scholarly journals Cytomegalovirus-Infected Primary Endothelial Cells Trigger NKG2C+ Natural Killer Cells

2016 ◽  
Vol 8 (4) ◽  
pp. 374-385 ◽  
Author(s):  
Zakia Djaoud ◽  
Raphaëlle Riou ◽  
Pierre-Jean Gavlovsky ◽  
Souad Mehlal ◽  
Céline Bressollette ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Activation ◽  
Nk Cell ◽  
Expression Patterns ◽  
Cell Subset ◽  
Human Leukocyte ◽  
Hla Class I ◽  
Cmv Infection

Among innate cells, natural killer (NK) cells play a crucial role in the defense against cytomegalovirus (CMV). In some individuals, CMV infection induces the expansion of NKG2C+ NK cells that persist after control of the infection. We have previously shown that KIR2DL+ NK cells, in contrast to NKG2C+ NK cells, contribute to controlling CMV infection using a CMV-infected monocyte-derived dendritic cell (MDDC) model. However, the nature of CMV-infected cells contributing to the expansion of the NKG2C+ NK cell subset remains unclear. To gain more insight into this question, we investigated the contribution of NKG2C+ NK cell activation by CMV-infected primary human aortic endothelial cells (EC) isolated from kidney transplant donors, which constitutively express the human leukocyte antigen (HLA)-E molecule. Here, we show that, although classic HLA class I expression was drastically downregulated, nonclassic HLA-E expression was maintained in CMV-infected EC. By comparing HLA expression patterns in CMV-infected EC, fibroblasts and MDDC, we demonstrate a cell-dependent modulation of HLA-E expression by CMV infection. NKG2C+ NK cell degranulation was significantly triggered by CMV-infected EC regardless of the nature of the HLA-E allele product. EC, predominantly present in vessels, may constitute a privileged site for CMV infection that drives a ‘memory' NKG2C+ NK cell subset.

scholarly journals Dynamic changes in natural killer cell subset frequencies in the absence of cytomegalovirus infection

2019 ◽  
Author(s):  
Ivayla E. Gyurova ◽  
Heinrich Schlums ◽  
Heidi Sucharew ◽  
Lilliam Ambroggio ◽  
David E. Ochayon ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Critical Role ◽  
Killer Cell ◽  
Cell Subset ◽  
Functional Differentiation ◽  
Cmv Infection ◽  
Antiviral Responses ◽  
Efficacious Vaccine

Individuals lacking functional natural killer (NK) cells suffer severe, recurrent infections with cytomegalovirus (CMV), highlighting the critical role of NK cells in antiviral defense. Therefore, ongoing attempts to develop an efficacious vaccine to prevent CMV infection should potentially aim to elicit NK-cell antiviral responses as an accessory to conventional T- and B-cell based approaches. In this regard, CMV infection provokes marked phenotypic and functional differentiation of the NK-cell compartment, including development of adaptive NK cells that exhibit enhanced antiviral activity. We examined longitudinal blood samples collected from 40 CMV-seronegative adolescents to ascertain whether a CMV glycoprotein B (gB) vaccine in the absence of CMV infection can stimulate differentiation or expansion of CMV-associated subsets of NK cells. Study participants uniformly lacked the CMV-dependent NKG2C+ subset of NK cells, suggesting that an adjuvanted CMV gB vaccine alone is an inadequate stimulus for sustained expansion of these cells. In contrast, we observed unexpected dynamic fluctuations in the frequency of NK cells lacking FcRγ, EAT-2, and SYK, which were independent of vaccination or CMV infection. Whereas FcRγ neg NK cells in CMV infection are reported to express increased levels of the maturation marker CD57, the FcRγneg NK cells observed in our CMV-negative vaccine cohort express less CD57 than their FcRγ+ counterparts. The FcRγneg NK cells in CMV-negative individuals were also functionally distinct from this subset in CMV infection, exhibiting comparable IFN-γ production and degranulation as FcRγ+ NK cells in response to cytokine or antibody-dependent stimuli. These results suggest that frequencies of some NK cell subsets may increase in response to unknown environmental or inflammatory cues distinct from that which occurs after CMV infection. Greater understanding of the nature of the signals driving CMV-independent accumulation of these subsets should permit development of mechanisms to facilitate vaccine-driven expansion of CMV-reactive NK cells.

