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ABSTRACT: Notch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, the role of Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO messenger RNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-sequencing analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR), a heterodimer of asialoglycoprotein receptor 1 [ASGR1] and ASGR2, physically associates with Notch1, and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Delta-like 4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation, and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.
Subject(s)
Hepatocytes , Janus Kinase 2 , Liver , Receptor, Notch1 , Thrombopoietin , Animals , Receptor, Notch1/metabolism , Receptor, Notch1/genetics , Thrombopoietin/metabolism , Thrombopoietin/genetics , Mice , Liver/metabolism , Hepatocytes/metabolism , Janus Kinase 2/metabolism , Janus Kinase 2/genetics , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , Mice, Knockout , Signal Transduction , Phosphorylation , Blood Platelets/metabolism , Mice, Inbred C57BL , Thrombocytopenia/metabolism , Thrombocytopenia/genetics , Thrombocytopenia/pathologyABSTRACT
Efficient homing of infused hematopoietic stem and progenitor cells (HSPCs) into the bone marrow (BM) is the prerequisite for successful hematopoietic stem cell transplantation. However, only a small part of infused HSPCs find their way to the BM niche. A better understanding of the mechanisms that facilitate HSPC homing will help to develop strategies to improve the initial HSPC engraftment and subsequent hematopoietic regeneration. Here, we show that irradiation upregulates the endomucin expression of endothelial cells in vivo and in vitro. Furthermore, depletion of endomucin in irradiated endothelial cells with short interfering RNA (siRNA) increases the HSPC-endothelial cell adhesion in vitro. To abrogate the endomucin of BM sinusoidal endothelial cells (BM-SECs) in vivo, we develop a siRNA-loaded bovine serum albumin nanoparticle for targeted delivery. Nanoparticle-mediated siRNA delivery successfully silences endomucin expression in BM-SECs and improves HSPC homing during transplantation. These results reveal that endomucin plays a critical role in HSPC homing during transplantation and that gene-based manipulation of BM-SEC endomucin in vivo can be exploited to improve the efficacy of HSPC transplantation.
ABSTRACT
Transforming growth factor ß1 (TGFB1) refers to a pleiotropic cytokine exerting contrasting roles in hematopoietic stem cells (HSCs) functions in vitro and in vivo. However, the understanding of hematopoiesis in vivo, when TGFB1 is constantly deactivated, is still unclear, mainly due to significant embryonic lethality and the emergence of a fatal inflammatory condition, which makes doing these investigations challenging. Our study aims to find the specific role of TGFB1 in regulating hematopoiesis in vivo. We engineered mice strains (Vav1 or Mx1 promoter-driven TGFB1 knockout) with conditional knockout of TGFB1 to study its role in hematopoiesis in vivo. In fetal and adult hematopoiesis, TGFB1 KO mice displayed deficiency and decreased self-renewal capacity of HSCs with myeloid-biased differentiation. The results were different from the regulating role of TGFB1 in vitro. Additionally, our results showed that TGFB1 deficiency from fetal hematopoiesis stage caused more severe defect of HSCs than in the adult stage. Mechanistically, our findings identified TGFB1-SOX9-FOS/JUNB/TWIST1 signal axis as an essential regulating pathway in HSCs homeostasis. Our study may provide a scientific basis for clinical HSC transplantation and expansion.
Subject(s)
Hematopoiesis , Hematopoietic Stem Cells , Transforming Growth Factor beta1 , Animals , Mice , Cell Differentiation , Cytokines/metabolism , Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Transforming Growth Factor beta1/metabolismABSTRACT
INTRODUCTION: Celastrol is an active pentacyclic triterpenoid extracted from Tripterygium wilfordii and has anti-inflammatory and anti-tumor properties. Whether Celastrol modulates platelet function remains unknown. Our study investigated its role in platelet function and thrombosis. METHODS: Human platelets were isolated and incubated with Celastrol (0, 1, 3 and 5 µM) at 37 °C for 1 h to measure platelet aggregation, granules release, spreading, thrombin-induced clot retraction and intracellular calcium mobilization. Additionally, Celastrol (2 mg/kg) was intraperitoneally administrated into mice to evaluate hemostasis and thrombosis in vivo. RESULTS: Celastrol treatment significantly decreased platelet aggregation and secretion of dense or alpha granules induced by collagen-related peptide (CRP) or thrombin in a dose-dependent manner. Additionally, Celastrol-treated platelets showed a dramatically reduced spreading activity and decreased clot retraction. Moreover, Celastrol administration prolonged tail bleeding time and inhibited formation of arterial/venous thrombosis. Furthermore, Celastrol significantly reduced calcium mobilization. CONCLUSION: Celastrol inhibits platelet function and venous/arterial thrombosis, implying that it might be utilized for treating thrombotic diseases.
