scholarly journals Production of hematopoietic colony-stimulating factors by human natural killer cells.

1989 ◽  
Vol 169 (2) ◽  
pp. 569-583 ◽  
Author(s):  
M C Cuturi ◽  
I Anegón ◽  
F Sherman ◽  
R Loudon ◽  
S C Clark ◽  
...  
Keyword(s):  
T Cells ◽  
Biological Activity ◽  
Nk Cells ◽  
Negative Selection ◽  
Lymphoblastoid Cell Lines ◽  
Ionophore A23187 ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Human Natural Killer Cells ◽  
Kinetics Of

We have analyzed the ability of highly purified preparations of human NK cells to produce CSF. NK cells, purified by negative selection from 10-d cultures of PBMC incubated with irradiated B-lymphoblastoid cell lines, were stimulated with rIL-2, FcR(CD16) ligands (particulate immune complexes or anti-CD16 antibodies bound to Sepharose), a combination of CD16 ligands and rIL-2, or the phorbol diester phorbol dibutyrate (PDBu) together with the Ca2+ ionophore A23187. Both rIL-2 and CD16 ligands induce accumulation of GM-CSF mRNA in NK cells and the combined effect of the two stimuli is synergistic. Maximal accumulation of GM-CSF mRNA is observed after PDBu/A23187 stimulation. The participation of contaminant T cells in the observed expression of the GM-CSF gene is excluded because CD16 ligands do not stimulate T cells and CD3 ligands, powerful stimulators of T cells, are inactive on NK cells. Accumulation of CSF-1 mRNA is observed only in NK cells stimulated with both CD16 ligands and rIL-2, whereas accumulation of IL-3 mRNA is observed only in NK cells stimulated with PDBu/A23187. Transcripts of the G-CSF, IL-1 alpha, and IL-1 beta genes were never detected in NK cells in these experiments. The kinetics of accumulation of GM-CSF and CSF-1 mRNA in NK cells stimulated with CD16 ligands and rIL-2 peaked at 2-4 h and was slower than that of TNF and IFN-gamma mRNA, which peak at 1 h. GM-CSF was precipitated from the supernatant fluids of NK cells stimulated with PDBu/A23187 and its biological activity was demonstrated by the ability of the supernatants to sustain proliferation of the TALL-101 cell line or CML blasts. Biological activity of IL-3 and CSF-1 was demonstrable in supernatant fluids of NK cells stimulated with PDBu/A23187 and CD16 ligands/rIL-2, respectively.

Phenotypic and Functional Effects of Novel Akt Inhibitor Perifosine on Immune System.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1555-1555
Author(s):  
Weihua Song ◽  
Teru Hideshima ◽  
Yu-Tzu Tai ◽  
Kenneth C. Anderson ◽  
Nikhil C. Munshi
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Immune System ◽  
Nk Cells ◽  
Negative Selection ◽  
Akt Inhibitor ◽  
Ifn Gamma ◽  
Alexa Fluor ◽  
Hla Dq ◽  
Dose Dependent

