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Strell, Phoebe, Anala Shetty, Clifford J. Steer et Walter C. Low. « Interspecies Chimeric Barriers for Generating Exogenic Organs and Cells for Transplantation ». Cell Transplantation 31 (janvier 2022) : 096368972211105. http://dx.doi.org/10.1177/09636897221110525.
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A growing need for organs and novel cell-based therapies has provided a niche for approaches like interspecies chimeras. To generate organs from one donor species in another host species requires techniques such as blastocyst complementation and gene editing to successfully create an embryo that has cells from both the donor and the host. However, the task of developing highly efficacious and competent interspecies chimeras is met by many challenges. These interspecies chimeric barriers impede the formation of chimeras, often leading to lower levels of chimeric competency. The barriers that need to be addressed include the evolutionary distance between species, stage-matching, temporal and spatial synchronization of developmental timing, interspecies cell competition and the survival of pluripotent stem cells and embryos, compatibility of ligand–receptor signaling between species, and the ethical concerns of forming such models. By overcoming the interspecies chimera barriers and creating highly competent chimeras, the technology of organ and cellular generation can be honed and refined to develop fully functioning exogenic organs, tissues, and cells for transplantation.
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Lacadena Calero, Juan Ramón. « BIOÉTIC AS MACAQUE-HUMAN CHIMERAS : SCIENTIFIC ASPECTS AND BIOETHICAL REFLECTIONS ». Anales de la Real Academia Nacional de Farmacia, no 87(02) (2021) : 117–21. http://dx.doi.org/10.53519/anaesranf.2021.87.02.01.
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The obtention by Izpisua and collaborators (2021) of macaque-human chimeric embryos by microinjection of human pluripotent stem cells into early blastocysts of cynomolgus monkey is described. They studied the competency of human pluripotent stem cells in macaque embryos cultured ex vivo until 19 days post-fertilization. A reflection on these experiments is made from the bioethical point of view.
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Afanassieff, Marielle, Florence Perold, Wilhelm Bouchereau, Antoine Cadiou et Nathalie Beaujean. « Embryo-derived and induced pluripotent stem cells : Towards naive pluripotency and chimeric competency in rabbits ». Experimental Cell Research 389, no 2 (avril 2020) : 111908. http://dx.doi.org/10.1016/j.yexcr.2020.111908.
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Hirabayashi, M., T. Goto, C. Tamura, M. Sanbo et S. Hochi. « 202 EFFECT OF LEUKEMIA INHIBITORY FACTOR AND FORSKOLIN ON ESTABLISHMENT OF RAT EMBRYONIC STEM CELL LINES ». Reproduction, Fertility and Development 26, no 1 (2014) : 215. http://dx.doi.org/10.1071/rdv26n1ab202.
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Rat embryonic stem (ES) cell lines can be established in culture medium containing inhibitors for glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK). We confirmed reproducibility of the 2i culture system in establishing rat ES cell lines (Hirabayashi et al. 2010 Mol. Reprod. Dev. 77, 94) and the likelihood of successful germline transmission (genuine) of rat ES cell lines established in leukemia inhibitory factor (LIF)- and forskolin (FK)-supplemented 2i medium (Hirabayashi et al. 2013 Transgenic Res. 22, 411–416). This study was designed to investigate whether LIF and/or FK supplemented to the 2i medium support establishment of germline-competent rat ES cell lines. E4.5 blastocysts were recovered from BLK rat females, and zona-free embryos were plated on mitomycin-treated mouse embryonic fibroblasts in N2B27 medium containing 1 mM MEK inhibitor PD0325901 and 3 mM GSK3 inhibitor CHIR99021, with rat 1000 U mL–1 LIF and/or 10 μM FK. Outgrowth rate of the blastocysts after 1 wk culture and establishment efficiency of ES cell lines after third passage were analyzed by Fisher's exact probability test. Arcsin-transformed percentage data on full-term development of ES cell-injected blastocysts, chimeric rat production, and germline-competent chimeras were analyzed by Fisher's least significant difference test after one-way ANOVA. Because of the higher outgrowth rates of blastocysts, supplementation of rat LIF, FK, or both contributed to the higher (P < 0.05) establishment efficiency of ES cell lines in BLK rat strain (76% to 92% v. 50% in LIF/FK-free 2i medium). Neither efficiency of producing chimeric rats (14% to 39% of blastocysts injected) nor germline transmission competency of the chimeric rats (67% to 100% of cell lines analyzed) was influenced by the pre-treatment of ES cell lines. When the LIF/FK-supplemented 2i medium was used, rat strain for blastocyst donor such as F344 or WI was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, however, all of them (9/9 in overall) were found to be germline-competent by progeny test of chimeric rats. In conclusion, both LIF and FK are not essential, but can play a beneficial role, for the establishment of genuine rat ES cell lines. This work was supported by a grant-in-aid for basic research from Japan Society for the Promotion of Science (No. 25290037; to M.H.).
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Tapponnier, Yann, Marielle Afanassieff, Irène Aksoy, Maxime Aubry, Anaïs Moulin, Lucas Medjani, Wilhelm Bouchereau et al. « Reprogramming of rabbit induced pluripotent stem cells toward epiblast and chimeric competency using Krüppel-like factors ». Stem Cell Research 24 (octobre 2017) : 106–17. http://dx.doi.org/10.1016/j.scr.2017.09.001.
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Whitaker, Neal, Trista M. Berry, Nathan Rosenthal, Jay E. Gordon, Christian Gonzalez-Rivera, Kathy B. Sheehan, Hilary K. Truchan et al. « Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine ». Journal of Bacteriology 198, no 19 (18 juillet 2016) : 2701–18. http://dx.doi.org/10.1128/jb.00378-16.
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ABSTRACTBacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that theEscherichia colipKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning inAgrobacterium tumefaciens,Anaplasma phagocytophilum, orWolbachia pipientis. A chimeric receptor assembled fromA. tumefaciensVirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) andA. tumefacienseffector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4Atand the TraJ/VirD4Atchimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4Atdomains bound this substratein vitro. We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains.IMPORTANCEBacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through theEscherichia colipKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.
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Kondoh, Gen, Yoichi Yamamoto, Kayo Yoshida, Yutaka Suzuki, Soh Osuka, Yuka Nakano, Takashi Morita et Junji Takeda. « Easy assessment of ES cell clone potency for chimeric development and germ-line competency by an optimized aggregation method ». Journal of Biochemical and Biophysical Methods 39, no 3 (mai 1999) : 137–42. http://dx.doi.org/10.1016/s0165-022x(99)00008-1.
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Fields, Chris, et Michael Levin. « Competency in Navigating Arbitrary Spaces as an Invariant for Analyzing Cognition in Diverse Embodiments ». Entropy 24, no 6 (12 juin 2022) : 819. http://dx.doi.org/10.3390/e24060819.
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One of the most salient features of life is its capacity to handle novelty and namely to thrive and adapt to new circumstances and changes in both the environment and internal components. An understanding of this capacity is central to several fields: the evolution of form and function, the design of effective strategies for biomedicine, and the creation of novel life forms via chimeric and bioengineering technologies. Here, we review instructive examples of living organisms solving diverse problems and propose competent navigation in arbitrary spaces as an invariant for thinking about the scaling of cognition during evolution. We argue that our innate capacity to recognize agency and intelligence in unfamiliar guises lags far behind our ability to detect it in familiar behavioral contexts. The multi-scale competency of life is essential to adaptive function, potentiating evolution and providing strategies for top-down control (not micromanagement) to address complex disease and injury. We propose an observer-focused viewpoint that is agnostic about scale and implementation, illustrating how evolution pivoted similar strategies to explore and exploit metabolic, transcriptional, morphological, and finally 3D motion spaces. By generalizing the concept of behavior, we gain novel perspectives on evolution, strategies for system-level biomedical interventions, and the construction of bioengineered intelligences. This framework is a first step toward relating to intelligence in highly unfamiliar embodiments, which will be essential for progress in artificial intelligence and regenerative medicine and for thriving in a world increasingly populated by synthetic, bio-robotic, and hybrid beings.
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Ugale, Amol Sanjay, Gudmundur Logi Norddahl, Martin Wahlestedt, Petter Säwén, Pekka Jaako, Cornelis J. H. Pronk, Shamit Soneji, Jorg Cammenga et David Bryder. « Hematopoietic Stem Cells Are Intrinsically Protected Against MLL-ENL Mediated Transformation ». Blood 124, no 21 (6 décembre 2014) : 839. http://dx.doi.org/10.1182/blood.v124.21.839.839.
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Abstract Studies on the developmental pathways of hematopoietic stem cells (HSCs) have led to roadmaps of differentiation and resulted in key information concerning lineage relationships and restriction points in the blood system. This knowledge is also central to understand the etiology of acute myeloid leukemia (AML), where recent work has proposed that the heterogeneity and aggressiveness of AML can associate with the developmental stage of transformation. Balanced chromosomal translocations that result in fusion proteins with aberrant transcriptional regulatory activities are frequent initiating events in acute myeloid leukemia, and a prototype family of such chimeric transcription factors is represented by fusions involving the mixed lineage leukemia-1 (MLL1) gene. Previous work using mouse models have suggested that at some stage of normal differentiation there is a loss of competence to induce AML. However discrepancies exists between these mouse models concerning the target cells of MLL fusion genes. While it is clear that cells can lose competence for leukemic transformation as part of their normal differentiation, the question remains whether the most primitive HSCs are always imbued with leukemogenic competency as part of their normal biology. To address this, we developed a Doxycycline inducible transgenic mouse model of the human chimeric transcription factor Mixed Lineage Leukemia-Eleven Nineteen Leukemia (MLL-ENL). Prospective isolations of candidate leukemia-initiating cells followed by adoptive transfers allowed us to detail leukemia-initiation and competence throughout the hematopoietic hierarchy. We show that AML can origin from multiple HPC subsets with intrinsic granulocytic/monocytic potential. Closely related myeloid progenitors displayed distinct leukemic- and functional capacity in response to physiological levels of MLL-ENL, highlighting the importance of a careful prospective isolation of progenitor populations. AML could also develop efficiently from common lymphoid progenitors, supporting a latent myeloid potential of these cells. By contrast, early commitment to the megakaryocytic/erythroid lineages was incompatible with leukemic development. By contrast, disease failed to arise from the most primitive progenitor subsets, including HSCs. Investigations of the immediate transcriptional responses to MLL-ENL showed evidence for a block in differentiation in both myeloid progenitors and HSCs, while MLL-ENL restricted cell cycle progression uniquely in HSCs. Our study highlights how an oncogene can exert unique functions depending on the developmental position of its cellular targets and demonstrate the existence of a mechanism, operational at the level of immature HSCs/progenitors, which act to prevent leukemic development. Figure 1 Graphical abstract Figure 1. Graphical abstract Disclosures No relevant conflicts of interest to declare.