Subject(s)
Platelet Activation , Thrombosis , Humans , Animals , Mice , Calcium/metabolism , Thrombin/metabolism , Hemostasis , Platelet Aggregation , Blood Platelets/metabolism , Pentacyclic Triterpenes , Thrombosis/metabolismABSTRACT
Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a protein tyrosine phosphatase that negatively regulates T-cell signaling. However, whether it is expressed and functions in platelets remains unknown. Here we investigated the expression and role of PTPN22 in platelet function. We reported PTPN22 expression in both human and mouse platelets. Using PTPN22-/- mice, we showed that PTPN22 deficiency significantly shortened tail-bleeding time and accelerated arterial thrombus formation without affecting venous thrombosis and the coagulation factors VIII and IX. Consistently, PTPN22-deficient platelets exhibited enhanced platelet aggregation, granule secretion, calcium mobilization, lamellipodia formation, spreading, and clot retraction. Quantitative phosphoproteomic analysis revealed the significant difference of phosphodiesterase 5A (PDE5A) phosphorylation in PTPN22-deficient platelets compared with wild-type platelets after collagen-related peptide stimulation, which was confirmed by increased PDE5A phosphorylation (Ser92) in collagen-related peptide-treated PTPN22-deficient platelets, concomitant with reduced level and vasodilator-stimulated phosphoprotein phosphorylation (Ser157/239). In addition, PTPN22 interacted with phosphorylated PDE5A (Ser92) and dephosphorylated it in activated platelets. Moreover, purified PTPN22 but not the mutant form (C227S) possesses intrinsic serine phosphatase activity. Furthermore, inhibition of PTPN22 enhanced human platelet aggregation, spreading, clot retraction, and increased PDE5A phosphorylation (Ser92). In conclusion, our study shows a novel role of PTPN22 in platelet function and arterial thrombosis, identifying new potential targets for future prevention of thrombotic or cardiovascular diseases.
Subject(s)
Hemostasis , Protein Tyrosine Phosphatase, Non-Receptor Type 22 , Thrombosis , Animals , Blood Platelets/metabolism , Humans , Mice , Mice, Knockout , Platelet Activation , Platelet Aggregation , Platelet Function Tests , Protein Tyrosine Phosphatase, Non-Receptor Type 22/metabolism , Thrombosis/geneticsABSTRACT
Acute myeloid leukemia (AML) is a malignant hematologic disease caused by gene mutations and genomic rearrangements in hematologic progenitors. The PHF6 (PHD finger protein 6) gene is highly conserved and located on the X chromosome in humans and mice. We found that PHF6 was highly expressed in AML cells with MLL rearrangement and was related to the shortened survival time of AML patients. In our study, we knocked out the Phf6 gene at different disease stages in the AML mice model. Moreover, we knocked down PHF6 by shRNA in two AML cell lines and examined the cell growth, apoptosis, and cell cycle. We found that PHF6 deletion significantly inhibited the proliferation of leukemic cells and prolonged the survival time of AML mice. Interestingly, the deletion of PHF6 at a later stage of the disease displayed a better anti-leukemia effect. The expressions of genes related to cell differentiation were increased, while genes that inhibit cell differentiation were decreased with PHF6 knockout. It is very important to analyze the maintenance role of PHF6 in AML, which is different from its tumor-suppressing function in T-cell acute lymphoblastic leukemia (T-ALL). Our study showed that inhibiting PHF6 expression may be a potential therapeutic strategy targeting AML patients.
