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  • 1
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 3380-3380
    Abstract: BAFF is a member of the tumor necrosis factor (TNF) family and is critical for the maintenance and homeostasis of normal B-cell development. Importantly, BAFF promotes the generation of rapidly dividing immunoglobulin secreting plasmablasts from activated memory B cells by enhancing their survival. Given that MM is a cancer of plasma cells and that the signaling cascades implicated in receptor ligand interactions of BAFF are crucial in MM cell biology, we hypothesized that this cytokine may play a critical role in MM cell development, survival, and proliferation. We performed gene expression profiling (GEP) on CD 138+ plasma cells isolated from 90 MM patients (45 newly diagnosed and 45 relapsed) and 11 healthy controls using the Affymetrix U133A arrays. Our data demonstrates increased expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), 2 receptors used by BAFF to exert its effects. Our data also shows an increased expression of a proliferation-inducing ligand (APRIL), another member of the TNF family with homology to BAFF. Expression levels of BAFF and BAFF-R could not be determined because of lack of these probe sets on the Affymetrix U133A arrays. GEP analysis shows increased BCMA expression (p & lt;0.0001, student T test) on newly diagnosed and relapsed MM versus normal plasma cells. Flow cytometry on MM cell lines demonstrated a differential expression of the three receptors of BAFF, with BCMA present on most cell lines but BAFF-R expressed at low levels only on LR5 cells and DOX40 MM cells. In contrast, flow cytometry performed on MM patient cells demonstrated the presence of all 3 receptors on CD 138+ cells. ELISA assays performed on 30 MM sera demonstrated a mean BAFF level of 618 pg/ml (range: 128–2126pg/ml) versus 235pg/ml (range: 158–326pg/ml) in 7 normal donor sera. Fifty six% (17/30) of MM patients had BAFF levels in excess of the highest value noted in normals. To understand the role BAFF might play in the biology of MM, we studied the effects of recombinant BAFF (rh-BAFF) on MM cells directly and in the context of its bone marrow microenvironment. (abstract # 554746) rh-BAFF conferred a survival advantage to MM cells and protected them against dexamethasone-induced cytotoxicity. Importantly, anti-apoptotic proteins Bcl2 and Mcl-1 were upregulated, as were growth and survival signals belonging to the JAK/STAT and MAPKinase pathways. Conversely, neutralizing antibody to BAFF blocked, at least in part, blocked the upregulation of anti-apoptotic proteins with associated growth and survival, confirming that these effects were due to BAFF. Importantly, all of these signals were downregulated even in the presence of bone marrow stromal cells (BMSCs). These data therefore show a role for BAFF mediating MM cell survival and provide the framework for inhibiting BAFF, either alone or in combination with dexamethasone, to improve patient outcome in MM.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 3454-3454
    Abstract: Either an overexpression or dysregulation of cyclin D1, D2, or D3, has been reported in the majority of multiple myeloma (MM) tumors, suggesting a possible early unifying event in MM pathogenesis. This proposed critical role of cyclin D dysregulation in myeloma pathogenesis makes the cyclins, specifically cyclin D1, an attractive therapeutic target. We have evaluated a specific small molecule cyclin D1 inhibitor, P276-00 in MM. Its specificity has been confirmed in an in vitro kinase assay by potent inhibitory activity for Cdk4-D1 as compared to Cdk2-E. Additionally in vitro kinase assays against a broad range of other kinases have also confirmed specificity for D1 and B cyclins at nanomolar concentrations. P276-00 has been tested against a wide range of cancer cell types in both in vitro and tumor xenograft models. Based on these data, it is undergoing phase I clinical testing in North America. We have observed both time and dose dependent in vitro activity against a broad range of MM cells sensitive and resistant to conventional agents like dexamethasone, doxorubicin, and melphalan with IC50 ranging from 400–800nM. Spectral karyotyping confirmed t(11;14) (q13;q32) in KMS 12 MM cells which were sensitive to P276-00. Importantly, it has demonstrated activity in primary patient derived tumor cells. Cell cycle analysis confirmed that P276-00 induced either growth arrest or apoptosis in MM cells depending on the cell