Prostate cancer associated natural killer cells show a pro-angiogenic and pro-inflammatory phenotype.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e17544-e17544
Author(s):  
Adriana Albini ◽  
Antonino Bruno ◽  
Matteo Gallazzi ◽  
Angelo Naselli ◽  
Lorenzo Mortara ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Endothelial Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Umbilical Vein ◽  
Macrophage Polarization ◽  
Cell Subset ◽  
Migration Assay ◽  
Macrophage Recruitment

e17544 Background: Prostate cancer (PCa) represents the second most common cancer worldwide in men. Angiogenesis has been shown to play a crucial role in PCa progression in several preclinical models. Production of pro-angiogenic factors can correlate with metastasis, Gleason score and prognosis in PCa and plasma levels of VEGF were shown to be higher in patients with metastatic PCa than those with localized disease. Natural killer (NK) cells, effector lymphocytes of the innate immunity, have been found to be immunosuppressed in solid cancers, including PCa. Methods: Here, we phenotypically and functionally characterize circulating NK cells from PCa patients and their interaction with endothelial cells and macrophages. NK cell subset distribution has been investigated by multicolor flow cytometry (FACS) for surface antigens on peripheral blood samples PCa patients (TANKs). Conditioned media (CM) from FACS-sorted TANKs were used for functional studies of angiogenesis, on human umbilical-vein endothelial cells (HUVEC), studies for macrophage recruitment (migration assay on Boyden chambers) and polarization. Molecular studies were performed by real time PCR (qPCR) on HUCECs and macrophages exposed to CM of TANKs. Protein arrays were performed to characterize the secretome on FACS-sorted TANKs. Results: We found that TANKs acquire a pro-angiogenic/ dNK-like CD56brightCD9+CD49a+CXCR4+ phenotype. The same phenotype was observed on cytolytic NK cells, from healthy donors, exposed to CMs of three different PCa cell lines (PC-3, DU-145, LNCaP), together with increased production of CXCL8, Angiogenin, Angiopoietin1 and reduced production of TNFa, IFNg and GranzymeA. CMs from TANKs support the formation on capillary-like structures on HUVEC, together with increased expression of VEGF, VEGR-2, CXCL8, ICAM-1 and VCAM-1. Secretome analysis revealed the ability of TANKs release pro-angiogenic pro-invasive factors (CLXL8, MMP-1, MMP-9; uPAR) and cytokines/chemokines involved in macrophage recruitment (CCL1, CCL2, CCL5, CCL7, CCL13, CXCL1, CXCL11) and M2-like polarization (IL-10). Finally, CMs from TANKs can recruit THP-1 monocyte and polarize THP-1 macrophage towards CD206, Arginase1, CXCL8-expressing M2-like/TAM phenotype. Conclusions: Our results place PCa TANKs as effector cells able in supporting angiogenesis in PCa by directly interaction with endothelial cells and via macrophage polarization.

scholarly journals Natural Killer Cells Are Present in Rag1−/− Mice and Promote Tissue Damage During the Acute Phase of Ischemic Stroke

2021 ◽  
Author(s):  
Leoni Rolfes ◽  
Tobias Ruck ◽  
Christina David ◽  
Stine Mencl ◽  
Stefanie Bock ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Immune Cell ◽  
Nk Cell ◽  
Cell Subset ◽  
Neurological Deficits ◽  
In Vitro Assays ◽  
Experimental Stroke ◽  
Transfer Model

AbstractRag1−/− mice, lacking functional B and T cells, have been extensively used as an adoptive transfer model to evaluate neuroinflammation in stroke research. However, it remains unknown whether natural killer (NK) cell development and functions are altered in Rag1−/− mice as well. This connection has been rarely discussed in previous studies but might have important implications for data interpretation. In contrast, the NOD-Rag1nullIL2rgnull (NRG) mouse model is devoid of NK cells and might therefore eliminate this potential shortcoming. Here, we compare immune-cell frequencies as well as phenotype and effector functions of NK cells in Rag1−/− and wildtype (WT) mice using flow cytometry and functional in vitro assays. Further, we investigate the effect of Rag1−/− NK cells in the transient middle cerebral artery occlusion (tMCAO) model using antibody-mediated depletion of NK cells and adoptive transfer to NRG mice in vivo. NK cells in Rag1−/− were comparable in number and function to those in WT mice. Rag1−/− mice treated with an anti-NK1.1 antibody developed significantly smaller infarctions and improved behavioral scores. Correspondingly, NRG mice supplemented with NK cells were more susceptible to tMCAO, developing infarctions and neurological deficits similar to Rag1−/− controls. Our results indicate that NK cells from Rag1−/− mice are fully functional and should therefore be considered in the interpretation of immune-cell transfer models in experimental stroke. Fortunately, we identified the NRG mice, as a potentially better-suited transfer model to characterize individual cell subset-mediated neuroinflammation in stroke.