ABSTRACT
BACKGROUND AIMS: Chimeric antigen receptor-T (CAR-T) cells have exhibited remarkable efficacy in treating refractory or relapsed multiple myeloma (R/R MM). Although obesity has a favorable value in enhancing the response to immunotherapy, less is known about its predictive value regarding the efficacy and prognosis of CAR-T cell immunotherapy. METHODS: We conducted a retrospective study of 111 patients with R/R MM who underwent CAR-T cell treatment. Using the body mass index (BMI) classification, the patients were divided into a normal-weight group (73/111) and an overweight group (38/111). We investigated the effect of BMI on CAR-T cell therapy outcomes in patients with R/R MM. RESULTS: The objective remission rates after CAR-T cell infusion were 94.7% and 89.0% in the overweight and normal-weight groups, respectively. The duration of response and overall survival were not significant difference between BMI groups. Compared to normal-weight patients, overweight patients had an improved median progression-free survival. There was no significant difference in cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome between the subgroups. In terms of hematological toxicity, the erythrocyte, hemoglobin, platelet, leukocyte and neutrophil recovery was accelerated in the overweight group. Fewer patients in the overweight group displayed moderate percent CD4 and CD4/CD8 ratios compared to the normal-weight group. Furthermore, the percent CD4 ratios were positively correlated with the levels of cytokines [interleukin-2 (IL-2) (day 14), interferon gamma (IFN-γ) (day 7) and tumor necrosis factor alpha (TNF-α) (days 14 and 21)] after cells infusion. On the other hand, BMI was positively associated with the levels of IFN-γ (day 7) and TNF-α (days 14 and 21) after CAR-T cells infusion. CONCLUSIONS: Overall, this study highlights the potential beneficial effect of a higher BMI on CAR-T cell therapy outcomes.
Subject(s)
Body Mass Index , Immunotherapy, Adoptive , Multiple Myeloma , Humans , Multiple Myeloma/therapy , Multiple Myeloma/immunology , Multiple Myeloma/mortality , Male , Female , Middle Aged , Immunotherapy, Adoptive/methods , Aged , Retrospective Studies , Adult , Receptors, Chimeric Antigen/immunology , Treatment Outcome , PrognosisABSTRACT
NLRP6 plays a crucial role in maintaining intestinal homeostasis by regulating the interaction between the intestinal mucosa and the microbiota. However, the impact of NLRP6 deficiency on intestinal damage following hematopoietic stem cell transplantation (HSCT) remains poorly understood. In this study, we established a syngeneic HSCT mouse model using C57BL/6 mice as donors and NLRP6-/- or C57BL/6 mice as recipients. Our findings revealed that NLRP6 deficiency had minimal influence on peripheral blood cell counts and splenic immune cell proportions in transplanted mice. However, it exacerbated pathological changes in the small intestine on day 14 post-transplantation, accompanied by increased proportions of macrophages, dendritic cells, and neutrophils. Furthermore, the NLRP6 deficiency resulted in elevated expression of MPO and CD11b, while reducing the levels mature caspase-1 and mature IL-1ß in the intestine. Moreover, the NLRP6 deficiency disturbed the expression of apoptosis-related molecules and decreased the tight junction protein occludin. Notably, recipient mice with NLRP6 deficiency exhibited lower mRNA expression levels of antimicrobial genes, such as Reg3γ and Pla2g2a. The short-term increase in inflammatory cell infiltration caused by NLRP6 deficiency was associated with intestinal damage, increased apoptosis, reduced expression of antimicrobial peptides, and impaired intestinal repair. Taken together, our findings demonstrate that the loss of NLRP6 exacerbates post-transplantation intestinal damage in recipient mice.
