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1.
Cancer Immunol Immunother ; 72(10): 3323-3335, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37477653

ABSTRACT

Allogeneic natural killer (NK) cell-based immunotherapy is a promising, well-tolerated adjuvant therapeutic approach for acute myeloid leukemia (AML). For reproducible NK cell immunotherapy, a homogenous, pure and scalable NK cell product is preferred. Therefore, we developed a good manufacturing practice (GMP)-compliant, cytokine-based ex vivo manufacturing process for generating NK cells from CD34+ hematopoietic stem and progenitor cells (HSPC). This manufacturing process combines amongst others IL15 and IL12 and the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) to generate a consistent and active NK cell product that fits the requirements for NK cell immunotherapy well. The cell culture protocol was first optimized to generate NK cells with required expansion and differentiation capacity in GMP-compliant closed system cell culture bags. In addition, phenotype, antitumor potency, proliferative and metabolic capacity were evaluated to characterize the HSPC-NK product. Subsequently, seven batches were manufactured for qualification of the process. All seven runs demonstrated consistent results for proliferation, differentiation and antitumor potency, and preliminary specifications for the investigational medicinal product for early clinical phase trials were set. This GMP-compliant manufacturing process for HSPC-NK cells (named RNK001 cells) is used to produce NK cell batches applied in the clinical trial 'Infusion of ex vivo-generated allogeneic natural killer cells in combination with subcutaneous IL2 in patients with acute myeloid leukemia' approved by the Dutch Ethics Committee (EudraCT 2019-001929-27).


Subject(s)
Immunotherapy, Adoptive , Leukemia, Myeloid, Acute , Humans , Immunotherapy, Adoptive/methods , Killer Cells, Natural/metabolism , Leukemia, Myeloid, Acute/genetics , Antigens, CD34/metabolism , Hematopoietic Stem Cells
4.
Br J Pharmacol ; 172(4): 1148-63, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25363319

ABSTRACT

BACKGROUND AND PURPOSE: The clinical effects of anti-angiogenic agents remain controversial. Therefore, elucidating the pharmacological properties of these compounds is a pivotal issue. EXPERIMENTAL APPROACH: The effects of treatment with sunitinib on tumour and normal tissues of mice bearing C-26 adenocarcinoma cells were analysed by matrix-assisted laser desorption ionization MS imaging (MALDI-MSI). Expression of the key targets of sunitinib--angiogenic receptors--was studied by immunofluorescent labelling. KEY RESULTS: MALDI-MS assays showed that sunitinib and its fragment ions were present throughout tumour and normal tissues. Major metabolites were identified in blood and solid tissues, while minor drug metabolites were detectable only in blood. Tumour growth and intratumour VEGF receptor-2 expressions were significantly reduced in sunitinib-treated mice, while the expression of the other targeted receptors, PDGF receptor -α or -ß and fibroblast growth factor receptor-1, remained unaffected. Within tumour tissue, the close proximity of sunitinib metabolites to the precursor ion suggested in situ metabolism of the administered drug. There were intratumour areas where the signal intensity of sunitinib correlated with expression of VEGF receptor-2. CONCLUSIONS AND IMPLICATIONS: This is the first study that demonstrates MALDI-MSI is a versatile platform to study the intratumour localization of an unlabelled anti-angiogenic drug. The combination of MALDI-MSI and immunofluorescence analysis can provide further insights into the molecular interaction of drug compounds and their targets within tumour tissue.


Subject(s)
Adenocarcinoma/metabolism , Angiogenesis Inhibitors/pharmacokinetics , Indoles/pharmacokinetics , Pyrroles/pharmacokinetics , Adenocarcinoma/drug therapy , Adenocarcinoma/pathology , Angiogenesis Inhibitors/blood , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Animals , Cell Line, Tumor , Female , Indoles/blood , Indoles/pharmacology , Indoles/therapeutic use , Kidney/metabolism , Liver/metabolism , Mice, Inbred BALB C , Pyrroles/blood , Pyrroles/pharmacology , Pyrroles/therapeutic use , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sunitinib , Tumor Burden/drug effects , Vascular Endothelial Growth Factor Receptor-2/metabolism
8.
Cytometry ; 45(4): 285-93, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11746098

ABSTRACT

BACKGROUND: The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. METHODS: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. RESULTS: From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. CONCLUSIONS: As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process.


