Journal articles on the topic 'Dead cell recognition'
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Shiotani, Shigetoshi, Toshio Fukuda, Fumihito Arai, Naokazu Takeuchi, Kyosuke Sasaki, and Tatsuyuki Kinosita. "Cell Recognition by Image Processing : Recognition of Dead or Living Plant Cells by Neural Network." JSME international journal. Ser. C, Dynamics, control, robotics, design and manufacturing 37, no. 1 (1994): 202–8. http://dx.doi.org/10.1299/jsmec1993.37.202.
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Hochreiter-Hufford, A., and K. S. Ravichandran. "Clearing the Dead: Apoptotic Cell Sensing, Recognition, Engulfment, and Digestion." Cold Spring Harbor Perspectives in Biology 5, no. 1 (January 1, 2013): a008748. http://dx.doi.org/10.1101/cshperspect.a008748.
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FUKUDA, Toshio, Shigetoshi SHIOTANI, Fumihito ARAI, Naokazu TAKEUCHI, Kyosuke SASAKI, and Tatsuyuki KINOSHITA. "Cell Recognition by Image Processing. 1st Report. Recognition of Dead or Alive Plant Cells by Neural Network." Transactions of the Japan Society of Mechanical Engineers Series C 57, no. 542 (1991): 3189–96. http://dx.doi.org/10.1299/kikaic.57.3189.
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Cao, Longxing, Haishuang Chang, Xiangyi Shi, Chao Peng, and Yongning He. "Keratin mediates the recognition of apoptotic and necrotic cells through dendritic cell receptor DEC205/CD205." Proceedings of the National Academy of Sciences 113, no. 47 (November 7, 2016): 13438–43. http://dx.doi.org/10.1073/pnas.1609331113.
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Clearance of dead cells is critical for maintaining homeostasis and prevents autoimmunity and inflammation. When cells undergo apoptosis and necrosis, specific markers are exposed and recognized by the receptors on phagocytes. DEC205 (CD205) is an endocytotic receptor on dendritic cells with antigen presentation function and has been widely used in immune therapies for vaccine generation. It has been shown that human DEC205 recognizes apoptotic and necrotic cells in a pH-dependent fashion. However, the natural ligand(s) of DEC205 remains unknown. Here we find that keratins are the cellular ligands of human DEC205. DEC205 binds to keratins specifically at acidic, but not basic, pH through its N-terminal domains. Keratins form intermediate filaments and are important for maintaining the strength of cells and tissues. Our results suggest that keratins also function as cell markers of apoptotic and necrotic cells and mediate a pH-dependent pathway for the immune recognition of dead cells.
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Taschuk, Frances, and Sara Cherry. "DEAD-Box Helicases: Sensors, Regulators, and Effectors for Antiviral Defense." Viruses 12, no. 2 (February 5, 2020): 181. http://dx.doi.org/10.3390/v12020181.
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DEAD-box helicases are a large family of conserved RNA-binding proteins that belong to the broader group of cellular DExD/H helicases. Members of the DEAD-box helicase family have roles throughout cellular RNA metabolism from biogenesis to decay. Moreover, there is emerging evidence that cellular RNA helicases, including DEAD-box helicases, play roles in the recognition of foreign nucleic acids and the modulation of viral infection. As intracellular parasites, viruses must evade detection by innate immune sensing mechanisms and degradation by cellular machinery while also manipulating host cell processes to facilitate replication. The ability of DEAD-box helicases to recognize RNA in a sequence-independent manner, as well as the breadth of cellular functions carried out by members of this family, lead them to influence innate recognition and viral infections in multiple ways. Indeed, DEAD-box helicases have been shown to contribute to intracellular immune sensing, act as antiviral effectors, and even to be coopted by viruses to promote their replication. However, our understanding of the mechanisms underlying these interactions, as well as the cellular roles of DEAD-box helicases themselves, is limited in many cases. We will discuss the diverse roles that members of the DEAD-box helicase family play during viral infections.
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Turkyilmaz, Serhan, Douglas R. Rice, Rachael Palumbo, and Bradley D. Smith. "Selective recognition of anionic cell membranes using targeted liposomes coated with zinc(ii)-bis(dipicolylamine) affinity units." Org. Biomol. Chem. 12, no. 30 (2014): 5645–55. http://dx.doi.org/10.1039/c4ob00924j.
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Liposomes containing phospholipid-PEG conjugates with terminal zinc(ıı)-bis(dipicolylamine) affinity units selectively target anionic membrane surfaces including the exterior of bacterial and dead/dying mammalian cells.
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Tanner, N. Kyle. "The Newly Identified Q Motif of DEAD Box Heicases IS Involved in Adenine Recognition." Cell Cycle 2, no. 1 (January 2003): 18–19. http://dx.doi.org/10.4161/cc.2.1.296.
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Li, Qiong, Xin Sun, Junyu Dong, Shuqun Song, Tongtong Zhang, Dan Liu, Han Zhang, and Shuai Han. "Developing a microscopic image dataset in support of intelligent phytoplankton detection using deep learning." ICES Journal of Marine Science 77, no. 4 (September 20, 2019): 1427–39. http://dx.doi.org/10.1093/icesjms/fsz171.
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Abstract Phytoplankton plays an important role in marine ecological environment and aquaculture. However, the recognition and detection of phytoplankton rely on manual operations. As the foundation of achieving intelligence and releasing human labour, a phytoplankton microscopic image dataset PMID2019 for phytoplankton automated detection is presented. The PMID2019 dataset contains 10 819 phytoplankton microscopic images of 24 different categories. We leverage microscopes to collect images of phytoplankton in the laboratory environment. Each object in the images is manually labelled with a bounding box and category of ground-truth. In addition, living cells move quickly making it difficult to capture images of them. In order to generalize the dataset for in situ applications, we further utilize Cycle-GAN to achieve the domain migration between dead and living cell samples. We built a synthetic dataset to generate the corresponding living cell samples from the original dead ones. The PMID2019 dataset will not only benefit the development of phytoplankton microscopic vision technology in the future, but also can be widely used to assess the performance of the state-of-the-art object detection algorithms for phytoplankton recognition. Finally, we illustrate the performances of some state-of-the-art object detection algorithms, which may provide new ideas for monitoring marine ecosystems.
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Cocco, Regina E., and David S. Ucker. "Distinct Modes of Macrophage Recognition for Apoptotic and Necrotic Cells Are Not Specified Exclusively by Phosphatidylserine Exposure." Molecular Biology of the Cell 12, no. 4 (April 2001): 919–30. http://dx.doi.org/10.1091/mbc.12.4.919.
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The distinction between physiological (apoptotic) and pathological (necrotic) cell deaths reflects mechanistic differences in cellular disintegration and is of functional significance with respect to the outcomes that are triggered by the cell corpses. Mechanistically, apoptotic cells die via an active and ordered pathway; necrotic deaths, conversely, are chaotic and passive. Macrophages and other phagocytic cells recognize and engulf these dead cells. This clearance is believed to reveal an innate immunity, associated with inflammation in cases of pathological but not physiological cell deaths. Using objective and quantitative measures to assess these processes, we find that macrophages bind and engulf native apoptotic and necrotic cells to similar extents and with similar kinetics. However, recognition of these two classes of dying cells occurs via distinct and noncompeting mechanisms. Phosphatidylserine, which is externalized on both apoptotic and necrotic cells, is not a specific ligand for the recognition of either one. The distinct modes of recognition for these different corpses are linked to opposing responses from engulfing macrophages. Necrotic cells, when recognized, enhance proinflammatory responses of activated macrophages, although they are not sufficient to trigger macrophage activation. In marked contrast, apoptotic cells profoundly inhibit phlogistic macrophage responses; this represents a cell-associated, dominant-acting anti-inflammatory signaling activity acquired posttranslationally during the process of physiological cell death.
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Hood, M. E., and H. D. Shew. "Initial Cellular Interactions Between Thielaviopsis basicola and Tobacco Root Hairs." Phytopathology® 87, no. 3 (March 1997): 228–35. http://dx.doi.org/10.1094/phyto.1997.87.3.228.
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Cellular events that occur during the initial interactions between Thielaviopsis basicola and root hairs of tobacco (Nicotiana tabacum) were examined microscopically. Time-course documentation of the infection process indicated a dynamic interaction between T. basicola and the living host cell. Upon root hair contact and recognition, the vegetative apex of T. basicola rapidly differentiated to form infection structures, and the host cell responded cytologically. Penetration was achieved by threadlike hyphae that subsequently developed distal swellings, and intracellular hyphae of sickle-shaped morphology advanced from the distal swelling and colonized the cell. Streaming of the host cytoplasm became aggregated near the infection site prior to penetration and accumulated around the infecting hyphae as long as the host cell was viable. Substantial callose deposition, in the form of a bell-shaped collar around infection structures, resulted from the cytological activity at the infection site. Penetration of dead root hairs was common, but did not lead to the development of infection structures or to a sustained association with the host tissue; T. basicola exited dead root hairs and resumed vegetative growth. The establishment of the parasitic relationship by T. basicola was characteristic of hemibiotrophic fungi in that, initially, biotrophic infection led to tissue colonization, and host cell survival was limited under parasitism.
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Brungs, Sonja, Waldemar Kolanus, and Ruth Hemmersbach. "Impact of altered gravity on the ROS-signalling in macrophages (P1293)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 63.4. http://dx.doi.org/10.4049/jimmunol.190.supp.63.4.
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Abstract Astronauts suffer from a higher susceptibility to infections during spaceflights. Different stressors could lead to a diminished immune defense. Macrophages present the first line of defense of the innate immune system. Their main challenge is the recognition, engulfment and destruction of pathogenic bacteria, viruses and dead cells. The killing of bacteria is accomplished by the production of Reactive Oxygen Species (ROS) during oxidative burst. We investigated the effects of altered gravity on the pattern recognition and ROS production of macrophages. Our results show that real microgravity (parabolic flight), simulated microgravity (2D fast-rotating clinostat) as well as hypergravity (centrifuge) alter the production of ROS after stimulation of cell surface receptors Dectin, TLR2/6, complement and Fcγ. We also find that phosphorylation of the tyrosine kinase Syk, an essential link between pattern recognition, cytoskeleton and ROS production, is sensitive to microgravity. We can conclude that both microgravity and hypergravity cause very fast changes in ROS production, characterising the oxidative burst as a gravisensitive processes. Altered gravity might have impact on the interaction of different pattern recognition signalling pathways, which are crucial for the bacteria clearance in terms of ROS production. Therefore, the study of ROS signalling under altered gravity is not only of interest for humans in space, but also for human health on Earth.
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Arias-Arias, Jorge L., Eugenia Corrales-Aguilar, and Rodrigo A. Mora-Rodríguez. "A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging." Viruses 13, no. 7 (June 22, 2021): 1193. http://dx.doi.org/10.3390/v13071193.
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Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.
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Olsen, Johan Pelck, Jeppe Kari, Michael Skovbo Windahl, Kim Borch, and Peter Westh. "Molecular recognition in the product site of cellobiohydrolase Cel7A regulates processive step length." Biochemical Journal 477, no. 1 (January 8, 2020): 99–110. http://dx.doi.org/10.1042/bcj20190770.
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Cellobiohydrolase Cel7A is an industrial important enzyme that breaks down cellulose by a complex processive mechanism. The enzyme threads the reducing end of a cellulose strand into its tunnel-shaped catalytic domain and progresses along the strand while sequentially releasing the disaccharide cellobiose. While some molecular details of this intricate process have emerged, general structure-function relationships for Cel7A remain poorly elucidated. One interesting aspect is the occurrence of particularly strong ligand interactions in the product binding site. In this work, we analyze these interactions in Cel7A from Trichoderma reesei with special emphasis on the Arg251 and Arg394 residues. We made extensive biochemical characterization of enzymes that were mutated in these two positions and showed that the arginine residues contributed strongly to product binding. Specifically, ∼50% of the total standard free energy of product binding could be ascribed to four hydrogen bonds to Arg251 and Arg394, which had previously been identified in crystal structures. Mutation of either Arg251 or Arg394 lowered production inhibition of Cel7A, but at the same time altered the enzyme product profile and resulted in ∼50% reduction in both processivity and hydrolytic activity. The position of the two arginine residues closely matches the two-fold screw axis symmetry of the substrate, and this energetically favors the productive enzyme-substrate complex. Our results indicate that the strong and specific ligand interactions of Arg251 and Arg394 provide a simple proofreading system that controls the step length during consecutive hydrolysis and minimizes dead time associated with transient, non-productive complexes.
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Stone, Amy E. L., Ahna B. Thompson, Abby W. Jamieson, Shelby Feliciano, Ali Said, and Michael J. Gale. "DDX39A interacts with LGP2 to inhibit RLR responses." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 68.3. http://dx.doi.org/10.4049/jimmunol.204.supp.68.3.