Subject(s)
Hematopoietic Stem Cell Transplantation , Intestinal Mucosa , Mice, Inbred C57BL , Mice, Knockout , Animals , Hematopoietic Stem Cell Transplantation/adverse effects , Mice , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Transplantation, Isogeneic , Apoptosis , Intestine, Small/pathology , Intestine, Small/metabolism , Caspase 1/genetics , Caspase 1/metabolism , Caspase 1/deficiency , Neutrophils/metabolism , Neutrophils/pathology , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , CD11b Antigen/metabolism , Pancreatitis-Associated Proteins/genetics , Macrophages/metabolism , Macrophages/pathology , Receptors, Cell SurfaceABSTRACT
A novel Gram-stain-negative, facultatively anaerobic and heterotrophic bacterium, designated strain ZH257T, was isolated from in situ enrichment samples incubated on the seamount floor of the Western Pacific Ocean. Cells were rod-shaped, oxidase- and catalase- positive, and motile by means of polar flagella. Strain ZH257T grew at 4-37â°C (optimum, 28-32â°C), pH 6.0-9.0 (optimum, pH 7.0) and with 2.0-9.0â% (w/v) NaCl (optimum, 3.0-4.0â%). Strain ZH257T was most closely related to members of the genus Pseudophaeobacter, sharing 99.13, 98.27 and 96.89â% 16S rRNA gene sequence identities with Pseudophaeobacter flagellatus GDMCC 1.2988T, Pseudophaeobacter arcticus DSM 23566T and Pseudophaeobacter leonis DSM 25627T, respectively. The DNA G+C content was 59.2 mol%. The estimated average nucleotide identity and digital DNA-DNA hybridization values between strain ZH257T and its closely related species were 79.61-93.04â% and 23.10-50.20â%, respectively. Strain ZH257T harboured complete denitrification and nitrate assimilation pathways. Strain ZH257T contained summed feature 8 (C18â:â1 ω7c and/or C18â:â1 ω6c) as major fatty acids (>5â%), and Q-10 as the major respiratory quinone. The polar lipid profile contained phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, an unidentified phospholipid, an unidentified aminolipid and four unidentified lipids. The combined phenotypic, genotypic and chemotaxonomic data showed that strain ZH257T represents a novel species of the genus Pseudophaeobacter, for which the name Pseudophaeobacter profundi sp. nov. is proposed, with the type strain ZH257T (=MCCC M29024T=KACC 23147T).
Subject(s)
Fatty Acids , Phospholipids , Fatty Acids/chemistry , Pacific Ocean , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Phylogeny , Phospholipids/chemistryABSTRACT
BACKGROUND: Hematopoietic stem cell transplantation involves irradiation preconditioning which causes bone marrow endothelial cell dysfunction. While much emphasis is on the reconstitution of hematopoietic stem cells in the bone marrow microenvironment, endothelial cell preservation is indispensable to overcome the preconditioning damages. This study aims to ascertain the role of Roundabout 4 (Robo4) in regulating irradiation-induced damage to the endothelium. METHODS: Microvascular endothelial cells were treated with γ-radiation to establish an endothelial cell injury model. Robo4 expression in the endothelial cells was manipulated employing lentiviral-mediated RNAi and gene overexpression technology before irradiation treatment. The permeability of endothelial cells was measured using qPCR, immunocytochemistry, and immunoblotting to analyze the effect on the expression and distribution of junctional molecules, adherens junctions, tight junctions, and gap junctions. Using Transwell endothelial monolayer staining, FITC-Dextran permeability, and gap junction-mediated intercellular communication (GJIC) assays, we determined the changes in endothelial functions after Robo4 gene manipulation and irradiation. Moreover, we measured the proportion of CD31 expression in endothelial cells by flow cytometry. We analyzed variations between two or multiple groups using Student's t-tests and ANOVA. RESULTS: Ionizing radiation upregulates Robo4 expression but disrupts endothelial junctional molecules. Robo4 deletion causes further degradation of endothelial junctions hence increasing the permeability of the endothelial cell monolayer. Robo4 knockdown in microvascular endothelial cells increases the degradation and delocalization of ZO-1, PECAM-1, occludin, and claudin-5 molecules after irradiation. Conversely, connexin 43 expression increases after silencing Robo4 in endothelial cells to induce permeability but are readily destroyed when exposed to 10 Gy of gamma radiation. Also, Robo4 knockdown enhances Y731-VE-cadherin phosphorylation leading to the depletion and destabilization of VE-cadherin at the endothelial junctions following irradiation. However, Robo4 overexpression mitigates irradiation-induced degradation of tight junctional proteins and stabilizes claudin-5 and ZO-1 distribution. Finally, the enhanced expression of Robo4 ameliorates the irradiation-induced depletion of VE-cadherin and connexin 43, improves the integrity of microvascular endothelial cell junctions, and decreases permeability. CONCLUSION: This study reveals that Robo4 maintains microvascular integrity after radiation preconditioning treatment by regulating endothelial permeability and protecting endothelial functions. Our results also provided a potential mechanism to repair the bone marrow vascular niche after irradiation by modulating Robo4 expression.