Subject(s)
Antigen-Antibody Reactions/immunology , Binding Sites, Antibody/immunology , CD56 Antigen/immunology , Flow Cytometry/methods , Immunomagnetic Separation/methods , Killer Cells, Natural/cytology , Dose-Response Relationship, Immunologic , Humans , Immunoglobulin G/blood , Immunomagnetic Separation/instrumentation , Phycoerythrin/immunology
9.
Trends Immunol ; 22(11): 633-40, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11698225

ABSTRACT

Human natural killer (NK) cells comprise approximately 15% of all circulating lymphocytes. Owing to their early production of cytokines and chemokines, and ability to lyse target cells without prior sensitization, NK cells are crucial components of the innate immune system. Human NK cells can be divided into two subsets based on their cell-surface density of CD56--CD56(bright) and CD56(dim)--each with distinct phenotypic properties. Now, there is ample evidence to suggest that these NK-cell subsets have unique functional attributes and, therefore, distinct roles in the human immune response. The CD56(dim) NK-cell subset is more naturally cytotoxic and expresses higher levels of Ig-like NK receptors and FCgamma receptor III (CD16) than the CD56(bright) NK-cell subset. By contrast, the CD56(bright) subset has the capacity to produce abundant cytokines following activation of monocytes, but has low natural cytotoxicity and is CD16(dim) or CD16(-). In addition, we will discuss other cell-surface receptors expressed differentially by human NK-cell subsets and the distinct functional properties of these subsets.


Subject(s)
CD56 Antigen/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Animals , CD56 Antigen/immunology , Cell Adhesion Molecules/metabolism , Cytokines/biosynthesis , Cytokines/metabolism , Cytotoxicity, Immunologic , Humans , Killer Cells, Natural/classification , Killer Cells, Natural/cytology , Lymphocyte Subsets/classification , Lymphocyte Subsets/cytology , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Models, Immunological , Receptors, Chemokine/immunology , Receptors, Chemokine/metabolism , Receptors, Cytokine/immunology , Receptors, Cytokine/metabolism , Receptors, Immunologic/immunology , Receptors, Immunologic/metabolism
10.
Am J Respir Cell Mol Biol ; 25(1): 60-8, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11472976

ABSTRACT

Changes in the levels of transforming growth factor (TGF)-beta cytokines or receptors observed during the progression of several inflammatory and fibrotic disorders have been used to implicate these cytokines in the pathophysiology of these diseases. Although correlative, these studies were inconclusive because they were unable to demonstrate actual continuous TGF-beta-mediated signaling in the involved tissues. We reasoned that the phosphorylation state and subcellular localization of Smad2, the intracellular effector of TGF-beta/activin-mediated signaling, could be used as a marker of active signaling mediated by these cytokines in situ. We therefore used an experimental model of ovalbumin-induced allergic airway inflammation and were able to demonstrate a dramatic increase in the numbers of bronchial epithelial, alveolar, and infiltrating inflammatory cells expressing nuclear phosphorylated Smad2 within the allergen-challenged lungs. This was accompanied by strong upregulation of the activin receptor ALK-4/ActR-IB and redistribution of the TGF-beta responsive ALK-5/TbetaR-I. Although levels of TGF-beta1, TGF-beta2, and TGF-beta3 messenger RNA (mRNA) were marginally altered, the level of activin mRNA was strongly upregulated during the inflammatory response. Our data illustrate the usefulness of antiphosphorylated Smad antibodies in demonstrating active TGF- beta/activin-mediated signaling in vivo and strongly suggest that activin/Smad-mediated signaling could be a critical contributor in the pathophysiology of allergic pulmonary diseases.


Subject(s)
Bronchitis/metabolism , DNA-Binding Proteins/metabolism , Hypersensitivity/metabolism , Inhibins/metabolism , Trans-Activators/metabolism , Transforming Growth Factor beta/metabolism , Activins , Animals , Base Sequence , Blotting, Western , Bronchitis/etiology , DNA Primers , Female , Hypersensitivity/complications , Immunohistochemistry , Mice , Mice, Inbred BALB C , Phosphorylation , Protein Transport , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Smad2 Protein
11.
Blood ; 97(10): 3146-51, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11342442

ABSTRACT

During the innate immune response to infection, monocyte-derived cytokines (monokines), stimulate natural killer (NK) cells to produce immunoregulatory cytokines that are important to the host's early defense. Human NK cell subsets can be distinguished by CD56 surface density expression (ie, CD56(bright) and CD56(dim)). In this report, it is shown that CD56(bright) NK cells produce significantly greater levels of interferon-gamma, tumor necrosis factor-beta, granulocyte macrophage-colony-stimulating factor, IL-10, and IL-13 protein in response to monokine stimulation than do CD56(dim) NK cells, which produce negligible amounts of these cytokines. Further, qualitative differences in CD56(bright) NK-derived cytokines are shown to be dependent on the specific monokines present. For example, the monokine IL-15 appears to be required for type 2 cytokine production by CD56(bright) NK cells. It is proposed that human CD56(bright) NK cells have a unique functional role in the innate immune response as the primary source of NK cell-derived immunoregulatory cytokines, regulated in part by differential monokine production.