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Abstract RIG-I-like receptors (RLRs) are DEAD-box helicases and cytoplasmic pathogen recognition receptors that recognize and bind to nonself/viral RNA and trigger innate antiviral immunity. RLRs are essential for the detection of RNA virus infection and include RIG-I, MDA5, and LGP2. LGP2 has been implicated as RIG-I or MDA5 co-factor to regulate their activity. We conducted a yeast 2-hybrid (Y2H) screen (using LGP2 as bait) of a human hepatic cDNA library to identify protein binding partners of LGP2. Identified proteins were validated as LGP2 interactors through overexpression and co-immunoprecipitation assays of LGP2 binding. DDX39A, a DEAD Box helicase protein with known roles in RNA splicing, was identified as a binding partner. DDX39A overexpression was found to inhibit interferon-beta production from RLR-signaling in a cell-based ligand-free model in a dose-dependent manner. Further, in a cell-based interferon beta-promoter-luciferase assay model of Sendai virus (SenV) infection, DDX39A and LGP2 synergistically inhibited promoter activity. Similar results were seen in a cell-based NFkB-luciferase assay of the same system. Endogenous expression of DDX39A was assayed through the Huh7 human hepatoma cell line system with various stimuli including viral infection with SenV and Interferon-beta treatment showing that DDX39A is constitutively expressed in those cells. Structure-function mutants of DDX39A and LGP2 were used to determine the protein domains and functions required for interaction via co-immunoprecipitation assays. Through these studies, we will define the interactions between LGP2 and DDX39A, and reveal the roles these proteins have as co-factors in RLR signaling.
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Thompson, Ahna B., Amy EL Stone, and Michael J. Gale. "Identifying the interactome of the RIG-I-like Receptor LGP2." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 203.18. http://dx.doi.org/10.4049/jimmunol.196.supp.203.18.
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Abstract RIG-I-like receptors (RLRs) are DEAD box helicases and cytoplasmic pathogen recognition receptors that recognize and bind to nonself/viral RNA and trigger innate antiviral immunity. RLRs are essential for detection of RNA virus infection, and include RIG-I, MDA5, and LGP2. LGP2 has been implicated as RIG-I or MDA5 cofactor to regulate their activity. We conducted a yeast 2 hybrid (Y2H) screen (using LGP2 as bait) of a human hepatic cDNA library to identify protein binding partners of LGP2. Identified proteins were validated as LGP2 interactors through overexpression and co-immunoprecipitation assays of LGP2 binding. The validated proteins were then submitted to a cell-based interferon beta-promoter-luciferase assay model of Sendai virus (SenV) infection as well as a cell based NFkB-luciferase assay of the same system. Endogenous expression of the validated proteins was then assayed through the Huh7 human hepatoma cell line system with various stimuli including viral infection with SenV and Interferon-beta treatment. Through these studies we will define the LGP2 interactome and identify proteins that act as co-factors in RLR signaling.
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Barik, Sailen. "What Really Rigs Up RIG-I?" Journal of Innate Immunity 8, no. 5 (2016): 429–36. http://dx.doi.org/10.1159/000447947.
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RIG-I (retinoic acid-inducible gene 1) is an archetypal member of the cytoplasmic DEAD-box dsRNA helicase family (RIG-I-like receptors or RLRs), the members of which play essential roles in the innate immune response of the metazoan cell. RIG-I functions as a pattern recognition receptor that detects nonself RNA as a pathogen-associated molecular pattern (PAMP). However, the exact molecular nature of the viral RNAs that act as a RIG-I ligand has remained a mystery and a matter of debate. In this article, we offer a critical review of the actual viral RNAs that act as PAMPs to activate RIG-I, as seen from the perspective of a virologist, including a recent report that the viral Leader-read-through transcript is a novel and effective RIG-I ligand.
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Leri, Annarosa, Jan Kajstura, and Piero Anversa. "Cardiac Stem Cells and Mechanisms of Myocardial Regeneration." Physiological Reviews 85, no. 4 (October 2005): 1373–416. http://dx.doi.org/10.1152/physrev.00013.2005.
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This review discusses current understanding of the role that endogenous and exogenous progenitor cells may have in the treatment of the diseased heart. In the last several years, a major effort has been made in an attempt to identify immature cells capable of differentiating into cell lineages different from the organ of origin to be employed for the regeneration of the damaged heart. Embryonic stem cells (ESCs) and bone marrow-derived cells (BMCs) have been extensively studied and characterized, and dramatic advances have been made in the clinical application of BMCs in heart failure of ischemic and nonischemic origin. However, a controversy exists concerning the ability of BMCs to acquire cardiac cell lineages and reconstitute the myocardium lost after infarction. The recognition that the adult heart possesses a stem cell compartment that can regenerate myocytes and coronary vessels has raised the unique possibility to rebuild dead myocardium after infarction, to repopulate the hypertrophic decompensated heart with new better functioning myocytes and vascular structures, and, perhaps, to reverse ventricular dilation and wall thinning. Cardiac stem cells may become the most important cell for cardiac repair.
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Fortenberry, James D., Marilyn L. Owens, and Lou Ann S. Brown. "S-nitrosoglutathione enhances neutrophil DNA fragmentation and cell death." American Journal of Physiology-Lung Cellular and Molecular Physiology 276, no. 3 (March 1, 1999): L435—L442. http://dx.doi.org/10.1152/ajplung.1999.276.3.l435.
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Enhancing the clearance of neutrophils by enhancing apoptotic cell death and macrophage recognition may be beneficial in acute lung injury. Exogenous nitric oxide gas depresses neutrophil oxidative functions and accelerates cell death (A. H. Daher, J. D. Fortenberry, M. L. Owens, and L. A. Brown. Am. J. Respir. Cell Mol. Biol. 16: 407–412, 1997). We hypothesized that S-nitrosoglutathione (GSNO), a physiologically relevant nitric oxide donor, could also enhance neutrophil DNA fragmentation. Neutrophils were incubated for 2–24 h in the absence and presence of GSNO (dose range 0.1–5 mM) and evaluated for cell death by a fluorescent viability/cytotoxicity assay. Neutrophil DNA fragmentation was assessed by cell death detection ELISA and by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling assay. Neutrophil oxidative function was also determined. Incubation with GSNO increased cell death at 2, 4, and 24 h. GSNO incubation for 24 h significantly increased DNA fragmentation in a dose-dependent fashion at 0.5 (median 126% of control value; P = 0.002) and 5 mM (185% of control value; P = 0.002) by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling and at 0.5 mM by ELISA (164% of control value; P = 0.03). The apoptosis-to-total cell death ratio increased with increasing GSNO concentration ( P < 0.05). Effects were mitigated by coincubation with superoxide dismutase. Five millimolar GSNO decreased overall superoxide generation and O2consumption but not when adjusted for dead neutrophils. GSNO significantly enhances cell death and neutrophil DNA fragmentation in a dose-dependent fashion.
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Qiu, Jin, Michal A. Olszewski, and Peter R. Williamson. "Cryptococcus neoformans Growth and Protection from Innate Immunity Are Dependent on Expression of a Virulence-Associated DEAD-Box Protein, Vad1." Infection and Immunity 81, no. 3 (December 21, 2012): 777–88. http://dx.doi.org/10.1128/iai.00821-12.
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ABSTRACTThe fungusCryptococcus neoformanshas emerged as a major cause of meningoencephalitis worldwide. Host response to the fungus involves both innate and adaptive immunity, but fungal genes that modulate these processes are poorly understood. Previous studies demonstrated attenuated virulence of a mutant of avirulence-associatedDEAD-box protein (VAD1) in mice, despite normal growth at host temperatures, suggesting modulation of the immune response. In the present study, theΔvad1mutant demonstrated progressive clearance from lung and was unable to induce pathological lesions or to cause extrapulmonary disease, despite retaining its ability to grow in mouse serum and a J774.16 macrophage cell line. Pulmonary clearance occurred with a minimal cellular infiltrate, marked by reduced CD4 cells, CD11b+Ly6Chighmonocytes, and F4/80+macrophages, but the mutant strain retained recruitment of CD8 cells, compared to infections with wild-type fungi. Adaptive cytokine responses were reduced, including Th1, Th2, and Th17 cytokines; however, early gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) responses were retained while nonprotective interleukin 4 (IL-4) and IL-5 were diminished. Furthermore, theΔvad1mutant was controlled in lungs despite CD4/CD8 cell depletion. These data, along with improved phagocytosis by macrophages and increases in early/innate IL-1α, IFN-γ, and chemokines elicited in the lungs within 3 days of infection with theΔvad1mutant, indicate thatVAD1expression reduces innate recognition ofC. neoformans, rendering the yeast resistant to elimination by the innate mechanisms of host defense. Thus, our studies define a novel role of the cryptococcal Vad1 protein as a central regulator of cryptococcal virulence and illustrate that Vad1 promotes microbe resistance to innate host defenses.
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Henderson, J. Nathan, Agnieszka M. Kuriata, Raimund Fromme, Michael E. Salvucci, and Rebekka M. Wachter. "Atomic Resolution X-ray Structure of the Substrate Recognition Domain of Higher Plant Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase." Journal of Biological Chemistry 286, no. 41 (August 31, 2011): 35683–88. http://dx.doi.org/10.1074/jbc.c111.289595.
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The rapid release of tight-binding inhibitors from dead-end ribulose-bisphosphate carboxylase/oxygenase (Rubisco) complexes requires the activity of Rubisco activase, an AAA+ ATPase that utilizes chemo-mechanical energy to catalyze the reactivation of Rubisco. Activase is thought to play a central role in coordinating the rate of CO2 fixation with the light reactions of photosynthesis. Here, we present a 1.9 Å crystal structure of the C-domain core of creosote activase. The fold consists of a canonical four-helix bundle, from which a paddle-like extension protrudes that entails a nine-turn helix lined by an irregularly structured peptide strand. The residues Lys-313 and Val-316 involved in the species-specific recognition of Rubisco are located near the tip of the paddle. An ionic bond between Lys-313 and Glu-309 appears to stabilize the glycine-rich end of the helix. Structural superpositions onto the distant homolog FtsH imply that the paddles extend away from the hexameric toroid in a fan-like fashion, such that the hydrophobic sides of each blade bearing Trp-302 are facing inward and the polar sides bearing Lys-313 and Val-316 are facing outward. Therefore, we speculate that upon binding, the activase paddles embrace the Rubisco cylinder by placing their hydrophobic patches near the partner protein. This model suggests that conformational adjustments at the remote end of the paddle may relate to selectivity in recognition, rather than specific ionic contacts involving Lys-313. Additionally, the superpositions predict that the catalytically critical Arg-293 does not interact with the bound nucleotide. Hypothetical ring-ring stacking and peptide threading models for Rubisco reactivation are briefly discussed.
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Kamal, Samia. "Non-surgical Removal of Basal Cell Carcinoma by Apis Mellifera L Venom." Clinical Medical Reviews and Reports 2, no. 9 (December 26, 2020): 01–06. http://dx.doi.org/10.31579/2690-8794/056.
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Background: Honeybee’s venom is potent anticancer drug without exhibiting any side effects. Skin Basal Cell Carcinoma (SBCC) is a common malignancy. It can cause significant local destructions depending on affected site. The diagnosis of SBCC can be suspected from clinical findings and confirmation of diagnosis histopathology. The present SBCC is recurrent and aggressive in the skin of head [upper right, in front of the right ear]; the patient was 65 years old in time of first intervention. Materials & Methods: Following the lesions primarily surgically excised, the malignant growths recurrent and visual recognition occurred after 1 year from first operation, then another (the second) surgical removal of SBCC from affected skin with removal of all skin layers near the affected site but another recurrence visually occurs after about 4 months, in the form of malignant growth in the skin of right ear. Dry honeybee’s venom 1 mg was dissolved in 1 ml dist. water as injectable solution. Moreover, ointment contains 2% bee venom was prepared to be used topically inside affected ear as injection is not possible. Results: Before this novel intervention, the desperate patient situation seems very dangerous; as the new growths appear as continuous spread near the removed skin, so that patient’s family decided to apply more noninvasive and non-surgical intervention. The only precaution was testing the patients to assure she is not hypersensitive to honeybees’ venom. The treatment performed by subcutaneous injection of 0.3 ml from prepared Honeybees venom (0.1 % conc.) in the skin of affected part of the ear. Subcutaneous infiltration was applied around the lesions of about 0.5 ml as well, topical application of the ointment inside inner part of affected ear. This process repeated daily with cleaning of the ear every time by suitable safe and sterile saline solutions. Management of healing process was enhanced by ascorbic acid solution as topical application on dead cancer cells and to help in exudates debris removal. The complete removal of malignant growths and recovery obtained after 1 month from first bee venom injections. No recurrence of SBCC seen for 3 yrs. Conclusions: Honeybees venom is highly effective and safe anticancer drug that can be used for all patients’ categories of all ages. Regarding the present case invasive surgical intervention was not the good choice from the beginnings, as recurrence and giving chance for spreading following the time lapse between every surgery.