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Zaslavsky, Alexander, Mackenzie Adams, Sandra Wissmueller, Douglas Campbell, Hans Klingemann, Brad Walsh et Ganesh S. Palapattu. « Glypican-1 as a novel immunotherapeutic target in prostate cancer. » Journal of Clinical Oncology 36, no 6_suppl (20 février 2018) : 174. http://dx.doi.org/10.1200/jco.2018.36.6_suppl.174.
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174 Background: New effective therapies for men with prostate cancer are desperately needed. Recently, cancer immunotherapy has emerged as an important new treatment strategy for prostate cancer and for castrate resistant prostate cancer (CRPC). Multiple studies have identified the heparan sulfate proteoglycan-1 Glypican 1 (GPC-1) as being overexpressed in different cancers, and also as being a possible marker of poor prognosis in several solid tumor cancers. GPC-1 has been recently identified as a potential marker for prostate cancer. The MIL-38 monoclonal antibody detects GPC-1 and an IgG1 chimeric version of this antibody has been developed for preclinical studies. Here we sought to examine MIL-38 binding to a panel of prostate cancer cell lines and examine its feasibility as a novel immunotherapeutic agent targeting GPC-1 in prostate cancer Methods: Expression of GPC-1 in CRPC cell lines was examined by Flow cytometry and Western Blotting using MIL-38 as the detector antibody. The competency of GPC-1 as an immunotherapeutic target was assessed via chimeric MIL-38 induced Antibody Dependent Cell Cytotoxicity (ADCC) using high affinity Natural Killer cells (haNKs) in vitro . Results: Flow cytometry and Western blot assessments of normal prostatic epithelial cells (i.e. RWPE-1) and cells from prostate cancer cell lines (i.e. PC-3, 22RV1, DU-145, VCaP, LNCaP, CWR-R1, and LAPC-4) revealed that only cancer cells expressed GPC-1. Enzalutamide resistant cell lines demonstrated higher expression of GPC-1 than their respective parental line. ADCC assays demonstrated enhanced haNK – prostate cancer cell cytotoxicity in the presence of chimeric MIL-38 anti-GPC-1 antibody, while the IgG1 isotype control had no effect. Conclusions: GPC-1 protein was expressed by most prostate cancer cell lines, including enhanced expression by enzalutamide resistant cells. Preliminary in vitro ADCC assay results revealed the potential utility of GPC-1 as an immunotherapeutic target in prostate cancer.
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Nakano, K., M. Watanabe, H. Matsunari, T. Matsuda, K. Honda, M. Maehara, T. Kanai et al. « 297 PRODUCTION OF CHIMERIC PORCINE FETUSES BY AGGREGATION METHOD USING PARTHENOGENETIC EMBRYOS ». Reproduction, Fertility and Development 25, no 1 (2013) : 296. http://dx.doi.org/10.1071/rdv25n1ab297.
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Porcine induced pluripotent stem (iPS) cells are considered to be an invaluable research tool in translational research with pigs as a large animal model. Pluripotency of the iPS cells needs to be verified by their competence to contribute to chimera formation. The aim of the present study is to establish feasible system to create chimeric pig fetuses using parthenogenetic embryos. In Experiment 1, inner cell mass (ICM) was isolated by immunosurgery from Day 6 blastocysts obtained by parthenogenetic activation of in vitro matured (IVM) oocytes. Isolated ICM were used as the donor cells after staining with fluorescent carbocyanine dye (DiI). Using parthenogenetic morulae or 4- to 8-cell embryos as the host embryos, chimeric embryos were prepared by injection or aggregation method. Injection of ICM was performed by micromanipulation: a single ICM was directly injected into the centre portion of the host morulae. In the aggregation method, a single ICM was aggregated with blastomeres isolated from 2 host embryos at the morula or 4- to 8-cell stage in a micro-well (400 µm diameter, 300 µm deep). The chimeric embryos were cultured in PZM-5 (Yoshioka et al. 2008) for 2 to 3 days to examine development to blastocysts and incorporation of donor ICM cells into the resultant blastocysts ICM (ICM chimerism). In Experiment 2, donor blastomeres isolated from a parthenogenetic morula or 4- to 8-cell embryo were stained by DiI and aggregated with a parthenogenetic host embryo at the morula or 4- to 8-cell stage, and the in vitro development to the blastocyst stage and the ICM chimerism were examined. In Experiment 3, ICM isolated from IVF blastocysts harboring humanized Kusabira-Orange (huKO) gene were used as donor cells. Donor ICM were aggregated with the host embryos at the morula or 4- to 8-cell stage, and the resultant blastocysts were transferred to 4 recipient gilts to collect fetuses on Day 18. Results of Experiments 1 and 2 are summarised in Table 1. Combination of the donor ICM and host morulae yielded high rates of blastocyst formation (~95%) and ICM chimerism (~85%), regardless of the method used (injection or aggregation). Transfer of 73 blastocysts developed from host morulae to 2 recipients (Experiment 3) gave rise to 25 (34.2%) fetuses, of which 6 (24.0%) were confirmed to be chimeric by their clear orange fluorescence and immunostaining by anti-huKO antibody. Of 22 (40.7%) fetuses obtained after transfer of 54 blastocysts derived from 4- to 8-cell host embryos to 2 recipients, 3 (13.6%) were chimeric. Contribution of the donor cells in the tissues of the chimeric fetuses measured by image analysis software (ImageJ, NIH, Bethesda, MD, USA) ranged between 16.1 and 65.2%. These results demonstrate that the aggregation method using parthenogenetic host embryos is an efficient means to produce chimeric pig fetuses, and thereby feasible for verification of pluripotent cells such as iPS cells. Table 1.In vitro development of injected or aggregated porcine embryos
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Wei, Hairong, et Michael Brown. « MHC Class I Dk expression in hematopoietic and non-hematopoietic cells is essential to NK cell licensing and murine CMV resistance (P6308) ». Journal of Immunology 190, no 1_Supplement (1 mai 2013) : 182.6. http://dx.doi.org/10.4049/jimmunol.190.supp.182.6.
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Abstract Ly49G2 inhibitory receptor-bearing NK cells are essential to viral control in murine (M)CMV infected MA/My and MHC class I Dk-expressing C57L mice. In bone marrow chimeric (BMT) mice, highly effective NK cell-mediated viral control requires Dk expression in both hematopoietic and non-hematopoietic cells. Because Dk is a licensing ligand for Ly49G2, we reasoned that Dk expression in discrete cell lineages modulates NK responsiveness and viral control. To address the question, we generated additional BMT mice and measured the effect of Dk expression on NK effector competency and virus control. As expected, G2+ NK cells in Dk-transgenic (Dk-Tg) mice reconstituted with Dk-Tg bone marrow were fully licensed and conferred MCMV resistance. Conversely, in BMT recipients with Dk expression restricted to only hematopoietic or non-hematopoietic cells, G2+ NK cells displayed reduced activation receptor stimulation and significantly less viral control. These data demonstrate that NK cell licensing is regulated by MHC class I expression in hematopoietic and non-hematopoietic cells and this corresponds to the extent of G2+ NK cell-mediated viral control. Whereas recent data has shown that licensing of adoptively transferred mature NK cells is sensitive to MHC I expression on non-hematopoietic recipient cells, ongoing adoptive transfer experiments with Dk-Tg and non-Tg mature NK cells further examines the relationship between NK cell licensing and their capacity for viral control.
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Matsunari, H., K. Nakano, T. Kanai, T. Matsuda, M. Maehara, M. Watanabe, K. Umeyama, M. Nagaya, H. Nakauchi et H. Nagashima. « 26 IN VIVO EXOGENIC ORGAN GENERATION WITH ORGANOGENESIS-DISABLED CLONED PIGS AS A PLATFORM ». Reproduction, Fertility and Development 26, no 1 (2014) : 127. http://dx.doi.org/10.1071/rdv26n1ab26.
