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BACKGROUND: Anti-programmed cell death 1 antibodies (anti-PD-1 Abs) are widely used for advanced melanoma, but the efficacy of an anti-PD-1 Abs is limited in the Asian population. There remains an unmet need to improve the therapeutic effects of anti-PD-1 Abs treatment, particularly in melanoma patients who are refractory to anti-PD-1 Abs. The aim was to evaluate anti-PD-1 Abs treatment in combination with TM5614 (plasminogen activator inhibitor-1: PAI-1 inhibitor) in patients with unresectable melanoma. METHODS: The TM5614-MM study was a multicentre, open-label, single-arm, phase 2 clinical trial to evaluate the efficacy and safety of nivolumab in combination with TM5614 in patients with advanced, unresectable malignant melanoma recruited at 7 Japanese institutes between 13 September 2021 and 31 March 2023. Patients with metastatic or unresectable melanoma previously treated with anti-PD-1 Abs were enrolled. Nivolumab 480 mg was administered intravenously every 4 weeks for 8 weeks, while TM5614 was administered orally at a dose of 120 mg (0-4 weeks) and 180 mg once daily (5-8 weeks). The primary endpoint was the overall response rate after 8 weeks of concomitant use of TM5614. RESULTS: Thirty nine patients were enrolled, and 34 patients in the anti-PD-1 Abs-refractory cohort. The overall response rate at 8 weeks was 25.9% (95% CI: 12.9-44.9%; P = .027) in 27 anti-PD-1-Abs refractory patients by investigator assessment in the protocol per set cohort. Seven patients discontinued treatment due to progressive disease or adverse events. Treatment-related grade 3 or higher adverse events occurred in 3 of 39 patients (7.7%) in the intention-to-treat cohort. CONCLUSIONS: TM5614 in combination with nivolumab is well-tolerated and effective in anti-PD-1 Abs-refractory, unresectable melanoma. TRIAL REGISTRATION: This trial was registered with Clinical Trial gov, jRCT2021210029.
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Background: Increased levels of plasminogen activator inhibitor-1 (PAI-1) in tumors have been found to correlate with poor clinical outcomes in patients with cancer. Although abundant data support the involvement of PAI-1 in cancer progression, whether PAI-1 contributes to tumor immune surveillance remains unclear. The purposes of this study are to determine whether PAI-1 regulates the expression of immune checkpoint molecules to suppresses the immune response to cancer and demonstrate the potential of PAI-1 inhibition for cancer therapy. Methods: The effects of PAI-1 on the expression of the immune checkpoint molecule programmed cell death ligand 1 (PD-L1) were investigated in several human and murine tumor cell lines. In addition, we generated tumor-bearing mice and evaluated the effects of a PAI-1 inhibitor on tumor progression or on the tumor infiltration of cells involved in tumor immunity either alone or in combination with immune checkpoint inhibitors. Results: PAI-1 induces PD-L1 expression through the JAK/STAT signaling pathway in several types of tumor cells and surrounding cells. Blockade of PAI-1 impedes PD-L1 induction in tumor cells, significantly reducing the abundance of immunosuppressive cells at the tumor site and increasing cytotoxic T-cell infiltration, ultimately leading to tumor regression. The anti-tumor effect elicited by the PAI-1 inhibitor is abolished in immunodeficient mice, suggesting that PAI-1 blockade induces tumor regression by stimulating the immune system. Moreover, combining a PAI-1 inhibitor with an immune checkpoint inhibitor significantly increases tumor regression. Conclusions: PAI-1 protects tumors from immune surveillance by increasing PD-L1 expression; hence, therapeutic PAI-1 blockade may prove valuable in treating malignant tumors.
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Antígeno B7-H1 , Inhibidor 1 de Activador Plasminogénico , Escape del Tumor , Animales , Femenino , Humanos , Ratones , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Evasión Inmune , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Transducción de Señal , Microambiente Tumoral/inmunologíaRESUMEN
mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo . Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.
