotyped lentiviral transduction of hACE2.293T was confirmed with increase in BL from baseline across diminishing viral titer (n=3; Fig. 2). Control 293T cells without hACE2 expression were not transduced, confirming specificity of viral binding and entry dependent on hACE2 (n=3; Fig. 2). S-protein pseudoviral infectivity of hACE2.293T cells was inhibited by both H84T-BanLec CAR-NK and unmodified NK cells, with enhanced inhibition observed in the CAR-NK condition (mean % pseudovirus infectivity +/- SEM of hACE2.293T in co-cultures with unmodified NK vs. H84T-BanLec CAR-NK; 65 +/-11% vs 35%+/- 6% for 1:1 effector-to-target ratio, p=0.05; 78 +/-3% vs 68%+/- 3% for 1:2.5 effector-to-target ratio, p=0.03; n=6; Fig.3). Both unmodified and H84T-BanLec CAR-NK cells were stimulated to secrete inflammatory mediators when co-cultured with pseudoviral particles and virally infected cells. CAR-NK cells showed overall higher cytokine secretion both at baseline and with viral stimulation. Conclusions: A glycoprotein binding H84T-BanLec CAR was stably expressed on the surface of NK cells. CAR-NK cells are activated by SARS-CoV-2 S-pseudovirus and virally infected cells. Viral entry into hACE2 expressing cells was inhibited by H84T-BanLec CAR-NK cells. Translation of H84T-BanLec CAR-NK cells to the clinic may have promise as an effective cellular therapy for SARS-CoV-2 infection. Figure 1 Figure 1. Disclosures Markovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct. Bonifant: Merck, Sharpe, Dohme: Research Funding; BMS: Research Funding; Kiadis Pharma: Research Funding.
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Maciocia, Paul, Patrycja Wawrzyniecka, Leo Kassimatis et Martin Pule. « Chimeric Antigen Receptor T-Cells for the Treatment of Gamma-Delta T-Cell Malignancies ». Blood 132, Supplement 1 (29 novembre 2018) : 3338. http://dx.doi.org/10.1182/blood-2018-99-119774.
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Abstract Introduction Cancers derived from the malignant transformation of gamma delta T-cells are rare but carry very poor prognosis. Hepatosplenic T-cell lymphoma is a highly aggressive condition characterised by hepatosplenic and bone marrow involvement. It has among the worst outcomes of all lymphoma subtypes, with a median survival of only 6-8 months. 95% of cases express the gamma delta T-cell receptor (GDTCR), which is also expressed on a proportion of cases of T-ALL. Treatment for these cancers is based on cytotoxic chemotherapy, with no tumour-specific therapies including immunotherapy available. We have developed a novel chimeric antigen receptor targeting GDTCR and here demonstrate specific in vitro and in vivo efficacy against gamma delta T-cell malignancies. Results We cloned anti-GDTCR antibody as a single chain variable fragment (ScFv), and confirmed specific binding to GDTCR-positive T-cell cell lines and primary GD cells. Next, we cloned anti-GDTCR ScFv into a 2ndgeneration chimeric antigen receptor (CAR) format, including a spacer derived from CD8-stalk, CD28 transmembrane domain and 41BB-zeta endodomain. This construct was stably introduced to primary alpha-beta T-cells by retroviral transduction and surface expression was confirmed by flow cytometry. We established 48-hour co-cultures of anti-GDTCR CAR T-cells or control anti-CD19 CAR T-cells with T-cell lines positive (Loucy, BE13, MOLT-13) or negative for surface GDTCR (Jurkat, SupT1-CD19). While control anti-CD19 CAR killed only SupT1-CD19 cells, specific cytotoxicity was seen by anti-GDTCR CAR T-cells only against GDTCR-positive cell lines. In addition, anti-GDTCR CAR T-cells demonstrated specific secretion of cytokines including interferon gamma and IL-2, and robust antigen-specific proliferation only in co-culture with GDTCR-positive cells. Expression of exhaustion, activation and differentiation markers in long term co-cultures with target cells was similar to that seen with control anti-CD19 CAR. To assess the in vivo potency of anti-GDTCR CAR T-cells, we established a murine model of disseminated GDTCR-positive leukaemia. NSG mice were intravenously injected with 4x10^6 Loucy cells, engineered to stably express Firefly luciferase. Tumour engraftment in bone marrow was confirmed at D7 following injection, and mice were treated with 0.8x10^6 anti-GDTCR or control anti-CD19 CAR T-cells. Disease burden was monitored by bioluminescence imaging. Mice receiving anti-GDTCR CAR demonstrated substantial reduction of tumour burden, increased expansion of CAR T-cells and prolonged survival compared to control-CAR treated animals. Conclusions We have developed a novel chimeric antigen receptor T-cell treatment for gamma-delta TCR-positive malignancies, including hepatosplenic T-cell lymphoma and some cases of T-ALL. Our approach is, to our knowledge, the first immunotherapeutic strategy proposed for these conditions. Given the restricted expression of GD-TCR on a small subset (0.5-5%) of peripheral T-cells and the absence of a clear human phenotype associated with GD T-cell deficiency, we suggest that this therapy may be well tolerated. Given the very poor prognosis and lack of effective therapies for GD-TCR-positive malignancies, as well as the considerable efficacy of CAR T-cell therapy in analogous B-cell disorders, our approach could bring much needed benefit to patients suffering these conditions. Disclosures Maciocia: Autolus: Equity Ownership, Patents & Royalties: UCLB. Pule:UCLB: Patents & Royalties; Autolus: Employment, Equity Ownership.