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The generation of organs from pluripotent stem cells (PSC) is one of the ultimate goals of regenerative medicine. We have demonstrated that functional organs can be generated in vivo from xenogenic PSC in the body of organogenesis-disabled mice using blastocyst complementation. To apply this principle in generating human organs, a technical platform using large non-rodent mammals is essential. The aim of the present study was to establish a blastocyst complementation system using cloned pig embryos. We generated transgenic-cloned pigs with an apancreatic phenotype via the overexpression of Hes1 (hairy and enhancer of split-1) under the Pdx1 promoter (pancreatic and duodenal homeobox-1). Cloned embryos of apancreatic pigs (host embryos, male) were complemented (i.e. chimerized) by blastomeres of cloned embryos (donor cells, female) with normal developmental competence. Chimeric embryos were cultured for 1 or 2 days before being transferred into the uteri of oestrus-synchronized gilts. The complementation of 292 Pdx1-Hes1 cloned embryos gave rise to 260 (89.0%) blastocysts. The transfer of these blastocysts resulted in 5 male chimeric pigs. Chimerism was confirmed by the detection of host embryo-derived Pdx1-Hes1 and marker transgenes of the donor cells, such as humanized Kusabira-Orange (huKO) or Pdx1-Venus. Chimeric pigs possessed normally formed pancreata entirely derived from the exogenous donor cells. We thus established a blastocyst complementation system in the pig using cloned embryos that would otherwise give rise to apancreatic animals. Chimeric pigs obtained developed normally, maintaining normal serum glucose concentrations up to maturity, and became fertile boars. Mating the chimeric boars with 7 wild-type sows gave rise to 72 fetuses/piglets of which 37 (51.4%) exhibited the apancreatic phenotype. These results indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize with a cloned dysorganogenetic embryo. Blastocyst complementation using cloned porcine embryos may permit the use of a large animal for the generation of functional organs from xenogenic PSC, including human iPSC. The chimeric boar produced by blastocyst complementation sired fetuses/offspring with the apancreatic phenotype in a Mendelian fashion. Porcine fetuses with an organogenesis-disabled phenotype may provide a useful platform for organ regeneration research. Table 1.Production of chimeric pigs by complementation and of Pdx1-Hes1 cloned embryos
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Haqshenas, G., X. Dong, H. Netter, J. Torresi et E. J. Gowans. « A chimeric GB virus B encoding the hepatitis C virus hypervariable region 1 is infectious in vivo ». Journal of General Virology 88, no 3 (1 mars 2007) : 895–902. http://dx.doi.org/10.1099/vir.0.82467-0.
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Two GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.
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Kim, Kee-Pyo, You Wu, Juyong Yoon, Kenjiro Adachi, Guangming Wu, Sergiy Velychko, Caitlin M. MacCarthy et al. « Reprogramming competence of OCT factors is determined by transactivation domains ». Science Advances 6, no 36 (septembre 2020) : eaaz7364. http://dx.doi.org/10.1126/sciadv.aaz7364.
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OCT4 (also known as POU5F1) plays an essential role in reprogramming. It is the only member of the POU (Pit-Oct-Unc) family of transcription factors that can induce pluripotency despite sharing high structural similarities to all other members. Here, we discover that OCT6 (also known as POU3F1) can elicit reprogramming specifically in human cells. OCT6-based reprogramming does not alter the mesenchymal-epithelial transition but is attenuated through the delayed activation of the pluripotency network in comparison with OCT4-based reprogramming. Creating a series of reciprocal domain-swapped chimeras and mutants across all OCT factors, we clearly delineate essential elements of OCT4/OCT6-dependent reprogramming and, conversely, identify the features that prevent induction of pluripotency by other OCT factors. With this strategy, we further discover various chimeric proteins that are superior to OCT4 in reprogramming. Our findings clarify how reprogramming competences of OCT factors are conferred through their structural components.
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Geiger, B., D. Salomon, M. Takeichi et R. O. Hynes. « A chimeric N-cadherin/beta 1-integrin receptor which localizes to both cell-cell and cell-matrix adhesions ». Journal of Cell Science 103, no 4 (1 décembre 1992) : 943–51. http://dx.doi.org/10.1242/jcs.103.4.943.
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To study the molecular mechanisms involved in formation of cell contacts, we have transfected cultured cells with a chimeric cDNA encoding the cytoplasmic and transmembrane domains of beta 1 integrin and the extracellular region of N-cadherin and determined the subcellular distribution of the chimeric molecule. We show that the chimeric receptor associates preferentially with cell-matrix focal contacts, suggesting that its distribution is directed by its beta 1 integrin segment, presumably via interactions of the cytoplasmic domain with cytoskeletal elements characteristic of focal contacts. Transfected cells which expressed relatively high levels of the cadherin/integrin chimera underwent an apparent epithelialization and contained the molecule both in cell-matrix and cell-cell contacts. Location in cell-cell contacts indicates competence of the cadherin extracellular domain to participate in formation of cell-cell junctions using a foreign cytoplasmic domain. Labeling of these cultures for talin, which is normally associated only with matrix adhesions, revealed specific labeling along the newly formed intercellular junctions. This suggests that the local association of talin with these sites is induced by the cytoplasmic tail of beta 1 integrin receptor presented by the chimeric protein. These results suggest that the formation of adherens-type junctions is driven by the cooperative interactions of the relevant adhesion molecules (cadherins and integrins) both with the respective extracellular ligands and with the cytoskeleton.
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Sánchez-Moguel, Ignacio, Carmina Montiel et Ismael Bustos-Jaimes. « Therapeutic Potential of Engineered Virus-like Particles of Parvovirus B19 ». Pathogens 12, no 8 (2 août 2023) : 1007. http://dx.doi.org/10.3390/pathogens12081007.
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Virus-like particles (VLPs) comprise one or many structural components of virions, except their genetic material. Thus, VLPs keep their structural properties of cellular recognition while being non-infectious. VLPs of Parvovirus B19 (B19V) can be produced by the heterologous expression of their structural proteins VP1 and VP2 in bacteria. These proteins are purified under denaturing conditions, refolded, and assembled into VLPs. Moreover, chimeric forms of VP2 have been constructed to harbor peptides or functional proteins on the surface of the particles without dropping their competence to form VLPs, serving as presenting nanoparticles. The in-vitro assembly approach offers exciting possibilities for the composition of VLPs, as more than one chimeric form of VP2 can be included in the assembly stage, producing multifunctional VLPs. Here, the heterologous expression and in-vitro assembly of B19V structural proteins and their chimeras are reviewed. Considerations for the engineering of the structural proteins of B19V are also discussed. Finally, the construction of multifunctional VLPs and their future potential as innovative medical tools are examined.
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Leventhal, Joseph, Larry D. Bozulic, Mark D. Badder, Mary Jane Elliott, Michael N. Issa, James Mathew, Iwona Konieczna et Suzanne T. Ildstad. « Evaluation Of Immunocompentence In Tolerant Chimeric Recipients Of Hematopoietic Stem Cell/Renal Transplants ». Blood 122, no 21 (15 novembre 2013) : 4483. http://dx.doi.org/10.1182/blood.v122.21.4483.4483.
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A phase 2 protocol was developed to attempt to induce donor-specific tolerance to renal allografts in related and unrelated donor/recipient combinations (IND 13947 phase 2). Subjects were conditioned with fludarabine (days -5, -4, -3), cyclophosphamide (50 mg/ky days -3 and +3), and 200 cGy total body irradiation. The kidney transplant was performed day 0. G-CSF mobilized peripheral blood stem cells processed to remove GVHD-producing cells and retain graft facilitating cells (FC) was administered on day +1. The conditioning was well tolerated and the subjects were managed as outpatients after post-operative day 2. This has resulted in high levels of durable chimerism and immunosuppression-free graft survival without GVHD or engraftment syndrome in mismatched related and unrelated recipients of living donor FC/hematopoietic stem cell/kidney allografts. Nine subjects are completely off immunosuppression from 2 months to 3 years. A number of others are in the process of tapering. In the present study, we have prospectively analyzed recovery of immune function, persistence of vaccination memory, and response to vaccination in subjects who exhibit high levels of chimerism. Chimerism testing was performed using molecular short tandem report (sensitivity ±5%). Three of four subjects who had been vaccinated to hepatitis B prior to transplantation and whose donors had not been vaccinated retained their immunity following transplantation. All subjects tested exhibited memory for varicella and the majority did as well for measles (9/11), mumps (8/11), and rubella (5/10). A blood group disparity was present in 9 chimeric donor/recipient pairs. One chimeric subject converted to donor blood type, 3 exhibited mixed donor/host RBC chimerism, and 5 retained their own blood type. Six chimeric subjects have been immunized with pneumococcal vaccine after transplantation. All generated an immune response to vaccination, confirming immunocompetence to generate an antibody response to antigen. Notably, recovery of CD8+ and CD4+ central memory, naïve, and effector memory T cells occurred within one year post-transplantation to levels that were not significantly different from pre-transplantation. In addition, CD31+/CD45RA+ CD8+ and CD4+ T cells representative of recent thymic emigrants were present by 3 months, demonstrating de novo thymic production of T cells after transplantation. Four patients were randomly selected for study of Tcell repertoire (TCR) generation after transplantation. Two had achieved durable full donor chimerism and the other two did not have durable chimerism. Peripheral blood samples freshly obtained from donors and recipients and T cell subsets were isolated using MACs microbead system. DNA was extracted and sequenced by ImmunoSeq (Adaptive Biotech, Seattle, WA) to evaluate TCR clonal repertoires in recipients. Although clonal diversity in TCR repertoire was reduced in post-Tx recipients (0.9 ± 0.05 pre-Tx vs. 0.79 ± 0.09 post-Tx), the repertoire was diverse enough to suggest recovery of immune competence. Interestingly, at least 97% of the unique sequences observed in post-Tx recipient were not present in either donor or recipient pre-Tx. Within the pool of shared sequences, full chimerism correlated with a shift towards homology with the donor, while loss of chimerism correlated with recipient pre-Tx. In addition, the chimeric patients also exhibited reduced diversity of TCR sequences and increased clonality. Top 20 “high frequency” clones are most stably expressed. CD8+ cells had the highest number of “high frequency” clones. Notably, the pattern of “low frequency” clones was highest in the CD127-CD4+CD25+ regulatory T cell subset, indicating an extensive and rapidly changing TCR repertoire. Taken together, these data suggest that immunologic recovery is robust in these nonmyeloablatively conditioned tolerant chimeric subjects. Disclosures: Bozulic: Regenerex, LLC: Employment. Badder:Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.
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Matatall, Katie, Ching-Chieh Shen, Yayun Zheng et Katherine Y. King. « Chronic Mycobacterium Avium Infection Leads to Cell Autonomous Exhaustion of Hematopoietic Stem Cells ». Blood 124, no 21 (6 décembre 2014) : 2947. http://dx.doi.org/10.1182/blood.v124.21.2947.2947.