Subject(s)
Connexin 43 , Endothelial Cells , Receptors, Cell Surface , Animals , Mice , Cadherins/metabolism , Cells, Cultured , Claudin-5 , Connexin 43/genetics , Endothelial Cells/metabolism , Gamma Rays , Permeability/radiation effects , Receptors, Cell Surface/metabolismABSTRACT
Herein, we report the synthesis of bimetal-organic frameworks (BMOFs) with both Brønsted and Lewis acidities, in which phosphotungstic acid (PTA) was encapsulated in BMOFs. It is efficient in converting starch to 5-hydroxymethyl-furfural (HMF) in deep eutectic solvents (DESs) such as choline chloride and formic acid. The highest yield of HMF (37.94%) was obtained using P0.5/BMOFs1.0 to catalyze starch in a mixed solvent system comprising DESs and ethyl acetate (EAC) (v/v; 2:3) at 180 °C and a reaction time of 10 min. Employing a DES as a cocatalyst and solvent reduced the use of organic solvents. The catalyst showed adequate reusability, and the HMF yield only decreased by 2.88% after six cycles of reuse compared with that of the initial catalyst. This study demonstrates the application potential of BMOFs in the conversion of biomass to useful molecules with commercial and/or research value.
Subject(s)
Deep Eutectic Solvents , Furaldehyde , Phosphotungstic Acid , Carbohydrates , Solvents , Hexoses , Starch , CatalysisABSTRACT
Liposomes are the most widely used nanocarrier platform for the delivery of therapeutic and diagnostic agents, and a number of liposomes have been approved for use in clinical practice. After systemic administration, most liposomes are cleared by macrophages in the mononuclear phagocyte system, such as the liver and bone marrow (BM). However, the majority of studies have focused on investigating the therapeutic results of liposomal drugs, and too few studies have evaluated the potential side effects of empty nanocarriers on the functions of macrophages in the mononuclear phagocyte system. Here, we evaluate the potential effects of empty liposomes on the functions of BM niche macrophages. Following liposome administration, we observed lipid droplet (LD) accumulation in cultured primary macrophages and BM niche macrophages. We found that these LD-accumulating macrophages, similar to foam cells, exhibited increased expression of inflammatory cytokines, such as IL-1ß and IL-6. We further provided evidence that liposome deposition and degradation induced LD biogenesis on the endoplasmic reticulum membrane and subsequently disturbed endoplasmic reticulum homeostasis and activated the inositol-requiring transmembrane kinase/endoribonuclease 1α/NF-κB signaling pathway, which is responsible for the inflammatory activation of macrophages after liposome engulfment. Finally, we also showed the side effects of dysfunctional BM niche macrophages on hematopoiesis in mice, such as the promotion of myeloid-biased output and impairment of erythropoiesis. This study not only draws attention to the safety of liposomal drugs in clinical practice but also provides new directions for the design of lipid-based drug carriers in preclinical studies.
Subject(s)
Bone Marrow , Liposomes , Mice , Animals , Liposomes/metabolism , NF-kappa B/metabolism , Interleukin-6/metabolism , Macrophages/metabolism , Hematopoiesis , Drug Carriers/metabolism , Drug Carriers/pharmacology , Cytokines/metabolism , Endoribonucleases , Inositol/metabolism , LipidsABSTRACT
Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.