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Tarţa, Corina Paula, Ioana Plăiaş, Luis F. Martinez et Luisa M. Martinez. « The Role of Car Aesthetics on Consumers’ Decisions : An Example from Romania ». Scientific Annals of Economics and Business 67, no 1 (mars 2020) : 33–43. http://dx.doi.org/10.47743/saeb-2020-0003.
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Visual impact is essential when consumers are assessing car preferences. The purpose of this paper is to present a holistic model focusing on car aesthetic dimensions and its impact on consumers’ purchase decisions. Our findings are based on a questionnaire completed by 388 participants and analyzed with SPSS and AMOS. The results show not only that the aesthetic dimensions such as color, shape and sound influence the processing stimuli, but also the significant and positive relationships established between the stimuli and the purchase decision. Also, this study can guide marketers when developing an effective marketing strategy. As our research focuses on the Romanian market, testing our framework in different cultures is strongly encouraged.
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Griffith, J. W., W. J. White, P. J. Danneman et C. M. Lang. « Cilia-associated Respiratory (CAR) Bacillus Infection of Obese Mice ». Veterinary Pathology 25, no 1 (janvier 1988) : 72–76. http://dx.doi.org/10.1177/030098588802500110.
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Cilia-associated respiratory (CAR) bacillus was identified in respiratory tract lesions of obese mice dying of chronic respiratory disease. Neither Mycoplasma pulmonis nor pathogenic bacteria were isolated from cultures of the lesions at necropsy, but there was serologic and histologic evidence of respiratory virus infection. Cranial-ventral areas of lung were firm and demarcated from unaffected lung at gross examination, and representative tissue sank in water. Microscopically, there was suppurative bronchopneumonia with extensive peribronchiole lymphocyte and plasma cell proliferation. The affected bronchiole epithelium was covered with a sheet of slightly basophilic, filamentous, gram negative bacteria. Bronchioles with lesser amounts of lymphocyte accumulations contained lesser amounts of filamentous bacteria. Bronchioles without filamentous bacteria lining the respiratory epithelium lacked peribronchiole lymphocyte accumulations. There was a high correlation between CAR bacillus-positive serology and the identification of diagnostic histologic lesions. CAR bacillus was readily stained using immunohistochemical methods, and the ultrastructural features were similar to that described in rat infections.
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Diener, L., N. Goedert, T. Schulz, C. Monoranu, R. Ernestus, C. Hagemann, M. Löhr, T. Nerreter et V. Dufner. « P06.04.B Efficacy of Podoplanin-CAR-T Cells inex vivo Patient Derived Glioblastoma Organoids ». Neuro-Oncology 24, Supplement_2 (1 septembre 2022) : ii38. http://dx.doi.org/10.1093/neuonc/noac174.128.
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Abstract Background Glioblastoma (GBM) is the most prevalent malignant brain tumor in adults. Recent immunotherapeutic approaches led to promising effects both in vitro and in preclinical animal models. However, tumor heterogeneity, antigen escape mechanisms and complex interactions with the tumor microenvironment (TME) hinder their clinical breakthrough. Thus, patient derived ex vivo models are needed to test new therapeutical approaches. Genetically modified T cells expressing a chimeric antigen receptor (CAR) hold enhanced affinity for tumor associated antigens. As Podoplanin (PDPN) shows stable and increased expression in GBM, it is a suitable target antigen. When activated, CAR-T cells initiate the triad of cytokine release, T cell proliferation and target cell apoptosis. We evaluated the performance of PDPN-CAR-T cells in GBM patient derived organoids (PDO). Material and Methods PDOs were generated from freshly resected GBM tissue and could be cultured successfully up to several months. We assessed PDPN expression of PDOs via immunohistochemistry (IHC) prior to treatment. PDPN-CAR T cells were generated from peripheral blood mononuclear cells of healthy donors via lentiviral transduction and expansion. The transduction rate was assessed by flow cytometry prior to application. PDOs treated with untransduced T cells served as controls. PDOs were incubated with a preset number of PDPN-CAR T cells at a E:T ratio of 1:4 and were examined microscopically to register morphological disintegration after 48h. Immunofluorescence staining was conducted to detect proliferating CD4+ CART cells (CD4+/ Ki67+) after 72h and cytokine release of IFN-γ was determined via ELISA after 20h. Results In total, PDOs from three patients were treated with PDPN-CART cells. All of them expressed the antigen according to IHC staining. All incubated PDOs presented clear disintegration of the circular organoid shape up to total dissolving, whereas control PDOs stayed intact. CD4+ CART cells showed extensive proliferation at a mean rate of 79.8% ± 6,59% which was significant for two PDOs in comparison to control PDOs (p = 0.48, p < 0.01). PDPNCART cells exhibited significantly elevated IFN-γ release in all PDOs (p < 0.01). Conclusion We here describe PDPN as a promising target and proved effectiveness of PDPN-CAR-T cells in an ex vivo 3D model. Additional ex vivo models like tumor slice cultures might be crucial to evaluate effectiveness of CAR-T cell therapy. PDPN-CAR-T cells should be tested in these ex vivo as well as in vivo GBM models alone and in a combined approach with CARs targeting additional antigens in order to overcome tumor heterogeneity.