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Wood, W., M. Turmaine, R. Weber, V. Camp, R. A. Maki, S. R. McKercher, and P. Martin. "Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU.1 null mouse embryos." Development 127, no. 24 (December 15, 2000): 5245–52. http://dx.doi.org/10.1242/dev.127.24.5245.
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Apoptosis is one of the key tools used by an embryo to regulate cell numbers and sculpt body shape. Although massive numbers of cells die during development, they are so rapidly phagocytosed that very few corpses are ever seen in most embryonic tissues. In this paper, we focus on the catastrophic cell death that occurs as the developing footplate is remodelled to transform webbed regions into free interdigital spaces. In the wild-type embryo, these dead cells are rapidly engulfed and cleared by macrophages. We show that in a macrophageless mouse embryo, null for the haemopoetic-lineage-specific transcription factor, PU.1, the task of phagocytosis is taken over by ‘stand-in’ mesenchymal neighbours in a clear example of cell redundancy. However, it takes three times as many of these mesenchymal phagocytes to complete the task and, at each stage of the clearance process - in the recognition of apoptotic debris, its engulfment and finally its digestion - they appear to be less efficient than macrophages. A molecular explanation for this may be that several of the engulfment genes expressed by macrophages, including the ABC1 transporter (believed to be part of the phagocytic machinery conserved from Caenorhabditis elegans to mouse), are not upregulated by these ‘stand-in’ phagocytes.
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XU, LUPING, XINGJIAN BAI, and ARUN K. BHUNIA. "Current State of Development of Biosensors and Their Application in Foodborne Pathogen Detection." Journal of Food Protection 84, no. 7 (March 12, 2021): 1213–27. http://dx.doi.org/10.4315/jfp-20-464.
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ABSTRACT Foodborne disease outbreaks continue to be a major public health and food safety concern. Testing products promptly can protect consumers from foodborne diseases by ensuring the safety of food before retail distribution. Fast, sensitive, and accurate detection tools are in great demand. Therefore, various approaches have been explored recently to find a more effective way to incorporate antibodies, oligonucleotides, phages, and mammalian cells as signal transducers and analyte recognition probes on biosensor platforms. The ultimate goal is to achieve high specificity and low detection limits (1 to 100 bacterial cells or piconanogram concentrations of toxins). Advancements in mammalian cell–based and bacteriophage-based sensors have produced sensors that detect low levels of pathogens and differentiate live from dead cells. Combinations of biotechnology platforms have increased the practical utility and application of biosensors for detection of foodborne pathogens. However, further rigorous testing of biosensors with complex food matrices is needed to ensure the utility of these sensors for point-of-care needs and outbreak investigations. HIGHLIGHTS
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Lee, Soon Goo, Kate Harline, Orchid Abar, Sakirat O. Akadri, Alexander G. Bastian, Hui-Yuan S. Chen, Michael Duan, et al. "The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase." Journal of Biological Chemistry 295, no. 40 (August 12, 2020): 13914–26. http://dx.doi.org/10.1074/jbc.ra120.014747.
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Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the “aromatic box” that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.
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Jin, Jing, Ruijun Tian, Adrian Pasculescu, Anna Yue Dai, Kelly Williton, Lorne Taylor, Mikhail M. Savitski, et al. "Mutational Analysis of Glycogen Synthase Kinase 3β Protein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90." Molecular and Cellular Biology 36, no. 6 (January 11, 2016): 1007–18. http://dx.doi.org/10.1128/mcb.01045-15.
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The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms. Using model cell lines stably expressing these two GSK3β forms, we observed no interaction between WT GSK3β and HSP90, in stark contrast to KD GSK3β forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome.
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Balaji, Kithiganahalli N., and W. Henry Boom. "Processing of Mycobacterium tuberculosisBacilli by Human Monocytes for CD4+ αβ and γδ T Cells: Role of Particulate Antigen." Infection and Immunity 66, no. 1 (January 1, 1998): 98–106. http://dx.doi.org/10.1128/iai.66.1.98-106.1998.
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ABSTRACT Mycobacterium tuberculosis readily activates both CD4+ and Vδ2+ γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented byM. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosisantigens to human CD4 and γδ T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2+ γδ T cells. For γδ T cells, recognition ofM. tuberculosis-infected monocytes was dependent on Vδ2+ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4+ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vδ2+ γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4+ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosisbacilli for CD4+ and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4+ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4+ and γδ T cells.
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Santagostino, Sara Francesca, Marco Spinazzi, and Enrico Radaelli. "Restricted Sensitivity of FJ-C Staining to Assess Neuronal Degeneration and Death in Preclinical Mouse Studies." Veterinary Pathology 58, no. 4 (January 5, 2021): 643–49. http://dx.doi.org/10.1177/0300985820985290.
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Fluorescein-derived fluorochromes and anionic dyes such as Fluoro-Jade (FJ) stains have been introduced to facilitate recognition of dying neurons in tissue sections. However, the definition of what is really detected by FJ-based stains and its sensitivity in the detection of neuronal cell death is unclear. In our work, we evaluated the outcome of FJ-C staining in mouse brains from 4 different well-characterized models of neurodegeneration. Neuronal degeneration and loss were highlighted with high sensitivity by FJ-C stain in mice with dysfunctional γ-secretase in the glutamatergic neurons and in mice affected by acute cerebral ischemia. Histopathologically, acute eosinophilic necrosis or “red dead” neurons were associated with FJ-C staining in both settings. Conversely, in mice affected by chronic cerebral microinfarcts due to tumor lysis syndrome as well as in a model of mitochondrial encephalopathy, FJ-C staining failed to detect neuronal death. Histopathologically, these models were characterized by extensive neuronal vacuolation associated with fading neurons (“ghost cells”). Therefore, contrary to the widespread belief that FJ-C stain has high affinity for all degenerating neurons regardless of the underlying cell death mechanism, we observed restricted sensitivity of the technique to specific conditions of neuronal cell death. As such, complementary techniques are essential to evaluate the presence of neurodegeneration in the absence of a positive FJ-C signal.
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Fernandez, Myriam R., and Michèle C. Heath. "Interactions of the nonhost French bean plant (Phaseolus vulgaris) with parasitic and saprophytic fungi. III. Cytologically detectable responses." Canadian Journal of Botany 67, no. 3 (March 1, 1989): 676–86. http://dx.doi.org/10.1139/b89-091.
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Cytologically detectable responses of the nonhost French bean (Phaseolus vulgaris L. cv. Pinto) to saprophytic and parasitic fungi were examined when fungal spores were introduced into heated or unheated leaves via wounds or by injection. Although similar types of responses were observed in interactions with all the fungi, some of these responses were characteristic of each group (saprophytes vs. parasites) in the frequency and (or) extent with which they were elicited. Differences in responses between and within each of these groups of fungi were more related to their degree of adaptation for parasitism than to their taxonomic relationships. Certain responses that were typically elicited by the saprophytes occurred to a lesser extent in tissue responding to the parasites, suggesting that the ability to not trigger, or suppress, these responses may be a general feature of parasitic fungi. None of the fungi elicited significant levels of plant cell necrosis, and for two of the saprophytes, dead spores elicited a lower frequency of responses than live ones. The data indicate that many of the responses of a nonhost plant to living fungi may be the result of reactions to fungal activity rather than to constitutive recognition molecules such as components of the fungal cell wall.
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Checa, Javier, and Josep M. Aran. "Airway Redox Homeostasis and Inflammation Gone Awry: From Molecular Pathogenesis to Emerging Therapeutics in Respiratory Pathology." International Journal of Molecular Sciences 21, no. 23 (December 7, 2020): 9317. http://dx.doi.org/10.3390/ijms21239317.
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As aerobic organisms, we are continuously and throughout our lifetime subjected to an oxidizing atmosphere and, most often, to environmental threats. The lung is the internal organ most highly exposed to this milieu. Therefore, it has evolved to confront both oxidative stress induced by reactive oxygen species (ROS) and a variety of pollutants, pathogens, and allergens that promote inflammation and can harm the airways to different degrees. Indeed, an excess of ROS, generated intrinsically or from external sources, can imprint direct damage to key structural cell components (nucleic acids, sugars, lipids, and proteins) and indirectly perturb ROS-mediated signaling in lung epithelia, impairing its homeostasis. These early events complemented with efficient recognition of pathogen- or damage-associated recognition patterns by the airway resident cells alert the immune system, which mounts an inflammatory response to remove the hazards, including collateral dead cells and cellular debris, in an attempt to return to homeostatic conditions. Thus, any major or chronic dysregulation of the redox balance, the air–liquid interface, or defects in epithelial proteins impairing mucociliary clearance or other defense systems may lead to airway damage. Here, we review our understanding of the key role of oxidative stress and inflammation in respiratory pathology, and extensively report current and future trends in antioxidant and anti-inflammatory treatments focusing on the following major acute and chronic lung diseases: acute lung injury/respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and cystic fibrosis.
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Andrews, J. C., and S. Winters-Hilt. "7UTILIZATION OF CELL PROFILING TO EVALUATE BOVINE SPERMATOZOA IN NORMAL AND SIMULATED MICROGRAVITY." Reproduction, Fertility and Development 16, no. 2 (2004): 126. http://dx.doi.org/10.1071/rdv16n1ab7.
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We developed a method to evaluate bovine sperm membranes in normal (1G) and simulated microgravity (Sim-μG). Bovine spermatozoa are used as a model system because they have cellular membranes analogous to those of other cell types, and yet are much simpler because they have no cytoplasm and do not participate in DNA transcription or mRNA translation. They can be cultured as single cells and are easily evaluated for membrane characteristics using flow cytometry. These features make the mammalian spermatozoon a useful model for exploring the principles of membrane structure/function in the presence of a variety of environmental challenges such as simulated microgravity. Cryopreserved, washed beef bull sperm (4–8×106mL−1)were incubated under non-capacitating conditions (modified glucose-free Tyrode’s medium containing low bicarbonate, HEPES buffer, pyruvate and 3mgmL−1 BSA V; 23°C in air), and these spermatozoa remained alive for 24–48h at 1G. To simulate μG, spermatozoa were incubated under the same conditions, in a HARV 10 rotating wall vessel (RWV, Synthecon, Inc, Houston, TX, USA) at 9rpms. Spermatozoa were incubated in 1G and Sim-μG environments for 2.5–4.5h and subsequently exposed to 0, 60 or 80μgmL−1 LC for 0, 4, 8, 12, 16 and 20min. Three fluorochrome combinations were used as probes at each [LC]/time point: (1) propidium Iodide (dead status)+SYBR 14 (live status); (2) PI+FITC-PSA (acrosome reactions [ARs]); (3) PI+MitoTracker Deep Red (mitochondrial activity). Approximately 1million spermatozoa from 3 bulls were evaluated over 4 days. Data were acquired on a FACSVantage SE flow cytometer, and initially analyzed (quality control) using the bundled FACSVantage SE software package (Cell Quest, BD BioSciences, San Jose, CA, USA). This provided graphics of simple cell relations (fluorescence v. LC exposure time). For further statistical analysis, and incorporation of non-parametric statistical tools (including pattern recognition using Support Vector Machines), the data were processed using a collection of Perl scripts and C programs. Results: Live/dead status: When Sim-μG+60μgmL−1 LC sperm were compared to 1G+60μgmL−1LC, and 80μgmL−1 LC sperm, their profiles were more similar to the 1G 80μgmL−1 LC profiles. AR status: the Sim-μG+60μgmL−1 LC profiles were similar to the 1G+60μgmL−1 LC profiles. Mitochondrial Status: the Sim-μG+60μgmL−1LC profiles were more similar to 1G+80μgmL−1 LC profiles. Summary: although Sim-μG sperm lost their motility within 3h, they were alive. Cell profiles indicate that Sim-μG sperm nuclear membranes are less stable and their mitochondria are less functional than the 1G controls, but their acrosomes are intact indicating that fertilizing potential may remain. Additional experiments are needed to determine the time course for Sim-μG, induced changes, and whether Sim-μG sperm can penetrate eggs. Funding: NASA (2002)-Stennis-24 and The University of New Orleans.
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Alserihi, Read F., Mohammed Razeeth Shait Mohammed, Mohammed Kaleem, Mohammad Imran Khan, Mario Sechi, Vanna Sanna, Torki A. Zughaibi, Adel M. Abuzenadah, and Shams Tabrez. "Development of (−)-epigallocatechin-3-gallate-loaded folate receptor-targeted nanoparticles for prostate cancer treatment." Nanotechnology Reviews 11, no. 1 (December 28, 2021): 298–311. http://dx.doi.org/10.1515/ntrev-2022-0013.