scholarly journals The molecular basis of how buried human leukocyte antigen polymorphism modulates natural killer cell function

2020 ◽  
Vol 117 (21) ◽  
pp. 11636-11647 ◽  
Author(s):  
Philippa M. Saunders ◽  
Bruce J. MacLachlan ◽  
Phillip Pymm ◽  
Patricia T. Illing ◽  
Yuanchen Deng ◽  
...  
Keyword(s):  
Human Leukocyte Antigen ◽  
Natural Killer ◽  
Nk Cell ◽  
Killer Cell ◽  
Peptide Binding ◽  
Human Leukocyte ◽  
Hla Class I ◽  
Cell Recognition ◽  
Leukocyte Antigen ◽  
Binding Cleft

Micropolymorphisms within human leukocyte antigen (HLA) class I molecules can change the architecture of the peptide-binding cleft, leading to differences in peptide presentation and T cell recognition. The impact of such HLA variation on natural killer (NK) cell recognition remains unclear. Given the differential association of HLA-B*57:01 and HLA-B*57:03 with the control of HIV, recognition of these HLA-B57 allomorphs by the killer cell immunoglobulin-like receptor (KIR) 3DL1 was compared. Despite differing by only two polymorphic residues, both buried within the peptide-binding cleft, HLA-B*57:01 more potently inhibited NK cell activation. Direct-binding studies showed KIR3DL1 to preferentially recognize HLA-B*57:01, particularly when presenting peptides with positively charged position (P)Ω-2 residues. In HLA-B*57:01, charged PΩ-2 residues were oriented toward the peptide-binding cleft and away from KIR3DL1. In HLA-B*57:03, the charged PΩ-2 residues protruded out from the cleft and directly impacted KIR3DL1 engagement. Accordingly, KIR3DL1 recognition of HLA class I ligands is modulated by both the peptide sequence and conformation, as determined by the HLA polymorphic framework, providing a rationale for understanding differences in clinical associations.

scholarly journals miR-1224 contributes to ischemic stroke-mediated natural killer cell dysfunction by targeting Sp1 signaling

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yan Feng ◽  
Yan Li ◽  
Ying Zhang ◽  
Bo-Hao Zhang ◽  
Hui Zhao ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Nk Cells ◽  
Natural Killer ◽  
Brain Ischemia ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Activity ◽  
Passive Transfer ◽  
Immune Defense ◽  
Nk Cell Activity

Abstract Background Brain ischemia compromises natural killer (NK) cell-mediated immune defenses by acting on neurogenic and intracellular pathways. Less is known about the posttranscriptional mechanisms that regulate NK cell activation and cytotoxicity after ischemic stroke. Methods Using a NanoString nCounter® miRNA array panel, we explored the microRNA (miRNA) profile of splenic NK cells in mice subjected to middle cerebral artery occlusion. Differential gene expression and function/pathway analysis were applied to investigate the main functions of predicted miRNA target genes. miR-1224 inhibitor/mimics transfection and passive transfer of NK cells were performed to confirm the impact of miR-1224 in NK cells after brain ischemia. Results We observed striking dysregulation of several miRNAs in response to ischemia. Among those miRNAs, miR-1224 markedly increased 3 days after ischemic stroke. Transfection of miR-1224 mimics into NK cells resulted in suppression of NK cell activity, while an miR-1224 inhibitor enhanced NK cell activity and cytotoxicity, especially in the periphery. Passive transfer of NK cells treated with an miR-1224 inhibitor prevented the accumulation of a bacterial burden in the lungs after ischemic stroke, suggesting an enhanced immune defense of NK cells. The transcription factor Sp1, which controls cytokine/chemokine release by NK cells at the transcriptional level, is a predicted target of miR-1224. The inhibitory effect of miR-1224 on NK cell activity was blocked in Sp1 knockout mice. Conclusions These findings indicate that miR-1224 may serve as a negative regulator of NK cell activation in an Sp1-dependent manner; this mechanism may be a novel target to prevent poststroke infection specifically in the periphery and preserve immune defense in the brain.