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Mitochondria are indispensable in maintaining hematopoietic stem cells (HSCs), and mitochondrial complex II (MCII) has been recognized as a key component of HSCs. However, the physiological role of MCII on long-term hematopoiesis and hematopoietic reconstitution capacity remains unknown. Hence, this study evaluated the impact of MCII dysfunctions on long-term HSC maintenance and hematopoietic homeostasis among conditional transgenic mice with a missense mutation in the succinate dehydrogenase complex subunit C gene (SdhcV69E). HSCs collected from SdhcV69E mice had a higher reactive oxygen species (ROS) accumulation and DNA damage in response to mitochondrial activation. Via the aging stress response, MCII dysfunctions caused decreased white blood cell count with myeloid-skewing property, macrocytic anemia, and thrombocytosis. Moreover, the HSCs of aged SdhcV69E mice exhibited greater ROS accumulation and lower membrane potential. Transplantation-induced replicative stress also caused premature senescent hematopoiesis. Furthermore, accelerated ROS accumulation and profound DNA damage in HSCs were observed in the SdhcV69E-derived cell recipients. The long-term hematopoietic reconstitution capacity was remarkably impaired in HSCs from the SdhcV69E-derived cell recipients. Taken together, MCII plays an essential role in long-term hematopoiesis, and MCII dysfunctions with aging or replicative stresses caused excessive ROS accumulation and DNA damage in HSCs, leading to premature senescence.
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Envejecimiento Prematuro , Ratones , Animales , Especies Reactivas de Oxígeno/metabolismo , Envejecimiento Prematuro/genética , Envejecimiento Prematuro/metabolismo , Transporte de Electrón , Células Madre Hematopoyéticas/metabolismo , Envejecimiento/genética , Ratones Transgénicos , Hematopoyesis/genética , Ratones Endogámicos C57BLRESUMEN
T-cell development in the thymus is dependent on Notch signaling induced by the interaction of Notch1, present on immigrant cells, with a Notch ligand, delta-like (Dll) 4, on the thymic epithelial cells. Phylogenetic analysis characterizing the properties of the Dll4 molecule suggests that Dll4 emerged from the common ancestor of lobe- and ray-finned fishes and diverged into bony fishes and terrestrial organisms, including mammals. The thymus evolved in cartilaginous fishes before Dll4, suggesting that T-cell development in cartilaginous fishes is dependent on Dll1 instead of Dll4. In this study, we compared the function of both Dll molecules in the thymic epithelium using Foxn1-cre and Dll4-floxed mice with conditional transgenic alleles in which the Dll1 or Dll4 gene is transcribed after the cre-mediated excision of the stop codon. The expression of Dll1 in the thymic epithelium completely restored the defect in the Dll4-deficient condition, suggesting that Dll1 can trigger Notch signaling that is indispensable for T-cell development in the thymus. Moreover, using bone marrow chimeras with Notch1- or Notch2-deficient hematopoietic cells, we showed that Dll1 is able to activate Notch signaling, which is sufficient to induce T-cell development, with both the receptors, in contrast to Dll4, which works only with Notch1, in the thymic environment. These results strongly support the hypothesis that Dll1 regulates T-cell development via Notch1 and/or Notch2 in the thymus of cartilaginous fishes and that Dll4 has replaced Dll1 in inducing thymic Notch signaling via Notch1 during evolution.
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Proteínas de Unión al Calcio , Péptidos y Proteínas de Señalización Intracelular , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Epitelio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ligandos , Mamíferos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , FilogeniaRESUMEN
In this study, we examined the antileukemic effects of pterostilbene, a natural methylated polyphenol analog of resveratrol that is predominantly found in berries and nuts, using various human and murine leukemic cells, as well as bone marrow samples obtained from patients with leukemia. Pterostilbene administration significantly induced apoptosis of leukemic cells, but not of non-malignant hematopoietic stem/progenitor cells. Interestingly, pterostilbene was highly effective in inducing apoptosis of leukemic cells harboring the BCR/ABL fusion gene, including ABL tyrosine kinase inhibitor (TKI)-resistant cells with the T315I mutation. In BCR/ABL+ leukemic cells, pterostilbene decreased the BCR/ABL fusion protein levels and suppressed AKT and NF-κB activation. We further demonstrated that pterostilbene along with U0126, an inhibitor of the MEK/ERK signaling pathway, synergistically induced apoptosis of BCR/ABL+ cells. Our results further suggest that pterostilbene-promoted downregulation of BCR/ABL involves caspase activation triggered by proteasome inhibition-induced endoplasmic reticulum stress. Moreover, oral administration of pterostilbene significantly suppressed tumor growth in mice transplanted with BCR/ABL+ leukemic cells. Taken together, these results suggest that pterostilbene may hold potential for the treatment of BCR/ABL+ leukemia, in particular for those showing ABL-dependent TKI resistance.