Subject(s)
Lipid Droplets , Macrophages , Adipose Tissue , Animals , Inflammation , Mice , Mice, Inbred C57BL , PhenotypeABSTRACT
Protein kinases are therapeutic targets for many human diseases, but the lack of user-friendly quantitative assays limits the ability to follow the activities of numerous kinases at once (multiplexing). To develop such an assay, we report an array of sulfonamido-oxine (SOX)-labeled peptides showing cross-reactivity to different mitogen-activated protein kinases (MAPKs) for use in a differential sensing scheme. We first verified using linear discriminant analysis that the array could differentiate MAPK isoforms. Then, using principal component analysis, the array was optimized based on the discrimination imparted by each SOX-peptide. Next, the activity of individual MAPK families in ternary mixtures was quantified by support vector machine regression. Finally, we multiplexed the quantification of three MAPK families using partial least squares regression in A549 cell lysates, which has possible interference from other kinase classes. Thus, our method simultaneously quantifies the activity of multiple kinases. The technique could be applied to other protein kinase families and the monitoring of diseases.
Subject(s)
MAP Kinase Signaling System , Mitogen-Activated Protein Kinases , Humans , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Peptides/metabolism , Phosphorylation , Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
CD19-targeted chimeric antigen receptor T (CAR-T) cells using murine single-chain variable fragment (scFv) has shown substantial clinical efficacy in treating relapsed/refractory acute lymphoblastic leukemia (R/R ALL). However, potential immunogenicity of the murine scFv domain may limit the persistence of CAR-T cells. In this study, we treated 52 consecutive subjects with R/R ALL with humanized CD19-specific CAR-T cells (hCART19s). Forty-six subjects achieved complete remission (CR) (N = 43) or CR with incomplete count recovery (CRi) (N = 3) within 1 month post infusion. During the follow-up with a median time of 20 months, the 1-year cumulative incidence of relapse was 25% (95% confidence interval [CI] 13-46), and 1-year event-free survival was 45% (95% CI 29-60). To the cutoff date, 20 patients presented CD19+ relapse and 2 had CD19- relapse. Among the 22 relapsed patients, 14 had treatment-mediated and treatment-boosted antidrug antibodies (ADA) as detected in a sensitive and specific cell-based assay. ADA positivity was correlated with the disease relapse risk. ADA-positive patients had a significantly lower CAR copy number than ADA-negative patients at the time of recurrence (p < .001). In conclusion, hCART19s therapy is safe and highly active in R/R ALL patients, and the hCART19s treatment could induce the emergence of ADA, which is related to the recurrence of the primary disease.
Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma , Receptors, Chimeric Antigen , Single-Chain Antibodies , Adaptor Proteins, Signal Transducing , Animals , Antigens, CD19 , Cell Count , Humans , Mice , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Single-Chain Antibodies/genetics , Single-Chain Antibodies/therapeutic useABSTRACT
Burkitt lymphoma (BL) is an aggressive Epstein-Barr virus (EBV)-driven B-cell lymphoma characterized by the translocation and rearrangement of the c-Myc proto-oncogene. High-intensity multidrug chemotherapy regimens have a limited effect on the survival of refractory or relapsed BL patients, mainly owing to the high EBV load and drug resistance. l-asparaginase ( l-Asp) and etoposide (VP-16) play a beneficial role in EBV-related lymphoproliferative diseases; however, their roles and mechanisms in BL remain unclear. In this study, we found that VP-16 inhibited BL cell proliferation and arrested the cell cycle at the G2 /M phase. It also induced autophagy and activated the extrinsic and intrinsic apoptotic signaling pathways in BL cells. Mechanistically, VP-16 inhibited c-Myc expression and regulated the PI3K/Akt/mTOR signaling pathway. Notably, VP-16 also showed a specific synergistic effect with l-Asp to induce apoptosis in EBV-positive BL cells but not in EBV-negative BL cells. VP-16 combined with l-Asp further inhibited c-Myc expression and downregulated the PI3K/Akt/mTOR signaling pathway. Additionally, we found that VP-16 inhibited the expression of latent membrane protein 1 (LMP1), and in combination with l-Asp further decreased LMP1 expression in Raji cells. Our in vivo data also showed that the dual-drug combination significantly inhibited the growth of BL tumors and prolonged the survival of mice compared to VP-16 alone. In conclusion, this study provides new evidence that l-Asp may enhance the antitumor effect of VP-16 by inhibiting the PI3K/Akt/mTOR signaling pathway in EBV-positive BL cells.