scholarly journals 789 Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule to enhance targeted tumor cytotoxicity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A824-A824
Author(s):  
Fay Dufort ◽  
Christopher Leitheiser ◽  
Gemma Mudd ◽  
Julia Kristensson ◽  
Alexandra Rezvaya ◽  
...  
Keyword(s):  
Phage Display ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Antigen ◽  
Cell Activation ◽  
Immune Cell ◽  
Nk Cell ◽  
High Affinity ◽  
Tumor Cytotoxicity ◽  
Immune Oncology

BackgroundNatural killer (NK) cells are immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Tumor specific activation of NK cells is thus an area of active investigation in immune oncology, but to date has relied on complex biologic modalities (e.g., antibodies, fusion proteins, or cell therapies), each of which has inherent disadvantages in this application. Thus, alternative approaches are warranted. Bicycle® are small (ca. 1.5 kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have now applied this technology to identify Bicycles that bind specifically to the key activating receptors, NKp46 and CD16a. When chemically coupled to tumor antigen binding Bicycles this results in highly potent, antigen-dependent receptor activation and NK cell activation. We term this new class of fully synthetic molecules Bicycle® natural killer- tumor-targeted immune cell agonists (NK-TICAs™) and we will describe their discovery and evaluation in this presentation.MethodsUsing our unique phage display screening platform, we have identified high affinity, selective binders to NKp46 and CD16a. By conjugating the Bicycle® NK cell-engaging binders to a model tumor antigen EphA2-binding Bicycle®, we have developed a bifunctional Bicycle NK-TICA™ molecule. In in vitro functional assays, we evaluated the ability of the Bicycle NK-TICAs™ to induce NK cell activation as well as cell-mediated cytotoxicity and cytokine production in NK-tumor co-culture assays.ResultsWe have developed a novel modular compound with high affinity and selectivity to NK cell receptors with specific tumor targeting capability. We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function. With Bicycle's novel NK-TICA™ compound, we demonstrate engagement of NK cells, specific activation and function of NK cells, and enhanced EphA2-expressing tumor cytotoxicity, in a dose dependent manner.ConclusionsBicycle NK-TICAs™ are novel therapeutic agents capable of enhancing the landscape of immune oncology. We hypothesize that utilization of Bicycle NK-TICA™ as a multifunctional immune cell engager will promote modulation of NK cells, and infiltration and anti-tumor activity of NK cells in solid tumors. The data presented here provide initial proof of concept for application of the Bicycle technology to drive NK cell-mediated tumor immunity.

scholarly journals Natural Killer Cells: Potential Biomarkers and Therapeutic Target in Autoimmune Diseases?

2021 ◽  
Vol 12 ◽  
Author(s):  
Elena Gianchecchi ◽  
Domenico V. Delfino ◽  
Alessandra Fierabracci
Keyword(s):  
Systemic Sclerosis ◽  
Autoimmune Diseases ◽  
Cytotoxic Activity ◽  
Nk Cells ◽  
Natural Killer ◽  
T Cell Activation ◽  
Cell Activation ◽  
Nk Cell ◽  
Killer Cell

Autoimmune diseases recognize a multifactorial pathogenesis, although the exact mechanism responsible for their onset remains to be fully elucidated. Over the past few years, the role of natural killer (NK) cells in shaping immune responses has been highlighted even though their involvement is profoundly linked to the subpopulation involved and to the site where such interaction takes place. The aberrant number and functionality of NK cells have been reported in several different autoimmune disorders. In the present review, we report the most recent findings regarding the involvement of NK cells in both systemic and organ-specific autoimmune diseases, including type 1 diabetes (T1D), primary biliary cholangitis (PBC), systemic sclerosis, systemic lupus erythematosus (SLE), primary Sjögren syndrome, rheumatoid arthritis, and multiple sclerosis. In T1D, innate inflammation induces NK cell activation, disrupting the Treg function. In addition, certain genetic variants identified as risk factors for T1D influenced the activation of NK cells promoting their cytotoxic activity. The role of NK cells has also been demonstrated in the pathogenesis of PBC mediating direct or indirect biliary epithelial cell destruction. NK cell frequency and number were enhanced in both the peripheral blood and the liver of patients and associated with increased NK cell cytotoxic activity and perforin expression levels. NK cells were also involved in the perpetuation of disease through autoreactive CD4 T cell activation in the presence of antigen-presenting cells. In systemic sclerosis (SSc), in addition to phenotypic abnormalities, patients presented a reduction in CD56hi NK-cells. Moreover, NK cells presented a deficient killing activity. The influence of the activating and inhibitory killer cell immunoglobulin-like receptors (KIRs) has been investigated in SSc and SLE susceptibility. Furthermore, autoantibodies to KIRs have been identified in different systemic autoimmune conditions. Because of its role in modulating the immune-mediated pathology, NK subpopulation could represent a potential marker for disease activity and target for therapeutic intervention.