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Apoptosis/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Leucemia/tratamiento farmacológico , Leucemia/genética , Mutación/genética , Estilbenos/farmacología , Administración Oral , Animales , Caspasas/metabolismo , Estrés del Retículo Endoplásmico , Células Madre Hematopoyéticas/patología , Humanos , Leucemia/patología , Ratones , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estilbenos/administración & dosificación , Células Tumorales CultivadasRESUMEN
Notch signaling primarily determines T-cell fate. However, the molecular mechanisms underlying the maintenance of T-lineage potential in pre-thymic progenitors remain unclear. Here, we established two murine Ebf1-deficient pro-B cell lines, with and without T-lineage potential. The latter expressed lower levels of Lmo2; their potential was restored via ectopic expression of Lmo2. Conversely, the CRISPR/Cas9-mediated deletion of Lmo2 resulted in the loss of the T-lineage potential. Introduction of Bcl2 rescued massive cell death of Notch-stimulated pro-B cells without efficient LMO2-driven Bcl11a expression but was not sufficient to retain their T-lineage potential. Pro-B cells without T-lineage potential failed to activate Tcf7 due to DNA methylation; Tcf7 transduction restored this capacity. Moreover, direct binding of LMO2 to the Bcl11a and Tcf7 loci was observed. Altogether, our results highlight LMO2 as a crucial player in the survival and maintenance of T-lineage potential in T-cell progenitors via the regulation of the expression of Bcl11a and Tcf7.
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Proteínas Adaptadoras Transductoras de Señales/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Proteínas con Dominio LIM/genética , Linfocitos T/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Femenino , Proteínas con Dominio LIM/metabolismo , Masculino , RatonesRESUMEN
The long-term prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) is still unsatisfactory even after the emergence of tyrosine kinase inhibitors (TKIs) against chimeric BCR-ABL, and this is associated with the high incidence of genetic alterations of Ikaros family zinc finger 1 (IKZF1), most frequently the hemi-allelic loss of exons 4-7 expressing a dominant-negative isoform Ik6. We found that lenalidomide (LEN), a representative of immunomodulatory drugs (IMiDs), which have been long used for the treatment of multiple myeloma, specifically induced accumulation of Ik6 with the disappearance of functional isoforms within 24 h (i.e., abrupt and complete shut-down of the IKZF1 activity) in Ik6-positive Ph+ALL cells in a neddylation-dependent manner. The functional IKZF3 isoforms expression was also abruptly and markedly downregulated. The LEN treatment specifically suppressed proliferation of Ik6-positive-Ph+ALL cells by inducing cell cycle arrest via downregulation of cyclins D3 and E and CDK2, and of importance, markedly upregulated their apoptosis in synergy with the TKI imatinib (IM). Apoptosis of IM-resistant Ph+ALL cells with T315I mutation of BCR-ABL was also upregulated by LEN in the presence of the newly developed TKI ponatinib. Analyses of flow cytometry, western blot, and oligonucleotide array revealed that apoptosis was caspase-/p53-dependent and associated with upregulation of pro-apoptotic Bax/Bim, enhanced dephosphorylation of BCR-ABL/Akt, and downregulation of oncogenic helicase genes HILLS, CDC6, and MCMs4 and 8. Further, the synergism of LEN with IM was clearly documented as a significant prolongation of survival in the xenograft mice model. Because this synergism was further potentiated in vitro by dexamethasone, a key drug for ALL treatment, the strategy of repositioning IMiDs for the treatment of Ik6-positive Ph+ALL patients certainly shed new light on an outpatient-based treatment option for achieving their long-term durable remission and higher QOL, particularly for those who are not tolerable to intensified therapeutic approaches.