Abstract Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agent which targets cell membranes and inhibits Akt activation. Perifosine inhibits multiple myeloma (MM) cell growth in vitro and in vivo. Currently perifosine is under phase II clinical evaluation in MM. Although perifosine has shown significant direct antitumor effects, its effect on immune system has not yet been clarified. The objective of this study was to investigate the effects of perifosine on immune system including the activity of antigen presenting cells (APCs), T cells and NK cells. Perifosine was used at a clinically relevant concentrations of 2.5 μM, 5 μM and 10 μM. Monocyte-derived dendritic cells (DCs) from healthy donors were used as the APCs. We observed that the perifosine up to 48 hours had no effect on viability (>90%), as assessed by annexin V and PI staining. Alteration of DC phenotype by perifosine was further examined by flow cytometry. Our results demonstrated that perifosine treatment led to a dose-dependent downregulation of surface antigen expression, associated with costimulation (CD40, CD80 and CD86), antigen presentation (HLA-ABC, HLA-DQ) and maturation (CD83) on immature DCs at 24 and 48 hours. The significant downregulation of CD40, CD83, HLA-ABC and HLA-DQ was also observed on mature DCs. Perifosine also inhibited immature DC uptake of antigens (40-kDa Dextran-FITC, 45-kDa protein A-Alexa Fluor 488 and 20-kDa protein G-Alexa Fluor 488) in a dose-dependent manner. Since DCs play a crucial regulatory role via cytokine production, we next determined IL-12p70 and IL-10 secretion by LPS-induced DCs with and without perifosine treatment. Compared to controls, perifosine treatment at 24 hours significantly inhibited LPS-induced-IL-12p70 production by DCs (trt vs. untrt = 166 pg/ml (2.5μM), 111 pg/ml (5μM) and 45 pg/ml (10μM) vs. 192 pg/ml), as well as inhibited IL-10 production (trt vs. untrt = 371 pg/ml (2.5μM), 306 pg/ml (5μM) and 179 pg/ml (10μM) vs. 472 pg/ml). These data suggest that perifosine is able to affect both immature and mature DCs and could contribute to inhibition of DC-mediated immune responses. Furthermore, we evaluated effect of perifosine on T cells obtained from healthy donor peripheral blood mononuclear cells (PBMCs) by negative selection. Although perifosine treatment, up to 10μM, had no effect on T cell survival, it inhibited IFN-gamma production by T cells stimulated with PMA and ionomycin compared to that of the control (intracellular flow cytometry, trt vs. untrt= 11.7% vs. 21.3%). The IFN-gamma inhibition by perifosine treatment (10μM) was further confirmed by ELISA upon stimulation with anti- CD2/CD3/CD28 activation beads (trt vs. untrt =11340 pg/ml vs. 18150 pg/mL). Similarly, viability of NK cells obtained from PBMCs by negative selection was not significantly affected by perifosine; however, decreased cytotoxicities were observed by NK cells treated with perifosine (10μM) against the U266 myeloma cell line (trt vs. untrt=30% vs. 51% (E:T=10:1) and 17% vs 30% (E:T=5:1) respectively). In addition, the IFN-gamma production by IL-2 and IL-12- induced NK cells was significantly inhibited following the perifosine treatment (trt vs. untrt=286 pg/ml (2.5μM), 210 pg/ml (10μM) vs. 439 pg/ml). These studies demonstrate that perifosine treatment significantly affects phenotype and function of human DCs, T cells and NK cells. Our pre-clinical data therefore indicates the need to monitor immune functions in patients under the Akt inhibitor treatment.

scholarly journals Immune Monitoring during Therapy Reveals Activitory and Regulatory Immune Responses in High-Risk Neuroblastoma

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2096
Author(s):  
Celina L. Szanto ◽  
Annelisa M. Cornel ◽  
Sara M. Tamminga ◽  
Eveline M. Delemarre ◽  
Coco C. H. de Koning ◽  
...  
Keyword(s):  
T Cells ◽  
High Risk ◽  
Nk Cells ◽  
Immune Cell ◽  
High Dose Chemotherapy ◽  
Immune Monitoring ◽  
High Dose ◽  
Effector Cells ◽  
Gm Csf ◽  
Immune Cell Subsets

Despite intensive treatment, including consolidation immunotherapy (IT), prognosis of high-risk neuroblastoma (HR-NBL) is poor. Immune status of patients over the course of treatment, and thus immunological features potentially explaining therapy efficacy, are largely unknown. In this study, the dynamics of immune cell subsets and their function were explored in 25 HR-NBL patients at diagnosis, during induction chemotherapy, before high-dose chemotherapy, and during IT. The dynamics of immune cells varied largely between patients. IL-2- and GM-CSF-containing IT cycles resulted in significant expansion of effector cells (NK-cells in IL-2 cycles, neutrophils and monocytes in GM-CSF cycles). Nonetheless, the cytotoxic phenotype of NK-cells was majorly disturbed at the start of IT, and both IL-2 and GM-CSF IT cycles induced preferential expansion of suppressive regulatory T-cells. Interestingly, proliferative capacity of purified patient T-cells was impaired at diagnosis as well as during therapy. This study indicates the presence of both immune-enhancing as well as regulatory responses in HR-NBL patients during (immuno)therapy. Especially the double-edged effects observed in IL-2-containing IT cycles are interesting, as this potentially explains the absence of clinical benefit of IL-2 addition to IT cycles. This suggests that there is a need to combine anti-GD2 with more specific immune-enhancing strategies to improve IT outcome in HR-NBL.