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Abstract Background: Chronic infections and inflammatory conditions can cause bone marrow suppression and pancytopenia, a dangerous condition that impairs recovery from infection. In some chronic infections such as tuberculosis, marrow suppression has been ascribed to myelofibrosis. However, inflammatory conditions also affect hematopoietic stem cells (HSCs) themselves. HSCs are activated to proliferate and differentiate in the setting of a Mycobacterium avium infection. These responses are mediated by interferon gamma (IFNg) signaling. We hypothesized that sustained interferon signaling during a chronic infection would impair self-renewal in HSCs and lead to premature exhaustion of the stem cell compartment. Objective: To ascertain whether persistent interferon exposure in an animal model of chronic Mycobacterium avium infection leads to cell autonomous reduction in HSC number and competency. Methods: We conducted sustained infection of C57Bl/6 wild type mice with M. avium over an 8-month period. At monthly intervals, we assessed HSC number and pluripotency in a mouse model of bone marrow transplantation. In order to determine whether suppression of HSC counts was due to a cell autonomous or environmental effect, we performed Mycobacterium avium infection in chimeric mice containing mixed WT and IFNg-receptor-deficient (Ifngr1-/-) marrow. Results: After 6 months of repeated infection with M. avium, mice became anemic and leukopenic. There was a gradual decrease in the number of committed hematopoietic progenitors and a depletion of HSCs in the bone marrow by 6 months following initial infection. The bone marrow of infected animals was hypercellular and not fibrotic. Despite the overall reduction in HSC number, HSCs that remained in chronically infected animals retained full self-renewal potential even across multiple rounds of transplantation. Whereas WT marrow was suppressed in M. avium-infected chimeras, Ifngr1-/- marrow demonstrated enhanced engraftment, indicating an IFNg-dependent cell autonomous reduction in self-renewal potential. Transcriptional profiling of HSCs from M. avium-infected and control animals by RNAseq indicated that genes involved in the interferon gamma-mediated inflammatory response and genes associated with aging were differentially upregulated. Conclusions: We find that chronic interferon gamma exposure can deplete the HSC pool without inducing myelofibrosis. Further, we demonstrate that IFNg-mediated impairment in HSC self-renewal is cell-autonomous. Despite a reduction in HSC number, a subset of HSCs is protected from exhaustion and maintains self-renewal potential. Serial M. avium infection may be utilized as a mouse model for bone marrow suppression associated with inflammatory conditions, as is seen in some patients with chronic infections or autoimmune disorders. In future studies, candidate genes identified by transcriptome analysis will be assessed for their contribution to HSC exhaustion in the setting of chronic inflammation. Disclosures No relevant conflicts of interest to declare.
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Willis, Lauren, Sara R. Fagerlie et Sattva S. Neelapu. « Evaluating Hematologist's Knowledge of CAR T-Cell Therapy in Hematologic Malignancies ». Blood 132, Supplement 1 (29 novembre 2018) : 2269. http://dx.doi.org/10.1182/blood-2018-99-115036.
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Abstract Background: The objective of this study was to assess current clinical practices of hematologist/oncologist (hem/onc) specialists related to chimeric antigen receptor (CAR) T-cell therapy in hematologic malignancies, in order to identify knowledge, competency, and practice gaps and barriers to optimal care. Methods: A continuing medical education (CME)-certified clinical practice assessment consisting of 25 multiple choice questions was developed to measure knowledge, skills, attitudes, and competence of hem/onc specialists regarding CAR T-cell therapy. The survey instrument was made available online to physicians without monetary compensation or charge. Respondent confidentiality was maintained, and responses were de-identified and aggregated prior to analyses. The activity launched on December 22, 2017 with global distribution, and participant responses are still being collected at the time of abstract submission. Results: At the time of this report there are 192 hem/onc activity participants, collection is on-going. Demographics are listed in Table 1 and levels of confidence and barriers to incorporating CAR T-cell therapy are listed in Table 2.Foundational KnowledgeSub-optimal knowledge was demonstrated in the area of CAR components, dosing, and FDA-approved indications.Over half (61%) could not correctly identify the components of a CAR construct (antigen-specific domain and the signaling domain).Almost half (45%) of the participants did not recognize that currently approved CAR T-cell therapies are dosed as a single infusion.25% demonstrated inaccurate knowledge by recommending patients wait 4 weeks after CAR T-cell infusion before driving.Over half (62%) of participants could not identify the FDA-approved indication for axicabtagene ciloleucel.Knowledge of Clinical Trial DataVery low awareness of efficacy data seen with various CAR T-cell products used to treat R/R B-cell ALL (ELIANA trial), R/R DLBCL (ZUMA-1, JULIET, TRANSCEND trials).Only 32% identified the correct CR/CRi rate seen with tisagenlecleucel in the ELIANA trial.Only 25% correctly identified the CR rate seen with axicabtagene ciloleucel in the ZUMA-1 trial.Only 32% demonstrated knowledge of the 6-month DFS rate for patients in the JULIET trial that had a CR at 3 months.Only 25% identified the association between the dose of JCAR017 and response rates from the TRANSCEND trial.Knowledge and Competence Managing Adverse EventsLack of competence recognizing and treating CAR T-cell associated adverse events such as cytokine release syndrome (CRS) and neurotoxicity.Almost half (44%) could not identify signs of CRS associated with CAR T-cell therapy and 43% lack knowledge that elevated serum C-reactive protein (CRP) is associated with the highest level of CRS (in patients with lymphoma receiving axicabtagene ciloleucel).41% could not identify that the mechanism of tocilizumab is to block IL-6 signaling.Over a third (35%) were unable to identify signs/symptoms/causes of neurotoxicity associated with CAR T-cell therapy.More than half of the learners (54%) could not identify the appropriate role of corticosteroid therapy after CAR T-cell administration in managing CRS and neurotoxicity. Conclusions: This activity found knowledge and competence deficits for hem/onc practitioners related to using CAR T-cell therapy for the treatment of patients with hematologic malignancies. Additionally, the activity demonstrated large gaps in confidence discussing CAR T-cell therapy with patients/families and managing adverse events. There is sub-optimal awareness of CAR T-cell foundational knowledge, clinical trial data, and recognition of common therapy related adverse events and management strategies. Additional education is needed to improve the knowledge, competence, and confidence of academic and community hem/onc specialists who care for patients with hematologic malignancies receiving CAR T-cell therapy as well as strategies for integrating novel agents into clinical practice. Disclosures Neelapu: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Karus: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Gatenbee, Chandler D., Mark Robertson-Tessi, Maximilian Strobl, Ryan O. Schenck, Bachisio Ziccheddu, Francesco Maura, Frederick Locke et Alexander R. A. Anderson. « Abstract A011 : Modeling the coevolution of native and CAR T-cells in large B cell lymphoma reveals a potential biomarker for response to therapy ». Cancer Research 84, no 3_Supplement_2 (1 février 2024) : A011. http://dx.doi.org/10.1158/1538-7445.canevol23-a011.
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Abstract Application of CD19 targeted Chimeric Antigen Receptor (CAR) T-cell therapy to large B cell lymphoma (LBCL) has yielded unprecedented clinical outcomes in a cancer largely resistant to conventional chemotherapy. This is highlighted in the recent ZUMA-7 trial, where axicabtagene ciloleucel (axi-cel), a CAR T-cell therapy, yielded a complete response (CR) rate in 65% of patients, an improvement over the 32% CR rate when using standard chemotherapy. The ZUMA-7 trial highlights the potential of CAR T-cell therapy to treat traditionally resistant cancers, but work remains to be done to identify biomarkers predictive of response. Operating under the assumption that CAR T not only directly targets CD19+ tumor cells, but also enhances tumor kill by amplifying the native T-cell populations, we hypothesized that neoantigen burden (NAB) may serve as a useful biomarker to make predictions of response to CAR T. We explore this hypothesis by building upon traditional predator-prey mathematical models to simulate the evolution of LBCL under immune predation and treatment with CAR T-cell therapy. More specifically, in our model native T-cells prey upon tumor cells that carry their cognate neoantigen, and each T-cell has variable search rate proportional to the cognate neoantigen’s recognition potential, modeled using a type III functional response. Tumor cells may mutate during division, always gaining a neoantigen, but possibly also losing HLA function, paving the way for immune escape. In addition to modeling the direct killing of tumor by CAR T, we also explore the synergistic effect of CAR T on native T-cells by modeling increased amplification of, and killing by, native T-cells based on interactions with the CAR T-cell population. Preliminary simulations suggest a high NAB can indeed be predictive of CAR T response, although only if the tumor has not evolved an escape mechanism. Indeed, simulations suggest that high NAB can also be a signature of an immune escaped tumor, as neoantigens become neutral traits once the immune system is removed as a selective force. These results suggest that knowledge of a patients’ NAB and immune competency can be used to guide CAR T treatment decisions. Citation Format: Chandler D. Gatenbee, Mark Robertson-Tessi, Maximilian Strobl, Ryan O. Schenck, Bachisio Ziccheddu, Francesco Maura, Frederick Locke, Alexander R.A. Anderson. Modeling the coevolution of native and CAR T-cells in large B cell lymphoma reveals a potential biomarker for response to therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A011.
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Sawaisorn, Piamsiri, Korakot Atjanasuppat, Usanarat Anurathapan, Somchai Chutipongtanate et Suradej Hongeng. « Strategies to Improve Chimeric Antigen Receptor Therapies for Neuroblastoma ». Vaccines 8, no 4 (11 décembre 2020) : 753. http://dx.doi.org/10.3390/vaccines8040753.