Subject(s)
Burkitt Lymphoma , Epstein-Barr Virus Infections , Animals , Apoptosis , Asparaginase/pharmacology , Asparaginase/therapeutic use , Burkitt Lymphoma/drug therapy , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/drug therapy , Etoposide/pharmacology , Etoposide/therapeutic use , Herpesvirus 4, Human/metabolism , Mice , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
Dermestid beetles (Coleoptera: Dermestidae) are important pests of various stored products, posing potential threats to international trade. Their detailed characterization on molecular basis is a pre-requisite for proper identification and for understanding of their phylogenetic relationships. In this work, the whole mitochondrial genomes (mitogenomes) of Trogoderma granarium, Dermestes lardarius, D. ater, Attagenus augustatus augustatus and Attagenus unicolor japonicus were firstly sequenced to update the database using the next-generation sequencing technique. Based on the selected model species, a comparative analysis of four Dermestidae genera was performed. The mitochondrial genomes of these five species above showed high similarity in nucleotide composition, base composition and gene order, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and a non-coding control region, which was similar to most of Coleoptera species. The phylogenetic analysis based on the PCGs and two rRNAs indicated that the relationships within Dermestidae were reconstructed as (((Trogoderma + Anthrenus) + Attagenus) + Dermestes) using both Maximum Likelihood (ML) and Bayesian Inference (BI) analysis. However, more mitogenomes should be sequenced to obtain a more holistic view of the whole family. This study not only showed the mitogenomes of five Dermestidae species and their high conservativeness, but also discussed its implications for reconstructing a more comprehensive phylogeny of dermestids.
Subject(s)
Coleoptera/genetics , Genome, Mitochondrial , Phylogeny , Animals , Coleoptera/classification , Genes, Insect , Molecular Sequence Annotation , Open Reading Frames , RNA, Ribosomal/genetics , RNA, Transfer/geneticsABSTRACT
CD19-target chimeric antigen receptor (CAR)-T cell therapy is highly effective for relapsed/refractory (R/R) acute lymphoblastic leukaemia (ALL), but is often complicated by cytokine release syndrome (CRS), which is potentially life-threatening. Endothelial cells are the core regulator of CRS in many infectious diseases and may also play a key role after CAR-T cell therapy. We provided a detailed clinical, laboratory description and endothelial cell activation biomarkers in patients with CRS. Endothelial cell activation was associated with occurrence, development and severity of CRS, manifested by decreased serum angiopoietin (Ang)-1 levels and increased levels of von Willebrand Factor (VWF), Ang-2, Ang-2:Ang-1, sE-selectin, soluble intercellular adhesion molecule (sICAM-1) and soluble vascular cell adhesion molecule (sVCAM)-1. Besides, the endothelial activation was correlated with the hepatic, kidney and hematopoietic dysfunction in CRS patients. After infusion of CAR-T cells, we detected changes of endothelial activation-related biomarkers within 36 hours in patients who subsequently developed CRS, especially severe CRS. Using top tree models, we could predict which patients would develop CRS, especially severe CRS, or identify the severity of CRS by certain biomarkers of endothelial activation. These data provide a new idea and will help identify new targets for early intervention and prevention of CRS.