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Bone marrow adipocytes (BMAs) have recently been recognized as a niche component with a suppressive function. Obese individuals with abundant BMAs exhibit impaired hematopoietic regeneration after hematopoietic stem cell transplantation (HSCT). We hypothesized that plasminogen activator inhibitor type-1 (PAI-1), an adipokine that regulates the fibrinolytic system, contributes to impaired hematopoiesis in bone marrow (BM) microenvironment with abundant BMAs. We demonstrated that BMAs differentiated in vitro could secrete PAI-1 and were positive for PAI-1 in vivo. In addition, the abundance of BMAs was associated with high levels of PAI-1 expression. The BMA-rich microenvironment exhibited impaired hematopoietic regeneration after HSCT when compared with a BMA-less microenvironment. The impaired hematopoietic regeneration in BMA-rich microenvironment was significantly alleviated by PAI-1 knockout or PAI-1 inhibitor treatment. Obese mice with abundant BMAs, compared with normal-weight mice, exhibited higher bone marrow PAI-1 concentrations, increased fibrinolytic system suppression, and lower stem cell factor (SCF) concentrations after HSCT. PAI-1 inhibitor administration significantly activated the fibrinolytic system in obese mice, contributing to the higher SCF concentration. Moreover, PAI-1 inhibitor treatment significantly alleviated the impaired hematopoietic regeneration in obese mice both after 5-fluorouracil injection and HSCT. These results indicate that PAI-1 hinders hematopoietic regeneration in BMA-rich microenvironments. The blockade of PAI-1 activity could be a novel therapeutic means of facilitating hematopoietic reconstitution in BMA-rich patients.
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Adipocitos/metabolismo , Médula Ósea/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Obesidad/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/farmacología , Animales , Antimetabolitos/farmacología , Médula Ósea/metabolismo , Fluorouracilo/farmacología , Técnicas de Inactivación de Genes , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/genética , Obesidad/terapia , Inhibidor 1 de Activador Plasminogénico/genética , Regeneración/efectos de los fármacos , Factor de Células Madre/metabolismo , Nicho de Células Madre/efectos de los fármacosRESUMEN
Therapeutic strategies that target leukemic stem cells (LSCs) provide potential advantages in the treatment of chronic myeloid leukemia (CML). Here, we show that selective blockade of plasminogen activator inhibitor-1 (PAI-1) enhances the susceptibility of CML-LSCs to tyrosine kinase inhibitor (TKI), which facilitates the eradication of CML-LSCs and leads to sustained remission of the disease. We demonstrated for the first time that TGF-ß-PAI-1 axis was selectively augmented in CML-LSCs in the bone marrow (BM), whereby protecting CML-LSCs from TKI treatment. Furthermore, the combined administration of TKI plus a PAI-1 inhibitor, in a mouse model of CML, significantly enhanced the eradication of CML cells in the BM and prolonged the survival of CML mice. The combined therapy of imatinib and a PAI-1 inhibitor prevented the recurrence of CML-like disease in serially transplanted recipients, indicating the elimination of CML-LSCs. Interestingly, PAI-1 inhibitor treatment augmented membrane-type matrix metalloprotease-1 (MT1-MMP)-dependent motility of CML-LSCs, and the anti-CML effect of PAI-1 inhibitor was extinguished by the neutralizing antibody for MT1-MMP, underlining the mechanistic importance of MT1-MMP. Our findings provide evidence of, and a rationale for, a novel therapeutic tactic, based on the blockade of PAI-1 activity, for CML patients.