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Abstract In continuation of our previous studies, we developed polymeric epigallocatechin 3-gallate (EGCG)-loaded nanoparticles (NPs) coupled with folic acid (FA), able to dually bind the human folate receptor alpha (FOLR1), and prostate-specific membrane antigen (PSMA+) in prostate cancer (PCa) model. After a preliminary computational molecular recognition of NP′ ligand binding on the FOLR1 active site, we synthesized the biocompatible block-copolymer PLGA–PEG–FA to prepare EGCG-targeted NPs (EGCG-T-NPs). The obtained NPs were characterized by various analytical techniques, and anticancer efficacy was determined by different sets of experiments in a 3D culture of PCa using PC3 and 22Rv1 cell lines. Results showed a significant reduction in spheroid size by EGCG-T-NPs, especially in PSMA+ (22Rv1) cells. The targeted NPs significantly enhanced the antiproliferative activity of EGCG against PCa cell lines, especially toward the PSMA+ cells, known to have higher FOLR1 expression. We did not observe any changes in the reactive oxygen species formation in both studied cell lines. However, significant changes in mitochondrial depolarization (15%) and polarization (18%) were recorded in response to EGCG-T-NP compared to control in 22Rv1. Similarly, EGCG-T-NP treatment also showed an increase in the number of dead apoptotic cells in 22Rv1 spheroids. Collectively, the obtained results support our hypothesis about the role of these targeted nanoprototypes in the increasing cellular uptake of EGCG payload into PCa cells, thus enhancing its antitumor efficacy.
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Nagata, Shigekazu. "Flippases and Scramblases at Plasma Membranes that Regulate Phosphatidylserine Exposure." Blood 126, no. 23 (December 3, 2015): SCI—31—SCI—31. http://dx.doi.org/10.1182/blood.v126.23.sci-31.sci-31.
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Abstract One of the hallmarks of apoptosis is the caspase-dependent exposure of phosphatidylserine (PtdSer) on cell surface, which is recognized by macrophages for engulfment of dead cells (1). How PtdSer is exposed to the cell surface had been elusive for a long time. We recently identified two membrane proteins (TMEM16F and Xkr8) that are involved in scrambling of phospholipids in plasma membrane (2, 3). TMEM16F carries 8 transmembrane regions, and requires Ca2+ to mediate phospholipid scrambling. It plays a role in the PtdSer-exposure in activated platelets for blood clotting, and patients of Scott Syndrome who suffer bleeding disorder carry a mutation in TMEM16F gene. Xkr8 is a protein carrying 6 transmembrane regions. Caspase 3 and 7 cleave off the C-terminal tail of Xkr8, and the cleaved Xkr8 promotes the PtdSer-exposure. In addition to the activation of scramblase, the flippase that translocates PtdSer from outer to inner leaflets was thought to be inactivated during apoptosis. In fact, we recently found that a pair of molecules, ATP11C of a P4-type ATPase and its chaperon CDC50A work as a flippase at plasma membrane (4, 5). ATP11C carries three caspase recognition sites in the middle of the molecule, and is cleaved during apoptosis. When ATP11C gene is mutated, the cells lose most of the flippase activity, but the asymmetrical distribution of PtdSer was still maintained at plasma membrane. Whereas, the cells lacking CDC50A completely lost the flippase activity and constitutively exposed PtdSer. The PtdSer-exposing living CDC50A-null cells were engulfed by thioglycollate-elicited macrophages, indicating that PtdSer exposed on the cell surface is necessary and sufficient to be recognized by macrophages for engulfment. Several molecules such as MFG-E8, Tim-4, Gas6, and Protein S specifically bind to PtdSer with high affinity, and promote the engulfment of PtdSer-exposing cells. However, how they work for the engulfment of apoptotic cells in certain macrophages has not been clear. We recently found that that resident peritoneal macrophages require both Tim4 and Protein S for engulfment, and Tim4, PtdSer-receptor, was involved in tethering of apoptotic cells, while Protein S promoted the engulfment of apoptotic cells by binding to MerTK, a tyrosine kinase receptor (6, 7). Here, I discuss how PdtSer is exposed during apoptotic cell death, and how dead cells are engulfed by macrophages. 1. Nagata S, Hanayama R, Kawane K. Autoimmunity and the clearance of dead cells. Cell. 2010;140:619-630. 2. Suzuki J, Umeda M, Sims PJ, Nagata S. Calcium-dependent phospholipid scrambling by TMEM16F. Nature. 2010;468:834-838. 3. Suzuki J, Denning DP, Imanishi E, Horvitz HR, Nagata S. Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science. 2013;341:403-406. 4. Segawa K, Suzuki J, Nagata S. Flippases and scramblases in the plasma membrane. Cell Cycle. 2014;13:2990-2991. 5. Segawa K, Kurata S, Yanagihashi Y, Brummelkamp T, Matsuda F, Nagata S. Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure. Science. 2014;344:1164-1168. 6. Nishi C, Toda S, Segawa K, Nagata S. Tim4- and MerTK-mediated engulfment of apoptotic cells by mouse resident peritoneal macrophages. Mol Cell Biol. 2014;34:1512-1520. 7. Toda S, Segawa K, Nagata S. MerTK-mediated engulfment of pyrenocytes by central macrophages in erythroblastic islands. Blood. 2014;123:3963-3971. Disclosures No relevant conflicts of interest to declare.
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Borrego, Francisco. "The CD300 molecules: an emerging family of regulators of the immune system." Blood 121, no. 11 (March 14, 2013): 1951–60. http://dx.doi.org/10.1182/blood-2012-09-435057.
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Abstract The CD300 family of molecules modulates a broad and diverse array of immune cell processes via their paired activating and inhibitory receptor functions. The description that CD300 molecules are able to recognize lipids, such as extracellular ceramide, phosphatidylserine, and phosphatidylethanolamine, that are exposed on the outer leaflet of the plasma membrane of dead and activated cells has opened a new field of research. Through their binding to lipids and other ligands, this family of receptors is poised to have a significant role in complex biological processes and in the host response to severe pathological conditions. Indeed, published data have demonstrated their participation in the pathogenesis of several disease states. Moreover, this family of receptors has great potential as targets for diagnosis and therapeutic purposes in infectious diseases, allergies, cancer, and other pathological situations. For instance, one member of the family, CD300a, has been studied as a possible biomarker. Here, a review is provided on the cellular distribution of the human and mouse families of receptors, the stimuli that regulate their expression, their ability to tune leukocyte function and immune responses, their signaling pathways, ligand recognition, and their clinical relevance.
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Corey, Daniel, and Irving L. Weissman. "Super Cross-Presentation of Tumor Antigens to Elicit Anti-Lymphoma Immunity By Synthetic Design of an Anti-Phosphatidylserine Bridge Protein." Blood 128, no. 22 (December 2, 2016): 1844. http://dx.doi.org/10.1182/blood.v128.22.1844.1844.
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Abstract Cell loss by apoptosis is a common feature in tumors. Dying tumor cells induce immune tolerance within the tumor microenvironment largely through highly conserved homeostatic clearance programs that restore tissue immune homeostasis and contribute to the formation of an immunosuppressive niche. The translocation of phosphatidylserine (PS) on cellular membranes, during the initial phases of apoptosis, functions as a recognition and removal signal that limits the immunogenicity of cell death. We examined whether altering clearance of dying cancer cells to elicit inflammatory turnover potentiates immune responses against lymphoma cells. To remove inhibitory signals in the homeostatic clearance pathway we utilized a molecular bridge scaffold to engineer a modified phosphatidylserine bridge protein (FA58C2-hIgG1 or C2-hIgG1) that works as a bridge between apoptotic cells expressing aminophospholipids and phagocytes bearing Fc receptors. In vitro administration of C2-hIgG1 to murine bone marrow derived macrophages promotes engulfment of apoptotic murine lymphoma cells (38C13 cell line) and ablates the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10) and suppression of pro-inflammatory cytokines tumor necrosis factor (TNF-α) and IL-12p40 to the presence of apoptotic cells. Similarly, uptake of C2-hIgG1 treated lymphoma cells triggers upregulation of the costimulatory markers CD80, CD86, and MHC class II on macrophages and promotes secretion of Th1-recruiting lymphocyte chemokines CXCL9, CXCL10, and CCL5. Accordingly, in vivo administration of C2-hIgG1 partially restores immune responses to dead lymphoma cells in antigen cross presentation assays and promotes recruitment and retention of tumor antigen specific CD8+ T cells, dendritic cells, and natural killer cells into tumors. These effects combine to elicit anti-lymphoma immunity, improve responses to immune checkpoint inhibitors, and enhance the effectiveness of adoptive T cell transfers using engineered T Cell Receptors (TCRs) but not CD19-directed chimeric antigen receptor engineered (CAR-T) T cells. Disclosures No relevant conflicts of interest to declare.
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Song, Gwonhwa, Jo-Ann G. W. Fleming, Jinyoung Kim, Thomas E. Spencer, and Fuller W. Bazer. "Pregnancy and interferon τ regulate DDX58 and PLSCR1 in the ovine uterus during the peri-implantation period." REPRODUCTION 141, no. 1 (January 2011): 127–38. http://dx.doi.org/10.1530/rep-10-0348.
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Interferon τ (IFNT), the pregnancy recognition signal in ruminants, abrogates the luteolytic mechanism for maintenance of the corpus luteum for production of progesterone (P4). This study examined the expression of DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58) and phospholipid scramblase 1 (PLSCR1) mRNAs in the ovine uterus as these genes were increased most in 2fTGH (STAT1 positive) cells by IFNT. The results of this study indicated that IFNT regulates expression ofDDX58andPLSCR1mRNAs in the ovine uterus, which confirmed the results of thein vitrotranscriptional profiling experiment with the 2fTGH (parental STAT1 positive) and U3A (STAT1 null) cell lines. Steady-state levels ofDDX58andPLSCR1mRNAs increased in cells of the ovine uterus between days 12 and 20 of pregnancy, but not between days 10 and 16 of the estrous cycle. The expression ofDDX58andPLSCR1mRNAs was greatest in endometrial stromal cells, but there was transient expression in uterine luminal and superficial glandular epithelial cells. P4alone did not induce expression ofDDX58andPLSCR1mRNAs; however, intrauterine injections of IFNT did induce expression ofDDX58andPLSCR1mRNAs in the endometria of nonpregnant ewes independent of effects of P4. These results indicate that IFNT induces expression ofDDX58andPLSCR1in ovine endometrial cells via the classical STAT1-mediated cell signaling pathway. Based on their known biological effects,DDX58andPLSCR1are IFN-stimulated genes, which may increase the antiviral status of cells of the pregnant uterus to protect against viral infection and/or enhance secretion of type I IFNs that inhibit viral replication.
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Gu, Lili, Anthony Fullam, Niamh McCormack, Yvette Höhn, and Martina Schröder. "DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to co-ordinate assembly of signalling complexes downstream from MAVS." Biochemical Journal 474, no. 4 (February 3, 2017): 571–87. http://dx.doi.org/10.1042/bcj20160956.
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The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon (IFN) induction downstream from antiviral pattern recognition receptors. It binds to TANK-binding kinase 1 and IκB-kinase-ε (IKKε), the two key kinases mediating activation of IFN regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKKε activation downstream from RIG-I and then links the activated kinase to IRF3. In the present study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TNF receptor-associated factor 3 (TRAF3) mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai virus (SeV) infection, both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the mitochondrial antiviral signalling (MAVS) adaptor, DDX3, IκB-kinase-ε (IKKε), TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting that it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented the formation of TRAF3–MAVS and TRAF3–IKKε complexes. Based on our data, we propose that early TRAF3 ubiquitination is required for the formation of a stable MAVS–TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that co-ordinates assembly of different TRAF3, IKKε and IRF3-containing signalling complexes downstream from MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling.
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Brooks, Pascal, Talke zur Bruegge, Erin C. Boyle, Stefan Kalies, Santiago Nahuel Villarreal, Andrea Liese, André Bleich, and Manuela Buettner. "CD14 and ALPK1 Affect Expression of Tight Junction Components and Proinflammatory Mediators upon Bacterial Stimulation in a Colonic 3D Organoid Model." Stem Cells International 2020 (February 1, 2020): 1–11. http://dx.doi.org/10.1155/2020/4069354.
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Cd14 and Alpk1 both encode pathogen recognition receptors and are known candidate genes for affecting severity in inflammatory bowel diseases. CD14 acts as a coreceptor for bacterial lipopolysaccharide (LPS), while ALPK1 senses ADP-D-glycero-beta-D-manno-heptose, a metabolic intermediate of LPS biosynthesis. Intestinal barrier integrity can be influenced by CD14, whereas to date, the role of ALPK1 in maintaining barrier function remains unknown. We used colon-derived 3D organoids, first characterised for growth, proliferation, stem cell markers, and expression of tight junction (TJ) components using qPCR and immunohistochemistry. They showed characteristic crypt stem cells, apical shedding of dead cells, and TJ formation. Afterwards, organoids of different genotypes (WT, Il10-/-, Cd14-/-, and Alpk1-/-) were then stimulated with either LPS or Escherichia coli Nissle 1917 (EcN). Gene expression and protein levels of cytokines and TJ components were analysed. WT organoids increased expression of Tnfα and tight junction components. Cd14-/- organoids expressed significantly less Tnfα and Ocln after LPS stimulation than WT organoids but reacted similarly to WT organoids after EcN stimulation. In contrast, compared to WT, Alpk1-/- organoids showed decreased expression of different TJ and cytokine genes in response to EcN but not LPS. However, Western blotting revealed an effect of ALPK1 on TJ protein levels. These findings demonstrate that Cd14, but not Alpk1, alters the response to LPS stimulation in colonic epithelial cells, whereas Alpk1 is involved in the response upon bacterial challenge.