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Chimeric antigen receptors (CARs) are among the curative immunotherapeutic approaches that exploit the antigen specificity and cytotoxicity function of potent immune cells against cancers. Neuroblastomas, the most common extracranial pediatric solid tumors with diverse characteristics, could be a promising candidate for using CAR therapies. Several methods harness CAR-modified cells in neuroblastoma to increase therapeutic efficiency, although the assessment has been less successful. Regarding the improvement of CARs, various trials have been launched to overcome insufficient capacity. However, the reasons behind the inadequate response against neuroblastoma of CAR-modified cells are still not well understood. It is essential to update the present state of comprehension of CARs to improve the efficiency of CAR therapies. This review summarizes the crucial features of CARs and their design for neuroblastoma, discusses challenges that impact the outcomes of the immunotherapeutic competence, and focuses on devising strategies currently being investigated to improve the efficacy of CARs for neuroblastoma immunotherapy.
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Bosch, Berend Jan, Cornelis A. M. de Haan et Peter J. M. Rottier. « Coronavirus Spike Glycoprotein, Extended at the Carboxy Terminus with Green Fluorescent Protein, Is Assembly Competent ». Journal of Virology 78, no 14 (15 juillet 2004) : 7369–78. http://dx.doi.org/10.1128/jvi.78.14.7369-7378.2004.
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ABSTRACT Due to the limited ultrastructural information about the coronavirion, little is known about the interactions acting at the interface between nucleocapsid and viral envelope. Knowing that subtle mutations in the carboxy-terminal endodomain of the M protein are already lethal, we have now probed the equivalent domain of the spike (S) protein by extending it terminally with a foreign sequence of 27 kDa: the green fluorescent protein (GFP). When expressed individually in murine cells, the S-GFP chimeric protein induced the formation of fluorescent syncytia, indicating that it was synthesized and folded properly, trimerized, and transported to the plasma membrane, where it exhibited the two key S protein functions, i.e., interaction with virus receptor molecules and membrane fusion. Incorporation into virus-like particles demonstrated the assembly competence of the chimeric spike protein. The wild-type S gene of mouse hepatitis coronavirus (MHV) was subsequently replaced by the chimeric construct through targeted recombination. A viable MHV-SGFP was obtained, infection by which could be visualized by the fluorescence induced. The efficiency of incorporation of the chimeric protein into particles was, however, reduced relative to that in wild-type particles which may explain, at least in part, the reduced infectivity produced by MHV-SGFP infection. We conclude that the incorporation of spikes carrying the large GFP moiety is apparently impaired by geometrical constraints and selected against during the assembly of virions. Probably due to this disadvantage, deletion mutants, having lost the foreign sequences, rapidly evolved and outcompeted the chimeric viruses during virus propagation. The fluorescent MHV-SGFP will now be a convenient tool to study coronaviral cell entry.
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Immidisetti, Amanda, Sean Munier et Nitesh Patel. « COVD-18. POTENTIAL TO HARNESS SARS-COV-2 NEUROTROPISM IN THE DELIVERY OF ONCOLYTIC VIROTHERAPY FOR THE TREATMENT OF HIGH-GRADE GLIOMA ». Neuro-Oncology 22, Supplement_2 (novembre 2020) : ii24—ii25. http://dx.doi.org/10.1093/neuonc/noaa215.101.
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Abstract BACKGROUND High-grade gliomas (HGG) pose therapeutic challenges stemming from blood brain barrier, infiltrative growth, suppressed immune function, and tumor heterogeneity. Oncolytic viruses (OVs) are gaining traction for addressing these challenges. There is evidence that the SARS-CoV-2 glycoprotein spike binds the ACE-2 receptor in nasal epithelium and reaches the brainstem and thalamus via axonal transport through the olfactory pathway, making it an attractive candidate for OV therapy. Prior studies on chimerization of pathogenic virus-derived glycoprotein spikes with non-pathogenic strains exploit neurotropism of a wild-type virus while improving the safety profile of the resulting OV. We review, 1) the engineering of chimeric OVs used in the treatment of HGG; 2) potential for a novel chimeric virotherapy in which the SARS-CoV-2 glycoprotein spike can be used to deliver OV therapy intranasally; and 3) areas which warrant further investigation to develop this approach for clinical use. METHODS We performed an extensive review of chimeric OVs and specific modifications engineered to optimize safety and efficacy. Additionally, we assessed potential to use these principals to engineer the SARS-CoV-2 glycoprotein spike onto a non-pathogenic, replication competent virus to yield a novel chimeric for noninvasive, intranasal delivery. RESULTS Viruses with pathogenic properties in wild-type have been successfully used as components of OVs and have demonstrated potential in both preclinical and clinical trials. Outcomes show that despite wild-type virulence, notable toxicities were not observed in clinical trials, highlighting the potential of viral pseudotyping as a safe therapeutic approach. CONCLUSIONS The proposed method to utilize the SARS-CoV-2 glycoprotein in a novel chimeric poses advantages including 1) potential for non-invasive delivery, 2) therapy without need for maximal or uniform tumor coverage due to replication competence, 3) ability to reach infiltrative glioma cells, 4) potential to reach the brainstem, and 5) stimulation of host immunity through tumor cell lysis and antigen presentation
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Jeong, Pil-Soo, Seung-Bin Yoon, Mun-Hyeong Lee, Hee-Chang Son, Hwal-Yong Lee, Sanghoon Lee, Bon-Sang Koo et al. « Embryo aggregation regulates in vitro stress conditions to promote developmental competence in pigs ». PeerJ 7 (13 décembre 2019) : e8143. http://dx.doi.org/10.7717/peerj.8143.
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Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in in vitro developmental competence and the relationship of stress conditions in porcine early embryogenesis. We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.
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MCCARTHY, SUSAN A., IRWIN J. GRIFFITH, PHILLIP GAMBEL, LOUIS H. FRANCESCUTTI, ARUN FOTEDAR, ERWIN DIENER et THOMAS G. WEGMANN. « IMMUNOLOGICAL COMPETENCE AND HOST-SPECIFIC TOLERANCE OF ANTIBODY-FACILITATED BONE MARROW CHIMERAS ». Transplantation 44, no 1 (juillet 1987) : 97–105. http://dx.doi.org/10.1097/00007890-198707000-00021.
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Yang, Yifang, Jingjun Lin, Anthony Harrington, Gabriel Cornilescu, Gee W. Lau et Yftah Tal-Gan. « Designing cyclic competence-stimulating peptide (CSP) analogs with pan-group quorum-sensing inhibition activity in Streptococcus pneumoniae ». Proceedings of the National Academy of Sciences 117, no 3 (8 janvier 2020) : 1689–99. http://dx.doi.org/10.1073/pnas.1915812117.
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Streptococcus pneumoniae is an opportunistic human pathogen that utilizes the competence regulon, a quorum-sensing circuitry, to acquire antibiotic resistance genes and initiate its attack on the human host. Interception of the competence regulon can therefore be utilized to study S. pneumoniae cell−cell communication and behavioral changes, as well as attenuate S. pneumoniae infectivity. Herein we report the design and synthesis of cyclic dominant negative competence-stimulating peptide (dnCSP) analogs capable of intercepting the competence regulon in both S. pneumoniae specificity groups with activities at the low nanomolar range. Structural analysis of lead analogs provided important insights as to the molecular mechanism that drives CSP receptor binding and revealed that the pan-group cyclic CSPs exhibit a chimeric hydrophobic patch conformation that resembles the hydrophobic patches required for both ComD1 and ComD2 binding. Moreover, the lead cyclic dnCSP, CSP1-E1A-cyc(Dap6E10), was found to possess superior pharmacological properties, including improved resistance to enzymatic degradation, while remaining nontoxic. Lastly, CSP1-E1A-cyc(Dap6E10) was capable of attenuating mouse mortality during acute pneumonia caused by both group 1 and group 2 S. pneumoniae strains. This cyclic pan-group dnCSP is therefore a promising drug lead scaffold against S. pneumoniae infections that could be administered individually or utilized in combination therapy to augment the effects of current antimicrobial agents.
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KITO, Seiji, Yoshiko NOGUCHI, Yuki OHTA, Tatsuya OHHATA, Masumi ABE, Naoko SHIOMI et Tadahiro SHIOMI. « Evaluation of Developmental Competence of Vitrified-warmed Early Cleavage Stage Embryos and their Application for Chimeric Mouse Production. » Experimental Animals 52, no 2 (2003) : 179–83. http://dx.doi.org/10.1538/expanim.52.179.
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Lee, Joohyeong, Lian Cai, Mirae Kim, Hyerin Choi, Dongjin Oh, Ali Jawad, Eunsong Lee et Sang-Hwan Hyun. « Developmental competence of chimeric porcine embryos through the aggregation of parthenogenetic embryos and somatic cell nuclear transfer embryos ». Korean Journal of Veterinary Research 63, no 1 (31 mars 2023) : e3. http://dx.doi.org/10.14405/kjvr.20230003.
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The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.
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Świerczek-Lasek, Barbara, Jacek Neska, Agata Kominek, Łukasz Tolak, Tomasz Czajkowski, Katarzyna Jańczyk-Ilach, Władysława Stremińska, Katarzyna Piwocka, Maria A. Ciemerych et Karolina Archacka. « Interleukin 4 Moderately Affects Competence of Pluripotent Stem Cells for Myogenic Conversion ». International Journal of Molecular Sciences 20, no 16 (13 août 2019) : 3932. http://dx.doi.org/10.3390/ijms20163932.