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Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva , Animales , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Ratones , Células Madre Neoplásicas , Inhibidor 1 de Activador Plasminogénico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Serpina E2RESUMEN
BACKGROUND: The transmembrane glycoprotein podoplanin (PDPN) is upregulated in some tumors and has gained attention as a malignant tumor biomarker. PDPN molecules have platelet aggregation-stimulating domains and, are therefore, suggested to play a role in tumor-induced platelet activation, which in turn triggers epithelial-to-mesenchymal transition (EMT) and enhances the invasive and metastatic activities of tumor cells. In addition, as forced PDPN expression itself can alter the propensity of certain tumor cells in favor of EMT and enhance their invasive ability, it is also considered to be involved in the cell signaling system. Nevertheless, underlying mechanisms of PDPN in tumor cell invasive ability as well as EMT induction, especially by platelets, are still not fully understood. METHODS: Subclonal TE11A cells were isolated from the human esophageal squamous carcinoma cell line TE11 and the effects of anti-PDPN neutralizing antibody as well as PDPN gene knockout on platelet-induced EMT-related gene expression were measured. Also, the effects of PDPN deficiency on cellular invasive ability and motility were assessed. RESULTS: PDPN-null cells were able to provoke platelet aggregation, suggesting that PDPN contribution to platelet activation in these cells is marginal. Nevertheless, expression of platelet-induced EMT-related genes, including vimentin, was impaired by PDPN-neutralizing antibody as well as PDPN deficiency, while their effects on TGF-ß-induced gene expression were marginal. Unexpectedly, PDPN gene ablation, at least in either allele, engendered spontaneous N-cadherin upregulation and claudin-1 downregulation. Despite these seemingly EMT-like alterations, PDPN deficiency impaired cellular motility and invasive ability even after TGF-ß-induced EMT induction. CONCLUSIONS: These results suggested that, while PDPN seems to function in favor of maintaining the epithelial state of this cell line, it is indispensable for platelet-mediated induction of particular mesenchymal marker genes as well as the potentiation of motility and invasion capacity.
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BACKGROUND/AIMS: Intestinal fibrosis is a major complication of Crohn's disease (CD). The profibrotic protein transforming growth factor-ß (TGF-ß) has been considered to be critical for the induction of the fibrotic program. TGF-ß has the ability to induce not only the expression of extracellular matrix (ECM) including collagen, but also the production of plasminogen activator inhibitor-1 (PAI-1) that prevents enzymatic degradation of the ECM during the onset of fibrotic diseases. However, the significance of PAI-1 in the developing intestinal fibrosis has not been fully understood. In the present study, we examined the actual expression of PAI-1 in fibrotic legion of intestinal inflammation and its correlation with the abnormal ECM deposition. METHODS: Chronic intestinal inflammation was induced in BALB/c mice using 8 repeated intrarectal injections of 2,4,6-trinitrobenzene sulfonic acid (TNBS). TM5275, a PAI-1 inhibitor, was orally administered as a carboxymethyl cellulose suspension each day for 2 weeks after the sixth TNBS injection. RESULTS: Using a publicly available dataset (accession number, GSE75214) and TNBS-treated mice, we observed increases in PAI-1 transcripts at active fibrotic lesions in both patients with CD and mice with chronic intestinal inflammation. Oral administration of TM5275 immediately after the onset of intestinal fibrosis upregulated MMP-9 (matrix metalloproteinase 9) and decreased collagen accumulation, resulting in attenuation of the fibrogenesis in TNBS-treated mice. CONCLUSIONS: PAI-1-mediated fibrinolytic system facilitates collagen degradation suppression. Hence, PAI-1 inhibitor could be applied as an anti-fibrotic drug in CD treatment.
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Epstein-Barr virus (EBV) is associated with several disorders. EBV is known to modulate the proliferation and survival of hematopoietic cells such as B cells and T cells in human. However, the effects of EBV on hematopoiesis itself have not been investigated. To study EBV infection in murine models, their hematopoiesis must be humanized, since EBV infection is limited only in primates. To engraft the human hematopoiesis, NOD/Shi-scid-IL2rγnull (NOG) mice were used. Usually, the hematopoiesis humanized mice reconstitute only lymphoid cells, but myeloid cells are not. However, we revealed human macrophages (hMφ) and their precursor monocytes were increased in peripheral tissues of EBV-infected mice. Furthermore, our previous report indicated Mφ accumulation in spleen was essential for development of EBV-positive tumors, suggesting that EBV modulates human hematopoiesis in order to thrive. Interestingly, we revealed a dramatic increase of immature granulocytes only in bone marrow of EBV-infected mice. In addition, GM-CSF, a cytokine that is essential for differentiation of the myeloid lineage, was significantly increased in EBV-infected mice. These results were also reproduced in patients with EBV-related disorders. We suggest that the hematopoietic alterations during EBV-infection might contribute immune suppression to the development and exacerbation of EBV-related disorders.