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Dozmorov, I. M., V. V. Kalinichenko, I. A. Sidorov et R. A. Miller. « Antagonistic interactions among T cell subsets of old mice revealed by limiting dilution analysis. » Journal of Immunology 154, no 9 (1 mai 1995) : 4283–93. http://dx.doi.org/10.4049/jimmunol.154.9.4283.
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Abstract When CD4 spleen cells from old (but not young) mice are tested for Con A-induced proliferation in limiting dilution assays, the dose response curve shows a nonlinear relationship. We interpret these observations using a two-cell model, in which proliferation of one cell type (LPC1) can be blocked by a second cell type (LPC2), which can itself generate detectable proliferation only at high multiplicities. The two-cell model accounts for several observations: 1) the variation in curve shape as a function of incubation time; 2) the skewed distribution of wells scored as "negative" in cultures of old splenocytes; and 3) the initially antagonistic effects of old splenocytes titrated into cultures containing fixed numbers of young responders. To provide a further test of the two-cell model, ionomycin-resistant (CaR) and ionomycin-sensitive (CaS) cells were separated using a Percoll/ionomycin gradient. The CaR preparation, shown previously to consist largely of memory T cells, showed the dose curve predicted for the LPC2 cell type, whereas the CaS (naive) cells showed the single-hit kinetics postulated for LPC1 cells. Furthermore, mixtures of CaR and CaS cells from young mice reproduced the zigzag dose curve characteristically produced by unseparated cells from old mice. These data suggest that the spleens of both young and old mice contain two kinds of Con A-responsive CD4 cell: one that proliferates vigorously, and a second, calcium ionophore-resistant type that proliferates less well, that can interfere with proliferation of the first cell type, and whose frequency increases with age.
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Collins, McKensie, Weimin Kong, Inyoung Jung, Stefan M. Lundh et J. Joseph Melenhorst. « B-CLL Mediated Resistance to CAR T Cell Therapy Via Insufficient Activation Is CAR-Independent ». Blood 136, Supplement 1 (5 novembre 2020) : 44. http://dx.doi.org/10.1182/blood-2020-137638.
Texte intégralRésumé :
Chronic Lymphocytic Leukemia (CLL) is a B cell malignancy that accounts for nearly 1/3rd of adult leukemia diagnoses in the Western world. Conventional chemo-immunotherapies initially control progression, but in the absence of curative options patients ultimately succumb to their disease. Chimeric Antigen Receptor (CAR) T cell therapy is potentially curative, but only 26% of CLL patients have a complete response. CLL-stimulated T cells have reduced effector functions and B-CLL cells themselves are believed to be immunosuppressive. Our work demonstrates that insufficient activation of CAR T cells by CLL cells mediates some of these effects and that the results are conserved between ROR1- and CD19-targeting CARs. Results: In this study we used an in vitro system to model the in vivo anti-tumor response in which CAR T cells serially engage with CLL cells. Multiple stimulations of CD19 or ROR1-targeting CAR T cells with primary CLL cells recapitulated many aspects of known T cell dysfunction including reduced proliferation, cytokine production, and activation. While the initial stimulation induced low level proliferation, subsequent stimulations failed to elicit additional effector functions. We further found that these functional defects were not permanent, and that CAR T cell function could be restored by switching to a stimulus with an aAPC (artificial Antigen Presenting Cell) control cell line. The aAPCs are well-characterized as potent stimulators of CAR T cell effector responses. Flow cytometry revealed that CLL-stimulated CAR T cells retained a non-activated, baseline differentiation profile, suggesting that CLL cells fail to stimulate CAR T cells rather than rendering them non-functional. One mechanism that could dampen activation is immune suppression. We assessed this at a high level by stimulating CAR T cells with CLL cells and aAPCs mixed at known ratios. However, even cultures containing 75% CLL cells stimulated proliferation and cytokine production. Extensive immune-phenotyping revealed high level expression of the IL-2 Receptor on 90% (18/20) of the B-CLL cells tested. Since cytokine sinking via IL-2 receptor expression is a well-known mechanism of regulatory T cell suppression, we hypothesized that CLL cells similarly sink IL-2, blunting T cell activation. To test this, we supplemented IL-2 into CLL/CAR T cell co-cultures and showed that this rescued proliferation but only