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Pluripotent stem cells convert into skeletal muscle tissue during teratoma formation or chimeric animal development. Thus, they are characterized by naive myogenic potential. Numerous attempts have been made to develop protocols enabling efficient and safe conversion of pluripotent stem cells into functional myogenic cells in vitro. Despite significant progress in the field, generation of myogenic cells from pluripotent stem cells is still challenging—i.e., currently available methods require genetic modifications, animal-derived reagents, or are long lasting—and, therefore, should be further improved. In the current study, we investigated the influence of interleukin 4, a factor regulating inter alia migration and fusion of myogenic cells and necessary for proper skeletal muscle development and maintenance, on pluripotent stem cells. We assessed the impact of interleukin 4 on proliferation, selected gene expression, and ability to fuse in case of both undifferentiated and differentiating mouse embryonic stem cells. Our results revealed that interleukin 4 slightly improves fusion of pluripotent stem cells with myoblasts leading to the formation of hybrid myotubes. Moreover, it increases the level of early myogenic genes such as Mesogenin1, Pax3, and Pax7 in differentiating embryonic stem cells. Thus, interleukin 4 moderately enhances competence of mouse pluripotent stem cells for myogenic conversion.
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Loskutoff, N. M., et D. C. Kraemer. « Factors influencing the developmental competence of intraspecific murine chimeras produced by multiple embryo aggregation ». Theriogenology 33, no 1 (janvier 1990) : 276. http://dx.doi.org/10.1016/0093-691x(90)90700-4.
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Lee, Myeong S., Brian A. Dougherty, Anne C. Madeo et Donald A. Morrison. « Construction and Analysis of a Library for Random Insertional Mutagenesis in Streptococcus pneumoniae : Use for Recovery of Mutants Defective in Genetic Transformation and for Identification of Essential Genes ». Applied and Environmental Microbiology 65, no 5 (1 mai 1999) : 1883–90. http://dx.doi.org/10.1128/aem.65.5.1883-1890.1999.
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ABSTRACT To explore the use of insertion-duplication mutagenesis (IDM) as a random gene disruption mutagenesis tool for genomic analysis ofStreptococcus pneumoniae, a large mutagenic library of chimeric plasmids with 300-bp inserts was constructed. The library was large enough to produce 60,000 independent plasmid clones inEscherichia coli. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scattered widely over the genome; 80% of these plasmids had 240- to 360-bp inserts, and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evaluate the randomness of mutagenesis during recombination and to test the usefulness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a large pneumococcal transformant library derived from 20,000 mutagenic plasmids. After we screened the mutants exhaustively for transformation defects, 114 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as well as a new gene with high similarity to theBacillus subtilis competence gene comFA. Mapping of the mutation sites at these competence loci showed that the mutagenesis was highly random, with no apparent hot spots. The recovery of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is useful for finding various types of nonessential genes throughout the genome. Since a promoterless lacZ reporter vector was used for the construction of the mutagenic plasmid library, it also serves as a random transcriptional fusion library. Finally, use of a valuable feature of IDM, directed gene targeting, also showed that essential genes, which can be targets for new drug designs, could be identified by simple sequencing and transformation reactions. We estimate that the IDM library used in this study could readily achieve about 90% genome coverage.
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Gustems, Montse, Andreas Busche, Martin Messerle, Peter Ghazal et Ana Angulo. « In Vivo Competence of Murine Cytomegalovirus under the Control of the Human Cytomegalovirus Major Immediate-Early Enhancer in the Establishment of Latency and Reactivation ». Journal of Virology 82, no 20 (6 août 2008) : 10302–7. http://dx.doi.org/10.1128/jvi.01255-08.
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ABSTRACT The human cytomegalovirus (HCMV) major immediate-early enhancer has been postulated to play a pivotal role in the control of latency and reactivation. However, the absence of an animal model has obstructed a direct test of this hypothesis. Here we report on the establishment of an in vivo, experimentally tractable system for quantitatively investigating physiological functions of the HCMV enhancer. Using a neonate BALB/c mouse model, we show that a chimeric murine CMV under the control of the HCMV enhancer is competent in vivo, replicating in key organs of mice with acute CMV infection and exhibiting latency/reactivation features comparable for the most part to those of the parental and revertant viruses.
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Carstea, Ana Claudia. « Germline competence of mouse ES and iPS cell lines : Chimera technologies and genetic background ». World Journal of Stem Cells 1, no 1 (2009) : 22. http://dx.doi.org/10.4252/wjsc.v1.i1.22.
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Peters, Okimi, et W. Allan King. « The detection of female cell activity in male sex chromosome chimeric Rideau Arcott sheep, using the Xist gene product as a marker ». SURG Journal 1, no 2 (21 février 2008) : 20–25. http://dx.doi.org/10.21083/surg.v1i2.414.
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The detection of the SRY (Sex-determining region on the Y chromosome) gene is a popular method used for the identification of freemartins (XX/XY female chimeras). This method relies on the fact that the SRY gene is a Y chromosome specific gene and is thus normally only present in males therefore detecting its presence in a female indicates the presence of male cells (XY cells) within the female. This concept can be extrapolated to the male counterparts of freemartins with regards to the Xist gene. This gene is normally only widely expressed in females and can be used as a marker for identifying females. Therefore, detecting Xist gene expression in males (in tissues other than the testes, as the Xist gene is expressed exclusively in the testes of males) may indicate that these males contain transcriptionally competent female cells and thus necessarily labels them as sex-chromosome chimeras. In the present study four previously identified male sex chromosome chimeras were screened for the expression of the Xist gene using reverse transcription Polymerase Chain Reaction (PCR), and it was detected in three of the four chimeras. Xist expression was not detected in one of the chimeras because the proportion of female cells in its blood is significantly low and thus it is likely that the blood sample used in the study did not possess female cells. None-the-less it was concluded that the detection of Xist expression in male sex chromosome chimeras can be used as an indication of the presence and transcriptional competence of female cells within them.
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Klco, Jeffery M., Saurabh Sen, Jakob L. Hansen, Christina Lyngsø, Gregory V. Nikiforovich, Soren P. Sheikh et Thomas J. Baranski. « Complement factor 5a receptor chimeras reveal the importance of lipid-facing residues in transport competence ». FEBS Journal 276, no 10 (mai 2009) : 2786–800. http://dx.doi.org/10.1111/j.1742-4658.2009.07002.x.
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Rüedi, E., M. Sykes, S. T. Ildstad, C. H. Chester, A. Althage, H. Hengartner, D. H. Sachs et R. M. Zinkernagel. « Antiviral T cell competence and restriction specificity of mixed allogeneic (P1 + P2 → P1) irradiation chimeras ». Cellular Immunology 121, no 1 (juin 1989) : 185–95. http://dx.doi.org/10.1016/0008-8749(89)90016-6.
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Peranteau, William H., Masayuki Endo, Obinna O. Adibe et Alan W. Flake. « Evidence for an immune barrier after in utero hematopoietic-cell transplantation ». Blood 109, no 3 (5 octobre 2006) : 1331–33. http://dx.doi.org/10.1182/blood-2006-04-018606.
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Abstract The competence of the immune system of the developing fetus to act as a barrier to in utero hematopoietic-cell transplantation (IUHCT) has been a source of debate. Until now, comparisons of allogeneic and congenic engraftment have been inconclusive due to methodologic limitations resulting in minimal and inefficient engraftment. In this study, E14 fetal mice received transplants of either allogeneic or congenic bone marrow using a new intravascular technique that allows definitive administration of much higher doses of donor cells. Our results demonstrate that 100% of surviving recipients demonstrate engraftment at 1 week of age, but that 70% of allogeneic recipients lose engraftment by 1 month of age, and 80% ultimately fail to sustain long-term chimerism. In contrast, all congenic recipients maintain stable, long-term, multilineage chimerism. These results strongly support an immune barrier to allogeneic engraftment after IUHCT.
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Peranteau, William H., Masayuki Endo, Obinna O. Adibe et Alan W. Flake. « Evidence for an Adaptive Immune Barrier after in Utero Hematopoietic Cell Transplantation. » Blood 108, no 11 (16 novembre 2006) : 3179. http://dx.doi.org/10.1182/blood.v108.11.3179.3179.
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Abstract In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative approach that takes advantage of normal immunologic development to achieve donor specific tolerance. Despite the many potential advantages of the fetal recipient, IUHCT across MHC barriers has been limited by low levels of engraftment and the inability to consistently achieve allochimerism. Although the immature immune system of the developing fetus has long been appreciated as a principal advantage of IUHCT, the competence of the fetal immune system to act as a barrier to IUHCT has been a source of debate. Until now, comparisons of allogeneic and congenic engraftment have been inconclusive due to methodologic limitations resulting in minimal and inefficient engraftment. In this study, a new intravascular technique that allows definitive administration of much higher doses of donor cells was employed to directly compare the incidence and levels of engraftment following in utero transplantation of either congenic or allogeneic bone marrow (BM) or enriched hematopoietic stem cells (HSCs). 20E+06 B6 GFP BM cells (H2Kb+, GFP+) or 1E+05 cKit+Sca-1+Lin- B6 GFP cells (H2Kb+, GFP+) were intravenously injected via the vitelline vein into gestational day 14 Balb/c (H2Kd+, allogeneic) or C57Bl/6 (H2Kb+, congenic) fetal mice. The peripheral blood (PB) of recipients was serially analyzed by flow cytometry for GFP+ donor cells at 1, 2, 4 and 6 months of age. A separate group of animals was harvested at 1 week of age (2 weeks after injection) to assess donor chimerism in PB and BM. Our results demonstrate that 100% of surviving recipients of whole BM demonstrate engraftment at 1 week of age, but that 70% of allogeneic recipients lose engraftment by 1 month of age, and 80% ultimately fail to sustain long-term chimerism. In contrast, all congenic recipients maintain engraftment at 6 months of age (Table 1). Chimerism levels in allogeneic recipients drop significantly after 1 month of life while those in congenic recipients remain stable. This results in a significant difference in engraftment levels in allogeneic and congenic recipients beyond 1 month of life (Fig 1). Similar results were seen when enriched HSCs were the donor cell source. 100% (2/2) of congenic recipients of enriched HSCs demonstrated stable low level PB engraftment up to 6 months of life (0.14–0.55% GFP+ donor cells). In contrast, no allogeneic recipients (0/9) of enriched HSCs were chimeric from 1 to 6 months of life. In combination, these results demonstrating a 100% efficiency of long-term engraftment in congenic recipients and loss of engraftment by 1 month of age in the majority of allogeneic recipients strongly implicate an adaptive immune barrier to allogeneic engraftment after IUHCT. Better understanding of the immune mechanisms limiting allogeneic engraftment after IUHCT is required to allow the development of successful strategies for IUHCT. Efficiency of Engraftment after IUHCT in Congenic and Allogeneic Recipients 1 week of age (BM) 1 week of age (PB) 1 month of age (PB) 6 months of age (PB) congenic 100% (8/8) 100% (8/8) 100% (25/25) 100% (25/25) allogeneic 100% (8/8) 100% (8/8) 29% (9/31) 19% (6/31) Figure Figure
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Liu, J., M. P. Ashton, H. Sumer, T. C. Brodnicki, M. K. O'Bryan et P. J. Verma. « 221 GENERATION OF GERM-LINE COMPETENT EMBRYONIC STEM CELLS FROM NON-OBESE DIABETIC (NOD) MICE USING A SINGLE INHIBITOR ». Reproduction, Fertility and Development 24, no 1 (2012) : 222. http://dx.doi.org/10.1071/rdv24n1ab221.