scholarly journals Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 670-677 ◽  
Author(s):  
WJ Murphy ◽  
JR Keller ◽  
CL Harrison ◽  
HA Young ◽  
DL Longo
Keyword(s):  
Growth Factors ◽  
Nk Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Hematopoietic Growth Factors ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Growth Promoting

Abstract Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon- gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G- CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.

Effect of exogenous interferon-gamma (IFN-gamma) on peripheral blood immune markers as part of a phase I clinical trial of combined IFN-gamma with nivolumab (Nivo) in patients (pts) with select solid tumors.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 97-97 ◽  
Author(s):  
Matthew R. Zibelman ◽  
Alexander Macfarlane ◽  
R. Katherine Alpaugh ◽  
Essel Dulaimi ◽  
Kim Costello ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
Phase I ◽  
Nk Cells ◽  
Solid Tumors ◽  
Mhc Class Ii ◽  
Peripheral Blood ◽  
Induction Phase ◽  
Ifn Gamma ◽  
Post Induction

97 Background: IFN-gamma is a known activator of PD-L1 expression and plays a key role in immune activation. We present initial correlative peripheral blood findings after the first 2 cohorts of pts enrolled in a phase I trial of combined IFN-gamma/Nivo in pts with various solid tumors. Methods: Pts with advanced solid tumors who had progressed after at least 1 prior therapy were recruited. An induction phase of IFN-gamma (50 or 75 mcg/m2 subq every other day for 1 week) preceded the combination of Nivo (3 mg/kg IV every 2 weeks) with continuation of IFN-gamma at same dose and schedule for 3 months. Pts with clinical benefit could remain on Nivo alone for up to 1 year. Peripheral blood was analyzed via flow cytometry at baseline, after IFN-gamma induction (prior to Nivo start), and after 6 weeks of combo treatment. Results: 13 pts have been accrued. Pre and post induction samples were collected on all pts. Nine pts had available on-treatment samples at time of data analysis. Comparing blood collections from baseline to post-IFN-gamma induction revealed a substantial expansion of the CD14high, CD16+ monocyte population (p=0.001) and a statistically significant increase in MHC class II expression on all monocyte populations (p≤ 0.002 for all types). Natural killer (NK) cells demonstrated greater numbers of cells per ml of blood (p=0.03) and increased NKp30 expression, an important receptor mediating tumor cell lysis (p= 0.005 for CD56dim; p= 0.03 for CD56bright). With the addition of Nivo, evidence for activation of CD56dim NK cells was detected as increased CD69 expression compared to post-induction (p=0.02). PD-1 expression on T-cells showed reduced expression in most comparators after initiation of Nivo (p=0.055 CD8+; p=0.008 CD4+). Conclusions: Early correlative peripheral blood data demonstrate expected systemic IFN-gamma effect as measured by monocyte activation and increased MHC II expression. Evidence of NK cell activation and proliferation was also observed. Decreased PD-1 expression on T-cells may reflect down-regulation in response to PD-1 blockade. Correlations with biopsy specimens and clinical response are planned. Clinical trial information: NCT02614456.

scholarly journals Two essential regulatory elements in the human interferon gamma promoter confer activation specific expression in T cells.