partially restored cytokine production. In contrast to our hypothesis, analysis of cytokine production by flow cytometry showed that CLL-stimulated CAR T cells did not produce IL-2 following a 6- or 12-hour stimulus, but TNFα was expressed after 12-hours. Similarly, CAR T cell degranulation, a prerequisite for target cell lysis was triggered after CLL recognition. These data again suggested that CLL cells insufficiently stimulate CAR T cell cytokine production, but also showed that cytolytic activity against CLL cells is intact. We further proposed that CLL cells express insufficient levels of co-stimulatory and adhesion molecules to activate CAR T cells. Flow cytometry showed that most CLL cells expressed co-stimulatory and adhesion molecules at low levels; we hypothesized that up-regulating these molecules would enhance CAR T cell targeting of CLL cells. CLL cells were activated with CD40L and IL-4, which increased expression of CD54, CD58, CD80, and CD86. Stimulating CAR T cells with activated CLL cells enhanced CAR T cell proliferation and induced cell conjugate formation, indicating cell activation. Therefore, improving CLL stimulatory capacity can rescue T cell dysfunctions. To assess whether IL-2 addition and CD40 ligation were synergistic, we combined the two assays; however, we saw no additional improvement over IL-2 addition alone, suggesting that the two interventions may act upon the same pathway. Importantly, we also showed that rescue of CAR T cell function via IL-2 addition or CD40 ligation was not CAR-specific, as we observed the functional defects and subsequent rescue with both a ROR1-targeting CAR and the gold standard CD19-targeting CAR. Conclusions: Together, these data show that CAR T cell "defects" in CLL are actually insufficient activation, and improving the stimulatory capacity of CLL cells may enable better clinical responses. Further, this effect is not CAR-specific and these results may therefore be broadly applicable to multiple therapies for this disease. Disclosures Melenhorst: IASO Biotherapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Novartis: Other: Speaker, Research Funding; Johnson & Johnson: Consultancy, Other: Speaker; Simcere of America: Consultancy; Poseida Therapeutics: Consultancy.
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Gilbert, Amy, Stephen Santoro, Tiffany Tse, Tara Candelario-Chopra, Tina Gomes, Jose Campos, Michael Spelman et al. « Evaluating the reversible control of an engineered CAR T cell ON-switch. » Journal of Clinical Oncology 35, no 15_suppl (20 mai 2017) : e14550-e14550. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e14550.
Texte intégralRésumé :
e14550 Background: CAR T cell therapy holds enormous promise for many cancer types but its application may be limited by serious toxicities. To lower this hurdle, our aim is to engineer tunable cell therapies. One of our approaches includes a “ON-switch” chimeric antigen receptor (Wu et al., Science 2015) that requires the administration of a small molecule acting as a dimerizing agent between one polypeptide chain containing the antigen recognition domain and half of an inducible heterodimerization system and another polypeptide chain containing the second half of the inducible heterodimerization motif, the CD3ζ chain and a costimulatory motif. Using an FDA approved small molecule drug, we evaluate the reversibility of ON-switch CAR T cells in preclinical models. Methods: First, we evaluated the proliferation, cytotoxicity and cytokine production of several ON-switch constructs in human primary T cells. Next, to address the reversibility of the ON-switch (ON→OFF→ON), we performed a series of co-culture experiments where the small molecule drug was added to tumor cells and ON-switch CAR T cells, then washed out, and then re-introduced back into the co-cultures. We compared CAR T cell mediated killing and cytokine production from the On-switch CAR T cells relative to a canonical CAR T control. Results: Our On-switch CAR T cells were shown to proliferate, secrete cytokines as well as mediate dose dependent cytotoxicity in the presence of the small molecule drug. Importantly, in the presence of antigen but in absence of the small molecule drug we did not measure any significant functional activity in our ON-switch CARs. Additonally, following the removal of the small molecule drug over a period several days we did not observe any significant CAR mediated cytotoxicity. Following the subsequent re-addition of the small molecule, we observed further CAR T cell mediated cytotoxicity against tumor cells. Conclusions: These results show that the small molecule inducible On-switch CARs maintain functional activity as well as reversibility allowing for the tunable control of a CAR T cell.