Subject(s)
Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/metabolism , Endothelial Cells/metabolism , Immunotherapy, Adoptive/adverse effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/complications , Adolescent , Adult , Aged , Antigens, CD19/immunology , Biomarkers , Child , Cytokine Release Syndrome/diagnosis , Disease Susceptibility , Female , Hematopoiesis , Humans , Immunotherapy, Adoptive/methods , Male , Middle Aged , Organ Specificity , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Receptors, Chimeric Antigen/immunology , Severity of Illness Index , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Young AdultABSTRACT
An anaerobic, alkaliphilic, halotolerant, Gram-stain-positive and rod-shaped bacterium, designated Q10-2T, was isolated from mangrove sediment sampled at the Jiulong river estuary, PR China. The cells of strain Q10-2T were motile and 0.5×2-4 µm in size. Strain Q10-2T grew at 8-45 °C (optimum, 32 °C), at pH 7.0-10.5 (optimum, pH 8.5) and in the presence of 0-6â% (w/v) NaCl (optimum, 3â%). It could use complex organic compounds and carbohydrates including d-fructose, d-galactose, d-glucose, d-mannitol, d-xylose, trehalose, lactose, maltose, sucrose and starch as carbon sources and electron donors. It could reduce sulphate, thiosulphate and elemental sulphur to sulphide, but not sulphite. Fe (â ¢) citrate, ferrihydrite, haematite and goethite in the presence of glucose as the electron donor were also reduced. Acetate, butyrate, ethanol, CO2 and H2 were end products of glucose fermentation. The predominant cellular fatty acids were composed of C14â:â0, C16â:â0 and summed features containing C16â:â1 ω7c and/or iso-C15â:â0 2-OH and iso-C17â:â1 and/or anteiso-C17â:â1 B. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel strain was most closely related to Fusibacter paucivorans DSM 12116T (95.5â% sequence similarity). The genome size of strain Q10-2T was 5.0 Mb, with a G+C content of 37.4âmol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain Q10-2T and F. paucivorans DSM 12116T were 69.1 and 21.8â%, respectively. The combined genotypic and phenotypic data showed that strain Q10-2T represents a novel species of the genus Fusibacter, for which the name Fusibacter ferrireducens sp. nov. is proposed. The type strain is Q10-2T (=MCCC 1A16257T=KCTC 15906T).
Subject(s)
Clostridiales/classification , Geologic Sediments/microbiology , Phylogeny , Anaerobiosis , Bacterial Typing Techniques , Base Composition , China , Clostridiales/isolation & purification , DNA, Bacterial/genetics , Fatty Acids/chemistry , Ferric Compounds , Nucleic Acid Hybridization , Phospholipids/chemistry , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sulfur , Sulfur-Reducing Bacteria/classification , Sulfur-Reducing Bacteria/isolation & purification , WetlandsABSTRACT
BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune disease characterized as a low platelet count resulting from immune-mediated platelet destruction. Dimethyl fumarate (DMF) is widely applied for the treatment of several autoimmune diseases with immunosuppressive effect. However, whether it ameliorates ITP is unclear. This study aims to evaluate whether DMF has a preventive effect on ITP in mice. METHODS: DMF (30, 60 or 90 mg/kg body weight) was intraperitoneally injected into mice followed by injection of rat anti-mouse integrin GPIIb/CD41antibody to induce ITP. Peripheral blood was isolated to measure platelet count and spleen mononuclear cells were extracted to measure Th1 and Treg cells along with detecting the levels of IFN-γ, and TGFß-1 in plasma and CD68 expression in spleen by immuohistochemical staining. Additionally, macrophage cell line RAW264.7 was cultured and treated with DMF followed by analysis of cell apoptosis and cycle, and the expression of FcγRI, FcγRIIb and FcγRIV mRNA. RESULTS: DMF significantly inhibited antiplatelet antibody-induced platelet destruction, decreased Th1 cells and the expression of T-bet and IFN-γ, upregulated Treg cells and the expression of Foxp3 and TGF-ß1 as well as reduced CD68 expression in the spleen of ITP mouse. DMF-treated RAW264.7 cells showed S-phase arrest, increased apoptosis and downregulated expression of FcγRI and FcγRIV. Meanwhile, in vitro treatment of DMF also decreased the expression of cyclin D1 and E2, reduced Bcl-2 level and increased Bax expression and caspase-3 activation. CONCLUSIONS: In conclusion, DMF prevents antibody-mediated platelet destruction in ITP mice possibly through promoting apoptosis, indicating that it might be used as a new approach for the treatment of ITP.