1993 ◽  
Vol 178 (5) ◽  
pp. 1483-1496 ◽  
Author(s):  
L Penix ◽  
W M Weaver ◽  
Y Pang ◽  
H A Young ◽  
C B Wilson
Keyword(s):  
T Cells ◽  
Interferon Gamma ◽  
Regulatory Elements ◽  
T Cell Subsets ◽  
Jurkat T Cells ◽  
Intact Cells ◽  
Ifn Gamma ◽  
Promoter Function ◽  
Gm Csf ◽  
Nuclear Extracts

Like interleukin 2 (IL-2), interferon gamma (IFN-gamma) is an early response gene in T cells and both are prototypical T helper cell type 1 (Th-1) lymphokines. Yet IL-2 and IFN-gamma production are independently regulated, as demonstrated by their differential expression in certain T cell subsets, suggesting that the regulatory elements in these two genes must differ. To explore this possibility, the 5' flank of the human IFN-gamma gene was analyzed. Expression of IFN-gamma promoter-driven beta-galactosidase reporter constructs containing 538 bp of 5' flank was similar to that by constructs driven by the IL-2 promoter in activated Jurkat T cells; expression nearly as great was observed with the construct containing only 108 bp of IFN-gamma 5' flank. These IFN-gamma promoter constructs faithfully mirrored expression of the endogenous gene, in that expression required activation both with ionomycin and PMA, was inhibited by cyclosporin A, and was not observed in U937 or THP-1 cells. The region between -108 and -40 bp in the IFN-gamma promoter was required for promoter function and contained two elements that are conserved across species. Deletion of 10 bp within either element reduced promoter function by 70%, whereas deletions in nonconserved portions of this region had little effect on promoter function. The distal conserved element (-96 to -80 bp) contained a consensus GATA motif and a potential regulatory motif found in the promoter regions of the GM-CSF and macrophage inflammatory protein (MIP) genes. Factors binding to this element, including GATA-3, were found in Jurkat nuclear extracts by electromobility shift assays and two of the three complexes observed were altered in response to activation. One or both of these motifs are present in the 5' flank of multiple, other lymphokine genes, including IL-3, IL-4, IL-5, and GM-CSF, but neither is present in the promoter of the IL-2 gene. The proximal conserved element (-73 to -48 bp) shares homology with the NFIL-2A element in the IL-2 promoter; these elements compete for binding of factors in Jurkat nuclear extracts, although the NFIL-2A element but not the IFN-gamma element binds Oct-1. Factors binding to this element in the IFN-gamma gene were present in extracts from resting and activated Jurkat T cells. However, by in vivo footprinting of intact cells, this element was protected from methylation only with activation.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 670-677 ◽  
Author(s):  
WJ Murphy ◽  
JR Keller ◽  
CL Harrison ◽  
HA Young ◽  
DL Longo
Keyword(s):  
Growth Factors ◽  
Nk Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Hematopoietic Growth Factors ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Growth Promoting

Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon- gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G- CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.

scholarly journals Comparing the Kinetics of NK Cells, CD4, and CD8 T Cells in Murine Cytomegalovirus Infection

2011 ◽  
Vol 187 (3) ◽  
pp. 1385-1392 ◽  
Author(s):  
Timothy E. Schlub ◽  
Joseph C. Sun ◽  
Senta M. Walton ◽  
Scott H. Robbins ◽  
Amelia K. Pinto ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cytomegalovirus Infection ◽  
Murine Cytomegalovirus ◽  
Kinetics Of

scholarly journals Response of resting human peripheral blood natural killer cells to interleukin 2.

1984 ◽  
Vol 160 (4) ◽  
pp. 1147-1169 ◽  
Author(s):  
G Trinchieri ◽  
M Matsumoto-Kobayashi ◽  
S C Clark ◽  
J Seehra ◽  
L London ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Human Peripheral Blood ◽  
Interleukin 2 ◽  
Cell Activity ◽  
Experimental Conditions ◽  
Ifn Gamma