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Médula Ósea/virología , Infecciones por Virus de Epstein-Barr/sangre , Animales , Médula Ósea/fisiología , Infecciones por Virus de Epstein-Barr/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Hematopoyesis , Humanos , Leucocitos Mononucleares/virología , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Upon hematopoietic stem cell transplantation (HSCT), the availability of recipients' niches in the bone marrow (BM) is one of the factors that influence donor HSC engraftment and hematopoietic reconstitution. Therefore, myeloablative conditioning, such as irradiation and/or chemotherapy, which creates empty niches in the recipients' BM, is required for the success of HSCT. However, the conventional myeloablation causes extensive damages to the patients' BM, which results in the treatment-induced severe complications and even mortality. Thus, alternative and mild conditioning could fulfill the need for safer HSCT-based therapies for hematological and nonhematological disorders. Recently, we have demonstrated that pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1) activity increases cellular motility and cause detachment of HSCs from the niches. In this study, we performed HSCT using a PAI-1 inhibitor without any myeloablative conditioning. Donor HSCs were transplanted to recipient mice that were pretreated with saline or a PAI-1 inhibitor. Saline pretreated nonmyeloablative recipients showed no engraftment. In contrast, donor cell engraftment was detected in the PAI-1 inhibitor pretreated recipients. Multilineage differentiation, including lymphoid and myeloid cells, was observed in the PAI-1 inhibitor pretreated recipients. Donor-derived cells that exhibited multilineage reconstitution as well as the existence of stem/progenitor cells were detected in the secondary recipients, confirming the maintenance of donor HSCs in the BM of PAI-1 inhibitor pretreated primary recipients. The results indicate that the PAI-1 blockade vacates functional niches in the recipients' BM, which allows the engraftment of long-term multilineage HSCs without myeloablative conditioning.
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Médula Ósea/metabolismo , Trasplante de Células Madre Hematopoyéticas , Inhibidor 1 de Activador Plasminogénico/metabolismo , Nicho de Células Madre , Acondicionamiento Pretrasplante , Animales , Linaje de la Célula , Células Madre Hematopoyéticas/metabolismo , RatonesRESUMEN
Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.
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Infecciones por Virus de Epstein-Barr/complicaciones , Exosomas/virología , Herpesvirus Humano 4/genética , Inflamación/virología , Linfoma/virología , ARN Viral/genética , Animales , Carcinogénesis/genética , Carcinogénesis/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/virología , Exosomas/genética , Exosomas/inmunología , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Inflamación/etiología , Inflamación/genética , Inflamación/inmunología , Linfoma/etiología , Linfoma/genética , Linfoma/inmunología , Ratones , MicroARNs/análisis , MicroARNs/genética , ARN Viral/análisis , Análisis de Secuencia de ARN , Microambiente TumoralAsunto(s)
Bortezomib/farmacología , Resistencia a Antineoplásicos/fisiología , Proteína Jagged-1/metabolismo , Mieloma Múltiple/patología , Receptores Notch/metabolismo , Animales , Activación Enzimática/fisiología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/metabolismo , Transducción de Señal/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hematopoietic stem and progenitor cells (HSPCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but usually stay in the niche. We questioned what determines the motility of HSPCs. Here, we show that transforming growth factor (TGF)-ß-induced intracellular plasminogen activator inhibitor (PAI)-1 activation is responsible for keeping HSPCs in the BM niche. We found that the expression of PAI-1, a downstream target of TGF-ß signaling, was selectively augmented in niche-residing HSPCs. Functional inhibition of the TGF-ß-PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSPCs from the TGF-ß-expressing niche cells, such as megakaryocytes. Furthermore, consistently high motility in PAI-1-deficient HSPCs was demonstrated by both a transwell migration assay and reciprocal transplantation experiments, indicating that intracellular, not extracellular, PAI-1 suppresses the motility of HSPCs, thereby causing them to stay in the niche. Mechanistically, intracellular PAI-1 inhibited the proteolytic activity of proprotein convertase Furin, diminishing MT1-MMP activity. This reduced expression of MT1-MMP in turn affected the expression levels of several adhesion/deadhesion molecules for determination of HSPC localization, such as CD44, VLA-4, and CXCR4, which then promoted the retention of HSPCs in the niche. Our findings open up a new field for the study of intracellular proteolysis as a regulatory mechanism of stem cell fate, which has the potential to improve clinical HSPC mobilization and transplantation protocols.