Subject(s)
CD56 Antigen/analysis , Homeostasis , Immunity , Killer Cells, Natural/immunology , Lectins, C-Type , Antigens, CD/analysis , Cell Division , Cells, Cultured , Coculture Techniques , Cytotoxicity, Immunologic , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interleukin-2/pharmacology , Interleukins/biosynthesis , Ionomycin/pharmacology , Lipopolysaccharides/pharmacology , Lymphotoxin-alpha/biosynthesis , Macrophages/metabolism , Membrane Glycoproteins/analysis , NK Cell Lectin-Like Receptor Subfamily D , RNA, Messenger/analysis , Receptors, IgG/analysis , Receptors, Immunologic/analysis , Receptors, Interleukin-2/analysis , Receptors, Interleukin-2/physiology , Receptors, KIR , Tetradecanoylphorbol Acetate/pharmacology
12.
Blood Cells Mol Dis ; 27(1): 223-30, 2001.
Article in English | MEDLINE | ID: mdl-11358383

ABSTRACT

The role of inflammation in the early genesis of certain malignancies has recently been appreciated. Interleukin (IL)-15, a proinflammatory cytokine and growth factor, is required for lymphocyte homeostasis. Intriguingly, the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms, suggesting that inappropriate expression of IL-15 may be detrimental to the host. We recently engineered a transgenic mouse in which the normal posttranscriptional control of IL-15 is eliminated, thereby overexpressing the murine IL-15 protein. IL-15 transgenic mice have early expansions in NK and CD8+ T lymphocytes and later develop fatal lymphocytic leukemia with a T-NK phenotype. This article recapitulates the phenotype of these IL-15 transgenic mice and discusses the utility of this model as a tool to further our understanding of leukemogenesis.


Subject(s)
Disease Models, Animal , Interleukin-15/adverse effects , Leukemia, T-Cell/etiology , Animals , Cell Transformation, Neoplastic/drug effects , Humans , Interleukin-15/genetics , Killer Cells, Natural , Leukemia, T-Cell/mortality , Mice , Mice, Transgenic
13.
Scand J Immunol ; 53(3): 296-301, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11251888

ABSTRACT

The expression of the proliferation-associated nuclear antigen Ki-67 in peripheral blood mononuclear cells was studied in 30 patients with acute malarial illness and 11 healthy controls from Addis Ababa or Nazareth in Ethiopia. Seventeen patients had Plasmodium falciparum infections and 13 had Plasmodium vivax. Two-colour immunoenzymatic staining was developed in order to simultaneously detect the expression of the nuclear antigen Ki-67 and determine the surface phenotype of the cell. The median percentage of proliferating, Ki-67 positive lymphocytes was significantly higher in patients with acute P. falciparum (11.8%) and P. vivax (15.6%) illnesses compared to the controls (4.3%). The majority of Ki-67 positive cells were T cells (CD3+) while the relative increase of Ki-67 expressing cells was similar for both the CD4+ and CD8+ T-cell subsets. Our data show an increased number of activated cells driven to proliferation in the peripheral blood of patients during acute malaria illness.


Subject(s)
Ki-67 Antigen/blood , Lymphocyte Activation , Malaria/immunology , T-Lymphocytes/immunology , Acute Disease , CD3 Complex/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Case-Control Studies , Cell Division , Humans , Malaria/blood , Malaria, Falciparum/blood , Malaria, Falciparum/immunology , Malaria, Vivax/blood , Malaria, Vivax/immunology , T-Lymphocytes/pathology
14.
Eur J Immunol ; 31(3): 792-801, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11241284