The present study shows that recombinant interleukin 2 (IL-2) purified to homogeneity induces a rapid and potent enhancement of spontaneous cytotoxicity of human peripheral blood lymphocytes. The cells mediating cytotoxicity after 18-h treatment with IL-2 have surface markers of natural killer (NK) cells and are generated from the peripheral blood subset containing spontaneous cytotoxic cells. A parallel production of gamma interferon (IFN-gamma) is induced by recombinant IL-2 (rIL-2), and NK cells appear to be the major producer cells, whereas T cells are unable to produce IFN-gamma under these experimental conditions. However, the kinetics of the enhancement of cytotoxicity are faster than those of IFN-gamma production, and monoclonal anti-IFN-gamma antibodies do not suppress this effect, making it unlikely that the IFN-gamma produced is responsible for the enhancement. The enhancement of NK cell activity induced by rIL-2 precedes any proliferative response of the lymphocytes, which is instead observed in longer-term cultures of both NK and T cells.

scholarly journals Interferon gamma production by natural killer (NK) cells and NK1.1+ T cells upon NKR-P1 cross-linking.

1996 ◽  
Vol 183 (5) ◽  
pp. 2391-2396 ◽  
Author(s):  
H Arase ◽  
N Arase ◽  
T Saito
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Interferon Gamma ◽  
Nk Cell ◽  
Cell Receptor ◽  
Interleukin 4 ◽  
Cross Linking ◽  
Target Cells ◽  
Ifn Gamma

Natural killer (NK) cells play an important role in immune response by producing interferon gamma (IFN-gamma) as well as exhibiting cytotoxic function. IFN-gamma produced by NK cells has been suggested to be involved in differentiation of T helper cells. On the other hand, the NKR-P1 molecule was recently identified as one of the important NK cell receptors, and it recognizes certain kinds of oligosaccharides on target cells and triggers NK cells for cytotoxicity. In the present study, we found that NK cells produce great amounts of IFN-gamma upon cross-linking of the NKR-P1 molecule. In contrast, stimulation of NK cells with IL-2 induced proliferation without producing IFN-gamma. Similar to NK cells, NK1.1+ T cells also produced IFN-gamma upon NKR-P1 cross-linking. NK1.1+ T cells produced IFN-gamma but not interleukin 4 (IL-4) upon NKR-P1 cross-linking, whereas they secreted both IFN-gamma and IL-4 upon T cell receptor cross-linking. These results indicate that NKR-P1 is a receptor molecule on NK and NK1.1+ T cells that induces not only cytotoxicity but also IFN-gamma production. Our findings provide a new pathway for IFN-gamma production by NK and NK1.1+ T cells through NKR-P1 molecules; it may be essential for immune regulation.

scholarly journals Receptor-induced death in human natural killer cells: involvement of CD16.

1995 ◽  
Vol 181 (1) ◽  
pp. 339-344 ◽  
Author(s):  
J R Ortaldo ◽  
A T Mason ◽  
J J O'Shea
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Nk Cells ◽  
Natural Killer ◽  
Tyrosine Kinases ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Activated T Cells ◽  
Human Natural Killer Cells ◽  
Induced Apoptosis

Propriocidal regulation of T cells refers to apoptosis induced by interleukin 2 (IL-2) activation with subsequent antigen receptor stimulation. We examined whether natural killer (NK) cells exhibited cytokine- and ligand-induced death similar to activated T cells. Peripheral NK cells were examined for ligand-induced death using antibodies to surface moieties (CD2, CD3, CD8, CD16, CD56), with and without prior activation of IL-2. Only those NK cells stimulated first with IL-2 and then with CD16 exhibited ligand-induced death; none of the other antibody stimuli induced this phenomenon. Next we examined various cytokines (IL-2, IL-4, IL-6, IL-7, IL-12, IL-13, interferon alpha and gamma) that can activate NK cells and determined if CD16-induced killing occurred. Only IL-2 and IL-12 induced NK cell death after occupancy of this receptor by aggregated immunoglobulin or by cross-linking with antireceptor antibody. The CD16-induced death was inhibited by herbimycin A, indicating that cell death was dependent upon protein tyrosine kinases. Identical to T cells, the form of cell death for NK cells was demonstrated to be receptor-induced apoptosis. Overall these data indicate that highly activated NK cells mediate ligand-induced apoptosis via signaling molecules like CD16. Whereas the propriocidal regulation of T cells is antigen specific, this is not the case for NK cells due to the nature of the receptor. The clinical implications of this finding are considered.

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