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Cao, Xin-xin, Jian Sun, Jian Li, Ding-rong Zhong, Na Niu, Ming-hui Duan, Zhi-yong Liang, and Dao-bin Zhou. "Evaluation of Clinicopathologic Characteristics and the BRAF V600E Mutation in Erdheim-Chester Disease Among Chinese Adults." Blood 126, no. 23 (December 3, 2015): 3870. http://dx.doi.org/10.1182/blood.v126.23.3870.3870.
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Abstract Objectives Erdheim-Chester disease (ECD) is a rare form of histiocytosis with a broad, non-specific clinical spectrum. Here, we retrospectively evaluated the clinical and pathologic characteristics, presence of the BRAF V600E mutation, treatment options and outcomes of Chinese patients diagnosed with ECD at our center. Methods Patients diagnosed with ECD between January 2010 and April 2015 at Peking Union Medical College Hospital were included for study. We evaluated baseline characteristics, reviewed histological material, and tested for the presence of the BRAF V600E mutation using immunohistochemistry and polymerase chain reaction (PCR). Results Sixteen patients were diagnosed with ECD. Median age at diagnosis was 47 years (range, 22-61 years). Median disease duration (from the first symptom to diagnosis) was 22.5 months (range, 3-100 months). The main sites of involvement included bone (93.8%), cardiovascular region (43.8%), skin (31.3%), central nervous system (25.0%), and ¡°hairy kidney¡± (25%). Thirteen patients displayed characteristic histological features, including foamy histiocyte infiltration of polymorphic granuloma and fibrosis or xanthogranulomatosis, with CD68-positive and CD1-¦Á- negative immunostaining. Three patients (designated 3, 5 and 10) displayed CD68-positive and CD1¦Á- negative histiocyte infiltration, but not the above histological characteristics, and were thus initially misdiagnosed as Rosai-Dorfman disease. All three cases were BRAFV600E mutation-positive, leading to revision of diagnosis as ECD. Diagnosis of ECD in each case was additionally supported by typical radiographic findings. The BRAF V600E mutation was detected in 68.8% patients using PCR and 50.0% patients with immunohistochemistry. Ten patients (62.5%) received IFN-¦Á as first-line treatment, 3 patients showed improvement, 3 remained stable, 3 were too early for evaluation and 1 died. Three patients (5, 10 and 11) underwent transsphenoidal pituitary lesion surgery but were not subjected to systemic treatment, owing to the absence of symptoms and disease activity post-surgery and remained stable after a median of 16 months (range, 6-30 months) from diagnosis. Thirteen patients (81.3%) were still alive at median follow-up of 14.5 months. Conclusion ECD remains a largely overlooked disease, and increased recognition by clinicians and pathologists is necessary for effective diagnosis and treatment. The presence of the BRAF V600E mutation may facilitate discrimination of ECD from other non-Langerhans cell histiocytoses. Table 1. Characteristics and treatment of 16 patients with ECD Patient Sex/ age, years Disease duration, mo Main sites of involvement BRAF IH BRAF V600E Therapy Vital Status OS£¬mo 1 M/33 5 B N/A - IFN-6 MIU 3/wk Alive 15 2 M/22 43 S, B - - IFN-3 MIU 3/wk Alive 11 3 M/25 18 B, LN, CNS - + Pred Dead 13 4 F/28 3 S, B + + None Alive 16 5 M/60 27 B, PIT + + Surgery Alive 15 6 F/61 5 B, H, LV, R£¬CNS, MS, S N/A + IFN-6 MIU 3/wk Dead 25 7 F/23 67 S, B, H, LV - - IFN-3 MIU 3/wk Alive 19 8 M/60 43 B, P, LV, R N/A + IFN-6 MIU 3/wk Alive 14 9 M/46 84 CNS, B + + IFN-6 MIU 3/wk Alive 22 10 F/51 7 PIT + + Surgery Alive 6 11 F/36 72 PIT, B + + Surgery Alive 30 12 M/55 100 B, S, CNS, PIT - + IFN-6 MIU 3/wk Alive 3 13 F/50 11 B, H N/A + IFN-6 MIU 3/wk Alive 5 14 F/46 8 B, LV, P + + IFN-6 MIU 3/wk Alive 1 15 M/52 30 B, LV, R, P£¬E - - IFN-6 MIU 3/wk Alive 1 16 M/47 4 B, LV, R, LN - - None Dead 36 Age is at diagnosis£»disease duration is from the first symptom to diagnosis IH, immunohisochemistry; B, long bones; LN, lymph nodes; LV, large vessels; H, heart; S, skin; CNS, central nervous system; MS, maxillary sinus; PIT, pituitary gland; R, retroperitoneal; P, pericardial effusion; E, Exophthalmos; MIU, million international units; N/A, not available Disclosures No relevant conflicts of interest to declare.
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Ueda, Ryosuke, Kenta Narumi, Reina Miyakawa, Hisayoshi Hashimoto, and Kazunori Aoki. "Natural Killer Cell-Mediated Antitumor Effect of Syngeneic Hematopoietic Stem Cell Transplantation." Blood 124, no. 21 (December 6, 2014): 1104. http://dx.doi.org/10.1182/blood.v124.21.1104.1104.
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Abstract Introduction: Autologous hematopoietic stem cell transplantation (HSCT) has been thought to be important in tumor eradication induced by high-dose chemotherapy with hematopoietic stem cell rescue. It was reported recently that autologous HSCT can also induce a strong antitumor immunity by homeostatic proliferation (HP) of T cells following preconditioning-induced lymphopenia. It is known that T cell HP is driven by the recognition of self antigens, and that the availability of tumor antigens during HP leads to effective antitumor autoimmunity with specificity and memory. The effect is mediated by a reduction in the activation threshold of low-affinity tumor-specific T cells, leading to their preferential engagement and expansion. However, the mechanisms of antitumor immunity induced by the autologous HSCT are not fully understood. Especially, in addition to adaptive immunity, the role of innate immunity is unclear. In this study, we examined the role of natural killer (NK) cells on the antitumor effect in HSCT recipients using mouse xenograft models. Furthermore, since neutrophils have been reported to be required for homeostasis, maturation, and function of NK cells in human and mice (Jaeger et al. J. Eep. Med. 209:565, 2012), we focused on the interaction between neutrophils and NK cells in tumors after syngeneic HSCT. Results: BALB/c mice were injected subcutaneously with CT26 murine colon cancer cells shortly after lethal irradiation (9Gy), and then bone marrow cells (5 x 106 cells) and splenocytes (4 x 106 cells) from syngeneic donor mice were infused into the recipient mice. Tumor growth was significantly suppressed in the HSCT recipients as previously reported (Narumi et al. Gene Ther 19:34, 2012, Udagawa et al. J. Immunol 191:3440, 2013). To clarify the contribution of NK cells to antitumor effects after syngeneic HSCT, we depleted NK cells by intraperitoneal injection of anti-asialo GM1 antibody in the HSCT recipients. The tumor growth inhibition was largely cancelled by the depletion of NK cells especially during an early period after HSCT (Figure 1a: 2303 mm3 vs. 1383 mm3 at day26; P = 0.0042). Flow cytometry showed that the frequency of surface CD107a+ NK cells, which is an activation marker, was significantly higher in tumors of HSCT recipients (24.4% vs. 6.3%; P = 0.0022) than that in non-HSCT mice, indicating that syngeneic HSCT induces NK cell activation which contributes to the antitumor effect. Next, we examined what factor influences the activation of NK cells in tumors after syngeneic HSCT. We found that a large number of neutrophils accumulated in tumors after HSCT by immunostaining. Thus, we focused on the relationship between neutrophils and NK cells in tumors after HSCT. The cytokine antibody array showed that lungkine (CXCL15), which is one of mouse ELR+ CXC chemokine and recruits neutrophils to lung airspaces during pneumonia, was markedly up-regulated in tumor at day14 after HSCT as compared with non-HSCT tumor. The real-time quantitative polymerase chain reaction confirmed that the lungkine mRNA level was higher in HSCT-tumor at day 6 than that in non-HSCT tumor (Figure 1b: relative expression: 4.0 vs. 0.3; P = 0.0075). Then, to investigate the effects of neutrophils on NK cells in tumors, we depleted neutrophils by an intraperitoneal injection of anti-Ly6G antibody from days 6 to 12 after syngeneic HSCT. Neutrophils were almost depleted in the spleens and tumors. The tumor growth was significantly suppressed in HSCT recipients as previously shown, whereas the depletion of neutrophil significantly increased tumor volumes in HSCT recipients but not in non-HSCT mice at day 10 (Figure 1c: 143 mm3 vs. 84 mm3; P = 0.024). Flow cytometry revealed that the neutrophil-depletion did not change the frequency of NK cells within live cells in tumors at days 14 and 21, however, neutrophil-depletion increased the fraction of dead NK cells at day 14 (Figure 1d: 32.2% vs. 19.2%; P = 0.042). Thus, neutrophils in tumors may prevent NK cells from cell death induction during HP, leading to the significant antitumor effect. Conclusions: NK cells play a major role in the antitumor effect in the early period after syngeneic HSCT. The neutrophils in tumors may support a sustained antitumor effect of NK cells. This novel relationship reveals an important aspect of antitumor immunity induction in HSCT recipients and may contribute to the development of effective therapeutic strategy for cancer using HSCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Ferreira, Alexandra G., Olga Zimmermannova, Ervin Ascic, Ilia Kurochkin, Diego Soto-Cabrera, Ariane Eceiza, Hreinn Benonisson, et al. "Abstract A40: Restoring tumor immunogenicity with dendritic cell reprogramming." Cancer Immunology Research 10, no. 12_Supplement (December 1, 2022): A40. http://dx.doi.org/10.1158/2326-6074.tumimm22-a40.
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Abstract Immunotherapy is revolutionizing cancer treatment, but success is limited to a fraction of patients. Tumor immunosurveillance and immunotherapy relies on presentation of tumor-associated antigens by conventional dendritic cells type 1 (cDC1). However, tumors develop mechanisms to avoid immune recognition such as downregulation of antigen presentation and exclusion of cDC1. We have previously demonstrated that enforced expression of the transcription factors PU.1, IRF8 and BATF3 (PIB) imposes the lineage conversion of fibroblasts to cDC1 by direct cell reprogramming. Here, we hypothesize that PIB reprograms cancer cells directly into functional tumor-antigen presenting cells (tumor-APCs) with enhanced immunogenicity. First, we show that enforced expression of PIB in a wide range of murine and human cancer cells from different origins is sufficient to induce surface expression of hematopoietic and DC-lineage specific markers (CD45 and Clec9a). Moreover, reprogramming restored the expression of antigen presentation complexes (MHC-I and MHC-II) and activated the expression of the co-stimulatory molecules CD40, CD80 and CD86, required for productive T cell activation. Transcriptomic analysis using mRNA-sequencing showed that PIB imposes a global cDC1 gene signature and an antigen presentation program in tumor cells as early as day 3 of reprogramming, overriding the original cancer cell program. Furthermore, Assay for Transposase-Accessible Chromatin (ATAC) sequencing analysis revealed that PIB-mediated cDC1 reprogramming elicited rapid epigenetic remodeling followed by gradual rewiring of transcriptional program and stabilization of cDC1 identity. Functionally, tumor-APCs present endogenous antigens on MHC-I, prime naïve CD8+ T and become prone to CD8+ T cell mediated killing. Tumor-APCs secrete pro-inflammatory cytokines (IL-12) and chemoattractants (CXCL10), uptake and process exogenous antigens, phagocyte dead cells, and cross-present exogenous antigens to activate naïve T-cells. In addition, reprogrammed tumor cells harboring TP53, KRAS and PTEN mutations downregulated proliferation and showed impaired tumorigenicity in vitro and in vivo. Importantly, we show that intra-tumoral injection of reprogrammed tumor-APCs elicited tumour growth control in vivo alongside increasing infiltration of CD8+ T and NK cells in B16-OVA tumors. Finally, we showed that our approach can be employed to convert primary cancer cells derived from melanoma, lung, breast, pancreatic, urothelial, and head and neck carcinomas as well as cancer associated fibroblasts. In summary, we provide evidence for the direct reprogramming of tumor cells into immunogenic cDC1-like cells, with restored antigen presentation capacity and the ability to reinstate anti-tumor immunity. Our approach elicits the immune system against cancer and counteract major tumor evasion mechanisms including tumor heterogeneity and impaired antigen presentation, laying the foundation for developing immunotherapeutic strategies based on the cellular reprogramming of human cancer cells. Citation Format: Alexandra G. Ferreira, Olga Zimmermannova, Ervin Ascic, Ilia Kurochkin, Diego Soto-Cabrera, Ariane Eceiza, Hreinn Benonisson, Inês Caiado, Rita Silvério-Alves, Fábio F. Rosa, Cristiana F. Pires, David Gomez-Jimenez, Carina Bernardo, Monika Bauden, Roland Anderson, Mattias Höglund, Kenichi Miharada, Yukio Nakamura, Lennart Greiff, Malin Lindstedt, Carlos-Filipe Pereira. Restoring tumor immunogenicity with dendritic cell reprogramming [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A40.