ABSTRACT

Natural killer (NK) cells are an early source of immunoregulatory cytokines during the innate immune response to viruses, bacteria, and parasites. NK cells provide requisite IFN-gamma to monocytes for the elimination of obligate intracellular pathogens. IL-1beta is a pro-inflammatory cytokine produced by monocytes (i.e. a monokine) during the early immune response to infection, but its role in promoting human NK cell IFN-gamma production is unknown. The current study examines the ability of the monokine IL-1beta, plus IL-12, to costimulate IFN-gamma production by resting CD56(bright) and CD56(dim) human NK cell subsets. CD56(bright) NK cells stimulated with IL-1beta plus IL-12 produced abundant IFN-gamma protein, while little IFN-gamma was produced in identical cultures of CD56(dim) cells. In addition, upon activation with IL-1beta, CD56(bright) NK cells exhibited considerably greater phosphorylation of extracellular signal-regulated kinases p42/44 as compared to CD56(dim) NK cells. Quantitative PCR analysis showed brisk induction of IFN-gamma gene expression following costimulation with IL-1beta plus IL-12 in CD56(bright) NK cells, but intracellular flow cytometry revealed that only a fraction (42+/-2.3%) of CD56(bright) NK cells account for this high IFN-gamma production. These data suggest that the monokine IL-1beta is a potent costimulus of IFN-gamma production by a subset of NK cells following infectious insult.


Subject(s)
CD56 Antigen/analysis , Interferon-gamma/genetics , Interleukin-1/pharmacology , Killer Cells, Natural/immunology , Cells, Cultured , Humans , Interferon-gamma/biosynthesis , Interleukin-1 Receptor Accessory Protein , Interleukin-12/pharmacology , Killer Cells, Natural/classification , Killer Cells, Natural/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Protein Biosynthesis , Proteins/genetics , RNA, Messenger/biosynthesis , Receptors, Interleukin-1/biosynthesis , Receptors, Interleukin-1/genetics , Transcriptional Activation
15.
J Exp Med ; 193(2): 219-31, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11208862

ABSTRACT

Inflammation likely has a role in the early genesis of certain malignancies. Interleukin (IL)-15, a proinflammatory cytokine and growth factor, is required for lymphocyte homeostasis. Intriguingly, the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms. Here, we engineered a transgenic mouse to overexpress IL-15 by eliminating these posttranscriptional checkpoints. IL-15 transgenic mice have early expansions in natural killer (NK) and CD8+ T lymphocytes. Later, these mice develop fatal lymphocytic leukemia with a T-NK phenotype. These data provide novel evidence that leukemia, like certain other cancers, can arise as the result of chronic stimulation by a proinflammatory cytokine.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Interleukin-15/genetics , Killer Cells, Natural/immunology , Leukemia, Experimental/genetics , Leukemia, Experimental/immunology , Animals , Base Sequence , DNA Primers/genetics , Genetic Engineering , Immunologic Memory , Inflammation Mediators/immunology , Leukemia, Experimental/etiology , Lymphocytosis/genetics , Lymphocytosis/immunology , Lymphocytosis/pathology , Mice , Mice, Transgenic , Phenotype , Time Factors
16.
J Immunol Methods ; 249(1-2): 207-22, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11226478

ABSTRACT

Two techniques based upon flow cytometry (FCM) and in situ image analysis were developed for quantification of intracellular cytokine and chemokine protein expression at the single cell level in dendritic cells (DCs). The qualitative and quantitative differences between the two methods were evaluated. In vitro differentiated DCs were stimulated with lipopolysaccaride (LPS) and thereafter stained for either IL-8, which is secreted through the Golgi-organelle, or IL-1ra, which localises diffusely in the cytoplasm. Microscopic examination, both for fluorophore and enzymatically stained cells, showed that DCs expressed IL-8 and IL-1ra with two different staining patterns. FCM analysis showed high frequencies of IL-1ra producing cells (76+/-13%), which was similar to the frequency obtained by in situ imaging. However, in contrast to IL-1ra, the incidence of IL-8 expressing DCs showed high variability between the donors. The numbers of positive cells were 19+/-19% as measured by FCM. The detection of IL-8 analysed by in situ imaging revealed higher frequencies (26+/-14%). The addition of brefeldin-A, leading to cytoplasmic accumulation of proteins secreted through the Golgi endoplasmatic route, generated a significantly increased signal intensity and incidence of producer cells, resulting in similar frequencies for both methods. FCM has the advantage of being less time consuming than image analysis and is also able to facilitate multiple colour analysis. However, FCM is less accurate in detecting and quantifying cytokines and chemokines with a preserved juxtanuclear staining pattern. The correct choice of detection technique therefore depends on the study question.