line. Apoptosis was in part caspase dependent suggested by partial reversal of cytotoxicity by Z-VAD Fmk. P276-00 inhibited Rb-1 phosphorylation as early as 6 hours in most of the MM cell lines tested associated with a decrease in cdk4 suggesting a regulatory role of P276-00 in cell cycle progression. These changes preceeded growth arrest and apoptosis of MM cells on cell cycle analysis. Ongoing studies are using SiRNA to Cyclin D1 to confirm this regulatory role of P276-00. As cyclin D1 dysregulation or overexpression can render MM cells more susceptible to proliferative stimuli such as IL-6, IGF-1, and the bone marrow microenvironment, we tested the effects of P276-00 in the presence of these cytokines and bone marrow stromal cells (BMSCs). Our data confirms that P276-00 was able to overcome these proliferative signals and induce apoptosis in MM cells. Next we evaluated in vivo efficacy of P276-00 in NOD-SCID mice bearing GFP+ MM xenografts. Animals were treated with either control PBS or P276-00 intraperitoneally at 25 mg/kg three times a week for 3 weeks. Our data confirms in vivo anti-tumor activity of P276-00 as suggested by a significant decrease in biluminesence of GFP+ MM cells (p 〈 0.05) and a decrease in tumor volume. Immunohistochemistry on tumor tissue from P76.00 treated, and control animals validates our in vitro studies and will be presented. In vitro combination studies with bortezomib have been completed suggesting synergism. P276-00 and bortezomib combination is currently being tested in our in vivo model. These studies confirm cyclin D1 to be an important therapeutic target in MM and form the basis of a phase I/II study of P276-00 alone and in combination in the treatment of MM.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 5153-5153
    Abstract: Ribonucleotide reductase is the rate-limiting enzyme of deoxynucleoside triphosphate synthesis and is therefore an excellent target for cancer chemotherapy. Inhibition of ribonucleotide reductase results in inhibition of DNA synthesis and has anti-neoplastic effects. Several ribonucleotide reductase inhibitors (RRIs) are already in clinical practice, including Hydroxyurea and Gemcitabine. Here we examine the anti-MM activity of a novel RRI, Didox. Our data shows that Didox has potent cytotoxicity against MM cells in 48-hour cultures. Apoptosis induced in MM cell lines by Didox (200mmol/l), evidenced by increased sub-G1 cell fraction, is caspase dependent, confirmed by blocking experiments with Z-VAD-FMK. Unlike other RRIs which mainly target the pyrimidine metabolism pathway (hydroxyurea and gemcitabine), didox targets both the purine and pyrimidine metabolism pathways in MM, as demonstrated by transcriptional profiling using the Affymetrix U133A 2.0 gene chip. Specifically, our data shows a ≥ 2 fold downregulation of genes in these pathways as early as 12 hours after exposure to Didox. Importantly, ribonucleotide reductase (RR) M1 component transcript was downregulated, associated with downregulation of this enzyme by Western Blot analysis, and inhibition of DNA synthesis. Furthermore, genes involved in DNA repair mechanisms were also downregulated after exposure to Didox, demonstrated by heirachial clustering of gene chip data and further validation by western blotting. Specifically RAD 51 homologue, which is involved in nucleotide excision repair and recombination repair, was downregulated in MM cells both transcriptionally as well as at the protein level. This was accompanied by downregulation of the BCL family protein including Bcl-2, Bclxl, and XIAP both at the transcriptional and post transcriptional levels. Since Didox acts on MM cells by inhibiting DNA synthesis and repair, combination studies with melphalan, an alkylating agent commonly used in MM, were next performed. Our data shows a strong in vitro synergism, with combination indices of & lt;0.7 by the Chou Talahay method. When combined with DNA damaging agents like doxorubicin, additive cytotoxicity was noted. These studies therefore provide the preclinical rationale for evaluation of Didox, alone and in combination with alkylating agents and anthracyclines, to improve patient outcome in MM.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 4
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 3494-3494