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Perl, A., R. J. Looney, D. H. Ryan, and G. N. Abraham. "The low affinity 40,000 Fc gamma receptor and the transferrin receptor can be alternative or simultaneous target structures on cells sensitive for natural killing." Journal of Immunology 136, no. 12 (June 15, 1986): 4714–20. http://dx.doi.org/10.4049/jimmunol.136.12.4714.
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Abstract The role of the low avidity 40,000 dalton receptor for IgG (Fc gamma R) present on K562 and U937 cells in sensitivity to natural killing (NK) was studied by using a murine monoclonal antibody (mAb) specific for the 40,000 dalton Fc gamma R (alpha Fc gamma R mAb). Pretreatment of K562 target cells with intact alpha Fc gamma R mAb or its Fab fragment or anti-transferrin receptor (alpha TFR) mAb partially blocked in a dose-dependent manner, NK activity to K562 cells. However, combined pretreatment with alpha Fc gamma R and alpha TFR mAb completely blocked NK activity against K562 targets. As compared with K562 cells, lower levels of NK were elicited against Molt-4, U937, HL-60, and Daudi targets. Although NK activity to Molt-4 targets was not affected by alpha Fc gamma R mAb, it was fully prevented by pretreatment with alpha TFR mAb. In contrast, NK to U937 cells was not influenced by alpha TFR mAb, but it was strongly inhibited by alpha Fc gamma R mAb. The resistance of 3H-TdR-prelabeled adherent HEp-2 cells to natural cell-mediated cytotoxicity was not affected by either mAb. Lectin-dependent cell-mediated cytotoxicity (LDCC) against HEp-2 cells due to the presence of concanavalin A, and was completely abrogated by pretreatment of the targets with alpha TFR mAb, but was unaffected by alpha Fc gamma R mAb. By use of the flow cytometer, a significant correlation was detected between the relative expression of 40,000 dalton Fc gamma R and the susceptibility to NK, whereas the expression of TFR was discordant from NK sensitivity. As determined in the single cell cytotoxicity assay alpha Fc gamma R mAb reduced the frequency of target binding effector cells without affecting the number of dead bound targets. This pattern of inhibition was found against both K562 and U937 targets. Alternatively, alpha TFR mAb inhibited both binding and killing of K562 and Molt-4 targets. Because pretreatment of HEp-2 cells with alpha TFR mAb did not influence conjugate formation, the blocking of LDCC to HEp-2 cells by alpha TFR mAb can be related to post-binding events. These data show that although both the 40,000 dalton Fc gamma R and the TFR can be target structures for NK cell recognition, the TFR may also play an important role in the post-binding events.
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Li, XingJun, Charles Goodwin, Zhenyun Yang, Sarah C. Nabinger, Briana Richine, Gordon Chan, Helmut Hanenberg, et al. "Macrophage NADPH Oxidase Activation and ROS Production Is Positively Regulated By Shp2 Phosphatase Function." Blood 124, no. 21 (December 6, 2014): 1397. http://dx.doi.org/10.1182/blood.v124.21.1397.1397.
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Abstract Macrophages are professional phagocytic cells, and express pattern recognition receptors such as C-type lectins and integrins for the detection of invading pathogens. Both Dectin-1 (a C-type lectin) and complement receptor 3 (CR3, a β2-integrin) are expressed on innate immune cells including macrophages, neutrophils, and dendritic cells. Dectin-1 stimulation by b-glucan-containing particles (zymosan) and CR3 stimulation by serum opsonized zymosan (SOZ) activate Erk- and Akt-dependent signaling resulting in phagocytosis and production of an oxidative burst. Shp2, a protein tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt signaling, supports hematopoietic development, and is commonly mutated in juvenile myelomonocytic leukemia (JMML). However, no studies have examined the role of Shp2 in Dectin-1- or CR3-stimulated NADPH oxidase activation or ROS production. As activation of Erk and Akt stimulates NADPH oxidase by phosphorylating p47phox, we hypothesized that Shp2 positively regulates ROS production in response to Dectin-1 or CR3 stimulation. Using murine peritoneal exudate macrophages (PEMs), both zymosan and SOZ exposure induced maximal ROS production 10 minutes post-stimulation, which corresponded to maximal induction of Shp2 phosphorylation (Y580, proposed to promote Shp2 phosphatase activity) and Erk phosphorylation. Using bone marrow derived macrophages (BMMs) from mice bearing a conditionally deleted allele of Ptpn11 (Shp2flox/flox;Mx1Cre+), ROS production was significantly reduced in response to zymosan and SOZ in Shp2flox/flox;Mx1Cre+ BMMs compared to control Shp2flox/flox;Mx1Cre- BMMs. Notably, the phagocytic index of the Shp2flox/flox;Mx1Cre+ and Shp2flox/flox;Mx1Cre- BMMs was similar, and protein components of the NADPH oxidase complex (p40phox, p67phox, and p47phox) were expressed at similar levels. To define the biochemical role of Shp2 in ROS production, we generated yellow fluorescent protein (YFP)-tagged Shp2 constructs bearing mutation of the N-SH2 (R32K) or phosphatase (C463A) domain and retrovirally expressed these constructs in murine BMMs. When subjected to zymosan or SOZ stimulation, mutation of either the N-SH2 or phosphatase domain resulted in reduced ROS production. Using time-lapse confocal videomicroscopy, we found that Shp2-R32K-YFP failed to translocate to the phagosome in SOZ-stimulated BMMs; however, phosphatase dead Shp2-C463A-YFP strongly translocated to the phagosome despite producing lower ROS levels. These findings specifically pointed to Shp2 phosphatase function as crucial in positively regulating NADPH oxidase and ROS production. Accordingly, we reasoned that macrophages expressing JMML-associated gain-of-function (GOF) Shp2 mutants, characterized to have increased phosphatase activity, would produce elevated ROS levels. As anticipated, BMMs retrovirally expressing GOF Shp2-D61Y or GOF Shp2-E76K and PEMs from mice bearing a conditionally induced gain-of-function allele of Ptpn11 (Shp2D61Y/+;Mx1Cre+) similarly produced significantly elevated levels of zymosan- and SOZ-stimulated ROS compared to WT Shp2-expressing BMMs or PEMs, respectively. Given the positive role of Shp2 phosphatase in promoting zymosan- and SOZ-stimulated ROS production, we investigated putative Shp2 substrates in response to zymosan stimulation. SHPS-1 (SH2 domain-containing protein tyrosine phosphatase substrate 1) is a myeloid inhibitory immunoreceptor expressed on macrophages, requires tyrosine phosphorylation to exert its inhibitory effect, and has been shown to be de-phosphorylated by Shp2. Consistent with its potential function in regulation ROS production, SHPS-1 is strongly associated with phagosomes in zymosan-stimulated PEMs. In immunoblot analysis, reduced phospho-SHPS-1 levels kinetically correlated with maximal zymosan-stimulated Shp2 phosphorylation and ROS production, and increased levels of phospho-SHPS-1 were found in BMMs expressing phosphatase dead Shp2-C463A compared to cells expressing WT Shp2. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated NADPH oxidase activation and ROS production in macrophages, and that mechanistically, Shp2 may exert its positive effect by de-phosphorylating and thus negatively regulating the inhibitory function of SHPS-1. Disclosures No relevant conflicts of interest to declare.
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Yun, Hyun Don, Dawn Schirm, Martin Felices, Jeffrey S. Miller, and Craig E. Eckfeldt. "Cyclin-Dependent Kinases (CDK) Signaling Blockade Potentiates NK Cell Mediated Cytotoxicity Against Acute Myelogenous Leukemia." Blood 132, Supplement 1 (November 29, 2018): 4538. http://dx.doi.org/10.1182/blood-2018-99-113004.
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Abstract Dinaciclib, a CDK 1, 2, 5, and 9 inhibitor, has been shown to have potent anti-cancer activity against several malignancies. Our group previously demonstrated a direct RALB-dependent proapoptotic activity of dinaciclib against acute myeloid leukemia (AML) (Oncogene (2017) 36, 3263-3273). Furthermore, there is emerging evidence that dinaciclib can influence the recognition and killing of cancer cells by the immune system suggesting that it could have multimodal anti-cancer activities. As natural killer (NK) cells have a critical role in antineoplastic cytotoxicity, we hypothesized that dinaciclib may influence NK cell mediated cytotoxicity against human AML cells. To address this hypothesis, we evaluated the effects of dinaciclib on the NK cytotoxicity against human AML target cells and expression of known regulators of NK cell function. Specifically, we treated THP-1 and KG-1 cells with vehicle or dinaciclib at 5-20 nM for 24 hours, then washed the AML targets and added purified NK cells from PBMCs of healthy donors at an effector: target (E:T) ratio of 2:1. After 24 hours, viable AML target cells were enumerated using Fixable Aqua Dead Cell Stain and AccuCheck Counting Beads (Thermo Fisher Scientific) by flow cytometry. Relative NK cell target killing (%) was calculated by comparing the percentage of AML targets killed with drug treatment combined with donor NK cells relative to drug treatment alone in the absence of NK cells (Relative NK killing %). We assessed NK cell activation by measuring intracellular interferon-ɣ production and CD107a expression as well as NK ligand expression on dinaciclib-treated AML targets using flow cytometry. Dinaciclib treatment of AML targets enhanced the relative NK killing % for both KG-1 and THP-1 AML targets compared to control treatment (for KG-1, 45.6% vs 14.5%, p=0.05; for THP-1, 63.7% vs 31.7%, p=0.01) (Figure 1A, 1B) and was associated with enhanced NK cell degranulation as measured by CD107a expression (DMSO group, 8.3% vs dinaciclib 20nM group, 17.7%, p=0.002) and inflammatory cytokine production as measured by intracellular interferon-ɣ expression (DMSO group, 10.4% vs dinaciclib 20nM group 17.3%, p=0.01) (Figure 1C, 1D) supporting the ability of dinaciclib to sensitize AML targets for NK cell-based immunotherapy. To investigate potential mechanisms that promote NK cell activation by dinaciclib-treated AML targets, we assessed expression of several key NK cell ligands on AML targets and found that dinaciclib treatment led to decreased expression of inhibitory ligands including HLA class I, CD112, CD155 and increased expression in activating ligands such as TRAILR1, CD48 (Figure 1E, 1F) providing potential mechanistic insights. Notably, preliminary studies using a primary, patient-derived AML sample treated with dinaciclib resulted in a similar increase in the proportion of CD107a+ NK cells compared to the control group (44.7% increase, p=0.02, Figure 1G, 1H). While the detailed mechanisms for our findings remain to be determined, this is the first report to our knowledge that inhibiting CDK signaling can sensitize AML targets to NK cell based immunotherapy, a completely novel treatment approach. Further studies are ongoing to investigate the potential for combined targeted therapy with dinaciclib and adoptive NK cell therapy including patient derived xenograft (PDX) mice to validate its translational potential. Disclosures Felices: GT Biopharma: Research Funding.
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Glickman, Michael H., and Aaron Ciechanover. "The Ubiquitin-Proteasome Proteolytic Pathway: Destruction for the Sake of Construction." Physiological Reviews 82, no. 2 (April 1, 2002): 373–428. http://dx.doi.org/10.1152/physrev.00027.2001.
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Between the 1960s and 1980s, most life scientists focused their attention on studies of nucleic acids and the translation of the coded information. Protein degradation was a neglected area, considered to be a nonspecific, dead-end process. Although it was known that proteins do turn over, the large extent and high specificity of the process, whereby distinct proteins have half-lives that range from a few minutes to several days, was not appreciated. The discovery of the lysosome by Christian de Duve did not significantly change this view, because it became clear that this organelle is involved mostly in the degradation of extracellular proteins, and their proteases cannot be substrate specific. The discovery of the complex cascade of the ubiquitin pathway revolutionized the field. It is clear now that degradation of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a variety of basic pathways during cell life and death as well as in health and disease. With the multitude of substrates targeted and the myriad processes involved, it is not surprising that aberrations in the pathway are implicated in the pathogenesis of many diseases, certain malignancies, and neurodegeneration among them. Degradation of a protein via the ubiquitin/proteasome pathway involves two successive steps: 1) conjugation of multiple ubiquitin moieties to the substrate and 2) degradation of the tagged protein by the downstream 26S proteasome complex. Despite intensive research, the unknown still exceeds what we currently know on intracellular protein degradation, and major key questions have remained unsolved. Among these are the modes of specific and timed recognition for the degradation of the many substrates and the mechanisms that underlie aberrations in the system that lead to pathogenesis of diseases.