Subject(s)
Chemokines/immunology , Cytokines/immunology , Dendritic Cells/immunology , Immunologic Techniques , Chemokines/analysis , Cytokines/analysis , Flow Cytometry/methods , Humans , Sensitivity and Specificity
18.
Int Rev Immunol ; 20(3-4): 503-34, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11878513

ABSTRACT

Our knowledge of NK cells and their critical role in the innate immune system has increased enormously since their discovery several decades ago. However, it is only within the last 10 years that rational cytokine therapies, such as those utilizing low doses of IL-2, have been successful in expanding NK cells in patients with cancer and/or immunodeficiency. Such experiences in vivo have highlighted the importance of basing immunotherapeutic strategies on the known cellular and molecular properties of the targeted cell population. Recent advances in our understanding of the physiologic factors and events that orchestrate NK cell ontogeny, including IL-15 and receptor tyrosine kinase ligands to c-kit and flt3, provide novel therapeutic possibilities for cytokine therapy. This review summarizes our current understanding of human NK cell ontogeny, and links this knowledge to ongoing and future clinical strategies for the endogenous expansion of NK cells in patients with cancer and/or immunodeficiency.


Subject(s)
Immunotherapy , Killer Cells, Natural/immunology , Animals , CD56 Antigen/immunology , Growth Substances/therapeutic use , Homeostasis/immunology , Humans , Interleukin-15/immunology , Interleukin-2/immunology , Receptors, Interleukin-15 , Receptors, Interleukin-2/immunology
19.
J Clin Invest ; 106(1): 117-24, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10880055

ABSTRACT

The continuous, in vivo infusion of low-dose IL-2 selectively expands the absolute number of human natural killer (NK) cells after 4-6 weeks of therapy. The mechanism responsible for this expansion is unknown and was examined in this study. NK cells cultured at low concentrations of IL-2, comparable to those found during in vivo therapy, proliferate for 6 days and then exit the cell cycle. However, NK cells in vivo did not traverse the S/G(2)/M phase of the cell cycle during low-dose IL-2 therapy. Low concentrations of IL-2 delay programmed cell death of NK cells but have the same effect on resting T cells that do not expand in vivo. When CD34(+) bone marrow hematopoietic progenitor cells are cultured for 21 days with low concentrations of IL-2, they differentiate into CD56(+)CD3(-) NK cells, not T cells. Thus, the selective expansion of human NK cells during continuous in vivo infusion of low-dose IL-2 likely results from enhanced NK-cell differentiation from bone marrow progenitors, combined with an IL-2-dependent delay in NK-cell death, rather than proliferation of mature NK cells in the periphery.


Subject(s)
Interleukin-2/pharmacology , Killer Cells, Natural/drug effects , Animals , Antigens, CD34/analysis , CD56 Antigen/analysis , Humans , Interleukin-2/therapeutic use , Killer Cells, Natural/physiology , Lymphocyte Activation/drug effects , Rabbits , Recombinant Proteins/pharmacology
20.
J Biochem Biophys Methods ; 44(1-2): 41-57, 2000 Jul 10.
Article in English | MEDLINE | ID: mdl-10889275

ABSTRACT

Dynamic interactions between hammerhead ribozymes and RNA substrates were measured using the surface plasmon resonance (SPR) technology. Two in vitro transcribed substrates (non-cleavable and cleavable) were immobilised on streptavidin-coated dextran matrices and subsequently challenged with non-related yeast tRNA or two hammerhead ribozymes, both of which had previously been shown to exhibit functional binding and cleavage of complementary target RNAs. The target-binding domain of one of the ribozymes was fully complementary to a 16-ribonucleotide stretch on the immobilised substrates, while the other ribozyme had a nine-ribonucleotide complementarity. The two ribozymes could readily be differentiated with regard to affinity. Cleavage could be measured, using the ribozyme with full target complementarity to the cleavable substrate. In contrast, the ribozyme with lower affinity lacked cleavage activity. We suggest that SPR will be useful for investigations of ribozyme-substrate interactions.


Subject(s)
RNA, Catalytic/metabolism , RNA/metabolism , Surface Plasmon Resonance/methods , Base Pairing , Biotinylation , Dextrans/metabolism , Kinetics , Models, Genetic , RNA, Fungal/metabolism , RNA, Transfer/metabolism , Streptavidin/metabolism , Time Factors , Transcription, Genetic
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