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The use of inbred mouse strains, where selective mating and tissue availability can be used to study the role of specific genes during disease pathogenesis is a synergistic approach to human type 1 diabetes (T1D) studies, which are hindered by genetic heterogeneity and inability to obtain relevant tissue biopsies. An excellent model for T1D is the non-obese diabetic (NOD) mouse strain, which spontaneously develops autoimmunity with lymphocytic infiltration of pancreatic islets. Autoimmune disease in NOD mice is multigenic and more than 20 susceptibility loci have been identified. Thus, genetic manipulation of NOD mice via gene targeting in embryonic stem cells (ESC) is a key strategy for identifying the underlying genes for these loci and characterising their roles in disease pathogenesis. Derivation of ESCs from NOD mice has proved to be extremely challenging and the NOD mouse is classified as one of the non-permissive mouse strains for derivation of ESC (Nagafuchi et al. 1999 FEBS Lett. 455, 101–141; Nichols et al. 2009 Nat. Med. 15, 814–818). Using the conventional mouse ESC culture medium - Knockout DMED medium supplemented with 20% Knockout serum replacement (KSR), 2 mM glutamine, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol (all from Invitrogen) and 2000 U mL–1 LIF (Millipore), we efficiently derived robust NOD ESC lines by inhibiting either the ERK or the GSK3b signalling pathway. Nineteen NOD ESC lines following supplementation of the ERK signalling inhibitor SC1 (SC1-NOD ESC) and 10 lines with GSK3b signalling inhibitor BIO (designed as BIO-NOD ESC) from 22 and 12 embryos, respectively (86.3 and 83.3%; P = 0.8). Two SC1-NOD and 2 BIO-NOD ESC were selected for characterisation. All 4 lines expressed the pluripotency-specific genes OCT4, NANOG and SSEA1, showed euploid karyotypes and generated teratomas in SCID mice. Two BIO-NOD ESC lines contributed to chimeric mice after injection into C57BL/6 blastocysts and embryo transfer. Chimeras from the both BIO-NOD ESC lines produced progeny of pure NOD genetics after mating with wild-type NOD females, demonstrating germ-line competence. The NOD ESC lines generated provide a unique tool to study genes implicated in T1D.
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Chen, Song-Lin, Zhen-Xia Sha, Han-Qing Ye, Yang Liu, Yong-Sheng Tian, Yunhan Hong et Qi-Sheng Tang. « Pluripotency and Chimera Competence of an Embryonic Stem Cell Line from the Sea Perch (Lateolabrax japonicus) ». Marine Biotechnology 9, no 1 (30 novembre 2006) : 82–91. http://dx.doi.org/10.1007/s10126-006-6050-1.
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Diego-Mantecón, José Manuel, Elena Haro, Teresa F. Blanco et Avenilde Romo-Vázquez. « The chimera of the competency-based approach to teaching mathematics : a study of carpentry purchases for home projects ». Educational Studies in Mathematics 107, no 2 (17 avril 2021) : 339–57. http://dx.doi.org/10.1007/s10649-021-10032-5.
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Labosky, P. A., D. P. Barlow et B. L. Hogan. « Mouse embryonic germ (EG) cell lines : transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines ». Development 120, no 11 (1 novembre 1994) : 3197–204. http://dx.doi.org/10.1242/dev.120.11.3197.
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Primordial germ cells of the mouse cultured on feeder layers with leukemia inhibitory factor, Steel factor and basic fibroblast growth factor give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells can be induced to differentiate extensively in culture, form teratocarcinomas when injected into nude mice and contribute to chimeras when injected into host blastocysts. Here, we report the derivation of multiple embryonic germ cell lines from 8.5 days post coitum embryos of C57BL/6 inbred mice. Four independent embryonic germ cell lines with normal male karyotypes have formed chimeras when injected into BALB/c host blastocysts and two of these lines have transmitted coat color markers through the germline. We also show that pluripotent cell lines capable of forming teratocarcinomas and coat color chimeras can be established from primordial germ cells of 8.0 days p.c. embryos and 12.5 days p.c. genital ridges. We have examined the methylation status of the putative imprinting box of the insulin-like growth factor type 2 receptor gene (Igf2r) in these embryonic germ cell lines. No correlation was found between methylation pattern and germline competence. A significant difference was observed between embryonic stem cell and embryonic germ cell lines in their ability to maintain the methylation imprint of the Igf2r gene in culture. This may illustrate a fundamental difference between these two cell types.
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Boyd, Nicholas, Kellie Cartledge, Huimin Cao, Vera Evtimov, Aleta Pupovac, Alan Trounson et Richard Boyd. « ‘Off-the-Shelf’ Immunotherapy : Manufacture of CD8+ T Cells Derived from Hematopoietic Stem Cells ». Cells 10, no 10 (2 octobre 2021) : 2631. http://dx.doi.org/10.3390/cells10102631.
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Cellular immunotherapy is revolutionizing cancer treatment. However, autologous transplants are complex, costly, and limited by the number and quality of T cells that can be isolated from and expanded for re-infusion into each patient. This paper demonstrates a stromal support cell-free in vitro method for the differentiation of T cells from umbilical cord blood hematopoietic stem cells (HSCs). For each single HSC cell input, approximately 5 × 104 T cells were created with an initial five days of HSC expansion and subsequent T cell differentiation over 49 days. When the induced in vitro differentiated T cells were activated by cytokines and anti-CD3/CD28 beads, CD8+ T cell receptor (TCR) γδ+ T cells were preferentially generated and elicited cytotoxic function against ovarian cancer cells in vitro. This process of inducing de novo functional T cells offers a possible strategy to increase T cell yields, simplify manufacturing, and reduce costs with application potential for conversion into chimeric antigen receptor (CAR)-T cells for cancer immunotherapy and for allogeneic transplantation to restore immune competence.
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Sato, Hideaki, Ayumi Wakayama, Kyoko Ito, Ikuo Kashiwakura et Koichi Ito. « Functional Adaptive Immune Responses in Hematopoietic Chimeric Mice After Umbilical Cord Blood Cell Transplantation. » Blood 120, no 21 (16 novembre 2012) : 2995. http://dx.doi.org/10.1182/blood.v120.21.2995.2995.
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Abstract Abstract 2995 Introduction: An increasing number of clinical trials have demonstrated the usefulness of cord blood as a source of hematopoietic stem cells for reconstitution of the hematopoietic system. Nevertheless, due to a lack of convenient animal models, information about the immunological competence of umbilical cord blood cell (UCBC)-derived lymphocytes has been relatively limited. Recently, we have established a murine model of UCBC transplantation, which reconstitutes the hematopoietic system of immunodeficient mice, and studied the correct immunological functions of UCBC-derived lymphocytes generated in an allogeneic environment. Materials and Methods: UCBC prepared from C57BL/6 (H-2b) mice were transferred into lethally irradiated BALB/c (H-2d) mice lacking T- and B-lymphocytes (RAG2 knockout). In order to evaluate the adaptive immune response in the recipient mice, rejection of skin grafts from third-party C3H/He (H-2k) mice and antibody responses to immunization with a T-dependent antigen, 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH), were assessed. Additionally, the responsiveness of generated T cells was verified by mixed lymphocyte reaction (MLR) assay, cytotoxic T-lymphocyte (CTL) assay and phytohemagglutinin (PHA) blast formation assay. Results: In this UCBC transplantation model, the overall survival of the recipient mice was dose-dependent, and all surviving recipient mice were hematopoietic chimeras possessing all lineages. These allogeneic chimeras specifically rejected skin grafts from third-party C3H/He mice (rejection time; 9–16 days) while showing tolerance to skin grafts from both donor-type and recipient type, although with a significant delay in comparison to normal BALB/c mice (8–12 days) and C57BL/6 mice (9–11 days). Although accurate antibody responses of the allogeneic chimeras against TNP-KLH immunization were not anticipated, substantial amounts of TNP-specific IgG were detected in their sera. Interestingly, the level of TNP-specific IgM was significantly higher than that of normal BALB/c mice after TNP-KLH immunization, indicating retarded immunoglobulin class-switching in the allogeneic chimeras. The mechanism responsible for the antibody production by allogeneic chimeras still remains unclear. CTL and MLR assay revealed cell-mediated third-party-specific activity of killer and helper T cells, consistent with the tendency for the skin graft rejection and antibody production capability of the allogeneic chimeras to be inferior to those of normal mice. T cells of the allogeneic chimeras also proliferated non-specifically in response to PHA stimulation. The T-cell proliferation indices of the allogeneic chimeras when those of normal mice were taken as 100 were 21 in the MLR assay and 48 in the PHA blast formation assay, suggesting relative inferiority of the recognition and response system of T cells generated in an allogeneic environment. In any event, it may be important to note that T-dependent antigen-specific antibody production and alloantigen-specific rejection were substantial in the allogeneic chimeras. Conclusions: Overall, the present findings suggest that UCBC-derived lymphocytes generated in HLA-mismatched recipients are immunologically functional and competent. Regarding the relative inferiority of immune responses in the allogeneic chimeras, we predict the causes originate from narrowing of the T- and B-cell receptor repertoire by the allogeneic environment, and this possibility is now being examined. Disclosures: No relevant conflicts of interest to declare.