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Shah, Mrinal Y., Eva Martinez, Relja Popovic, Teresa Ezponda, Eliza C. Small, Christine Will, Paola Neri, Nizar J. Bahlis, and Jonathan D. Licht. "MMSET/WHSC1 Enhances DNA Damage Repair Leading To An Increase In Resistance To Chemotherapeutic Agents." Blood 122, no. 21 (November 15, 2013): 808. http://dx.doi.org/10.1182/blood.v122.21.808.808.
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Abstract MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, and is believed to be the driving factor in the pathogenesis of this subtype of MM. Overexpression of MMSET also occurs in solid cancers, including neuroblastoma, colon and prostate. MMSET overexpression in MM and prostate cells leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3). This altered epigenetic landscape is accompanied by changes in proliferation, gene expression, and chromatin accessibility. Prior work linked methylation of histones, including H3K36, to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET might play a role in DNA damage repair and response. To investigate the role of MMSET in DNA damage repair, we transfected U2OS cells with a linearized vector expressing a neomycin-resistant gene. In the presence of G418, only cells that are able to integrate this plasmid through non-homologous end joining (NHEJ) can survive. siRNA knockdown of MMSET led to a decrease in cell survival, suggesting that MMSET is necessary for efficient DNA repair. We also used U2OS cells engineered to express the AsiSI enzyme fused to an estrogen receptor hormone-binding domain. Upon tamoxifen treatment, double strand breaks (DSBs) are induced at multiple AsiSI recognition sites, accompanied by an increase in γH2AX foci. The extent of repair after AsiSI-induced damage was ascertained by the ability of a DNA fragment that spans a specific cut site to be PCR amplified. With MMSET knockdown, there was a >10 fold increase in unrepaired DNA. ChIP analysis showed that with the depletion of MMSET, γH2AX persisted at the cut site. ChIP for specific effectors of DNA damage showed a marked decrease of recruitment of CtIP and RAD51 to the DSB. However, immunoblot analysis showed that CtIP and RAD51 levels were drastically decreased with MMSET depletion, thus explaining the loss of their recruitment to DSBs. In contrast, XRCC4 levels were maintained with MMSET siRNA, but its recruitment to the DSB decreased. CtIP is important for both NHEJ and homologous recombination (HR), RAD51 is critical for HR, and XRCC4 is necessary for NHEJ, suggesting that MMSET is important in multiple pathways of DNA repair. To study the effect of MMSET in MM, we used the t(4;14)+ KMS11 cell line, NTKO, and genetically matched TKO cells in which the overexpressed MMSET allele was knocked out. NTKO cells have elevated levels of DNA damage at baseline, as measured by a comet assay and by the presence of elevated numbers of 53BP1-positive foci. Upon addition of the DNA damaging agent melphalan, NTKO cells showed increased damage as measured by an increase in the tail moment by the comet assay. Paradoxically, upon treatment of these cells with the DNA damaging agents, NTKO cells survived better than TKO cells. NTKO repaired DNA damage at an enhanced rate and continued to proliferate after a significant DNA damage insult, whereas TKO cells accumulated DNA damage and entered cell cycle arrest. We repleted TKO cells with constructs expressing either wild-type MMSET or an HMT-dead (Y1118A) isoform. Upon treatment, cells expressing the wild-type MMSET have showed enhanced DNA repair and continued proliferation after DNA damage, whereas cells expressing the HMT-dead protein repaired DNA damage more slowly and entered cell cycle arrest. The HMT activity of MMSET was critical for the induction of expression of genes required for multiple DNA repair pathways including CHEK2, DDB2, DDIT3, RAD51, and MRE11, again suggesting that MMSET modulates DNA repair by affecting expression of critical components of the repair machinery. The clinical relevance of these finds becomes more apparent in vivo. Luciferase-tagged KMS11 cells harboring doxycycline-inducible MMSET shRNA were injected into nude mice. After one week, mice were treated with doxycycline and injected with melphalan or saline. Knockdown of MMSET or melphalan treatment alone decreased tumor growth but eventually all mice had progressive disease. Only when MMSET was knocked down and chemotherapy given were the mice rendered tumor free. These findings indicate a new mechanism for the ability of MMSET to enhance DNA repair and identify the protein as a potential therapeutic target in MM and other cancers. Disclosures: No relevant conflicts of interest to declare.
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Koh, Won Kyun, Hamza Celik, Jacob Tao, Jake Fairchild, Ostap Kukhar, Christine R. Zhang, Paul Gontarz, and Grant A. Challen. "DNMT3A Regulates Hematopoietic Stem Cell Function Via DNA Methylation-Independent Functions." Blood 138, Supplement 1 (November 5, 2021): 24. http://dx.doi.org/10.1182/blood-2021-153759.
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Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is strictly regulated to sustain blood production throughout adult life. De novo DNA methyltransferase 3-alpha (DNMT3A) is one of the major epigenetic regulators that is essential for efficient HSC differentiation. DNMT3A mutations are prevalent in myeloid diseases that include acute myeloid leukemia (AML; ~22%) and myelodysplastic syndrome (MDS; ~10%) where they act as initiating events However, the precise molecular mechanisms of how DNMT3A regulates normal hematopoiesis and its mutations prime HSCs for leukemic formation are unclear. Although DNMT3A is described as a DNA methyltransferase enzyme, the lack of consistent correlation between changes in DNA methylation and differential gene expression in Dnmt3a-null HSCs in mouse models, and AML patients with DNMT3A mutations undermine the conventional understanding of DNMT3A's canonical role in hematopoietic cells. Hence, we hypothesized that DNMT3A may have novel functions outside of DNA methylation that regulate HSC fate decisions. To answer this question, we first ectopically expressed GFP-labeled Dnmt3a constructs (wild-type Dnmt3a, Dnmt3aE752A; complete DNA methylation dead, and Dnmt3aR832A; reduced DNA methylation target recognition) and empty vector (negative control) in Dnmt3a-null (Vav-Cre: Dnmt3afl/fl = Dnmt3a-/- in hematopoiesis) bone marrow (BM) cells. The result showed that similar to restoring wild-type Dnmt3a, ectopic expression of Dnmt3aE752A as well as Dnmt3aR832A showed a rescue effect of decreased engraftment of transduced cells in the peripheral blood as well as reduced HSC numbers in the BM. Analysis of DNA methylation by whole-genome bisulfite sequencing (WGBS) in transduced cells showed this phenotypic and functional rescue of the Dnmt3a-/- phenotype occurred in the absence of restored DNA methylation patterns. To study the importance of Dnmt3a-mediated DNA methyltransferase activity in a more physiological system, we generated knock-in mice that have one copy of either wild-type Dnmt3a, Dnmt3aE752A, or Dnmt3aR832A (CAGG-Cre-ER T2 = ER T2-Cre: Dnmt3afl/+, Dnmt3afl/E752A, and Dnmt3afl/R832A) to be compared to the Dnmt3a-null group (ER T2-Cre: Dnmt3afl/-). These mice contain one allele with loxP-flanked Dnmt3a that is deleted by tamoxifen-inducible Cre-mediated recombination and one allele of either wild-type Dnmt3a, Dnmt3aE752A, Dnmt3aR832A, or germline knockout Dnmt3anull. 5-weeks post-tamoxifen (~93% floxed allele recombination), competitive transplantation of 250 phenotypically defined test HSCs against with 2.5x10 5 congenic competitor BM cells was performed. Dnmt3a fl/R832A recipients had higher engraftment (35.6 % +/- 6.1) than Dnmt3afl/+ (28.5% +/- 7.2) and Dnmt3afl/- (10.7% +/- 2.79), while Dnmt3afl/E752A had slightly higherengraftment (12.5% +/- 3) than Dnmt3afl/-. Analysis of the BM 18 weeks post-transplant showed that Dnmt3afl/E752A and Dnmt3afl/R832A HSCs phenocopied the HSC self-renewal potential phenotype of heterozygous Dnmt3a fl/+HSCs (Fig. 1). The absolute count of donor-derived HSCs per mouse after the transplant were: ER T2-Cre control (675.7 +/- 299.3), Dnmt3afl/+ (1870 +/- 961.4), Dnmt3afl/- (3546 +/- 1019), Dnmt3afl/E752A (1130 +/- 362.7), and Dnmt3afl/R832A (1184 +/- 344.5) (mean +/- S.E.M.). While the described clonal expansion of Dnmt3a-null HSCs was observed, HSCs with one copy of full-length Dnmt3a but devoid of its methyltransferase capacity mimicked the heterozygous state rather than the homozygous loss-of-function. This is the first evidence to suggest that DNMT3A potentially regulates HSCs by non-canonical (DNA methylation independent) mechanisms. DNA methylation analysis by WGBS is ongoing to determine if Dnmt3afl/E752A and Dnmt3afl/R832A HSCs show a methylome comparable to Dnmt3a-null HSCs whilst having the functional potential of Dnmt3a-heterozygous HSCs, which will be complemented with other molecular analyses including gene expression. Our study opens new avenues for investigations into the molecular mechanisms of DNMT3A function in HSC biology, which could ultimately benefit clinical practice by identifying new therapeutic approaches for the patients with DNMT3A mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Siragusa, Sergio, William Morice, Morie A. Gertz, Robert Kyle, Philip R. Greipp, John A. Lust, Thomas E. Witzig, et al. "Asymptomatic Amyloidosis at the Time of Diagnostic Bone Marrow Biopsy in Newly Diagnosed Patients with Multiple Myeloma and Smoldering Multiple Myeloma." Blood 114, no. 22 (November 20, 2009): 2803. http://dx.doi.org/10.1182/blood.v114.22.2803.2803.
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Abstract Abstract 2803 Poster Board II-779 Background. The rate of asymptomatic amyloidosis (asym-amyloidosis) detected in patients with newly diagnosed multiple myeloma (MM) or smoldering multiple myeloma (SMM) is unknown. This topic is significant because unrecognized AL may be associated with increased mortality may change the patient's management. The objective of the present investigation was to evaluate the number and clinical significance of asym-amyloidosis in MM and SM patients at the time of the diagnostic bone marrow (BM) biopsy for MM. Materials and Methods. The study population was selected from the Mayo Clinic Dysproteinemia database and consisted of consecutive patients with an established diagnosis of MM or SMM without recognition of symptomatic AL. Bone marrow biopsies at diagnosis of MM or SMM were retrospectively stained with Congo Red and reviewed by a single pathologist. A patient was considered to have asym-amyloid if Congo Red staining with apple green birefringence was found. Results. Biopsies from 144 (M 84, F 59) patients were evaluated: 77 had a diagnosis of MM and 67 of SMM. The median age was 59 (range 26-84) years. No differences were found regarding hemoglobin, platelets, prothrombin time, serum and urine proteins, serum albumin, alkaline phosphate, creatinine and β2-microglobulin among MM and SMM patients. At a median follow-up 76 months (range 0-216), 32% patients were alive, 65% dead and 2.7% lost to follow-up. Immunoglobulin isotypes were as follows: 96/144 (67%) had IgG 23/144 (16%) IgA, 12/144 (8%) had light chain only, 1/77 (1%) had IgD, none had IgM and 12/144 (8%) had biclonal or indeterminate; 84/144 (58%) were κ restricted. The presence of amyloid was found in only 2 cases (1%, 95% CI – 0.6 to 3.2), 1 in MM and 1 in SMM group. Neither of these patients had or developed signs or symptoms suggestive of organ involvement by amyloid. Among the 142 other patients without amyloid deposition in their index bone marrow, 1 (0.7%, 95% CI -0.6 to 2.0) developed symptomatic AL after 119 months of follow-up. Characteristics of these three patients are shown in table 1. Conclusions. We found only 2 cases (1%) of amyloidosis in the 144 cases of MM or SMM. Our estimates are lower than the rates which have been reported by others, perhaps because of our high level of suspicion for amyloid at our Amyloidosis Center. These data do not support the need for searching for asym-amyloidosis in patients with newly diagnosed MM or SMM as long as they have no clinical features of AL. Disclosures: Off Label Use: Hydroxyurea use in myelofibrosis. Gertz:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Witzig:Novartis: Research Funding. Kumar:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Thiagarajan, D., R. Fiskesund, J. Steen, M. Rahman, S. Lundström, and J. Frostegård. "THU0230 IGG1 ANTIBODIES AGAINST PHOSPHORYLCHOLINE ARE NEGATIVELY ASSOCIATED WITH DISEASE ACTIVITY, DISEASE DAMAGE, CARDIOVASCULAR DISEASE AND ATHEROSCLEROSIS IN SLE: POTENTIAL UNDERLYING MECHANISMS." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 342.2–343. http://dx.doi.org/10.1136/annrheumdis-2020-eular.5779.