    Abstract: Multiple Myeloma (MM) is characterized by increased osteoclasts (OC) activity leading to severe bone disease. Several inducers of OC number and activity have been identified, including several key chemokines. In particular, macrophage inflammatory protein-1α (MIP-1α) and RANTES activate CCR1 and CCR5, resulting in increased osteoclastogenesis (Oba et al. Exp Hematol. 2005) and increased OC motility (Yu X. J Bone Miner Res. 2004) These studies underscore the role of CCR1 in promoting osteoclastogenesis. Here we demonstrate the effects of inhibition of CCR1 with a specific receptor antagonist (MLN3897, Millenium Pharmaceuticals) on normal mature OC. Mature OC were obtained by stimulating PBMC of healthy donors with RANKL and MCSF (50 ng/ml) for three weeks. OC expressed high levels of CCR1 and secreted both MIP-1α (1 ng/ml +/− 1.8) and RANTES (12 pg/ml +/− 0.66), suggesting an autocrine effect of these chemokines. Analyzing the bone resorptive ability with a pit formation assay, we observed that MLN3897 impaired OC function in a dose-dependent fashion (at 10 nM: 20% of reduction in resorbed area, at 100 nM: 50% of reduction). We then studied the effects of CCR1 inhibition on OC viability by analyzing nuclear integrity with Hoechst33258 staining. After 12 hours of cytokine-deprivation, MLN3897 enhanced OC nuclear fragmentation and condensation, suggesting a role for CCR1 ligands in OC survival. We next studied the interactions between OC and MM cells. Because OC secrete several chemoattractants, we studied their ability to stimulate RPMI8226 MM cell migration. We first confirmed high expression levels of CCR1 on RPMI8226 MM cells by flow cytometry (86% compared to isotype control). Inhibition of CCR1 with MLN3897 did not induce any direct cytotoxicity or growth arrest of these cells. We then studied the effects of OC on RPMI8226 MM cells. We observed that OC potently stimulated migration of RPMI8226 MM cells (8-fold increase), which was almost completely blocked by treatment with MLN3897. In contrast, MIP-1α alone induced only a modest effect on migration: the highest effective concentration (0.5 ng/ml) induced only a 2-fold increase. Neutralizing MIP-1α antibody partially reversed these effects, suggesting that other factors may contribute to this migratory effect. Our data therefore demonstrate that CCR1 inhibition by MLN3897 impairs mature OC survival and activity. Although MLN3897 does not have any direct antiMM activity, it affects MM-OC interactions and inhibits MM cell migration to OC. Ongoing studies are further characterizing the effects of CCR1 inhibition on MM-OC interactions in order to provide the framework for its clinical evaluation in MM.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 5
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 256-256
    Abstract: Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. We sought to identify the effects of targeting cyclin D1 for the treatment of MM. We first studied gene expression of cyclin D1 in MM cells from 64 patients and normal plasma cells from healthy volunteers as controls (n=24). Cyclin D1 was over-expressed in 42/64 MM patients (65%). To assess the importance of targeting cyclin D1, we next used shRNA lentiviral constructs to silence cyclin D1 in INA6 and MM.1S MM cells. Our data demonstrated & gt; 50% of apoptotic cell death in cyclin D1 shRNA versus control transfected MM cells, indicating that small molecule cyclin dependent kinase inhibitors may provide a valuable therapeutic tool to specifically target cyclin D1. We next evaluated a clinical grade small molecule cyclin D1 specific inhibitor P276-00 in MM. Its specificity has been confirmed in an in vitro kinase assay by potent inhibitory activity for Cdk4-D1 as compared to Cdk2-E. Additionally, in vitro kinase assays against a broad range of other kinases have also confirmed its specificity at nanomolar concentrations for D1 and B cyclins. P276-00 treatment of MM cell lines and patient derived cells induced both time and dose dependent in vitro growth inhibition in a broad range of MM cells sensitive and resistant to conventional agents including dexamethasone, doxorubicin, and melphalan, with IC50 ranging from 400–800nM. Cell cycle analysis confirmed that P276-00 induced either growth arrest or apoptosis in MM cells. Apoptosis was, at least in part, caspase-dependent, since cytotoxicity was partially reversed by Z-VAD Fmk. P276-00, in a dose and time dependent fashion inhibited Rb-1 phosphorylation as early as 6 hours associated with down-regulation of cdk4, suggesting a regulatory role of P276-00 in cell cycle progression. These changes preceded growth arrest and apoptosis in MM cells. Proliferative stimuli such as IL-6, IGF-1, and adherence to bone marrow stromal cells induced cyclin D1 and B