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Han, Jongsuk, Hyeongbin Son, Daun Jung, Ki-Yeon Kim, Chaeyeon Jin, Hyeonwook Hwang, Soon-Suk Kang et al. « Comparison of Natural Killer Cells Differentiated from Various Pluripotent Stem Cells ». International Journal of Molecular Sciences 25, no 15 (27 juillet 2024) : 8209. http://dx.doi.org/10.3390/ijms25158209.
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Allogeneic natural killer (NK) cell therapy has been effective in treating cancer. Many studies have tested NK cell therapy using human pluripotent stem cells (hPSCs). However, the impacts of the origin of PSC-NK cells on competence are unclear. In this study, several types of hPSCs, including human-induced PSCs (hiPSCs) generated from CD34+, CD3−CD56+, and CD56− cells in umbilical cord blood (UCB), three lines of human embryonic stem cells (hESCs, ES-1. ES-2 and ES-3) and MHC I knockout (B2M-KO)-ESCs were used to differentiate into NK cells and their capacities were analyzed. All PSC types could differentiate into NK cells. Among the iPSC-derived NK cells (iPSC-NKs) and ESC-derived NK cells (ES-NKs), 34+ iPSCs and ES-3 had a higher growth rate and cytotoxicity, respectively, ES-3 also showed better efficacy than 34+ iPSCs. B2M-KO was similar to the wild type. These results suggest that the screening for differentiation of PSCs into NK cells prior to selecting the PSC lines for the development of NK cell immunotherapy is an essential process for universal allotransplantation, including the chimeric antigen receptor (CAR).
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Windoffer, Reinhard, Monika Borchert-Stuhlträger et Rudolf E. Leube. « Desmosomes : interconnected calcium-dependent structures of remarkable stability with significant integral membrane protein turnover ». Journal of Cell Science 115, no 8 (15 avril 2002) : 1717–32. http://dx.doi.org/10.1242/jcs.115.8.1717.
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Desmosomes are prominent cell adhesion structures that are major stabilizing elements, together with the attached cytoskeletal intermediate filament network, of the cytokeratin type in epithelial tissues. To examine desmosome dynamics in tightly coupled cells and in situations of decreased adhesion, fluorescent desmosomal cadherin desmocollin 2a (Dsc2a) chimeras were stably expressed in human hepatocellular carcinoma-derived PLC cells (clone PDc-13) and in Madin-Darby canine kidney cells (clone MDc-2) for the continuous monitoring of desmosomes in living cells. The hybrid polypeptides integrated specifically and without disturbance into normal-appearing desmosomes that occurred in association with typical cytokeratin filament bundles. Tracking of labeled adhesion sites throughout the cell cycle by time-lapse fluorescence microscopy revealed that they were immobile and that they maintained their structural integrity for long periods of time. Time-space diagrams further showed that desmosomal positioning was tightly controlled, even during pronounced cell shape changes, although the desmosomal arrays extended and contracted, suggesting that they were interconnected by a flexible system with intrinsic elasticity. Double-fluorescence microscopy detecting Dsc2a chimeras together with fluorescent cytokeratin 18 chimeras revealed the association and synchronous movement of labeled desmosomes and fluorescent cytokeratin filaments. Only a minor destabilization of desmosomes was observed during mitosis, demonstrated by increased diffuse plasma membrane fluorescence and the fusion of desmosomes into larger structures. Desmosomes did not disappear completely at any time in any cell, and residual cytokeratin filaments remained in association with adhesion sites throughout cell division. On the other hand, a rapid loss of desmosomes was observed upon calcium depletion, with irreversible uptake of some desmosomal particles. Simultaneously, diffusely distributed desmosomal cadherins were detected in the plasma membrane that retained the competence to nucleate the reformation of desmosomes after the cells were returned to a standard calcium-containing medium. To examine the molecular stability of desmosomes, exchange rates of fluorescent chimeras were determined by fluorescence recovery after photobleaching, thereby identifying considerable Dsc2a turnover with different rates of fluorescence recovery for PDc-13 cells (36±17% recovery after 30 minutes) and MDc-2 cells (60±20% recovery after 30 minutes). Taken together, our observations suggest that desmosomes are pliable structures capable of fine adjustment to functional demands despite their overall structural stability and relative immobility.
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Wang, Wenliang, Maria Fasolino, Benjamin Cattau, Naomi Goldman, Weimin Kong, Megan A. Frederick, Sam J. McCright, Karun Kiani, Joseph A. Fraietta et Golnaz Vahedi. « Joint profiling of chromatin accessibility and CAR-T integration site analysis at population and single-cell levels ». Proceedings of the National Academy of Sciences 117, no 10 (24 février 2020) : 5442–52. http://dx.doi.org/10.1073/pnas.1919259117.
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Chimeric antigen receptor (CAR)-T immunotherapy has yielded impressive results in several B cell malignancies, establishing itself as a powerful means to redirect the natural properties of T lymphocytes. In this strategy, the T cell genome is modified by the integration of lentiviral vectors encoding CAR that direct tumor cell killing. However, this therapeutic approach is often limited by the extent of CAR-T cell expansion in vivo. A major outstanding question is whether or not CAR-T integration itself enhances the proliferative competence of individual T cells by rewiring their regulatory landscape. To address this question, it is critical to define the identity of an individual CAR-T cell and simultaneously chart where the CAR-T vector integrates into the genome. Here, we report the development of a method called EpiVIA (https://github.com/VahediLab/epiVIA) for the joint profiling of the chromatin accessibility and lentiviral integration site analysis at the population and single-cell levels. We validate our technique in clonal cells with previously defined integration sites and further demonstrate the ability to measure lentiviral integration sites and chromatin accessibility of host and viral genomes at the single-cell resolution in CAR-T cells. We anticipate that EpiVIA will enable the single-cell deconstruction of gene regulation during CAR-T therapy, leading to the discovery of cellular factors associated with durable treatment.
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Marchetti, Marta, Alain Jauneau, Delphine Capela, Philippe Remigi, Carine Gris, Jacques Batut et Catherine Masson-Boivin. « Shaping Bacterial Symbiosis With Legumes by Experimental Evolution ». Molecular Plant-Microbe Interactions® 27, no 9 (septembre 2014) : 956–64. http://dx.doi.org/10.1094/mpmi-03-14-0083-r.
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Nitrogen-fixing symbionts of legumes have appeared after the emergence of legumes on earth, approximately 70 to 130 million years ago. Since then, symbiotic proficiency has spread to distant genera of α- and β-proteobacteria, via horizontal transfer of essential symbiotic genes and subsequent recipient genome remodeling under plant selection pressure. To tentatively replay rhizobium evolution in laboratory conditions, we previously transferred the symbiotic plasmid of the Mimosa symbiont Cupriavidus taiwanensis in the plant pathogen Ralstonia solanacearum, and selected spontaneous nodulating variants of the chimeric Ralstonia sp. using Mimosa pudica as a trap. Here, we pursued the evolution experiment by submitting two of the rhizobial drafts to serial ex planta–in planta (M. pudica) passages that may mimic alternating of saprophytic and symbiotic lives of rhizobia. Phenotyping 16 cycle-evolved clones showed strong and parallel evolution of several symbiotic traits (i.e., nodulation competitiveness, intracellular infection, and bacteroid persistence). Simultaneously, plant defense reactions decreased within nodules, suggesting that the expression of symbiotic competence requires the capacity to limit plant immunity. Nitrogen fixation was not acquired in the frame of this evolutionarily short experiment, likely due to the still poor persistence of final clones within nodules compared with the reference rhizobium C. taiwanensis. Our results highlight the potential of experimental evolution in improving symbiotic proficiency and for the elucidation of relationship between symbiotic capacities and elicitation of immune responses.
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Buck, Amanda M., Tyler-Marie Deveau, Timothy J. Henrich et Amelia N. Deitchman. « Challenges in HIV-1 Latent Reservoir and Target Cell Quantification in CAR-T Cell and Other Lentiviral Gene Modifying HIV Cure Strategies ». Viruses 15, no 5 (9 mai 2023) : 1126. http://dx.doi.org/10.3390/v15051126.
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Gene-modification therapies are at the forefront of HIV-1 cure strategies. Chimeric antigen receptor (CAR)-T cells pose a potential approach to target infected cells during antiretroviral therapy or following analytical treatment interruption (ATI). However, there are technical challenges in the quantification of HIV-1-infected and CAR-T cells in the setting of lentiviral CAR gene delivery and also in the identification of cells expressing target antigens. First, there is a lack of validated techniques to identify and characterize cells expressing the hypervariable HIV gp120 in both ART-suppressed and viremic individuals. Second, close sequence homology between lentiviral-based CAR-T gene modification vectors and conserved regions of HIV-1 creates quantification challenges of HIV-1 and lentiviral vector levels. Consideration needs to be taken into standardizing HIV-1 DNA/RNA assays in the setting of CAR-T cell and other lentiviral vector-based therapies to avoid these confounding interactions. Lastly, with the introduction of HIV-1 resistance genes in CAR-T cells, there is a need for assays with single-cell resolution to determine the competence of the gene inserts to prevent CAR-T cells from becoming infected in vivo. As novel therapies continue to arise in the HIV-1 cure field, resolving these challenges in CAR-T-cell therapy will be crucial.