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Células Madre Hematopoyéticas/metabolismo , Espacio Intracelular/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Nicho de Células Madre , Factor de Crecimiento Transformador beta/metabolismo , Animales , Médula Ósea/metabolismo , Movimiento Celular , Espacio Extracelular/metabolismo , Furina/metabolismo , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/citología , Humanos , Metaloproteinasa 14 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre Multipotentes/metabolismo , Transducción de SeñalRESUMEN
The aim of this study is to evaluate the feasibility of NOD/Shi-scid-IL2Rγnull(NOG) mice transplanted with human CD34+/CD38-/Lin-/low hematopoietic cells from cord blood (CB) as an experimental model of the gene expression in human hematopoiesis. We compared the gene expressions of human CD34+/CD38-/Lin-/low cells from human bone marrow (BM) and in xenograft models. The microarray data revealed that 25 KEGG pathways were extracted from the comparison of human CD34+/CD38-/Lin-/low HSCs between CB and BM, and that 17 of them--which were mostly related to cellular survival, RNA metabolism and lymphoid development--were shared with the xenograft model. When the probes that were commonly altered in CD34+/CD38-/Lin-/low cells from both human and xenograft BM were analyzed, most of them, including the genes related hypoxia, hematopoietic differentiation, epigenetic modification, translation initiation, and RNA degradation, were downregulated. These alterations of gene expression suggest a reduced differentiation capacity and likely include key alterations of gene expression for settlement of CB CD34+/CD38-/Lin-/low cells in BM. Our findings demonstrate that the xenograft model of human CB CD34+/CD38-/Lin-/low cells using NOG mice was useful, at least in part, for the evaluation of the gene expression profile of human hematopoietic stem cells.
Asunto(s)
Expresión Génica , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas , Xenoinjertos , Transcriptoma , ADP-Ribosil Ciclasa 1 , Animales , Antígenos CD34 , Células de la Médula Ósea , Diferenciación Celular/genética , Estudios de Factibilidad , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Humanos , Ratones Endogámicos NOD , Ratones Endogámicos , Modelos AnimalesRESUMEN
Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta-like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast-specific manner, we investigated the ligand-specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1-induced Notch signaling was responsible for the expansion of the bone-forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1-Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast-specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast-osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1-mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand-specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569-2580, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.
Asunto(s)
Remodelación Ósea , Fémur/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Osteoblastos/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Células Madre/metabolismo , Tibia/metabolismo , Animales , Resorción Ósea/genética , Resorción Ósea/metabolismo , Proteínas de Unión al Calcio , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Femenino , Fémur/embriología , Fémur/crecimiento & desarrollo , Genotipo , Edad Gestacional , Homeostasis , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Osteoclastos/metabolismo , Osteogénesis , Fenotipo , Tibia/embriología , Tibia/crecimiento & desarrolloRESUMEN
Analysis of the hematopoiesis of non-human primates is important to clarify the evolution of primate-specific hematopoiesis and immune regulation. However, the engraftment and development of the primate hematopoietic system are well-documented only in humans and are not clear in non-human primates. Callithrix jacchus (common marmoset, CM) is a New World monkey with a high rate of pregnancy and small size that lives in closed colonies. As stem cell factor (SCF) is an essential molecule for hematopoietic stem cell development in mice and humans, we focused on CD117, the SCF receptor, and examined whether CD117-expressing cells possess the hematopoietic stem/progenitor cell characteristics of newborn marmoset-derived hematopoietic cells that can develop into T cells and B cells. When CD117(+) cell fractions of the bone marrow were transplanted into immunodeficient NOD (non-obese diabetic)/Shi-scid, common γc-null (NOG) mice, these cells engrafted efficiently in the bone marrow and spleens of the NOG mice. The CD117(+) cells developed into myeloid lineage cells, CD20(+) B cells and CD3(+) T cells, which could express CM cytokines in vivo. The development of B cells did not precede that of T cells. The development of CD8(+) T cells was dominant in NOG mice. The engraftment was comparable for both CD117(+)CD34(+) cells and CD117(+)CD34(-) cells. These results suggest that the CD117(+) cell fraction can differentiate into all three cell lineages, and the development of marmoset immunity in the xenogeneic environment follows diverse developmental pathways compared with human immunity.