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Background:Phosphorylcholine (PC) is an important component in cellular membranes and in lipoproteins that is exposed and recognized by the immune system, when cells undergo apoptosis or lipoproteins like LDL undergo oxidation. PC is also exposed in some microorganisms including nematodes and bacteria (non-self). We reported that IgM anti-PC is associated with protection in atherosclerosis, SLE, RA and other chronic inflammatory conditions.1We also reported potential underlying protective mechanisms: 1: increase in clearance of human dead cells,22: inhibition of uptake of oxLDL in macrophages, 3: inhibition of cell death.14: anti-inflammatory; 5: promotion of polarization of T regulatory cells in SLE-patients´ T cells from a low level and also in plaque T cells.3We generated in-house fully human IgG1 anti-PC clones for experimental studies to study anti-PC properties in humans. In contrast to mice, anti-PC are not germ-line encoded with a dominant clone.4Objectives:We here study IgG1 and IgG2 anti-PC, with focus on atherosclerosis and SLE and properties of fully human IgG1 clones, in relation to SLE.Methods:We determined anti-PC by ELISA in 116 SLE-patients and 110 age- and sex-matched controls. For functional studies, we used three in-house generated, fully human monoclonal IgG1 anti-PC (A01, D05, E01). Apoptosis was induced in Jurkat T-cells and pre-incubated with A01, D05, E01 or isotype control IgG1 and effects on efferocytosis by human macrophages studied. Anti-PC peptide/protein characterization was determined using a proteomics de novo sequencing approach.Results:IgG1 but not IgG2 anti-PC levels were higher among SLE patients (p=0.02). IgG1 anti-PC was negatively associated with SLICC and SLEDAI (OR: 2,978 CI: 0.876-10.098, OR: 5.108 CI 1.3 20.067 respectively) and negatively associated with CVD, atherosclerotic plaques and echolucent (potentially vulnerable plaques) but the association for the two former was not significant after controlling for confounders. D05 had maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. The monoclonal antibodies showed differential binding specificity to PC and PC associated neo-epitopes. Peptide analysis showed difference in the CDR3 region of the three anti-PC IgG1 clones which are crucial for recognition of PC on apoptotic cell surface and other neo-epitopes.Conclusion:Anti-PC IgG1 is negatively associated with disease activity, and disease damage in SLE, but the negative association with CVD is also dependent on confounding risk factors. One potential underlying mechanism could be increased clearance of dead cells.References:[1]Frostegard J. Immunity, atherosclerosis and cardiovascular disease.BMC Med. 2013;11:117.[2]Rahman M, Sing S, Golabkesh Z, Fiskesund R, Gustafsson T, Jogestrand T, Frostegard AG, Hafstrom I, Liu A and Frostegard J. IgM antibodies against malondialdehyde and phosphorylcholine are together strong protection markers for atherosclerosis in systemic lupus erythematosus: Regulation and underlying mechanisms.Clin Immunol. 2016;166-167:27-37.[3]Sun J, Lundstrom SL, Zhang B, Zubarev RA, Steuer J, Gillgren P, Rahman M, Ajeganova S, Liu A and Frostegard J. IgM antibodies against phosphorylcholine promote polarization of T regulatory cells from patients with atherosclerotic plaques, systemic lupus erythematosus and healthy donors.Atherosclerosis. 2018;268:36-48.[4]Fiskesund R, Steen J, Amara K, Murray F, Szwajda A, Liu A, Douagi I, Malmstrom V and Frostegard J. Naturally occurring human phosphorylcholine antibodies are predominantly products of affinity-matured B cells in the adult.J Immunol. 2014;192:4551-9.Disclosure of Interests: :Divya Thiagarajan: None declared, Roland Fiskesund: None declared, Johanna Steen: None declared, Mizanur Rahman: None declared, Susanna Lundström: None declared, Johan Frostegård Grant/research support from: Unconditional competitive grant from Amgen, related only to PCSK9, not the topic of this abstract
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McCollom, Joseph William, Stephanie A. Dublis, James Hoogeboom, Abigail Doyle, and Jacob Templin. "Catastrophic Multi-Organ Failure with Bone Marrow Necrosis in a Sickle Cell Beta Plus Thalassemia Patient." Blood 124, no. 21 (December 6, 2014): 4942. http://dx.doi.org/10.1182/blood.v124.21.4942.4942.
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Abstract The variant sickle cell hemoglobinopathies have a variety of phenotypic presentations. Sickle cell beta plus thalassemia is an uncommon variant with an incidence that is 1/10th of sickle cell trait. This phenotype usually is associated with a milder clinical course, however often the first clinical presentation of such patients can be fatal vaso-occlusive crisis. Our case involves a 47 year old African American male with a reported history of sickle cell trait presenting with acute on chronic lower back pain for which an organic cause could not be identified with plain Xrays. He was treated with benzodiazepines and narcotics and later admitted to the ICU after being found unresponsive at home. He required intubation for airway protection. He was started on a Narcan drip for presumed narcotic overdose, without improvement. Noncontrast CT imaging of the head was negative. Laboratory findings were significant for anemia, thrombocytopenia, leukocytosis, acute kidney injury and elevated liver enzymes. Hemolysis was suspected with a markedly elevated LDH. Peripheral smear showed mild microangiopathic changes with 0-1 schisctocytes per hpf without evidence of sickle cells. Hematology was consulted and the patient was started on plasma exchange for a presumed diagnosis of TTP, however it was discontinued when his ADAMTS13 returned at a low-normal 67%, and TTP was felt less likely. MRI of the brain showed multiple focal and patchy areas infarcts throughout white matter of both cerebral hemispheres. MRI of the lumbar and thoracic spine showed a heterogeneous appearance of the bone marrow, concerning for a marrow infiltrative process. Work up for infectious or vasculitic causes were unremarkable. Hemoglobin electrophoresis showed Hb A1 20.2 (L), A2 5.8 (H), Hb F 2.2 (H), Hb S 71.8 (H), identifying doubly heterozygous sickle cell beta plus thalassemia. Bone marrow biopsy was hyperplastic with areas of geographic necrosis with sickle cells causing sludging and congested sinusoidal spaces suggesting ischemic necrosis due to vaso-occlusion. He was treated with exchange transfusion, with repeat hemoglobin electrophoresis showing A1 73.2 (L), A2 3.5 (H), HbS 23.3 (H). His lupus anticoagulant was positive raising concern for APLAS contributing to his catastrophic clinical course and severe neurologic disease,so he was started on anticoagulation with Lovenox and high dose steroids at a dose of 1 mg/kg daily. When the anticardiolipin and beta2 glycoprotein antibodies returned negative, the steroids were tapered and eventually discontinued. The clinical significance of the lupus inhibitor remains unclear. Shortly after initiation of plasma exchange and simple transfusion, the patient did stabilize hematologically. Platelets returned to the normal range and there was no evidence to suggest ongoing hemolysis. Renal and liver function improved. However he made no neurological recovery. He continued to require ventilator support and underwent a tracheostomy. A repeat MRI head showed progressive infarcts of both cerebral hemispheres, with new cerebellar infarcts. He was declared brain dead after 23 days and care was withdrawn. This case demonstrates that sickle cell beta plus thalassemia can present with acute hemolysis and bone marrow necrosis in otherwise healthy adults. The literature supports bone marrow necrosis and subsequent fat emboli as the likely pathophysiologic nidus responsible for multiorgan system failure including the catastrophic neurologic insult in this patient. Prompt recognition of this uncommon and challenging disease presentation and timely treatment with exchange transfusion may lead to improved clinical outcomes. Illustration 1: Bone marrow necrosis with ghosted cells Illustration 2: T2 weighted MRI with diffuse white matter ischemia Disclosures No relevant conflicts of interest to declare.
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Chlebowska, Justyna, Pawel Gaj, Piotr Stawinski, Michal Lazniewski, Malgorzata Firczuk, Karolina Furs, Radoslaw Sadowski, et al. "SK053, an Inhibitor of Enzymes Involved in Allosteric Disulfide Bonds Formation, Targets Expression of Histone Genes and Induces Differentiation of Human AML Cell." Blood 124, no. 21 (December 6, 2014): 3503. http://dx.doi.org/10.1182/blood.v124.21.3503.3503.
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Abstract Targeting epigenetic modifiers, such as histone deacetylases, bromodomain and extraterminal (BET) domains or methyltransferases as well as the use of differentiation-inducing agents (all-trans retinoic acid and arsenic trioxide) are rising hopes for development of effective therapeutic strategies for acute myeloid leukemia (AML). However, despite significant advances the cure rates for patients aged sixty or more are still very low. Targeting allosteric disulfide bonds becomes a novel approach to cancer treatment. Several disulfide bonds involved in cancer development and progression have already been identified as potentially druggable targets (Hogg P, Nat Rev Cancer 2013). Our interest in this field focuses on proteins with thioredoxin fold such as protein disulfide isomerase (PDI). We have recently developed SK053, a small molecule inhibitor of thioredoxin/thioredoxin reductase system, that exerts anti-tumor effects both in vitro and in murine tumor models (Klossowski S et al., J Med Chem 2012). Our studies revealed that SK053 is not a target-specific, but mechanism-selective inhibitor of allosteric disulfide bonds formation that also blocks the enzymatic activity of PDI. Biotinylated form of SK053 precipitates both TRX as well as PDI from human acute myeloid leukemia cells. Mass spectrometry analysis as well as molecular docking simulations show that SK053 covalently binds to cysteine C397 localized in the fourth (catalytic) PDI domain. SK053 reduces proliferation and induces monocytic and granulocytic differentiation in various types of human AML cells (HL60, MV4-11, Fig. 1A). Accordingly, it up-regulates expression of selected differentiation-related genes and increases expression of cell membrane differentiation markers (CD11b, CD14 and CD15). SK053 induces differentiation of primary leukemic blasts (Fig. 1B). Our ongoing studies show that silencing of PDI, TXN or PRDX alone using lentiviral shRNA systems does not result in differentiation of HL60 cells (Fig. 1C). RNA-seq analysis revealed that incubation of HL60 cells with SK053 down-regulates mRNA for MYC and ID1 oncogenes, which are involved in the regulation of blast proliferation. Moreover, the transcripts for histone proteins: HIST1H3J, HIST1H2AB, HIST1H1B, HIST1H3H, HIST1H2AH were strongly down-regulated. Drugs that disrupt histone modifiers are in clinical trials with promising results but very little is known about direct targeting of histone proteins. Expression of other genes important for development of myeloid lineage such as adhesion molecules (collagene type XV, fibronectin I, MAC-1), hydrolytic enzymes (carboxypeptidase, proteinase 3, CA12 anhydrase, ADAM19 metalloprotease), proteoglycan 2 (core of eosinophilic granules) and PGLYRP3 (peptidoglycan recognition protein 3) was significantly up-regulated. In summary, SK053, an inhibitor of allosteric disulfide bonds that targets thioredoxin/thioredoxin reductase system, PDI and decreases histone expression has significant anti-leukemic activity and induces differentiation of various types of human AML cells. Thus, targeting allosteric disulfide bonds with small molecule inhibitors presents promising therapeutic strategy in acute myeloid leukemia. Fig. 1. (A) Cytostatic activity of SK053 in established human AML cell lines evaluated with trypan blue staining, the graph presents mean cell count ± SD, n=6 (B) Differentiation of AML primary blast after treatment with SK053. Cells pre-incubated for 3 days with SK053 were incubated with fluorochrome-conjugated anti-CD11b mAb for 30 min. at RT in the dark. Subsequently dead cells were stained with 7AAD. Graphs represent MFI only from 7AAD-CD33+population, n=3, *P<0.05 vs controls, one-way ANOVA with Dunett’s post test (C) Knock-down of PDI, TRX 1 and PRDX 1 in HL60 cells do not result in granulocytic differentiation. 10 days post transduction with lentiviral shRNA systems HL60 cells were stained for CD11b marker as described above. Graphs represent MFI only from 7AAD- cells. Fig. 1. (A) Cytostatic activity of SK053 in established human AML cell lines evaluated with trypan blue staining, the graph presents mean cell count ± SD, n=6 (B) Differentiation of AML primary blast after treatment with SK053. Cells pre-incubated for 3 days with SK053 were incubated with fluorochrome-conjugated anti-CD11b mAb for 30 min. at RT in the dark. Subsequently dead cells were stained with 7AAD. Graphs represent MFI only from 7AAD-CD33+population, n=3, *P<0.05 vs controls, one-way ANOVA with Dunett’s post test (C) Knock-down of PDI, TRX 1 and PRDX 1 in HL60 cells do not result in granulocytic differentiation. 10 days post transduction with lentiviral shRNA systems HL60 cells were stained for CD11b marker as described above. Graphs represent MFI only from 7AAD- cells. The research was supported by the Republic of Poland Ministry of Science and Higher Education [grant no IP2011038971 (DN)] and National Science Centre Poland [grant no NN405127640 (AM) and 2013/10/E/NZ5/00778 (DN)] and a grant from the European Commission 7th Framework Programme: FP7-REGPOT-2012-CT2012-316254-BASTION. Disclosures No relevant conflicts of interest to declare.