cyclins, contributing to the development of drug resistance; P276-00 was able to overcome these proliferative signals and induce apoptosis in MM cells. Next we evaluated in vivo efficacy of P276-00 in NOD-SCID mice bearing GFP+ MM xenografts. Mice were treated with either control PBS or P276-00 intraperitoneally at 25 mg/kg three times a week for 3 weeks. Our data confirmed in vivo anti-tumor activity of P276-00, evidenced by a significant decrease in biluminesence of GFP+ MM cells (p & lt;0.05) and a decrease in tumor volume, as well as an improvement in overall survival of treated mice. Finally, in vitro combination studies with bortezomib showed strong synergism associated with down-regulation of the anti-apoptotic protein MCL-1. These studies form the basis of an ongoing Phase I study in the treatment of relapsed/refractory MM.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 6
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 3520-3520
    Abstract: Zoledronic acid (ZA) is an amino-bisphosphonate with very potent antiresorptive activity widely used in the treatment of multiple myeloma (MM) bone disease. Recently, the increasing incidence of osteonecrosis of the jaw and its possible association with prolonged use of amino-bisphosphonates such as ZA has been reported. Therefore, we here studied the effects of ZA on bone remodeling in vitro and in vivo. Although ZA has been shown to inhibit osteoclastogenesis and increase bone mineralization, data on its effects on osteoblastic activity and on bone formation are conflicting. To study the effects of ZA on osteoblasts (OB), 5 week old C57BL/6 mice were treated with low and high doses of ZA intraperitoneally (IP) weekly. The dose ranged from 0.05-1mg/kg, with the highest dose recapitulating a lifetime dose of ZA over a 5 year period in an adult MM patient. Blood was collected at baseline and weekly thereafter. IP calcein injections were administered to study bone formation rates. Consistent with its known effects on bone mass and density, in vivo DXA scans in ZA-treated animals demonstrated an increase in whole body bone mineral density (BMD). ZA treatment was associated with a dose-related increase in trabecular bone at the distal femur, evaluated by microCT and confirmed by static histomorphometry. ELISA assays showed a decrease in TRACP5B (bone resorption), as expected due to the anti-osteoclastic activity of ZA. In addition to osteoclast (OC) inhibition, mice treated with ZA also showed alterations in OB activity. Specifically, serum osteocalcin (a marker for bone formation) levels were lower in ZA treated mice, and dynamic histomorphometry confirmed decreased bone formation rates. In order to study the mechanism of our in vivo observations, we tested the effects of ZA on OB, OC, and bone marrow stromal cells (BMSCs) treated in vitro with ZA (0.01μM to 100μM) for 7, 14, and 21 days. Decreased viability during differentiation of both OB and OC was observed, without any significant effects on BMSCs. This was associated with decreased alkaline phosphatase activity, suggesting functional impairment of OB activity. Our data therefore suggests that ZA impairs OB number and activity, in addition to effects on OC and may impair normal physiologic bone remodeling. Ongoing studies will determine the molecular mechanisms whereby ZA mediates these sequelae and inform future studies of ZA use in patients with MM bone disease.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 7
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 3591-3591
    Abstract: Osteonecrosis of the jaw (ONJ) is a recently described entity observed in patients with a history of aminobisphosphonate use. To date nearly 400 patients with ONJ have been reported in literature and this number continues to rise. However,prospective characterization of ONJ is lacking. We here characterize ONJ clinically and radiographically using multiple imaging modalities including panorex films, CAT scans, magnetic resonance imaging (MRI), FDG-PET scans, and NaF-PET bone scans in 11 patients with multiple myeloma (MM). Moreover, bone turnover and remodelling markers in these patients were analyzed prospectively in order to gain insights into the pathophysiology of ONJ. Eleven patients between the ages of 57 and 81 yrs were included. There were 8 men and 3 women, and 6 patients presented with Durie Salmon stage III disease. Patients were treated with various combinations of conventional and novel agents, including stem cell transplantation (4/11). Patients received either pamidronate (n=3), zolendronic acid (n=4), or both agents sequentially (n=4). The mean duration of bisphosphonate (BP) therapy was 38.7 months (range: 9–81 months). All patients were examined independently by 2 oral medicine physicians, and clinical data was validated on 2 separate dental visits. Six of 11 patients had mandibular lesions, 3 had lesions in the maxilla, and 2/11 patients had lesions in both the maxilla and mandible. Plain and panorex filmsdemonstrated a mottled appearance with increased radiolucency at the site of ONJ.This was associated with an increase in both glucose metabolism and mineralization at sites of ONJ, as measured with the maximum standardized uptake value (SUVmax) on FDG and NaF-PET scans, respectively. However, one patient with increased uptake on NaF-PET did not have increased glucose metabolism with FDG-PET. The target-to-background ratio of SUVmax for NaF-PET scans was significantly greater than FDG-PET suggesting that NaF-PET may have greater sensitivity than FDG-PET in confirming a diagnosis of ONJ. Several markers of bone turnover and remodelling were measured including serum calcium, vitamin D (25-OH), urinary N telopeptides, bone alkaline phosphatase (BAP), osteopontin, MIP 1a, RANK-L, osteoprotegrin, and DKK. Transcriptional profiling on peripheral blood mononuclear cells (PBMCs) using the Affymetrix U133Plus 2.0 gene chip was also performed in all 11 patients and compared with those of 10 MM patients on BP therapy without ONJ and 5 normal donors. These correlative studies will be presented and provide insights into the pathophysiology of ONJ. Importantly these studies both define clinical and radiographic features on ONJ, but also identify biomarkers to be evaluated in future prospective studies of BP therapy and ONJ.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 8
    Online Resource
    Online Resource
    Inderscience Publishers ; 2023
    In:  International Journal of Entrepreneurship and Small Business Vol. 50, No. 1 ( 2023), p. 66-93
    In: International Journal of Entrepreneurship and Small Business, Inderscience Publishers, Vol. 50, No. 1 ( 2023), p. 66-93
    Type of Medium: Online Resource
    ISSN: 1476-1297 , 1741-8054
    Language: English
    Publisher: Inderscience Publishers
    Publication Date: 2023
    SSG: 3,2
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  • 9
    In: Journal of Clinical Investigation, American Society for Clinical Investigation
    Type of Medium: Online Resource
    ISSN: 0021-9738
    Language: English
    Publisher: American Society for Clinical Investigation
    Publication Date: 2008
    detail.hit.zdb_id: 2018375-6
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  • 10
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 5005-5005
    Abstract: In a previous study we reported that R-enantiomer of etodolac (R-etodolac) which is under investigation in Phase II clinical trials in chronic lymphocytic leukemia, induces potent cytotoxicity at clinically relevant concentrations in both drug-sensitive and drug-resistant multiple myeloma (MM) cell lines, as well as in patient MM tumor cells. In this study, we demonstrated that SDX-308 (CEP-18082), a novel analog of etodolac, has more potent cytotoxicity than R-etodolac against MM cell lines and tumor cells from patients with refractory MM. It also induces cytotoxicity against bortezomib-resistant tumor cells. The molecular mechanisms whereby SDX-308 triggers MM cell cytotoxicity were next delineated. SDX-308 induces apoptosis via caspase-8/-9/-3 activation and poly(ADP-ribose) polymerase PARP cleavage. It strongly inhibits Wnt/β-catenin pathway by blockade of nuclear translocation of β-catenin, followed by significant inhibition of transcription and expression of target proteins. These target proteins include cell cycle regulatory c-myc molecules and anti-apoptotic survivin molecules. Neither interleukin-6 nor insulin-like growth factor-1, which induce MM cell growth and abrogate Dex-induced apoptosis, protect against growth inhibition triggered by SDX-308. Importantly, growth of MM cells adherent to bone marrow (BM) stromal cells is also significantly inhibited by SDX-308. Our data therefore indicate that the novel etodolac analog SDX-308 has more potent cytotoxicity in MM cells than R-etodolac even in the context with BM microenvironment, providing the preclinical rationale to conduct clinical trials of this agent to improve patient outcome in MM.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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