scholarly journals The Ly-49D Receptor Activates Murine Natural Killer Cells

1996 ◽  
Vol 184 (6) ◽  
pp. 2119-2128 ◽  
Author(s):  
L.H. Mason ◽  
S.K. Anderson ◽  
W.M. Yokoyama ◽  
H.R.C. Smith ◽  
R. Winkler-Pickett ◽  
...  
Keyword(s):  
Gene Family ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Subset ◽  
Class I ◽  
Target Cells ◽  
Color Flow ◽  
Functional Capabilities

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immunoprecipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I–bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of FcγR+ target cells in a reverse antibody-dependent cellular cytotoxicity–type assay and induces apoptosis in Ly49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/IxYxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

scholarly journals NT5E/CD73 as Correlative Factor of Patient Survival and Natural Killer Cell Infiltration in Glioblastoma

2019 ◽  
Vol 8 (10) ◽  
pp. 1526 ◽  
Author(s):  
Jiao Wang ◽  
Sandro Matosevic
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Activation ◽  
Patient Survival ◽  
Nk Cell ◽  
Killer Cell ◽  
Surface Protein ◽  
The Cancer Genome Atlas ◽  
Negative Prognostic Factor ◽  
A Cell

CD73, a cell-surface protein encoded by the gene NT5E, is overexpressed in glioblastoma (GBM), where it contributes to the tumor’s pathophysiology via the generation of immunosuppressive adenosine. Adenosinergic signaling, in turn, drives immunosuppression of natural killer (NK) cells through metabolic and functional reprogramming. The correlation of CD73 with patient survival in relation to GBM pathology and the intratumoral infiltration of NK cells has not been comprehensively studied before. Here, we present an analysis of the prognostic relevance of CD73 in GBM based on transcriptional gene expression from patient data from The Cancer Genome Atlas (TCGA) database. Utilizing bioinformatics data mining tools, we explore the relationship between GBM prognosis, NT5E expression, and intratumoral presence of NK cells. Our analysis demonstrates that CD73 is a negative prognostic factor for GBM and that presence of NK cells may associate with improved prognosis. Moreover, the interplay between expression of NT5E and specific NK genes hints to potential functional effects of CD73 on NK cell activation.

scholarly journals Enhancing network activation in natural killer cells: predictions from in silico modeling

2020 ◽  
Vol 12 (5) ◽  
pp. 109-121
Author(s):  
Sahak Z Makaryan ◽  
Stacey D Finley
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
In Silico ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
Nk Cell ◽  
Mechanistic Model ◽  
Cell Killing ◽  
Published Data ◽  
Network Activation

Abstract Natural killer (NK) cells are part of the innate immune system and are capable of killing diseased cells. As a result, NK cells are being used for adoptive cell therapies for cancer patients. The activation of NK cell stimulatory receptors leads to a cascade of intracellular phosphorylation reactions, which activates key signaling species that facilitate the secretion of cytolytic molecules required for cell killing. Strategies that maximize the activation of such intracellular species can increase the likelihood of NK cell killing upon contact with a cancer cell and thereby improve efficacy of NK cell-based therapies. However, due to the complexity of intracellular signaling, it is difficult to deduce a priori which strategies can enhance species activation. Therefore, we constructed a mechanistic model of the CD16, 2B4 and NKG2D signaling pathways in NK cells to simulate strategies that enhance signaling. The model predictions were fit to published data and validated with a separate dataset. Model simulations demonstrate strong network activation when the CD16 pathway is stimulated. The magnitude of species activation is most sensitive to the receptor’s initial concentration and the rate at which the receptor is activated. Co-stimulation of CD16 and NKG2D in silico required fewer ligands to achieve half-maximal activation than other combinations, suggesting co-stimulating these pathways is most effective in activating the species. We applied the model to predict the effects of perturbing the signaling network and found two strategies that can potently enhance network activation. When the availability of ligands is low, it is more influential to engineer NK cell receptors that are resistant to proteolytic cleavage. In contrast, for high ligand concentrations, inhibiting phosphatase activity leads to sustained species activation. The work presented here establishes a framework for understanding the complex, nonlinear aspects of NK cell signaling and provides detailed strategies for enhancing NK cell activation.

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